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Reviews and Research Journal of the Brazilian Society for Virology

Volume 19, September/October 2014, Supplement 2 Annals of the XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto Convention Center, Ribeirão Preto, São Paulo,

Editors Edson Elias da Silva Fernando Rosado Spilki

BRAZILIAN SOCIETY FOR VIROLOGY BOARD OF DIRECTORS (2013-2014)

Officers Area Representatives

President: Dr Eurico de Arruda Neto Basic Virology (BV) Vice-President: Dr Bergmann Morais Ribeiro Dra Paula Rahal, UNESP (2013 – 2014) First Secretary: Dr Mauricio Lacerda Nogueira Dr Davis F. Ferreira, UFRJ (2013 – 2014) Second Secretary: Dra Luciana Jesus da Costa First Treasurer: Dr Luis Lamberti Pinto da Silva Environmental Virology (EV) Second Treasurer: Dra Clarice Weis Arns Dra Célia Regina Barardi, UFSC (2013 – 2014) Executive Secretary: Dr Fabrício Souza Campos Dr Fernando Rosado Spilki, Feevale (2013 – 2014)

Human Virology (HV) Fiscal Councilors Dr Luiz Tadeu Figueiredo, USP-RP (2013 – 2014) Dra Maria Luisa Barbosa Dra Nancy Bellei, UNIFESP (2013 – 2014) Dra Viviane Fongaro Botosso Dra Maria Angela Orsi Immunobiologicals in Virology (IV) Dra Sílvia Cavalcanti, UFF (2013 – 2013) Dr Edson Elias da Silva, Fiocruz (2013 – 2014)

Plant and Invertebrate Virology (PIV) Dr Paulo Brioso, UFRJ (2013 – 2014) Dr Tatsuya Nagata, UNB (2013 – 2014)

Veterinary Virology (VV) Dr Paulo Brandão, USP (2013 – 2014) Dra Rita Cubel, UFF (2013 – 2014)

Address Universidade Feevale, Instituto de Ciências da Saúde Estrada RS-239, 2755 - Prédio Vermelho, sala 205 - Laboratório de Microbiologia Molecular Bairro Vila Nova - 93352-000 - Novo Hamburgo, RS - Brasil Phone: (51) 3586-8800 E-mail: F.R.Spilki - [email protected] http://www.vrrjournal.org.br Organizing Committee Bergmann Morais Ribeiro, UNB Célia R. M. Barardi, UFSC Clarice Weis Arns, UNICAMP Davis Fernandes Ferreira, UFRJ Edson Elias da Silva, Fiocruz ‘Chairman of the Committee’ Eurico de Arruda Neto, USP Fabrício Souza Campos, UFRGS Fernando Rosado Spilki, Universidade FEEVALE Francisco Murilo Zerbini, UFV Luciana Jesus Costa, UFRJ Luis Lamberti Pinto da Silva, USP Luiz Tadeu Figueiredo, USP Maria Angela Orsi, LANAGRO Maria Luisa Barbosa, Instituto Adolfo Lutz Maurício Lacerda Nogueira, FAMERP Nancy Bellei, UNIFESP Paula Rahal, UNESP Paulo Sergio Torres Brioso, UFRRJ Paulo Eduardo Brandão, USP Rita de Cássia Nasser Cubel Garcia, UFF Silvia Maria Baeta Cavalcanti, UFF Tatsuya Nagata, UNB Viviane Fongaro Botosso, Instituto Butantan

Hélio Gelli Pereira Award Committee Fernando Rosado Spilki Luciana Barros de Arruda Luis Lamberti Pinto da Silva Mauricio Lacerda Nogueira

XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology, Ribeirão Preto, São Paulo, Brazil Virus Reviews and Research, Volume 19, Supplement 2, 2014 Financial Support General Information

CAPES Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Secretary Office Hours September, 28st - 8am - 8pm CNPQ September, 29nd - 7am - 7:30pm September, 30rd - 7am - 7:30pm Tecnológico October, 01th - 7am - 6pm FAPESPConselho Nacional de Desenvolvimento Cientifico e Fundação de Amparo à Pesquisa do Estado de São Paulo Name Badge Name badges will be required for access in all activities, Exhibitors including lunch. QIAGEN SARSTEDT Media Desk (for lecturers only) SÍNTESE The media desk will be open as scheduled for the secretary SOTELAB VECOFLOW mediaoffice. desk at least 2 hours before the scheduled time for the Sponsors presentation.Data - files with Please presentations note that the - use must of bepersonal delivered computers at the Silver Sponsorship - LOBOV by presenters will not be allowed. Bronze Sponsorship - ROCHE and SIGMA ALDRICH Lounge Area A lounge area will be available for lecturers, invited persons Organizers and SBV staff. Office Marketing Eventos Certificates

org.br 15 days after the end of the meeting. Certificates of attendance will be available on line at www.sbv. Travel Agency

Congress of Virology. FLYTOUR is the official tour operator of the XXV Brazilian (16) 3024.3900 Booking and information: [email protected] Poster Presentations The posters must be exposed from 1pm until the end of the session, and then removed.

Poster Session 1: September 29nd, 6:30-8:30 pm Human Virology Immunobiologicals in Virology • Plant and Invertebrate Virology • Poster• Session 2: September 30nd, 6:30-8:30 pm Basic Virology Environmental Virology • Veterinary Virology • •

XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology, Ribeirão Preto, São Paulo, Brazil Virus Reviews and Research, Volume 19, Supplement 2, 2014 XXV Brazilian Congress of Virology Scientific Program

Room Turquesa Workshop 1 - Enteric 1. Replication and virus-cell interaction of - Ian Goodfellow, University of Cambridge, UK 2. (Aichivirus B) infection in Brazilian cattle herds - Amauri Alcindo Alfieri, Londrina State University, Londrina, Paraná, PR, Brazil 3. Norovirus and in day-care children – Divina das Dores de Paula Cardoso, Federal University of Goiás, Goiânia, GO, Brazil 4. Enteric viruses in drinking water, Southern Brazil – Fernando Rosado Spilki, Feevale University, Novo Hamburgo, RS, Brazil (Chair) Room Ágata Satellite Workshop 2 - Next-generation sequencing of viral 1. Discovery of novel viruses from insects and plants - Fernando Lucas de Melo, University of Brasília, Brasília, DF, Brazil 2. Next generation sequencing as a tool for discovering and monitoring arboviruses – Renata Dezengrini Slhessarenko, Federal University of Mato Grosso, Cuiabá, MT, Brazil 3. Next generation sequencing technologies for and transcriptome studies of Porcine - João Pessoa Araújo Junior, São Paulo State University, Botucatu, SP, Brazil 4. Next generation sequencing and metagenomic virome analysis from environmental samples – Tatsuya Nagata, University of Brasília, Brasília, DF, Brazil (Chair) Room Ônix Satellite Workshop 3 - Respiratory viruses: from bench to trench 1. Update on newcastle disease virus – Muhammad Munir - The Pirbright Institute, UK 2. Coronaviruses in bats: what is new? - Edison Luiz Durigon, University of São Paulo, São Paulo, SP, Brazil (Chair) 2: 00 pm - 4:00 pm 3. Ana Claudia Trocoli Torrecilhas, Federal University of São Paulo, São Paulo, SP, Brazil 4. RespiratoryTargeting cell viral division infections cycle 25 in homolog immunosuppressed B to regulate influenzapatients -virus Meri replication Bordignon - Nogueira, Federal University of Paraná, Curitiba, PR, Brazil Room Topázio Satellite Workshop 4 - Virology nucleus AUGM (Asociación de Universidades Grupo Montevideo) 1. An overview of the mission and priorities of the Molecular Virology Nucleus (AUGM) - Juan Daniel Claus, Coordinator of AUGM, Universidad Nacional del Litoral, Santa Fé, Argentina 2. Molecular biology of baculovirus: basic and applied studies - Víctor Romanowski, Universidad Sunday, September 28 September Sunday, Nacional de La Plata, La Plata, Argentina 3. State-of-art of viral vector-borne zoonoses in Argentina - Marta Contigiani, Universidad Nacional de Córdoba, Córdoba, Argentina 4. Mechanism of carcinogenesis associated with human papillomavirus infection: role of the domains of protein PDZ interaction - Daniela Gardiol, Universidad Nacional de Rosario, Rosario, Argentina 5. Detection of human papillomavirus and co-risk factors for the development of cervical cancer in Paraguay - Laura Mendoza Torres, Universidad Nacional de Asunción, Asunción, Paraguay 6. Development of molecular tools for identifying viral - Adriana Giri, Universidad Nacional de Rosario, Rosario, Argentina 7. Emerging viruses in Uruguay: some yes and others no - Juan Arbiza, Universidad de la República, Montevideo, Uruguay 8. Distribution of vaccinia virus in the South America - Erna Geessien Kroon, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil 9. Recent developments in recombinant viral vaccines for veterinary use in Brazil - Eduardo Furtado Flores, Federal University of Santa Maria, Santa Maria, RS, Brazil 10. Coronavirus in wild birds in Brazil - Clarice Arns, Campinas State University, Campinas, SP, Brazil (Chair)

4:00 pm - 4:30 pm Coffee break

Room Esmeralda Opening Ceremony Keynote conference: 7:00 pm - 9:00 pm Infections of neurons by herpesviruses: How do they do that? Lynn Enquist, Princeton University, Princeton, NJ, USA 9:00 pm - 11:00 pm Cocktail• reception and Visit to Exhibits

XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology, Ribeirão Preto, São Paulo, Brazil Virus Reviews and Research, Volume 19, Supplement 2, 2014 Room Ágata Mini-course 1: Introduction to viral replication Luciana Jesus da Costa, Federal University of , Rio de Janeiro, RJ, Brazil Room Turquesa Mini-course• 2: Proteomic applications in virology Alessandra Vidotto, Medical School of São José do Rio Preto, São José do Rio Preto, SP, Brazil 8:00 am - 9:00 am Room Topázio Mini-course• 3: Molecular diagnosis in virology Roberta Bronzoni, Federal University of Mato Grosso, Sinop, MT, Brazil, and Luciano Kleber de Souza Luna, University of São Paulo, Ribeirão Preto, SP, Brazil Room• Ônix Mini-course 4: Viral phylogeny and sequence analysis Camila Romano, University of São Paulo, São Paulo, SP, Brazil Oral presentations: session 1 to 4 • SESSION 1 – Human Virology I - Room Topázio 9:00 am - 10:30 am SESSION 2 – Plant and Invertebrate I - Room Turquesa • SESSION 3 – Veterinary Virology I - Room Ônix • SESSION 4 – Environmental Virology - Room Ágata • 10:30 am - 11:00 am Coffee-break• and Visit to Exhibits

Room Esmeralda Conference 1: 11:00 am - 12:00 noon Host cell restriction factors for HIV-1 replication Jeremy Luban, University of Massachusetts Medical School, Worcester, USA; Luciana• Jesus da Costa, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil (Chair)

12:00 pm- 2:00 pm Lunch-break and Visit to Exhibits

Monday, September 29 September Monday, Room Esmeralda Conference 2: 2:00 pm - 3:00 pm Emerging and re-emerging virus infections in pigs Tanja Opriessnig, University of Edinburgh, Scotland, UK and Iowa State University, USA; Paulo• Eduardo Brandão, University of São Paulo, São Paulo, SP, Brazil (Chair) Oral presentations: session 5 to 8 SESSION 5 – Basic Virology I - Room Ágata 3:00 pm - 4:30 pm SESSION 6 – Human Virology II - Room Topázio • SESSION 7 – Plant and Invertebrate Virology II - Room Turquesa • SESSION 8 – Veterinary Virology II - Room Ônix • 4:30 pm - 5:00 pm Coffee-break• and Visit to Exhibits

Oral presentations: session 9 to 12 SESSION 9 – Human Virology III - Room Topázio 5:00 pm - 6:30 pm SESSION 10 – Basic Virology II - Room Ágata • SESSION 11 – Veterinary Virology III - Room Ônix • SESSION 12 – Immunobiologicals in Virology - Room Turquesa • 6:30 pm - 8:30 pm Poster• Session 1 and Visit to Exhibits / Social Mixer Room Ágata Mini-course 1: Introduction to viral replication Luciana Jesus da Costa, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Room Turquesa Mini-course• 2: Proteomic applications in virology Alessandra Vidotto, Medical School of São José do Rio Preto, São José do Rio Preto, SP, Brazil 8:00 am - 9:00 am Room Topázio Mini-course• 3: Molecular diagnosis in virology Roberta Bronzoni, Federal University of Mato Grosso, Sinop, MT, Brazil, and Luciano Kleber de Souza Luna, University of São Paulo, Ribeirão Preto, SP, Brazil Room• Ônix Tuesday, September 30 September Tuesday, Mini-course 4: Viral phylogeny and sequence analysis Camila Romano, University of São Paulo, São Paulo, SP, Brazil

• XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology, Ribeirão Preto, São Paulo, Brazil Virus Reviews and Research, Volume 19, Supplement 2, 2014 Room Topázio Round Table 1 - Veterinary Virology - Challenges and progress in disease control 1. Helena Lage Ferreira, University of São Paulo, Pirassununga, SP, Brazil (Chair) 2. VirusesChallenges in wild in monitoring birds with avianspecial influenza reference virus to avian - paramyxoviruses – Muhammad Munir, The Pirbright Institute, UK 3. Porcine epidemic virus - Tanja Opriessnig, University of Edinburgh, Scotland, UK 4. Update on FMDV - Rossana Allende, Pan American Foot-and-Mouth Disease Center (Panaftosa), RJ, Brazil Room Ônix Round Table 2 - Plant and Invertebrate Virology - Viral metagenomics 1. : diversity, genome organization, ecological implications and biotechnological potential: CfPDV as a case study - Fernando Luis Cônsoli, University of São Paulo, São Paulo, SP, Brazil 2. Genomic analysis of new baculoviruses isolated in Brazil - Daniel Mendes Pereira Ardisson Araujo, University of Brasília, Brasília, DF, Brazil 3. and associated weeds ssDNA virome – Simone Ribeiro, EMBRAPA CENARGEN, Brasília, DF, Brazil 4. Cell modulation factors are carried along structural proteins in the AgMNPV-2D budded and occluded Round Tables 1 to 4 virions - Carla Torres Braconi, University of São Paulo, São Paulo, SP, Brazil 9:00 am - 10:30 am Chair: Bergmann Morais Ribeiro, University of Brasília, Brasília, DF, Brazil Room Ágata Round Table 3 - Environmental Virology - Advances in environmental virology 1. Standard Methods to concentrate viruses from freshwater samples - Garland Shay Fout, U. S. Environmental Protection Agency, Cincinnati, OH, USA 2. Avian parvoviruses as markers for environmental contamination – Silvia Bofill-Mass, University of Barcelona, Barcelona, Spain 3. Virus inactivation in sludge – Maria Elisa Magri, Federal University of Lavras, Lavras, MG, Brazil 4. Virus disinfection in drinking, reuse and sea waters - Célia Regina Monte Barardi, Federal University of Santa Catarina, Florianópolis, SC, Brazil Chair: Fernando Rosado Spilki, University Feevale, Novo Hamburgo, RS, Brazil Room Turquesa Round Table 4 - Basic Virology - HIV-host cell interaction 1. HIV replication mechanisms - Jeremy Luban, University of Massachusetts Medical School, USA 2. Diverse mechanisms involved in translation initiation of HIV-1 transcripts - Luciana Jesus da Costa, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Tuesday, September 30 September Tuesday, 3. Luis Lambert Pinto da Silva, University of São Paulo, Ribeirão Preto, SP, Brazil (Chair) Mechanisms by which the HIV-1 accessory protein Nef modifies protein trafficking in host cells – 10:30 am - 11:00 am Coffee break and Visit to Exhibits Room Esmeralda Conference 3: 11:00 am - 12:00 noon as symbionts and immunomodulatory gene delivery vehicles Michael R. Strand, University of Georgia, Athens, GA, USA Room• Ônix Evolution of canine parvovirus - a need for new vaccines? 12:30 pm- 13:30 pm Dr. Uwe Truyen, Editor-in-chief of Veterinary Microbiology, Institute for Animal Hygiene, University of Leipzig, Leipzig,• Germany 12:00 am - 2:00 pm Lunch break and Visit to Exhibits Room Esmeralda Conference 4: 2:00 pm - 3:00 pm Jolanda Smit, The University Medical center Groningen, Groningen, The Netherlands Room• TurquesaEarly events in infection Round Table 5 - Basic Virology - Interactions of with hosts 1. Effects of yellow fever virus infection on splicing – Mauricio Lacerda Nogueira, São José do Rio Preto Round Tables 5 to 8 Medical School, São José do Rio Preto, SP, Brazil (Chair) 3:00 pm - 4:30 pm 2. Structure of dengue virus proteins – Ronaldo Mohana Borges, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil 3. Rafael Maciel de Freitas, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil The influence of dengue vírus infection on Aedes aegypti feeding behaviour -

XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology, Ribeirão Preto, São Paulo, Brazil Virus Reviews and Research, Volume 19, Supplement 2, 2014 Room Topázio Round Table 6 - Human Virology - Human infections by emerging viruses 1. Central nervous system infections by arboviruses - Maria Paula Mourão, Heitor Vieira Dourado Foundation for Tropical Medicine, Manaus, AM, Brazil 2. Clinical pathogenesis of hantavírus pulmonar syndrome - Luiz Tadeu Moraes Figueiredo, University of São Paulo School of Medicine, Ribeirão Preto, SP, Brazil (Chair) 3. Chloroquine for dengue virus infections – Benedito Antônio Lopes da Fonseca, University of São Paulo School of Medicine, Ribeirão Preto, SP, Brazil Room Ônix Round Table 7 - Veterinary Virology - Molecular diversity of animal viruses 1. Reservoirs of vaccinia virus in Brazil – Erna Geessien Kroon, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil 2. Molecular epidemiology and evolution of porcine parvoviruses – André Felipe Streck, Federal Round Tables 5 to 8 University of Rio Grande do Sul, Porto Alegre, RS, Brazil 3:00 pm - 4:30 pm 3. Emergent viruses associated with in dogs and cats - Tatiana Xavier de Castro, Fluminense Federal University, Niterói, RJ, Brazil 4. Molecular diversity of canine distemper virus – Alice Fernandes Alfieri, Londrina State University, Londrina, PR, Brazil Chair: Rita de Cássia Nasser Cubel Garcia, Fluminense Federal University, Niterói, RJ, Brazil Room Ágata Round Table 8 - Human virology/Immunobiologic products - Computational virology applied to dengue virus studies 1. Genetic mosaicism in two strains of dengue type 3 - Paolo Marinho de Andrade Zanotto, University of São Paulo, São Paulo, SP, Brazil (Chair) 2. Molecular Dynamics of dengue NS3 protease conformations – Jaime Martins de Santana, University of Brasília, Brasília, DF, Brazil 3. Early budding stages of a Dengue Virus particle unveiled by Molecular Dynamics - Ricardo Oliveira dos Santos Soares, University of São Paulo, Ribeirão Preto, SP, Brazil 4:30 pm - 5:00 pm Coffee-break and Visit to the Exhibits Room Turquesa Round Table 9 – Canceled Tuesday, September 30 September Tuesday, Room Ônix Round Table 10 - Basic Virology - Virus-host cell interactions and virus assembly 1. Assembly of icosahedral virus capsids - Juliana Cortines, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil 2. Innate immunity against poxviruses – Daniel Mansur, Federal University of Santa Catarina, Florianópolis, SC, Brazil (Chair) 3. Molecular mechanisms of human assembly – Alberto Fraile Ramos, INMETRO, Rio de Janeiro, RJ, Brazil Round Tables 9 to 12 4. Cell interactions of human respiratory syncytial virus (HRSV) N, P and M proteins – Armando Morais 5:00 pm - 6:30 pm Ventura, University of São Paulo, São Paulo, SP, Brazil Room Ágata Round Table 11 - Immunobiologicals - Experimental animal models in virology 1. Experimental infection of Callithrix penicillata with dengue virus – Pedro Fernando Vasconcelos, Evandro Chagas Institute, Belém, PA, Brazil (Chair) 2. Experimental infection of Mus musculus with Rocio vírus – Juliana Helena Chávez, Federal University of Mato Grosso, Cuiabá, MT, Brazil 3. Experimental infection of Macaca fascicularis with human – Marcelo Alves Pinto, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil Room Topázio Round Table 12 - Helio Gelli Pereira Award Oral Presentations 6:30 pm - 8:30 pm Poster Session 2 and Visit to Exhibits 10:00 pm Social mixer and dance

XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology, Ribeirão Preto, São Paulo, Brazil Virus Reviews and Research, Volume 19, Supplement 2, 2014 Mini-course 1: Introduction to viral replication / Room Ágata Luciana Jesus da Costa, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Mini-course 2: Proteomic applications in virology / Room Turquesa • Alessandra Vidotto, Medical School of São José do Rio Preto, São José do Rio Preto, SP, Brazil 8:00 am – 9:00 am Mini-course 3: Molecular diagnosis in virology / Room Topázio • Roberta Bronzoni, Federal University of Mato Grosso, Sinop, MT, Brazil, and Luciano Kleber de Souza Luna, University of São Paulo, Ribeirão Preto, SP, Brazil Mini-course• 4: Viral phylogeny and sequence analysis / Room Ônix Camila Romano, University of São Paulo, São Paulo, SP, Brazil Room Esmeralda • Conference 5: 9:00 am - 10:00 am The cardiac innate response to viral infections Barbara Sherry, North Carolina State University, Raleigh, NC, USA • 10:00 am - 10:30 am Coffee-break and Visit to Exhibits Room Esmeralda 10:30 am - 12:30 pm SBV Business meeting and election of new board of directors 12:30 pm - 2:00 pm Lunch break and Visit to Exhibits Room Ônix 12:30 pm - 2:00 pm This week in virology Vincent Racaniello, Columbia University, New York, NY, USA Room Esmeralda Conference 6: 2:00 pm -3:00 pm The emergence of chikungunya virus in South America Xavier Nicolas de Lamballerie, Emerging Viruses Unit, University of Marseille, • Room Ônix Round Table 13 - Veterinary Virology - Antivirals for animal viruses 1. Viral activity of natural compounds and derivates against pet’s virus and animals herpesvirus – Márcia Rogéria Lamêgo Almeida, Federal University of Viçosa, Viçosa, MG, Brazil 2. In vitro and in vivo inhibition of rabies virus by RNAi – Paulo Eduardo Brandão, University of São Paulo, São Paulo, SP, Brazil (Chair) 3. Control of ruminant replication by small interfering RNA – Renata Servan de Almeida, Agricultural Research for Development (CIRAD), Montpellier, France 4. Antiherpes activity of Brazilian marine organisms - Claudia Maria Oliveira Simões, Federal

Wednesday, October 1 October Wednesday, University of Santa Catarina, Florianópolis, SC, Brazil Room Ágata Round Table 14 - Plant and Invertebrate Virology - Control of emerging plant viruses 1. Recent advances in the study of papaya sticky disease – Patricia Fernandes, Federal University of Espírito Santo, Vitória, ES, Brazil 2. : Biology and management in tropical crops – Andrew Geering, University of Queensland, Brisbane, Australia 3. Epidemiology and management of viruses transmitted by Bemisia tabaci in solanaceous crops – Round Tables 13 to 16 Jorge Alberto Marques Rezende, University of São Paulo, Piracicaba, SP, Brazil (Chair) 3:00 pm - 5:00 pm Room Turquesa Round Table 15 - Immunobiologicals - Advances in biotechnology products with applications in virology 1. as vectors – Diego Diel, University of Illinois, Urbana-Champaign, USA 2. Expression of viral vaccines in plants – Tatsuya Nagata, National University of Brasília, Brasília, DF, Brazil (Chair) 3. Advances in biotechnology products with applications in virology - Marcos Freire, Bio-Manguinhos/ Fiocruz, Rio de Janeiro, RJ, Brazi 4. Carbon nanotubes applied to point-of-care tesing for immunoassay – Rosa Amalia Fireman Dutra, Federal University of Pernambuco, Recife, PE, Brazil Room Topázio Round Table 16 - Human Virology - Advances in HTLV research 1. Effects of HTLV-1 infection on human mesenchymal stromal cells - Simone Kashima Haddad, University of São Paulo, Ribeirão Preto, SP, Brazil (Chair) 2. Lúcia Helena Faccioli, University of São Paulo, Ribeirão Preto, SP, Brazil 3. HTLV-1Leukotrienes reverse in geneticsHTLV-1-related and search neuroinflammatory for an intracellular diseases carrier - of ORF-1 gene product – Luiz Carlos Alcântara, Gonçalo Moniz Research Center, Fiocruz, Salvador, BA, Brazil

XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology, Ribeirão Preto, São Paulo, Brazil Virus Reviews and Research, Volume 19, Supplement 2, 2014 Hélio Gelli Pereira Award The evaluation of papers submitted for the Helio Gelli Pereira Award will take place on Tuesday, September 30, from 5:30 to 7:00 pm. Presenters will have 10 minutes for their presentation, followed by 5 minutes of questions from the examiners and, if time permits, from the audience.

Hélio Gelli Pereira Award A RESOURCEFUL GIANT: APMV IS ABLE TO INTERFERE WITH THE HUMAN TYPE I INTERFERON SYSTEM Silva, L.C.F.; de Almeida, G.M.; Oliveira, D.B.; Dornas, F.P.; Campos, R.K.; La Scola, B.; Kroon, E.G.; Ferreira, P.C.P.; Abrahão, J.S. THE MOVEMENT PROTEINS (NSM) OF DISTINCT TOSPOVIRUSES SELF-INTERACT, ASSOCIATE WITH THE COGNATE NUCLEOCAPSID (NP) AND WITH HETEROLOGOUS NSM AND NP PROTEINS AND WITH THE CELLULAR MEMBRANE IN A NON- TRANSMEMBRANE FASHION Leastro, M.O.; Pallás, V.; Resende, R.O.; Navarro, J.A.S. HIV-1 TRANSCRIPTS USE IRES-INITIATION UNDER CONDITIONS WHERE CAP-DEPENDENT TRANSLATION IS RESTRICTED 1 BY POLIOVIRUS 2A. PROTEAS - Amorim, R.; da Costa, S.M.; Cavaleiro, N.; da Silva, E.E.; da Costa, L.J. Room Topázio Room ACANTHAMOEBA POLYPHAGA INHIBITS TRANSCRIPTION OF AMOEBAL 2 SUBTILISIN-LIKE SERINE PROTEINASE MRNA AND CIRCUNVENTS CELL ENCYSTMENT Boratto, P.V.M.; Almeida, G.M.F.; Botelho, L.; Lima, A.; Costa, A.O.; Santos, D.A.; La Scola, B.; Kroon, E.G.; Ferreira, P.C.P.; Abrahão, J.S. HTLV-1 INFECTS HUMAN MESENCHYMAL STROMAL CELL IN VITRO AND MODIFIES THEIR PHENOTYPIC CHARACTERISTICS Rodrigues, E.S.; de Macedo, M.D.; Pinto, M.T.; Orellana, M.D.; Rocha Junior, M.C.; de Magalhães, D.A.R.; Tanaka, Y.; Takayanagui, O.M.; Tuesday, September 30 / 5:00 pm - 6:30 September Tuesday, Covas, D.T.; Kashima, S.

XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology, Ribeirão Preto, São Paulo, Brazil Virus Reviews and Research, Volume 19, Supplement 2, 2014 Oral Presentation

SESSION 1 – Human Virology I HV33 - DETECTION OF IMPORTED CASES OF CHIKUNGUNYA VIRUS IN BRAZIL: VIRUS ISOLATION, MOLECULAR AND SEROLOGICAL ASSAYS Cunha, M.S.; Maeda, A.Y.; Bisordi, I.; Rocco, I.M.; da Silva, F.G.; de Souza, R.P.; Coimbra, T.L.M.; Nogueira, J.S.; Kisielius, J.J.; Silveira, V.R.; Oliveira, A.L.R.; Suzuki, A. HV125 - ORAL HUMAN PAPILLOMAVIRUS INFECTION IN HIV-POSITIVE PEOPLE Silva, C.O.; Santos, L.S.; de Azevedo, K.M.L.; Pereira, O.M.D.; Oliveira, L. do H. dos S. HV147 - NEW REAL TIME PCR ASSAY FOR RAPID DETECTION OF TRICHODYSPLASIA SPINULOSA-ASSOCIATED POLYOMAVIRUS IN BIOLOGICAL SAMPLES Urbano, P.R.; Nali, L.H. da S.; Pannuti, C.S.; Pierrotti, L.C.; Neto, E.D.; Romano, C.M. HV213 - EXPRESSION PROFILE OF HUMAN ENDOGENOUS W (HERV-W) LOCI FROM MULTIPLE SCLEROSIS PATIENTS UNDER DISTINCT THERAPIES BY HIGH THROUGHPUT SEQUENCING Room Topázio Room Nali, L.H. da S.; Urbano, P.R.P.; Silva, D.F.; Olival, G.S. do; Penalva de Oliveira, A.C.; Romano, C.M. 9:00 am - 10:30 HV240 - HIGH FREQUENCE OF SAFFOLD VIRUS IN TONSIL TISSUES FROM PATIENTS WITH CHRONIC TONSILLAR HYPERTROPHY Luna, L.K. de S.; Valera, F.C.P.; Tamashiro, E.; Lima, W.T.A.; Arruda, E. HV247 - FIRST REPORT OF OCCURRENCE AMONG ALLOGENEIC STEM CELL TRANSPLANT RECIPIENTS IN BRAZIL: EVIDENCE FOR PROLONGED VIRAL EXCRETION AND LONG-TERM VIRAL RNA PRESENCE IN THE BLOOD Souza, M.; Lemes, L.N.; Correa, T. dos S.; Fiaccadori, F.S.; Souza, K.M.C. da Silva, L.P.; Arantes, A. de M.; Cardoso, D. das D. de P.; Souza, M. SESSION 2 – Plant and Invertebrate I PIV150 - TWO NOVEL SPECIES FROM THE NEW WORLD WITH FEATURES RECALLING OLD WORLD Godinho, M.T.; Lima, A.T.M.; Zerbini, F.M.

PIV160 - COMPLETE GENOMEXavier, SEQUENCE C.A.D.; OF TWO NEW VIRUSES ISOLATES ASSOCIATED TO COTTON BLUE DISEASE BREAKING RESISTANCE IN BRAZIL Vaslin, M.F.S.; Fausto, A.K. da S.; Romanel, E.; Silva, T. da F.; Schrago, C.G.; Galbieri, R.; Bélot, J.L.; Vasli, M.F.S. PIV210 - SYSTEMIC ACQUIRE RESISTANCE INDUCED BY CELL WALL PEPTIDOGALACTOMANNAN OF THE FUNGUS CLADOSPORIUM HERBARUM MEDIATES VIRUS PROTECTION IN TOBACCO PLANTS

Monday, September 29 September Monday, Vaslin, M.F.S.; Montebianco, C.B.; Mattos, B.B.B.; Silva, T. da F.; Romanel, E.; Bergter, E.B.; Vaslin, M.F.S. PIV221 - PROTEIN SYNTHESIS ANALYSIS OF INSECT CELL LINES INFECTED WITH SPODOPTERA FRUGIPERDA MULTIPLE

Room Turquesa Room NUCLEOPOLYHEDROVIRUS (SFMNPV)

9:00 am - 10:30 Marcio, M.S.; Sihler, W.; Souza, M.L. PIV295 - CHARACTERIZATION OF A BACULOVIRUS HOST-ACQUIRED PROTEASE INHIBITOR AND ITS ABILITY TO AUGMENT VIRAL VIRULENCE Ardisson, A.D.M.P.; Rohrmann, G.A.; Bergmann, M.R.; Rollie, J.C. PIV357 - IDENTIFICATION OF A NEW DCL3 GENE IN COTTON GOSSYPIUM RAIMONDII (ULBRICH) D-GENOME AND STUDY OF THE ROLE OF NEW ISOFORMS OF GENE SILENCING RELATED GENES IN COTTON VIRUS INFECTED PLANTS Vaslin, M.F.S.; Romanel, E.; Moura, M.; Lei, G.; Paterson, A. SESSION 3 – Veterinary Virology I VV27 - ANALYSIS OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE APOBEC3H GENE OF DOMESTIC CATS (FELIS CATUS) AND THEIR ASSOCIATION WITH THE SUSCEPTIBILITY TO FELINE IMMUNODEFICIENCY VIURUS AND FELINE LEUKEMIA VIRUS INFECTIONS Costenaro, J.G.; de Castro, F.L.; Junqueira, D.M.; de Medeiros, R.M.; da Silva, T.R.; Knak, M.B.; Almeida, S.E. de M.; Campos, F.S.; Roehe, P.M.; Franco, A.C. VV37 - PARTIAL CHARACTERIZATION OF AVIAN INFLUENZA VIRUS (H11N9) IN MIGRATORY BIRDS (ARENARIA INTERPRES) CAPTURED IN AMAZON REGION, BRAZIL Room Ônix Room de Araujo, J.; Júnior, S.M. de A.; Gaidet, N.; Hurtado, R.F.; Walker, D.; Thomazelli, L.M.; Ometto, T.; Seixas, M.M.M.; Galindo, D.B.;

9:00 am - 10:30 Webby, R.J.; Webster, R.G.; Durigon, E.L. VV96 - AVIAN CORONAVIRUS AND AVIAN BORNAVIRUS DETECTION IN FREE-RANGING PSITTACINES Lima Neto, D.F.; Barnabé, A.C.S.; Caserta, L.; Martini, M.C.; Simas, P.V.M.; Nagel, N.E.; Lierz, M.; Hafez, H.M.; Felippe, P.A.N.; Arns, C.W.

XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology, Ribeirão Preto, São Paulo, Brazil Virus Reviews and Research, Volume 19, Supplement 2, 2014 SESSION 3 – Veterinary Virology I VV113 - PRIMARY AND SECONDARY MUCOSAL IMMUNITY AFTER CHALLENGE WITH BRAZILIAN AVIAN INFECTIOUS BRONCHITIS VARIANT Okino, C.H.; Mores, M.A.Z.; Montassier, H.J.; Mattos, G.L.M.; Brentano, L.; Coldebella, A.; Ritterbusch, G.A.; Esteves, P.A.; Trevisol, I.M. VV172 - DETECTION IN THE BAT TADARIDA BRASILIENSIS (GEOFFROY, 1824: CHIROPTERA, MOLOSSIDAE) FROM RIO GRANDE DO SUL STATE, BRASIL Room Ônix Room Duppont, Priscilla Medeiros - Pacheco, Susi M. - Rosa, Júlio Cesar A. - Streck, André Felipe-Alves, Christian D. B. T. - Da Silva,

9:00 am - 10:30 Mariana S. - Budaszewski, Renata F. - Weber, Matheus N.-Canal, Cláudio W. VV207 - DETECTION OF AVIAN GROUP F IN FECAL SAMPLES OF BROILER IN PARA STATE, BRAZIL Bezerra, D.A.M.; Silva, M.J.M.; Bezerra, D.A.M.; Silva, R.R.; Soares, L.S.; Mascarenhas, J.D.P. SESSION 4 – Environmental Virology EV90 - PATHOGENS INACTIVATION BY UNIONIZED AMMONIA FOR THE PRODUCTION OF SAFE BIOFERTILIZER FROM SWINE EFFLUENT AND SLUDGE Fongaro, G.; Kunz, A.; Magri, M.E.; Zeredo, A.C.B.; Schissi, C.D.; Zaguini, J.; Barardi, C.R.M. EV140 - ANALYSIS OF MARINE VIRAL FAMILIES ASSOCIATED WITH SIDERASTREA STELLATA FESTIVAL IN ARRAIAL DO CABO AND BÚZIOS, RJ, BRAZIL Santana-Pereira, P.; Giongo, V.; Pedrosa, L.G.; Paula, J.E.F.B.; Amorim, L.; Paixão, I.C.N.P. EV255 - DETECTION OF HUMAN ADENOVIRUSES IN SEDIMENTS FROM STREAMS IN URBAN AREAS FROM RIO DOS SINOS WATERSHED Staggemeier, R.; Heck, T.M. da S.; Andriguetti, N.B.; Soliman, M.C.; Souza, F.G. de; Fabres, R.B.; Luz, R.B.; Zirbes, G.; Henzel, A.; Rigotto, C.; Spilki, F.R.; Almeida, S.E. de M. EV306 - ENVIRONMENTAL POLIOVIRUS SURVEILLANCE: DETECTION OF WILD AND VACCINE - DERIVED POLIOVIRUSES

Room Ágata Room IN SÃO PAULO SEWAGE Hachich, E.M.; Barbosa, M.RF.; Garcia, S.C.; Bonanno, V.M.S.; Eduardo, M.B.P.; Burlandy, F.M.; Costa, E.V.; da Silva, E.E.; Sato, M.I.Z. 9:00 am - 10:30 EV430 - DIFFERENT PHAGES ACTION PATHWAYS IN REDUCING BIOFILMS FORMED BY OIL REFINERIES ASSOCIATED BACTERIA Dias, R.S.; Fonseca, L.A.B.V.; Albanese, J.M.; Silva, L.C.F.; Suhette, R.; Torres, A.P.R.; Sousa, M.P.; Silva, C.C.; Oliveira, V.M.; de Paula, S.O. Monday, September 29 September Monday, EV457 - FIRST IDENTIFICATION OF ACANTHAMOEBA POLYPHAGA MIMIVIRUS IN LIMNOPERNA FORTUNEI FROM GUAIBA LAKE, RIO GRANDE DO SUL, BRAZIL dos Santos, R.N.; Arantes, T.; Correa, R.A.; de Albuquerque, N.R.M.; Kluge, M.; Santos, C.; dos Santos, C.P.; Campos, F.C.; Roehe, P.M.; Franco, A.C. SESSION 5 – Basic Virology I BV12 - OROPOUCHE ORTHOBUNYAVIRUS MINIGENOME SYSTEM REVEALS SIGNIFICANT ERRORS IN THE PUBLISHED GENOME SEQUENCES Acrani, G.O.; Lunel, N.L.T.; Spiegel, M.; Weidmann, D.M.D.S.; Nunes, M.R.T.; Ellott, R.M. BV86 - KROON VIRUS: A NEW AND HIGHLY RESISTANT WITH EXTRA SHELL LAYER Dornas, F.P.; Silva, L.C.F.; Boratto, P.V.M.; Rodrigues, R.A.; Freitas, G.M.; Assis, F.L.; Trindade, G.S.; Kroon, E.G.; Cortines, J.; La Scola, B.; Abrahão, J.S. BV95 - DYNAMIC COVALENT BONDS IN VIRAL CAPSIDS: AUTO PROTEOLYSIS INVOLVED IN CAPSID MATURATION CAN BE REVERSED UNDER PHYSIOLOGICAL CONDITIONS Domitrovic, T.; Matsui, T.; Johnson, J.E.

Room Ágata Room BV105 - ANALYSIS OF DIFFERENTIAL METHYLATION PROFILE IN PATIENTS WITH DENGUE USING METHYLATION-

3:00 pm - 4:30 SENSITIVE ARBITRARILY PRIMED POLYMERASE CHAIN REACTION Alessandra, V.B.T.G.; Morais, S.M. de S.; Ferreira, J.M.S.; Malaquias, L.C.C.; Coelho, L.F.L. BV171 - STUDY OF STOP CODONS RECURRENCE IN C VIRUS NS5A PROTEIN Campos, G.R.F.; Bittar, C.; Rahal, P. BV175 - CHARACTERIZATION OF THE INTERACTION BETWEEN THE METHYLOSSOME AND THE HUMAN RESPIRATORY SYNCYTIAL VIRUS NUCLEOCAPSID Ogawa, J.; Oliveira, A.; Simabuco, F.; Éleouët, J.F.; Ventura, A.

XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology, Ribeirão Preto, São Paulo, Brazil Virus Reviews and Research, Volume 19, Supplement 2, 2014 SESSION 6 – Human Virology II HV258 - DETECTION AND TYPING OF HUMAN PAPILLOMAVIRUS BY NESTED MULTIPLEX PCR (NMPCR) REVEALS NEW EPIDEMIOLOGICAL PROFILE Faria, M.G.; Matias, B.F.; Costa, E.P.; Julião, J.A.S.; Junior, P.C.F.; Goulart, L.R. HV262 - USE OF CHIMERIC PROTEINS EXPRESSED IN PROKARYOTIC SYSTEM IN THE DEVELOPMENT OF A SCREENING METHOD FOR THE EVALUATION OF ANTI-HTLV-1 ANTIBODIES Santos, D.M. da S.; do Carmo, A.P.; Martins, M.L.; da Fonseca, F.G.; Stancioli, E.F.B. HV311 - HEPATITIS C VIRUS QUASISPECIES ANALYSIS USING ULTRA-DEEP PYROSEQUENCING Yamasaki, L.H.T.; Khudyakov, Y.; Vaughan, G.; Raeva, L.M.G.; Diminitrova, Z.E.; Skums, P.; Rendon, D.S.C.; Jardim, A.C.G.; Bittar, C.;

Room Topázio Room Mello, I.M.V.G.C.; Rahal, P. 3:00 pm - 4:30 HV340 - EFFECTS OF HTLV-1 INFECTION ON BONE MARROW CELLS FROM HTLV-1 INFECTED INDIVIDUALS Rodrigues, E.S.; Favarin, M. do C.; Macedo, M.D. de; Otaguiri, K.K.; Orellana, M.D.; Takayanagui, O.M.; Slavov, S.N.; Covas, D.T.; Kashima, S. HV346 - EVIDENCE OF HEPATITIS E VIRUS CIRCULATION IN RURAL AMAZONIA Merlone, M.P.; Vitral, C.L.; Oliveira, J.M.; Silva, J.P.; Nunes, M.S.; Ferreira, M.U.; Pinto, M.A. SESSION 7 – Plant and Invertebrate Virology II PIV378 - THE FIRST COMPLETE GENOME SEQUENCE OF COFFEE RINGSPOT VIRUS; AN EMERGING THREAT TO COFFEE PRODUCTION AND QUALITY Figueira, A. dos R.; Ramalho, T.O.; Sotero, A. de J.; Duarte, P. de S.G.; Wang, R.; Farman, M.; Goodin, M.M. PIV383 - SUBCELLULAR LOCALIZATION OF COFFEE RINGSPOT VIRUS PROTEINS Ramalho, T.O.; Figueira, A.R.; Sotero, A.J.; Duarte, P.S.G.; Wang, R.; Farman, M.; Goodin, M.M. PIV423 - COMPLETE GENOME SEQUENCE OF A TOBACCO-INFECTING BLISTERING Fernandes, J.E.F.; Melo, F.L.; Ribeiro, B.M.; Ribeiro, G.S. PIV452 - COMPLETE GENOME SEQUENCE OF A NOVEL IFLAVIRUS ISOLATED FROM OPSIPHANES INVIRAE Silva, L.A.; Melo, F.L.; Tinôco, R.S.; Fernandes, O.A. Room Turquesa Room

3:00 pm - 4:30 PIV509 - THE FUNCTIONAL ANALYSIS OF DISTINCT TOSPOVIRUS MOVEMENT PROTEINS (NSM) REVEALS DIFFERENT BEHAVIOR FROM THE ALFALFA MOSAIC VIRUS (AMV) MODEL SYSTEM Leastro, M.O.; Peiró, A.; Pallás, V.; Navarro, J.A.S.; Resende, R.O. Monday, September 29 September Monday, PIV510 - THE MOVEMENT PROTEINS (NSM) OF DISTINCT TOSPOVIRUSES SELF-INTERACT, ASSOCIATE WITH THE COGNATE NUCLEOCAPSID (NP) AND WITH HETEROLOGOUS NSM AND NP PROTEINS AND WITH THE CELLULAR MEMBRANE IN A NON-TRANSMEMBRANE FASHION Leastro, M.O.; Pallás, V.; Resende, R. de O.; Navarro, J.S. SESSION 8 – Veterinary Virology II VV229 - LONG-TERM CIRCULATION OF INFLUENZA A VIRUSES IN SWINE IN BRAZIL Schaefer, R.; Nelson, M.; Gava, D.; Cantão, M.E.; Zanella, J.R.C. VV260 - EVIDENCE OF CIRCULATION AMONG URBAN DOMESTIC CATS, MINAS GERAIS, BRAZIL Miranda, J.B.; Costa, G.B.; Almeida, G.G.; Figueiredo, P. de O.; Bonjardim, C.A.; Ferreira, P.C.P.; Abrahão, J.S.; Kroon, E.G.; Trindade, G. de S. VV303 - DEVELOPMENT AND STANDARDIZATION OF A MULTIPLEX RT-PCR FOR THE DETECTION THE PRINCIPAL RESPIRATORY VIRUS IN BIRDS Sakata, S.T.; Rosales, C.A.R.; Reischak, D.; Orsi, M.A. VV316 - SEQUENCING AND PHYLOGENETIC ANALYSIS OF NUCLEOPROTEIN OF RABIES VIRUS ISOLATED FROM CATTLE Room Ônix Room IN THE STATE OF RIO GRANDE DO SUL 3:00 pm - 4:30 Fernandes, M.E.S.; Pereira, P.M.C.; Achkar, S.M.; Oliveira, R.N.; Ferreira, J.C.; Rosa, J.C.A.; Castilho, J.G.; Almeida, L.L.; Carnieli, Jr. P - Roehe PM - Batista HBCR VV321 - HERPESVIRUS SIMPLEX TYPE-1 OUTBREAK IN MARMOSETS (CALLITHRIX) Ullmann, L.S.; Kurissio, J.K.; Linhares, A.G.; Linhares, L.S.A.; Milanelo, L.; Araujo Jr, J.P. VV333 - ABC OF CANINE PARVOVIRUSES GENOGROUPS AND ANTIGENIC TYPES Streck, A.F.; Borchardt, A.; Daudt, C.; Canal, C.W.

XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology, Ribeirão Preto, São Paulo, Brazil Virus Reviews and Research, Volume 19, Supplement 2, 2014 SESSION 9 – Human Virology III HV353 - IMPLEMENTATION OF NEXT GENERATION SEQUENCING TECHNIQUES FOR PATHOGEN DISCOVERY IN CEREBROSPINAL FLUID OF PATIENTS WITH ENCEPHALITIS AND MENINGITIS Nunes, C.F.; Soares, A.C.; Urbano, P.R.; Gerhardt, D.; Romano, C.M. HV417 - CO-DETECTIONS OF RESPIRATORY VIRUSES BY QPCR IN THE ABSENCE OF SYMPTOMS OF ACUTE RESPIRATORY INFECTION (ARI): NEW INSIGHTS ON SOURCES OF VIRUS SHEDDING Criado, M.F.; Modena, J.L.P.; de Paula, F.E.; de Jesus, B.L.S.; Pestana, N.F.; Prates, M.C.; Silva, M.L.; Saturno, T.; Tamashiro, E.; Valera, F.C.P.; Lima, W.T.A.; Arruda, E. HV428 - ANALYSIS OF INFLUENZA A(H1N1)PDM09 VIRAL LOAD IN PATIENTS WITH DIFFERENT CLINICAL PRESENTATION

Room Topázio Room Perosa, A.H.; Bellei, N. 5:00 pm - 6:30 HV436 - HIV-1 ENV SUBTYPES AND DISEASE PROGRESSION IN A COHORT OF HIV-1 POSITIVE INDIVIDUALS FROM RIO DE JANEIRO, BRAZIL Leite, T.; Campos, D.; Coelho, A.; Teixeira, S.; Veloso, V.; Guimarães, M.; Morgado, M.G. HV491 - OPTIMIZATION OF A LOOP-MEDIATED ISOTHERMAL AMPLIFICATION ASSAY FOR NAKED-EYE DETECTION OF DENGUE VIRUS INFECTION Monteiro, D.C.S.; Carvalho, B.K.S.; Nascimento, V.A.; Souza, V.C.; Naveca, F.G. SESSION 10 – Basic Virology II BV225 - HIV-1 TRANSLATION INITIATION IS INHIBITED BY POLIOVIRUS 2A PROTEASE Amorim, R.; Costa, L.J. da BV280 - 2A PROTEASE FROM HUMAN C15 DOES NOT PROVIDE INTERFERON RESISTANCE AND TRAFD1 HAS AN ANTIVIRAL ACTIVITY AGAINST HUMAN AND ENCEPHALOMYOCARDITIS VIRUS Gagliardi, T.B.; Dabelic, R.; Racaniello, V.R. BV345 - IDENTIFICATION OF THE CELLULAR AND MOLECULAR DETERMINANTS OF FLAVIVIRUS FUSION Esposito, D.L.A.; Nguyen, J.B.; Modis, Y. BV379 - DEVELOPMENT OF TWO POTENTIAL TOOLS AGAINST THE REPLICATION OF THE DENGUE VIRUS Room Ágata Room Fujimura, P.T.; Vaz, E.R.; Cardoso Jr, C.A.M.; Carvalho, W.J.; Reis, C.F.; Santos Jr, C.D.; Freitas, G.R.O.; Yokosawa, J.; Carneiro, A.P.; 5:00 pm - 6:30 Goulart, L.R.; Vecchi, L.; Ueira,C.U.V. BV490 - IN VITRO MICROEVOLUTION OF DENGUE VIRUS 4 IN AEDES ALBOPICTUS CELLS

Monday, September 29 September Monday, Nascimento, V.A.; Souza, V.C.; Naveca, F.G. BV501 - ALIX IS A HOST CELL FACTOR REQUIRED FOR LYSOSOMAL TARGETING OF CD4 BY HIV-1 NEF Januário, M.E. da S.; da Silva, E.M.L.; de Castro, R.O.; Amorim, N.A.; da Costa, L.J.; da Silva, L.L.P. SESSION 11 – Veterinary Virology III VV351 - GENETIC CHARACTERIZATION OF CANINE CIRCOVIRUS DETECTED IN STOOL SAMPLES FROM DOGS IN THE SOUTH REGION OF BRAZIL Cruz, T.F.; Batista, T.N.; Baccarin, A.M.; Gradiz, J.J.; Vieira, E.M.; Tozato, C.C.; Kurissio, J.K.; Araujo Jr, J.P. VV365 - PHYLOGENETIC ANALYSIS OF AICHIVIRUS B IN FECAL SAMPLES FROM CALVES IN BRAZIL Rribeiro, J.; Lorenzetti, E.; Medeiros, T.N. da S.; Junior, J.C.R.; Bronkhorst, D.E.; Crespo, S.E.I.; Favero, L.M.;

VV393 - VIABILITY OF VACCINIA VIRUS IN EXPERIMENTALLY CONTAMINATED CHEESES AT DIFFERENTAlfieri, A.F.;TIMES Alfieri, OF THE A.A. MATURATION PROCESS Rehfeld, I.S.; Fraiha, A.L.S.; Matos, A.C.D.; Guedes, M.I.M.C.; Costa, A.G.; de Souza, M.R.; Lobato, Z.I.P. VV396 - MOLECULAR DETECTION AND PHYLOGENETIC RELATIONSHIPS OF AN AMPHIBIAN ASSOCIATED TO OUTBREAKS OF MORTALITY IN BRAZILIAN FISH FARM

Room Ônix Room Queiroz, S.R. de A.; de Pádua, S.B.; Filho, R.N. de M.; Araújo, A.P. de; Maganha, S.R. de L.; Navarro, J. de O.; Lima, M.P.; de Alencar,

5:00 pm - 6:30 A.L.F.; Arruda, E. de P.; Munin, F.S.; Fernandes, A.M.; de Sousa, R.L.M. VV470 - VIRUCIDAL ACTIVITY OF A PURIFIED COMPOUND ISOLATED FROM PRASIOLA CRISPA AGAINST EQUINE HERPESVIRUS TYPE 1 Marinho, R.S.S.; Ramos, C.J.B.; Leite, J.P.G.; Belo, C.A.D.; Pereira, A.B.; Teixeira, V.L.; Paixão, IC.N.P.; Pinto, A.M.V. VV473 - EMERGING VIRUSES MONITORING IN BATS, RODENTS AND MARSUPIALS FROM BRAZILIAN RAIN FOREST REGION AND AMAZON REGION COLLECTED BETWEEN 2008 AND 2013 Campos, A.C. de A.; Anthony, S.J.; de Araújo, J.; Ometto, T.; Hurtado, R.; Nardi, M.S.; Nava, A.; Solorio, M.R.; Souza, M.C.P.; Rostal, M.; Loh, E.; Torrelio, C.M.Z.; Aguirre, A.A.; Daszak, P.; Durigon, E.L.

XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology, Ribeirão Preto, São Paulo, Brazil Virus Reviews and Research, Volume 19, Supplement 2, 2014 SESSION 12 – Immunobiologicals in Virology IV100 - EVALUATION OF TETRAVALENT AND CONSERVED SYNTHETIC PEPTIDES VACCINES DERIVED FROM DENGUE VIRUS ENVELOPE DOMAIN I AND II Rocha, R.P.; Franco, I.R.; Livonesi, M.C.; Fumagalli, M.J.; Rodrigues, N.F.; da Costa, L.C.F.; dos Santos, M.C. da S.G.; Rocha, E.S. de O.; Kroon, E.G.; Malaquias, L.C.C.; Coelho, L.F.L. IV183 - THE IMPACT OF ADJUVANT IN WHOLE INACTIVATED VIRUS (WIV) VACCINES FOR INFLUENZA A INFECTION IN PIGS WITH VACCINE-ASSOCIATED ENHANCED DISEASE Souza, K.C.; Rajao, D.; Loving, L.C.; Gauger, C.P.; Vincent, L.A. IV218 - SELECTION AND CHARACTERIZATION OF MIMOTOPES THROUGH PHAGE DISPLAY FOR IMMUNOGLOBULIN A DETECTION IN HPV DIAGNOSIS Matias, B.F.; Lima, M.I.S.; Faria, M.G.; Costa, E.P.; Alves, P.T. Pereira, U.P.; Fernandes Jr, P.C.; Goulart, L.R. Room Turquesa Room 5:00 pm - 6:30 IV477 - INFLUENZA: DEVELOPMENT OF A REVERSE GENETICS SYSTEM BY YEAST-BASED HOMOLOGOUS Monday, September 29 September Monday, RECOMBINATION AND ITS APPLICATION IN THE IMPROVED VACCINE PRODUCTION IN CULTURE CELL Silva Jr, J.V.J.; Cruz, F. da S.P.; Bertani, G.R.; Machado, A. de M.V.; Gil, L.H.V.G. IV482 - RECONSTRUCTION OF LATENT AND REACTIVATED QUASISPECIES OF SIVMM251 INFECTING RHESUS MONKEYS AFTER TREATMENT WITH THE LATENCY ANTAGONIST INGENOL-B Gonçalves, G. dos S.; Abreu, C.M.; Price, S.L.; Gama, L.; Lewis, M.; Pianowski, L.F.; Tanuri, A.

XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology, Ribeirão Preto, São Paulo, Brazil Virus Reviews and Research, Volume 19, Supplement 2, 2014 HELIO GELLI PEREIRA AWARD XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

17 Helio Gelli Pereira Award

A RESOURCEFUL GIANT: APMV IS ABLE TO INTERFERE THE MOVEMENT PROTEINS (NSM) OF DISTINCT WITH THE HUMAN TYPE I INTERFERON SYSTEM TOSPOVIRUSES SELF-INTERACT, ASSOCIATE Silva, L.C.F.; de Almeida, G.M.; Oliveira, D.B.; Dornas, WITH THE COGNATE NUCLEOCAPSID (NP) AND F.P.; Campos, R.K.; La Scola, B.; Kroon, E.G.; Ferreira, WITH HETEROLOGOUS NSM AND NP PROTEINS P.C.P.; Abrahão, J.S. AND WITH THE CELLULAR MEMBRANE IN A NON- TRANSMEMBRANE FASHION Laboratório de Vírus, Departamento de Microbiologia, 1 2 1 Instituto de Ciências Biológicas, UFMG, Belo Horizonte, MG. Leastro, M.O. ; Pallás, V. ; Resende, R.O. ; Navarro, J.A.S.2 Acanthamoeba polyphaga mimivirus (APMV) is a giant, 1. UnB - Universidade de Brasília, Campus double-stranded virus of the family that was Universitário Darcy Ribeiro, Brasília - DF, 70910-900 discovered in 2003. Recent studies have shown that this 2. IBMCP/CISIC-UPV - Instituto de Biología virus is able to replicate in murine and human phagocytes Molecular y Celular de Plantas do Consejo Superior de and might be considered a putative human pathogen Investigaciones Científicas de la Universidad Politécnica de that causes pneumonia. However, there is little data , Ingeniero Fausto Elio, s/n 46022 Valencia - Espana. regarding APMV and its host defense relationship. In the present study, we investigated how some components Tospovirus most economically important plant viruses of the interferon (IFN) system are stimulated by APMV in human peripheral blood mononuclear cells (PBMC) production. The M RNA genomic segment that encodes worldwide with significant losses in agronomic and how APMV replication is affected by IFN treatment. a non-structural protein (NSm) involved in cell-to- Our results demonstrated that APMV is able to replicate cell and systemic movement commonly associates in human PBMC, inducing type I Interferons (IFN) but with ribonucleoprotein complexes (RNP) located in inhibiting interferon stimulated genes (ISG) induction the tubular structure of plasmodesmata and seems by viroceptor and STAT-1 and STAT-2 dephosphorylation to interact with cell periphery forming aggregates on independent mechanisms. We also showed that APMV the cell surface. Membrane-proteins topology can be is resistant to the antiviral action of interferon-alpha2 predicted based on amino acid sequence analysis or (IFNA2) but is sensitive to the antiviral action of experimentally determined by biochemical techniques, interferon-beta (IFNB1). Our results demonstrated the productive infection of professional phagocytes with such as alkaline enzymatic modifications, Tag APMVand showed that this virus is recognized by the One of the aims of this study was to understand the in modification by glycosylation and chemical modification. immune system of vertebrates and inhibits it. It provides vivo membrane association of the movement proteins of the tospoviruses species BeNMV, CSNV, TCSV and TSWV. interaction and raise new and relevant evolutional For this purpose, we used two different approaches in questionsthe first dataabout regardingthe relationship APMVand between the IFNAPMV system and vertebrate hosts. (BiFC) assay and the chemical treatments addressed toplanta: identify the the bimolecular type of MP-membrane fluorescence complementationassociation. Was proposed that the four MPs analyzed are peripherally associated to the cytosolic face of the ER membranes in living plant cells. Finally, dimerization analysis of homologous and heterologous tospoviurus proteins (NSm and N) generated information that can facilitate a better interpretation of viral proteins interactions, strengthening the understanding of mixed infection issues.

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Helio Gelli Pereira Award XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

18 Helio Gelli Pereira Award

HIV-1 TRANSCRIPTS USE IRES-INITIATION ACANTHAMOEBA POLYPHAGA MIMIVIRUS INHIBITS UNDER CONDITIONS WHERE CAP-DEPENDENT TRANSCRIPTION OF AMOEBAL 2 SUBTILISIN-LIKE TRANSLATION IS RESTRICTED 1 BY POLIOVIRUS 2A. SERINE PROTEINASE MRNA AND CIRCUNVENTS CELL PROTEAS - ENCYSTMENT Amorim, R.1; da Costa, S.M.1; Cavaleiro, N.1; da Silva, Boratto, P.V.M.1; Almeida, G.M.F.1; Botelho, L.1; Lima, E.E.2; da Costa, L.J.1 A.2; Costa, A.O.1; Santos, D.A.2; La Scola, B.3; Kroon, E.G.1; Ferreira, P.C.P.1; Abrahão, J.S.1 1. Instituto de Microbiologia, Departamento de Virologia. Universidade Federal do Rio de Janeiro. 5 Rio de 1. Universidade Federal de Minas Gerais, Janeiro. RJ – Brazil. Instituto de Ciências Biológicas, Laboratório de Vírus, Av. 2. Laboratório de Enterovírus. Instituto Antonio Carlos, 6627, Pampulha, Belo 10 Horizonte, MG, Oswaldo Cruz. Fundação Oswaldo Cruz. Rio de Janeiro. RJ – Brazil. Brazil. 2. Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Laboratório de Micologia, Av. The 30 different species of mRNAs synthesized Antonio Carlos, 6627, Pampulha, Belo Horizonte, MG, Brazil. during the HIV-1 replication cycle are all capped and 3. URMITE CNRS UMR 6236 – IRD 3R198, polyadenilated. Internal ribosome entry sites have Aix Marseille Université, Marseille, France been recognized in the 5’ untranslated region of some mRNA species of HIV-1, which would contribute to an Acanthamoeba is a genus of free-living amoebas alternative mechanism of initiation of mRNA translation. distributed worldwide known as agents of public However, the Cap-dependent translation is assumed to health concern for causing disease in humans. On be the main mechanism driving the initiation of HIV-1 natural environments, they can act as hosts for many protein synthesis. In this work, we describe a cell system microorganisms, including members of the family in which lower to higher levels of transient expression Mimiviridae, one of the largest and most complex group of the poliovirus 2A protease strongly inhibited cellular of viruses ever described. Many studies have investigated Cap-dependent translation with no toxic effect to the the interactions between these protozoa and some of its cells during a 72-hour time frame. In this system, the harbored microorganisms (mostly pathogenic bacteria) synthesis of HIV-1 proteins was inhibited in a temporal but few have explored the mechanisms involving their dose-dependent way. Higher levels of 2A protease interaction with giant viruses. In the present study, we expression severely inhibited HIV-1 protein synthesis explored the behavior presented by Acanthamoeba spp. and Mimivirus (APMV) in response to different inhibiting viral production and infectivity. Intermediate conditions of incubation and interaction. The results toduring lower the levels first of 24 2A hours Protease of infectionexpression consequently caused the obtained suggest that amoeba encystment is a crucial inhibition of viral protein synthesis only during the phenomenon for avoiding viral infection success, and therefore it is the target of an arm wrestling between protein synthesis and viral release were recovered to the the virus and amoeba. We demonstrate that once controlfirst 48 hourslevels. ofHowever, viral replication. the infectivity After ofthis viral period progeny both amoeba encystment is triggered, trophozoites become was still partially inhibited. These results indicate that two mechanisms of mRNA translation initiation APMV is able to interfere with the expression of the significantly less permissive to APMV and, remarkably, contribute to the synthesis of HIV-1 proteins; during serine proteinase mRNA related to amoebal encystment. These results may represent one of the most ancient synthesis is strongly dependent on Cap-initiation, while atthe later first time 24-48 points hours IRES-driven of viral replication translation HIV-1 initiation protein is eukaryotic cell. examples of fight for supremacy involving a virus and a sufficient to produce high amounts of viral particles.

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Helio Gelli Pereira Award XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

19 Helio Gelli Pereira Award

HTLV-1 INFECTS HUMAN MESENCHYMAL STROMAL CELL IN VITRO AND MODIFIES THEIR PHENOTYPIC CHARACTERISTICS RODRIGUES, E.S.1,2; DE MACEDO, M.D.1,2; PINTO, M.T.1,2; ORELLANA, M.D.1,2; ROCHA JUNIOR, M.C.1,2; DE MAGALHÃES, D.A.R.1; TANAKA, Y.4; TAKAYANAGUI, O.M.3; COVAS, D.T.1,3; KASHIMA, S.1,2 1. Regional Blood Center of Ribeirão Preto, University of São Paulo, Brazil 2. School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil 3. School of Medicine of Ribeirão Preto, University of São Paulo, Brazil 4. University of the Ryukyus, Okinawa, Japan The typical characteristics of mesenchymal stem microenvironment; however, the exact contribution of HTLV-1cells (MSCs) to MSC candysfunction be affected remains byto be inflammatory elucidated. In this study, we demonstrated that MSC cell surface molecules VCAM-1 and ICAM-1 are upregulated by contact with HTLV-1, and HLA-DR was most highly expressed in MSCs co-cultured with MT2 cells. The expression levels of VCAM-1 and HLA-DR were increased in MSCs cultured in the presence of PBMCs isolated from HTLV-1-infected symptomatic individuals compared with those cultured with cells from asymptomatic infected individuals or healthy subjects. HTLV-1 does not impair the MSC differentiation process into osteocytes and adipocytes.

1 in vitro through direct contact with HTLV-1-infected cells;In addition, however, MSCs cell-free were virus efficiently particles infected were not with capable HTLV- of causing infection. In summary, HTLV-1 can alter MSC function, and this mechanism may contribute to the pathogenesis of this viral infection

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Helio Gelli Pereira Award ORAL PRESENTATION XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

21 Oral Presentation

BV12 - OROPOUCHE ORTHOBUNYAVIRUS 958bp, with a longer 5’ UTR. The published UTR was MINIGENOME SYSTEM REVEALS SIGNIFICANT not functional in the minigenome system, whereas a ERRORS IN THE PUBLISHED GENOME SEQUENCES minigenome containing the recently discovered longer Acrani, G.O.1; Lunel, N.L.T.2; Spiegel, M.3; Weidmann, version was strongly active. Our results pave the way D.M.D.S.3; da Silva, D.4; Nunes, M.R.T.4; Ellott, R.M.2 for the establishment of a reverse genetics system to generate infectious virus from cloned cDNA. Financial 1. USP - Universidade de São Paulo, Av. Prof. support: Wellcome Trust and Medical Research Council Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 / FAPESP – Sao Paulo Research Foundation, grant 2. FACULTY OF VETERINARY, UNIVERSITY 2013/02798-0. OF GLASGOW, 464 Bearsden Road, Glasgow, G61 1QH, Scotland BV86 - KROON VIRUS: A NEW AND HIGHLY RESISTANT 3. UNIVERSITY MEDICAL CENTER GIANT VIRUS WITH EXTRA SHELL LAYER GOTTINGEN, Georg-August-Universität Robert-Koch- 1 1 1 Straße 40, 37075 Göttingen Briefpostadresse 37099 Göttingen Dornas, F.P. ; Silva, L.C.F. ; Boratto, P.V.M. ; Rodrigues, 1 1 1 1 4. IEC - Instituto Evandro Chagas, Rodovia R.A. ; Freitas, G.M. ; Assis, F.L. ; Trindade, G.S. ; 1 2 3 1 BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 Kroon, E.G. ; Cortines, J. ; La Scola, B. ; Abrahão, J.S. Oropouche virus (OROV, Bunyaviridae) is the second 1. UFMG - Universidade Federal de Minas most frequent arboviral febrile illness in Brazil. OROV Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, occurs predominantly in the North Amazon region, but Belo Horizonte - MG, 31270-901 has been isolated in the Southeast region of the country 2. UFRJ - Universidade Federal do Rio de Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de and also in Peru, Venezuela and Panama. OROV is a Janeiro - RJ, 21941-901 negative sense RNA virus with three genome segments 3. UNIVERSITÉ DE LA MÉDITERRANÉE, named S, M, and L. Complete sequences for the three Jardin du Pharo - 58, bd Charles Livon -13284 Marseille genomic segments are available at the NCBI Database. Cedex 07 The 3’ and 5’ untranslated regions (UTRs) of each viral segment function as important regulatory sequences for The members of the Mimiviridae have been isolated from various environments due mainly the host ubiquity, and are highly conserved among them. We report here although few studies have demonstrated biological and 5’replication and 3’ RACE and transcription,results and a minigenomeare specific for system each genususing structural assays of them. Therefore, this work aimed to the UTR of each segment controlling a Renilla luciferase characterize a new virus of the Mimiviridae, named Kroon reporter gene. The analysis of the function of the UTRs virus, isolated from a lake in Lagoa Santa city, MG, Brazil. of OROV strain BeAn19991 showed that the sequences For this, water samples were collected, were submitted published in the NCBI Database contain numerous to an enrichment process in rice medium and then errors. Our results show that the published UTRs are not functional, while changing the residue number 9 of viruses. Samples were eluted in PBS and then inoculated were filtered afterwards (in 200nm filters) to retain the the 3’ genomic UTR was necessary to regain its function. in amoebae (Acanthamoeba castellanii) cultures where The viral 3’ and 5’ UTR form a “panhandle” structure that were isolated. In parallel, the samples were submitted is necessary for proper replication and transcription of to a Real-Time PCR assay, targeting the helicase viral the viral genome, and the mismatch we generated at gene to be phylogenetics analyzed. The virus was also residue number 9 has been shown to be important in other bunyaviruses. We also show that the published L structural analyses, including cytopathic effect assays, purified for further characterization. Biological and ORF contains some deletions, insertions and mutations resistances tests, optical microscopy, SDS-PAGE and that alter the translated protein and should be revised. mass spectrometry analyses reveal unique features of Finally, we prove that the published sequences for the Kroon virus. Electron microscopy imaging revealed S segment are shorter than the real viral genome. The particles with dimensions similar to that described published sequences indicate that the viral S segment for other and some multiplication phases is 754bp long, but we prove that it has a genome of similar to giant viruses. The helicase gene sequencing

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

22 Oral Presentation and phylogenetic analyses indicated that Kroon virus reaction may be involved in structural transitions belongs to group A mimivirus, clustering together with important for infectivity. Nudaurelia capensis omega APMV and others. At the heat resistance test, the Kroon virus show 2 logs more resistant than APMV at the 75oC family that infects insects. This virus while at the resistance UV light assay the Kroon virus isvirus an accessible(NωV) is a system non-enveloped for the studies (+)ssRNA of non-enveloped virus from the show approximately 1.5 logs more than Kroon virus when virus maturation and entry in animal cells. In neutral submitted at 10 minutes. Details of the transmission electron microscopy images revealed a remarkable thepH, 240procapsid copies ofcollapses NωV capsid into proteina compact assemble capsid around with high resistance of this virus. To evidence this propose, distinctiveRNA, forming quasi-equivalent a round procapsid. contacts After (T=4 acidification, capsid). aextra SDS-PAGE shell layer,were performed suggesting after the the first virus evidence protein of heat the This structural transition activates auto-proteolysis of denaturation. The gel analyses show different proteins the capsid protein generating lytic peptides that stay approximately the same weight of the capsid APMV non-covalently associated to the capsid. The mature protein. Mass spectrometry shows the homolog proteins capsid presents lytic activity against membranes when of the APMV capsid and an extra putative capsid protein. exposed to alkaline pH, what correlates with the in- One of this proteins show a deleted region indicating vivo environment of infection inside caterpillars mid- the possibility to answer the reason of the extra shell gut. We found that the structural transition of mature layer. The number of the shell layers virus might be capsids on high pH not only induces the exposure of the involved as an environmental evolution resistance, as lytic peptides to the environment, but also, induces an UV incidence of a tropical area and the adaptation with unexpected reconstitution of the covalent bond between the high organic matters due the increase of the host the capsid protein and the peptides that were cleaved multiplication. So, the Kroon virus structural discovery during maturation. This reverse-reaction is restricted can show how complex giant viruses may be, opening to a subset of the peptides present in the capsid and does not affect the lytic activity against membranes. Possible functional implications of this reaction for waysBV95 to -new DYNAMIC research in COVALENT this field. BONDS IN VIRAL capsid disassembly and RNA exposure will be discussed. CAPSIDS: AUTO PROTEOLYSIS INVOLVED IN FINANCIAL SUPPORT: CNPQ, PEW CHARITABLE TRUST CAPSID MATURATION CAN BE REVERSED UNDER AND NATIONAL INSTITUTE OF HEALTH (NIH) PHYSIOLOGICAL CONDITIONS Domitrovic, T.1; Matsui, T.2; Johnson, J.E.3 BV105 - ANALYSIS OF DIFFERENTIAL METHYLATION PROFILE IN PATIENTS WITH DENGUE USING 1. INSTITUTO DE MICROBIOLOGIA METHYLATION-SENSITIVE ARBITRARILY PRIMED PAULO DE GÓES/UFRJ - Centro de Ciências da Saúde - POLYMERASE CHAIN REACTION Bloco I, Cidade Universitária - Ilha do Fundão, Rio de Janeiro 1 1 - RJ, 21941-590 Alessandra, V.B.T.G. ; Morais, S.M. de S. ; Ferreira, 2. STANFORD UNIVERSITY - 450 Serra J.M.S.2; Malaquias, L.C.C.1; Coelho, L.F.L.1 Mall, Stanford, CA 94305, Estados Unidos 1. UNIFAL - Universidade Federal de Alfenas, 3. THE SCRIPPS RESEARCH INSTITUTE, R. Gabriel Monteiro da Silva, 714 - Centro, Alfenas - MG, 10550 N Torrey Pines Rd, La Jolla, CA 92037, Estados Unidos 37130-000 Virtually all animal viruses transition from a procapsid 2. UFSJ - Universidade Federal de São João del- noninfectious state to a mature infectious state. Rei, Praça Frei Orlando, 170, Centro, São João del-Rei, Minas Auto proteolysis of the capsid protein is a common Gerais, 36307-352 event during maturation of Picorna, Birna, Noda and Dengue virus (DV) is an enveloped virus, positive single- Reoviruses and generally results in the generation of a stranded RNA, transmitted to humans through the bite membrane-active peptide that remains associated to of infected female Aedes aegypti mosquito. There are the capsid by non-covalent interactions. In this work, two main clinical manifestations caused by DV infection we show that self-cleavage is reversible and that this named Dengue Fever (DF) and Dengue Hemorrhagic

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

23 Oral Presentation

Fever (DHF). Several studies indicate that one of the BV171 - STUDY OF STOP CODONS RECURRENCE IN most important factor involved in the DHF development HEPATITIS C VIRUS NS5A PROTEIN is the presence of non-neutralizing antibodies. These Campos, G.R.F.; Bittar, C.; Rahal, P. antibodies can facilitate DV infection in susceptible cells in a secondary infection caused by a different UNESP/IBILCE - Universidade Estadual Paulista - Instituto de Biociências, Letras e Ciências Exatas, Rua serotype of DV. However, this hypothesis can not explain Cristóvão Colombo, 2265 Bairro Jardim Nazareth, São José the cases of primary infection with DHF outcome, do Rio Preto - SP, 15054-000 indicating that genetic or epigenetic host factors may be The occurrence of mutations that introduce TGA DNA methylation is the best characterized epigenetic stop codon in the genomic region of Hepatitis C Virus influence the predisposition and development of DHF. (HCV), recurrent in different patients, led to the search gene regulation. In eukaryotes, it is found predominantly of alternative explanations for these mutations. In a inmodification the 5 position exerting carbon great importance of cytosine in silencingfollowed andby conventional translation process, these stop codons a guanine in the CpG dinucleotide. Therefore, the would take premature stop of polyprotein translation, analysis of differential methylation between different therefore de following protein, the viral RNA dependent preventing the complete codification of NS5A and or hypomethylated regions may provide evidence to RNA polymerase NS5B. In order to investigate the elucidateclinical manifestations the pathogenesis and theof dengue. identification In this ofpresent hyper impact of this codon in the position 111 of NS5A protein, study, the DNA from patients with DF and DF with a site directed mutation was produced in the CMV_SGR_ hemorrhagic manifestations was extracted and used JFH1_GFP5A plasmid and in the subgenomic replicon SGR-JFH1-FEO, introducing the TGA codon in the Methylation-Sensitive Arbitrarily Primed Polymerase position 111 of NS5A protein of these constructions. The Chainfor analysis Reaction of differential(MS-AP-PCR). methylation After electrophoresis, profile by using one expression of GFP protein, set after codon 111 of CMV_ band showed a differential methylation pattern and SGR_JFH1_GFP5A, wild type and mutated, was analyzed in pGEM-T-Easy vector. The recombinant plasmid were hepatocellular carcinoma strains (Huh 7 and HepG2). by fluorescence microscopy 48h after transfection in extractedthis band wasand cutted,sequenced. purified, The re-amplifiedsequence data and showed cloned The results show the expression of GFP in both wild type 98% homology to a region of chromosome 13 present in and mutated plasmid, an indication of complete NS5A SMAD family member 9 gene, which transduces signals expression. A replication assay was carried out for the with the BLAST tool, we found out that it is located next luciferase protein expression, establishing the replication mutated and wild type replicons, by the quantification of tofrom a CpG TGF-β island, family according members. to Searching the MethPrimer for the sequenceprogram. level of these constructions, in Huh 7. The results show that up to 12h after electroporation the mutated replicon status in SMAD region in other samples by another has replication levels similar to wild type, corroborating Further studies are needed to confirm the methylation previous result, but these levels reduce over time. The Sequencing.FINANCIAL SUPPORT: FAPEMIG; CNPQ; present study suggests that TGA codon in position 111 CAPES.technique such as Methylation Specific PCR or Bisulfite of Hepatitis C Virus is not determinant of translation termination. New studies are necessary to determinate which mechanism is responsible for the readthrough

activity, in different steps of viral cycle and impact in host pathogenicity.FINANCIALof this codon, as well as its SUPPORT: influence FAPESP, in complete PROCESSO virus Nº 2013/02856-0

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

24 Oral Presentation

BV175 - CHARACTERIZATION OF THE INTERACTION potential target to block HRSV replication. FINANCIAL BETWEEN THE METHYLOSSOME AND THE HUMAN SUPPORT: FAPESP and CAPES RESPIRATORY SYNCYTIAL VIRUS NUCLEOCAPSID BV225 - HIV-1 TRANSLATION INITIATION IS Ogawa, J.; Oliveira, A.; Simabuco, F.; Éleouët, J.F.; INHIBITED BY POLIOVIRUS 2A PROTEASE Ventura, A. Amorim, R.; Costa, L.J. da 1. USP - Universidade de São Paulo, Av. Prof. Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 UFRJ - Universidade Federal do Rio de Janeiro, Av. 2. UNICAMP - Universidade Estadual de Pedro Calmon, 550 - Cidade Universitária, Rio de Janeiro - Campinas, Cidade Universitária Zeferino Vaz - Barão RJ, 21941-901 Geraldo, Campinas - SP, 13083-970 Translation initiation in eukaryotic cells is made mainly 3. INRA - Institut National de la Recherche via two mechanisms: the recognition and association Agronomique, Siège; 147 rue de l’Université 75338 of several eukaryotic initiation factors (eIFs) to the Cedex 07 5’ Cap structure present at eukaryotic messenger The Human respiratory syncytial virus (HRSV) is one of the most important pathogens of the respiratory tract, regions of highly structured regions of mRNAs named causing respiratory illness particularly in newborns, internalRNAs (mRNAs); ribosome or entry the associationsites (IRES). of IRES-dependent eIFs to specific babies, children and immunocompromised patients. translation occurs for some mammalian mRNAs under The genome of HRSV encodes eleven proteins, being certain metabolic conditions and for RNAs of some fundamental to understand its relationship with the host, to characterize the interactions between those Type 1 (HIV-1) uses cellular factors to start its protein proteins and cellular components. Viral nucleoprotein synthesis,viruses as which poliovirus. is done Human mainly Immunodeficiency by the cap structure Virus (N) associates with the viral RNA forming the basic of its mRNAs. However, there is still some controversy structure of the HRSV helical nucleocapsid. In a previous whether translation of retroviral mRNAs is strictly Cap- work of our laboratory it was found that N interacts with dependent, since some studies have demonstrated the cellular proteins Hsp70, PRMT5 and WDR77, utilizing a FLAG tagged N expression in HEK293 cells protocol and SIV. In this study, we used poliovirus 2A protease and polyclonal mouse sera against N. Association of (2APro),existence thatof IRES interrupts elements cap-dependent on the 5′UTR translation of HIV-1, FIV by two PRMT5 and two WDR77 polypeptides form a the cleavage of initiation factor eIF4G, and analyzed methylosome core unit and its interaction with the its interference on the replication of HIV-1 in Hek293T nucleocapsid might have a role in virus replication. cells. We made co-transfections by cationic liposomes N has arginine residues exposed on the surface that of the infectious clone of HIV-1 or a reporter plasmid are potential targets of methylation by PRMT5 and containing luciferase gene with an expression vector of 2Apro. The expression of luciferase was evaluated by between N and the viral RNA, enabling access of viral luminometry, expression of viral proteins by western- RNAthis modificationpolymerase during could bethe modulating processes of the transcription interaction blotting and production of HIV-1 infectious particles by and replication. We used Hep2 cells infected by HRSV We observed a strong reduction in the expression of and western blot, N-methylosome interaction utilizing luciferasethe infection in of2Apro TZM-bl co-transfected cells indicated cells by atβ-galactosidase. 24 hour post- commerciallyA2 strain extracts available to confirm, monoclonal by immunoprecipitation antibodies against N. In addition a HRSV minigenome system, functional in levels and a mild decrease in eIF4GII levels. Expression BSRT7 cells transfected with a set of vectors expressing oftransfection, the viral proteins along with GagPol a significant (and its decreasecleaved products)in eIF4GI viral replication proteins and a reporter gene, was and Nef was strongly reduced in cells co-transfected used to probe co-localization of N and PRMT5 during with 2APro and remained inhibited until 72 hours post- replication process in inclusion bodies. The results transfection. Moreover, in the presence of 2APro, the release of infectious viral particles was 90% reduced We conclude that the inhibition of PRMT5 activity is a compared to control cells. This decrease in protein confirmed N-methylosome interaction by both strategies. September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

25 Oral Presentation

viruses. Supernatant was collected at 24h post-infection as viral full-length mRNAs were detected in the cytoplasm and viral titres were normalized by RFP-expression, ofsynthesis 2APro transfected does not seem cells. to Together,be related our with results mRNA indicate traffic, greatly between plasmids, i.e., pPARP12 was expressed of viral structural and accessory proteins and therefore inquantified 19.3% of by the flow cells cytometry.while pTLR3 RFP-expression only in 0.33%. varied HRVs tothat inhibit 2Apro the is able production to reduce of significantly HIV-1 infectious the translation particles. are resistant to most ISGs used in this study, specially Studies are under way to precisely demonstrate that in HRV14 and HRV16, both members of the major Hek293T cells the synthesis of HIV-1 proteins is solely group. EMCV was sensitive to most ISGs, and the titre cap-dependent.FINANCIAL SUPPORT: CNPQ, CAPES/ decreased around 3 orders of magnitude (p<0.001). On the other hand, 2A protease from all HRV provided more resistance to all E2AHRV. “Tumor necrosis factor PROEX,BV280 FAPERJ. - 2A PROTEASE FROM HUMAN RHINOVIRUS receptor-associated factors domain 1” (TRAFD1) was C15 DOES NOT PROVIDE INTERFERON the ISG with antiviral activity against all viruses used RESISTANCE AND TRAFD1 HAS AN ANTIVIRAL in this study. In conclusion, 2A protease from HRV ACTIVITY AGAINST HUMAN RHINOVIRUSES AND improved EMCV replication and also resistance to IFN- ENCEPHALOMYOCARDITIS VIRUS treatment, except to E2AHRVC15 that was IFN-sensitive Gagliardi, T.B.1; Dabelic, R.2; Racaniello, V.R.2 as the wild type. 2A protease from all HRVs improved 1. USP - Universidade de São Paulo, Av. Prof. EMCV resistance to the ISGs used in this study. TRAFD1 Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 was the ISG with greatest antiviral activity against HRVs 2. CU and other viruses. BV345 - IDENTIFICATION OF THE CELLULAR AND when a virus infects the cell and that results in interferon MOLECULAR DETERMINANTS OF FLAVIVIRUS Innate immune response is the first one to be activated (IFN) production following transcription of interferon- FUSION stimulated genes (ISG). This study evaluated the activity Esposito, D.L.A.; Nguyen, J.B.; Modis, Y. of 2A protease from human rhinoviruses (HRVs) against IFN and ISG responses. This study was developed using YALE UNIVERSITY, New Haven, CT 06520, Estados HRV1A, 2, 14 and16; encephalomyocarditis virus (EMCV); Unidos Flaviviruses are major pathogens that are responsible for protease from HRV1A, 2, 14, 16 and C15. All experiments a number of important mosquito-borne diseases of both wereand five done EMCV in H1Hela recombinants cells, using (E2AHRV) multiplicity expressing of infection 2A humans and animals. Flavivirus infection begins with cellular entry of the virions, which occurs by a membrane Cells were pre-treated with 0, 1000, 2000 and 5000u fusion reaction. It is unclear whether virus-like particles of 1 and the viruses were quantified by plaque assay. (VLPs - noninfectious ordered structures that resemble Supernatant and cells were collected at 24h post- whole virions in terms of their structural organization, infectionof IFN-α2a. and After the viruses24h, they were were titrated. infected The with presence a virus. of cellular processing, and capacity for membrane fusion, 2A protease from all HRV improved EMCV replication and therefore serve as useful models for investigating mainly at the end of cycle (exponential phase), turning viral entry) have the same requirements for fusion and it 2h faster. In addition, the E2AHRV infectiousness was the same cell entry pathways as full-sized virions. In also improved (replicative burst increased 3 to 5 orders this study, we examined the functional requirements of magnitude). As expected, HRVs were IFN-resistant. for productive membrane fusion of West Nile (WN) All E2AHRV showed to be IFN-resistant, except for and Japanese encephalitis (JE) VLPs to liposomal target E2AHRVC15, which was IFN-sensitive like the wild membranes by monitoring membrane fusion using a type. We also analyzed the antiviral response of 31 ISGs. lipid-mixing assay. WN and JE VLPs were produced in mammalian cells with the prM-E region of the WN/ protein” (RFP) were transfected into H1Hela cells and, afterPlasmids 48h, that the express cultures ISGs were tagged infected with with“red fluorescentone of the JE virus genome and, after purification, were labeled September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posterswith - Oral Presentation the hydrophobic fluorescent indocarbocyanine XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

26 Oral Presentation

fever (DHF) or dengue shock syndrome (DSS). Although, performed by mixing the labeled VLPs with 100-nm much research has been done in Dengue, currently, liposomes,dye DiI (Invitrogen). and fusion Awas fluorescent observed plateafter assayacidifying was there is no licensed antiviral agent to defend against the the reaction (0.1 M sodium acetate pH 5.2). Fusion of JE dengue. Thus, development of therapeutic strategies and WN VLPs to liposomes occurred on the time scale selected by Phage Display an antibody fragment capable the human physiological temperature of 37 oC. Fusion tois neededrecognize to the fight viral against structural the virus. protein In thisNS1. studyNS1 is we a wasof minutes also enhanced with optimal with fusion increasing efficiencies acidity, observed with the at versatile nonstructural glycoprotein that is expressed threshold for fusion between pH 5.5-5.7. Importantly, on the cell surface and secreted into the extracellular the presence of anionic lipids, namely BMP (LBPA) space. The intracellular NS1 has been associated with and phosphatidylinositol 3-phosphate (PI3P), in the early steps of viral replication, since it can be found co- localized together whith dsRNA and other nonstructural fusion potential of VLPs, consistent with an emerging proteins like NS3 and NS5. Futhermore, we development liposome target membranes significantly enhanced the a mechanism to degrade the NS1 when scFv is fused to compartments. Financial Support: * Subcontract within the SEL1L molecule (scFv viral-degradin). SEL1L is a model of flavivirus fusion to mid-to-late endosomal component of HRD1 complex, which is involved on the 07/22/13 – 07/21/15 US Department of Defense Small Quality Control protein process of the endoplasmatic MoleculeContract Antiviral No. W81XWH-12-C-0028 Agents Against Flaviviruses (L2 Diagnostics) * P01 reticulum (ER). This molecule (SEL1L) is able to recognize GM022778-37 (Steitz, Project Director; Modis, Co-PI) and to translocate protein with misfolded luminal 04/01/09 – 06/30/14 NIH/NIGMS Program Project domains to proteasome. After three round of selection, Grant Structural bases of the functions of RNA-protein selected cDNAs encoding antibody fragments (scFv) machines were subcloned into an expression vector in mammalian cells to produce scFv in soluble form or to produce scFv BV379 - DEVELOPMENT OF TWO POTENTIAL TOOLS fused to the SEL1L molecule (scFv viral-degradin). In our AGAINST THE REPLICATION OF THE DENGUE VIRUS Fujimura, P.T.1; Vaz, E.R.1; Cardoso Jr, C.A.M.1; the soluble scFv with NS1 in the infected cell. In addition, Carvalho, W.J.1; Reis, C.F.2; Santos Jr, C.D.3; Freitas, study, we performed immunofluorescence to co-localize G.R.O.1; Yokosawa, J.1; Carneiro, A.P.4; Goulart, L.R.1; degradation and qPCR to quantify the new production of Vecchi, L.1; Ueira,C.U.V.1 viralwe also particle performed after inabation Western with blot toour verify soluble the scFv specific and 1. UFU - Universidade Federal de Uberlândia, scFv viral-degradin. Our results showed that the soluble Av. João Naves de Ávila, 2121 - Santa Mônica, Uberlândia - antibody scFv co-localizes with NS1 inside the cell, MG, 38408-100 probably in the viral replication complex, and it is able to 2. UNICAMP - Universidade Estadual de block the viral replication in more than 90%. Moreover, Campinas, Cidade Universitária Zeferino Vaz - Barão Geraldo, Campinas - SP, 13083-970 (NS1) and it was also able to inhibit the new viral particle 3. UFSCAR - Universidade Federal de São productionthe scFv viral-degradin in more than degraded 90%. Therefore, the specific this molecule soluble Carlos, Rodovia Washington Luís, Km 235 - SP 310 - Jardim scFv and scFv viral-degradin may be the potential tools Guanabara, São Carlos - SP, 13565-905 to combat dengue virus replication. Financial Support: 4. USP - Universidade de São Paulo, Av. Prof. Capes, CNPq, FAPEMIG, UFU. Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 Dengue is the most important mosquito-borne in tropical and subtropical areas and has become a global threat affecting around 100 countries in the world. Dengue virus infection can be manifested by the self-limited febrile illness known as dengue fever (DF) or by a severe complication caused by the hemorrhagic

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

27 Oral Presentation

BV490 - IN VITRO MICROEVOLUTION OF DENGUE 4248 (NS2B region). Further experiments are being VIRUS 4 IN AEDES ALBOPICTUS CELLS Nascimento, V.A.; Souza, V.C.; Naveca, F.G. provideperformed data in that order can to help verify to the a better significance understanding of these ILMD/Fiocruz - Instituto Leônidas e Maria Deane - ofmutations the mechanisms for viral fitness. of viral The adaptation results of thisin vitro, work thusmay Fiocruz Amazônia, Rua Terezina, 476 - Adrianópolis, Manaus contributing to evolution studies with RNA viruses. - AM, 69057-070 FINANCIAL SUPPORT: FIOCRUZ/CNPQ PAPES VI; POM - The Dengue virus (DENV) belongs to the FIOCRUZ; CAPES ÁREA family, genus Flavivirus and four distinct serotypes are recognized. Like other RNA viruses, DENV displays BV501 - ALIX IS A HOST CELL FACTOR REQUIRED genome variations due to error-prone RNA polymerase. FOR LYSOSOMAL TARGETING OF CD4 BY HIV-1 NEF These variants may be observed not only when samples Januário, M.E. da S.1; da Silva, E.M.L.1; de Castro, R.O.1; from different individuals are analyzed, but also within Amorim, N.A.1; da Costa, L.J.2; da Silva, L.L.P.1 the same host. Considering the need for a better 1. FMRP/USP - Faculdade de Medicina de understanding of DENV evolutionary process, and the Ribeirão Preto/ Universidade de São Paulo, Av. Bandeirantes, possible emergence of highly virulent lineages, this study 3900 - Monte Alegre, Ribeirão Preto - SP, 14049-900 aimed to evaluate the in vitro microevolution of DENV 2. IMPG/UFRJ - Instituto de Microbiologia serotype 4, genotype II, during the adaptation process in Paulo de Góes da Universidade Federal do Rio de Janeiro, insect cells culture. For this purpose, a sample of DENV- Av. Carlos Chagas Filho, 373 - Cidade Universitária, Rio de 4 collected in May 2011 (BrAM005/11) was inoculated Janeiro, Ilha do Fundão Rio de Janeiro - RJ, 21941-590 into C6/36 cells (P1), followed by serial passages in the Nef is an accessory protein of HIV-1 that enhances same cell line until the passage 25 (P25). Subsequently, production of infectious viral particles and promotes viral RNA was extracted and cDNA was generated using a progression to AIDS. The most studied function of Nef is the ability to downregulate the expression of The full-length genome of the BrAM005/2011 sample, specific primer targeting the last 21 nucleotides at 3’ UTR. type I transmembrane glycoprotein expressed on the in eight overlapping amplicons, which were sequenced specific cell surface proteins, such as CD4. CD4 is a as well P1, P5, P10, P15, P20 and P25 were amplified surface of helper T-cells and cells of the macrophage/ by Sanger’s based method. The same samples were monocyte lineage that plays important roles in adaptive also submitted to Next-Generation Sequencing (NGS) immune responses, and serves as the primary receptor for HIV-1, HIV-2 and SIV. Downregulation of CD4 by Nef after sequencing were further analyzed using Geneious using the Ion Torrent technology. The files generated is thought to prevent deleterious superinfection and to software version 7.1.7. The BrAM005/11 sequence was facilitate release of newly produced viral particles. Nef initially aligned with the reference sequence available in induces CD4 endocytosis via clathrin-coated vesicles GenBank (NC_002640.1) for annotation. Furthermore, (CCVs) through interaction with AP-2. CD4 that is the insect cells passages (P1 to P25) were aligned for internalized by Nef is then targeted to late endosomes/ the nucleotide variation comparison using annotated multivesicular bodies (MVBs) and subsequently to BrAM005/11 as reference. The data analysis provided lysosomes for degradation, but how this is achieved is by the Sanger’s sequencing showed two nucleotide mostly unknown. Our previous work showed that Nef mutations that occurred during the viral adaptation. One interacts with the middle (V) and the Bro1 domains of of these mutations occurred at position 1602 and results Alix, a protein involved in MVB biogenesis. Moreover, in a residue substitution (T222P) in the domain II of the overexpression of truncated Alix V-domain impairs viral envelope protein; whereas a silent mutation was CD4 degradation induced by Nef. Here we show that observed in position 3365 (NS1 region). The NGS results Nef-Alix interaction takes place in late endosomes corroborate those obtained by Sanger’s sequencing and that contain internalized CD4 and that depletion of showed two additional mutations: at nucleotide position Alix impairs delivery of CD4 to lysosomes induced by 1810, which results in a residue substitution K291R Nef. Alix depletion or V-domain overexpression does in the viral envelope and a silent mutation at position September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

28 Oral Presentation not compromise transmembrane protein degradation through the canonical MVB-pathway, suggesting that of treatments (T2 and T3, respectively). T1 was able to Nef uses a alternative mechanism, mediated by Alix, to 3 logs10 reduction in swine effluent at 80 and 45 days deliver cargo to MBVs. Together our results show that Considering the relative time of treatment to inactivate Nef interacts with Alix to facilitate delivery of CD4 to the 99.9%reduce (3log10)2 logs10 of of all ɸX-174 the studied after pathogens, 80 days of considering treatment. MVB pathway, thus promoting sustained depletion of this surface receptor in infected cells. the T2 treatment was the most promising for swine the amount of urea added and efficiency of inactivation, EV90 - PATHOGENS INACTIVATION BY UNIONIZED sludge. Hygienization by NH3 provides new possibilities AMMONIA FOR THE PRODUCTION OF SAFE foreffluent alternative and T3 treatments treatment of wasdifferent more types indicated of waste, for BIOFERTILIZER FROM SWINE EFFLUENT AND liquid as well as solid, at both small and large scale, SLUDGE being a viable method for reuse of agricultural waste as Fongaro, G.1; Kunz, A.2; Magri, M.E.1; Zeredo, A.C.B.1; biofertilizer, considering the animal and human health Schissi, C.D.1; Zaguini, J.1; Barardi, C.R.M.1 quality. FINANCIAL SUPPORT: CNPQ 472804/2013-8. 1. UFSC – Universidade Federal de Santa FINANCIAL SUPPORT: CNPQ 472804/2013-8. Catarina, Campus Universitário Reitor João David Ferreira EV140 - ANALYSIS OF MARINE VIRAL FAMILIES Lima - Trindade, Florianópolis - SC, 88040-900 ASSOCIATED WITH SIDERASTREA STELLATA 2. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica - PqEB s/nº.Brasília - DF, 70770- FESTIVAL IN ARRAIAL DO CABO AND BÚZIOS, RJ, 901 BRAZIL Santana-Pereira, P.; Giongo, V.; Pedrosa, L.G.; Paula, The biomass derived from swine manure has good J.E.F.B.; Amorim, L.; Paixão, I.C.N.P. potential to be used as biofertilizer due to its high nutrient concentration. However, the land application UFF - Universidade Federal Fluminense, Rua Miguel of manure should be based on safety parameters. de Frias, 9, Icaraí, Niterói - RJ, 24220-900 This work used ammonia for hygienization as an The reef ecosystem is considered as one of the oldest, additional method to produce a safe biofertilizer from stable and biodiverse on the planet. A complex network of interactions and interrelationships due to the high bioreactor. The microorganisms used as models were: diversity of organisms in this environment is sustained swine effluent and sludge after treatment in anaerobic by primary productivity, which is characterized as one 2) and Salmonella typhimurium. The enumerations of of the largest ocean. Studies have shown that viruses are somatic coliphage ɸX-174, Human Adenovirus (HAdV- highly dynamic components in the aquatic ecosystem, performed by integrated cell culture assay–preceded because they have a fundamental role in the chain bythe reverseHAdV, ɸX-174 transcription and S. tyfimurium(ICC-RT-qPCR), were double respectively layer agar method and by ISO 6579 (2002). The following abundance and diversity of bacteria and phytoplankton, whichto be significantare symbionts microbial of corals agents and inare controlling important thein 379 mM (T2) and 754 mM (T3) reaching pH 9.8 at controlling availability of nutrients. In this study we final concentrations of urea were used: 186 mM (T1), 23oC, providing, in this condition, unionized ammonia performed the molecular and morphological analysis (NH3), as inactivating agent. All experiments were performed in triplicate and with negative controls. The coralreef that didn’t have been described yet, being the results revealed that i) S. typhimurium either in swine for identification of viral groups in a scleractinian associated with marine organisms. For this we used to 3 days for all treatments; ii) HAdV had a 4 logs10 TEMfirst and work PCR in for Brazil determination studying the of marine presence virus of families viruses effluent or in sludge, had a 4 logs10 reduction in up as widely tools applied in marine virology studies. The the respective treatments T1, T2 and T3, and in swine samples were collected in two áreas (Arraial do cabo reduction in swine effluent at 27, 20 and 15 days with sludge the inactivation occurred at 15, 9 and 3 days with and Búzios) with the aid of chisel and hammer, kept on ice until the time of processing. After processed were theSeptember/October respective treatments 2014 Volume T1, 19 T2 – Supplement and T3; iii) 2 -ɸX-174 Abstracts/Posters had - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

29 Oral Presentation stored at -80 ° C ultrafreezer until the analysis. We in sediment samples from streams located in urban perform the transmission electron microscopy using areas of EstânciaVelha/Portão (EstânciaVelha and concentrated phage prepared in glutaraldehyde (2.5%) Portão), Schmidt (Campo Bom), Pampa and Luiz Rau (Novo Hamburgo), RS, Brazil. Samples (n=102) were collected from 17 different points, bimonthly, from meshwith addition coated collodion. of uranyl acetateAfter incubation (2% final concentration)the grids were September/12 to July/13. Samples were eluted 10% examinedfor performing in a transmission negative staining electron in grids microscope of Cu + (JEOL,+ 300 v./v. in E-MEM (pH 10,5). Viral DNA was extracted by a JEM1011 - Electron Microscopy) at 80 Kv. The images commercial kit and HAdV detection was performed by were observed in increases 18000-300000 times. The qPCR using primers that targeted a conserved region PCR analyses were performed using the DNA extraction (hexon gene) of the virus genome. Overall, 42.16% (43/102) samples were positive for the presence of belonging to and viral families HAdV: Arroio Schmidt (45.8%; 11/24), Arroios Pampa (CPS1,kit (ROCHE) CPS2, and P60, specific T3, T7 sequences and T4 ofprimers). oligonucleotides Through and Luis Rau (both 37.5%; 9/24), and EstânciaVelha/ these analyzes it was possible determine the presence Portão (36.7%; 11/30). There was a variation in viral of the viral groups belonging to Podoviridae, Myoviridae load among 4.52 x 102 a 1.30 x 105 genome copies per and viral families by TEM. In addition, it gram of sediment. These viral agents detected in these was possible to identify fragments belonging to the streams are related to various pathologies, such as groups Myoviridae and Podoviridae by PCR. Also, some gastroenteritis, respiratory infections and conjunctivitis. observed suggesting the presence of viral particles FINANCIAL SUPPORT: CAPES, FAPERGS, CNPQ, PROJETO withamplified different fragments, sequences having of the non target expected sequence. size, These were MAISThe results ÁGUA, suggest UNIVERSIDADE significant FEEVALE. anthropic contamination. cnidarians and thus more robust studies is required to EV306 - ENVIRONMENTAL POLIOVIRUS determineresults confirm the possible the interaction consequences between of this virusesassociation. and SURVEILLANCE: DETECTION OF WILD AND VACCINE FINANCIAL SUPPORT: CNPQ, FAPERJ, PROPI AGIR - DERIVED POLIOVIRUSES IN SÃO PAULO SEWAGE Hachich, E.M.1; Barbosa, M.RF.1; Garcia, S.C.1; EV255 - DETECTION OF HUMAN ADENOVIRUSES IN Bonanno, V.M.S.1; Eduardo, M.B.P.2; Burlandy, F.M.3; SEDIMENTS FROM STREAMS IN URBAN AREAS FROM Costa, E.V.3; da Silva, E.E.3; Sato, M.I.Z.1 RIO DOS SINOS WATERSHED 1. CETESB - Companhia Ambiental do Estado Staggemeier, R.; Heck, T.M. da S.; Andriguetti, N.B.; de São Paulo, Av. Prof. Frederico Hermann Jr.,345, Av. Prof. Soliman, M.C.; Souza, F.G. de; Fabres, R.B.; Luz, R.B.; Frederico Hermann Júnior, 345 - Alto de Pinheiros São Paulo Zirbes, G.; Henzel, A.; Rigotto, C.; Spilki, F.R.; Almeida, - SP, 05459-010 S.E. de M. 2. CVE/SES-SP - Centro de Vigilância FEEVALE - Universidade Feelave, Av. Dr. Maurício Epidemiológica da Secretaria de Saúde do Estado de São Cardoso, 510 | Bairro Hamburgo Velho, Novo Hamburgo - RS, Paulo, Av. Dr. Arnaldo, 351 - 6º andar — Pacaembu, São 93510-250 Paulo - SP, 01246-000 3. FIOCRUZ - Fundação Oswaldo Cruz, Av. Soil can act as an important reservoir of natural Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 resources, however, it may also allow the permanence The environmental poliovirus surveillance (ENV) is an of many potentially dangerous microorganisms. Enteric important complementary tool to the investigation of viruses in the soil may migrate through the successive adsorption-desorption phenomena, thus providing surveillance) for the Global Polio Eradication Initiative risk of contamination of groundwater. Among the (GPEI)acute flaccidof WHO. paralysis CETESB, (standard the Environmental approach Company for polio enteric viruses, adenoviruses (AdV) have been the focus of many studies, because of their persistence Virology for about 40 years, and since 1999 develops in the environment and their great impact on public aof ProgramSao Paulo of State, ENV worksin collaboration in the field with of Environmental the Center of health. The aim of this study is the detection of HAdV

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

30 Oral Presentation

Epidemiological Surveillance of Sao Paulo State. The 1. UFV - Universidade Federal de Viçosa, surveillance is based on the examination of sewage Avenida Peter Henry Rolfs, s/n - Campus Universitário, Viçosa and seawater samples from potential points of foreign - MG, 36570-000 people entrance, such as international airport and 2. CENPES seaports, and from wastewater treatment plants 3. UNICAMP - Universidade Estadual de (WWTP). Sampling is performed bimonthly through Campinas, Cidade Universitária Zeferino Vaz - Barão gauze pad (Moore swab) or grab sampling. Moore’s Geraldo, Campinas - SP, 13083-970 swab is transported to the laboratory in 3% dessecated whether biotic or abiotic, is an important survival The grab samples are concentrated using the PEG 8000. Biofilms occur spontaneously in different environments, beef extract and concentrated by organic flocculation. systems represents a drawback in the application of decontamination and tested for sterility. Poliovirus is thisstrategy. technology Biofilm by formation different on industries, reverse osmosisincluding (RO) oil isolatedSamples accordingare treated to with the WHO chloroform alternative to clarification test algorithm and (S1 Supplement to the WHO Polio Laboratory Manual), of microbial contamination and thus contributes for the refineries. In RO systems the feed water maybe a source inoculation into L20B and RD cell. Virus isolates are submitteda specific approach to intratypic comprising differentiation of several simultaneousby RT-PCR. formation of biofilm and consequent biofouling. In this All poliovirus isolated are sent to the Bacteriawork, the isolated influence from of non-specificfeed water sampled bacteriophage from a wasRO Laboratory of FIOCRUZ (WHO Reference Laboratory investigated in the biofilm formed by isolated bacteria. for Poliomyelitis) for sequencing, aiming to diagnose higher biomass, were selected to be used in the biocontrol the presence of vaccine derived poliovirus (VDPV) system that showed greater ability to form biofilm, i.e. phage suspension of a multiplicity of infection (MOI) of August 1999 to April 2014, 1,274 samples of sewage and 0.01assay was employing added into phages 96-well against microtiter biofilm plate formation. containing A seawaterand confirm were the tested. wild Apoliovirus WPV sorotype1 (WPV) withisolation. nucleotide From identity of 99,6% to the Equatorial Guinea recent isolates bacterial suspension, and biofilm formation was Viracopos Airport WWTP, Campinas, collected on 5th observedmeasured in using 7 of biomass 11 tested quantification bacteria, some with showed crystal violetdose- was detected in a sewage sample from the influent of the dependentstaining (CV). reduction Statistically and others, significant increasing reductions the MOI wereused rendered 50% CPE (5 days) and the second passage in RDMarch, rendered 2014. 100% The first CPE passage (48 h). On of theJanuary sample 20th, in at L20B the analysis, two bacteria which responded differently with Petrobras Pier in São Sebastião Coast, a VDPV was also MOIdid not increasing cause further were selectedreduction and in biofilm.analyzed After in MBEC® the CV devices by scanning electron microscopy (SEM) in order passage in L20B (50% CPE, 5 days) and a second one in RDisolated (100% from CPE, a seawater4 days). RT-PCR sample showed collected the after presence a first By SEM is possible to observe that phage interferes with of a Poliovirus Sabin 2. The sequencing of VP1 gene theto evaluate adherence the ofinfluence one tested of phages bacteria on and biofilm in the structure. stability (903 nucleotides) of this isolate, showed a nucleotide identity of 91% with the reference PV Sabin 2. These that the phage vB_AspP-UFV1 (Podoviridae) interfered of biofilm formed by other bacteria. The results suggest monitoring as a preventive action for the GPEI. enzymes or phage infection, without bacterial lysis. This findings reinforce the importance of the environmental in biofilm formation by two strategies, by action of EV430 - DIFFERENT PHAGES ACTION PATHWAYS IN control on membranes in reverse osmosis systems. REDUCING BIOFILMS FORMED BY OIL REFINERIES FINANCIALapproach may SUPPORT: represent PETROBRAS a good alternative in biofilm ASSOCIATED BACTERIA Dias, R.S.1; Fonseca, L.A.B.V.1; Albanese, J.M.1; Silva, L.C.F.1; Suhette, R.2; Torres, A.P.R.2; Sousa, M.P.2; Silva, C.C.1; Oliveira, V.M.3; de Paula, S.O.1

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

31 Oral Presentation

EV457 - FIRST IDENTIFICATION OF ACANTHAMOEBA Chikungunya virus (CHIKV) is a mosquito-borne POLYPHAGA MIMIVIRUS IN LIMNOPERNA FORTUNEI emerging pathogen that has a major health impact in FROM GUAIBA LAKE, RIO GRANDE DO SUL, BRAZIL humans causing a dengue-like illness characterized by dos Santos, R.N.; Arantes, T.; Correa, R.A.; de high fever, headaches, rash, nausea, vomiting, myalgia, Albuquerque, N.R.M.; Kluge, M.; Santos, C.; dos and arthralgia. Recent outbreaks have been reported in Santos, C.P.; Campos, F.C.; Roehe, P.M.; Franco, A.C. many parts of the world since 2004 after an outbreak at the coast of Kenya. More recently, the virus has reached UFRGS - Universidade Federal do Rio Grande do Sul, the Americas, where there is an ongoing outbreak at Avenida Paulo Gama, 110 - Farropilhas, Porto Alegre - RS, the Caribbean Countries, and positive cases have been 90040-060 The discovery of a complex group of viruses, known from these areas. Viremic travelers are a risk for as giant viruses, has aroused the interest of many spreadingconfirmed CHIKV in the to United non-endemic States in regions, travelers specially returning in Brazil, where there is a high vector incidence and a great this group, Acanthamoeba polyphaga mimivirus, was number of immunologically naïve people. Sera from 13 environmental virologists. The first representative of isolated from samples collected in a cooling tower of an Brazilian soldiers and one female missionary, suspected air conditioning system in a hospital in England in 2003. of Chikungunya fever, all coming from Haiti, were sent to This virus has a large size (750 nm in diameter), with Núcleo de Doenças de Transmissão Vetorial, Centro de a 1.2 mb genome and 911 protein-coding genes. This Virologia at the Instituto Adolfo Lutz, São Paulo, Brazil, for diagnosis. Blood samples were collected between days 2 mimiviruses in samples collected from golden mussel and 16 after onset of symptoms, and so the choice of each study aims the isolation and molecular identification of (Limnoperma fortunei) at Guaiba Lake, in Rio Grande laboratory method was based upon the time of sample do Sul. A total of 300 samples were collected from collection relative to symptom onset. Samples varying grids submerged in the lake, and transported to the from 2 and 7 days were innoculated in Vero and C6/36 laboratory under refrigeration. Next, these samples cell lines and in mice by intracerebral and subcutaneous were incubated with rice enrichment medium for 30 routes, while samples with 4 days or more were tested days at 25 °C and submitted to DNA extraction. Real- for Chikungunya IgM antibody capture enzyme-linked time PCR was performed using primers to the helicase immunsorbent assay (MAC-ELISA). All serum samples gene of mimiviruses. So far, in the 25 samples examined, as well as the supernatants of cells were tested by Real- time RT-PCR (RT-qPCR) for CHIKV detection. Viral RNA them. Next, we will perform a subsequent cloning and mimivirus genome segments were identified in all of molecular characterization of the helicase amplicons. In Four infected Vero cells showed cytophatic effect after addition, the samples will be submitted to isolation in 4was and amplified 5 days post-innoculation. in 10 serum samples All thesetested supernatants by RT-qPCR. cultures of Acanthamoeba castellani T4 to observe the citopatic effects. Although preliminary, these results without cytophatic effect. Plus, electron microscopy also indicate that the golden mussel can host and may play a were confirmed by RT-qPCR, as well as other two role in mimiviruses maintenance in nature. FINANCIAL RT-qPCR was positive after one passage in C6/36 cells. SUPPORT: CAPES. CHIKVconfirmed was viral also particles.isolated in One three serum mice sample groups negative inoculated for subcutaneously that showed intense signs of alopecia at HV33 - DETECTION OF IMPORTED CASES OF the inoculation site, while two animal groups inoculated CHIKUNGUNYA VIRUS IN BRAZIL: VIRUS ISOLATION, only by intracerebral route presented alopecia on the MOLECULAR AND SEROLOGICAL ASSAYS Cunha, M.S.; Maeda, A.Y.; Bisordi, I.; Rocco, I.M.; da Silva, F.G.; de Souza, R.P.; Coimbra, T.L.M.; Nogueira, head and occular inflamation. MAC-ELISA detected J.S.; Kisielius, J.J.; Silveira, V.R.; Oliveira, A.L.R.; specific immunoglobulin M for CHIKV in all four samples Suzuki, A. IgMtested. detection, Here we in report samples the fromfirst isolation patients ofreturning Chikungunya from IAL - Instituto Adolfo Lutz, Av. Dr. Arnaldo, 355 - Haiti.virus in Brazil, confirmed by real-time RT-PCR, as well as Cerqueira César, São Paulo - SP, 01246-000 September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

32 Oral Presentation

HV125 - ORAL HUMAN PAPILLOMAVIRUS INFECTION 1. IMT/FAMUSP - Intituto de Medicina IN HIV-POSITIVE PEOPLE Tropical da Faculdade de Medicina da Universidade de São Silva, C.O.; Santos, L.S.; de Azevedo, K.M.L.; Pereira, Paulo, Av. Dr. Arnaldo, 455 - Cerqueira César, São Paulo - SP, O.M.D.; Oliveira, L. do H. dos S. 01246903 2. DIVISÃO DE UROLOGIA DO SERVIÇO UFF - Universidade Federal Fluminense, Rua Miguel DE TRANSPLANTE RENAL/HC/FMUSP - Hospital das de Frias, 9, Icaraí, Niterói - RJ, 24220-900 Clínicas da Faculdade de Medicina da Universidade de São Human papillomavirus are associated with a variety of Paulo, Av. Dr. Enéas de Carvalho Aguiar, 155 - Cerqueira oral lesions and they have been detected in healthy oral César, São Paulo - SP, 05403-000 mucosa of HIV-infected individuals. The aim of this study A new polyomavirus, TSV was recently described was to survey information concerning HPV infection in a solid organ transplant recipient with rare and from randomly selected HIV infected individuals and serious skin disease, initially described as side-effect of to compare the results obtained with a control group. cyclosporine treatment. The disease, Trichodysplasia Methods: This cross-sectional study included oral spinulosa (TS) is characterized by the development of samples from 75 HIV positive and 120 HIV negative follicular papules and keratin spines known as spicules, individuals (control group) who were asymptomatic for which usually manifests on the face of the patient. oral mucosa lesions. To detect human papillomavirus TS has a similar pattern among affected patients, demonstrating hair follicle dilatation and keratotic performed. HPV typing was determined by restriction plugging of the infundibulum and keratin spicules. fragmentstatus, polymerase length polymorphism chain reaction analysis. amplification Demographic was Usually occurs proliferation of abnormal enlarged and data, life style and tobacco history was obtained through eosinophilic cells, containing trichohyaline irregular a questionnaire. Results: The HIV patients (n=75) aged granules. Although there is a strong association between from 21 to 75 years, mean 41, 96 years and standard TS disease and the virus, data regarding the mechanisms deviation 10,313. None of the variables studied was of pathogenesis and transmission as well as viral detection in HIV individuals. Of the total, 54,7% were on a skin cancer biopsy from an immunosuppressed womensignificantly and 25,3 associated % smokers. with They increased presented odds a ofgreater HPV patientdiversity using are still metagenomic unknown. Recently, approaches we identified(GenBank TSV ID frequency (70.7%) of HPV infection compared with the KM007161). In this work, we developed a Real Time PCR control group (45.83%). It was also observed a higher for TSV detection and investigated the virus in spicules rate of multiple HPV infection (18.9% vs 8, 82%) than in and serial sampled urine obtained from the same patient health people. The most commonly type detected in HIV six months before and after trichodysplasia spinulosa individuals was HPV-53 (38.88%), whereas in control diagnostic. We developed a novel Real Time PCR assay group it was HPV-6 (31.58%). Indeterminate types were directed to AgT gene using SyBR® Green chemistry. Our largely detected in both groups. Conclusions: The HIV positive population in this study had a high HPV oral polyomaviruses but TSV. Using plasmid as a control, the infection. The prevalence of indeterminate types can be limitmethod of detection was very was specific 1000 since copies do per not microliter detect any in waterother and 500 copies per microliter in urine. Intra-laboratory not detected by methodology utilized, or they can belong repeatability and reproducibility was also conducted. due to the presence of non-genital infectionFINANCIAL by α-HPV According to our test, TSV was detected in the spicules, biopsy and all urine samples tested. The viral load ranged to other genera as β- HPV and γ-HPV types. from 10E3 to more than 10E8 copies varying according SUPPORT:HV147 - NEWCNPQ, REAL PROPPI TIME - UFF. PCR ASSAY FOR RAPID to the time of sampling. In conclusion, we presented a DETECTION OF TRICHODYSPLASIA SPINULOSA- new and sensitive test to detect TSV, responsible for a ASSOCIATED POLYOMAVIRUS IN BIOLOGICAL SAMPLES time that virus is shed in urine at least 6 months before Urbano, P.R.1; Nali, L.H. da S.1; Pannuti, C.S.1; Pierrotti, therare tumorigenesisand aggressive skinand cancer.remains We detectable also show foreven the after first L.C.1; Neto, E.D.2; Romano, C.M.1 the treatment. We suggest that early detection of TSV September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

33 Oral Presentation in urine can be used to monitor immunocompromised chromosomes. All patients presented transcripts patients that are at risk to develop the disease. from different sources, however chromosomes with highest read count frequently found in all patients HV213 - EXPRESSION PROFILE OF HUMAN ENDOGENOUS RETROVIRUS W (HERV-W) LOCI FROM that these are the most frequently sources of ENV- MULTIPLE SCLEROSIS PATIENTS UNDER DISTINCT HERVWwere: chromosomes expression in 1,MS 6, patients. 9, 14 andPatients X, suggesting receiving THERAPIES BY HIGH THROUGHPUT SEQUENCING Natalizumab presented transcripts from average 16 Nali, L.H. da S.1; Urbano, P.R.P.1; Silva, D.F.4; Olival, G.S. different chromosomes in contrast to those treating do3; Penalva de Oliveira, A.C.2; Romano, C.M.1 1. IMT/FAMUSP - Intituto de Medicina with βInterferon, which presented transcripts from Tropical da Faculdade de Medicina da Universidade de São that patients under Natalizumab therapy, usually with Paulo, Av. Dr. Arnaldo, 455 - Cerqueira César, São Paulo - SP, higheraverage Expanded 8 different Disability sources. Status This finding Scale, aremight expressing suggest 01246903 HERV-W from more chromosomes than patients under 2. Instituto de Infectologia Emílio Ribas, Av. Dr. Arnaldo, 165 - Cerqueira Cesar, São Paulo - SP, 01246-900 study we showed that our method is useful to determine 3. Centro de Atendimento e Tratamento de theβInterferon source of therapy. HERV-W Although expression the smallin biological sampling samples. of this Esclerose Múltipla - Santa Casa De São Paulo Increasing the number of samples in this study may 4. Instituto de Ciências Biomédicas clarify if there is actually a distinct HERV-W expression HERVs are remaining viral elements, once exogenous, that HV240 - HIGH FREQUENCE OF SAFFOLD VIRUS IN through the generations. HERVs sequences compose 8% profile in MS patients under different therapies. TONSIL TISSUES FROM PATIENTS WITH CHRONIC ofinfected human germ genome line and cells are and found became in many fixed sites in host throughout genome TONSILLAR HYPERTROPHY 1 2 2 W family (HERV-W) may play a role in Multiple Sclerosis Luna, L.K. de S. ; Valera, F.C.P. ; Tamashiro, E. ; Lima, the genome. Several findings indicate that HERVs from the W.T.A.2; Arruda, E.1 HERV-W expression levels in MS patients than in healthy 1. Cell Biology Department And Virology controls(MS) pathogenesis. to ENV HERV-W Such protein findings detection vary from in MS higher brain Research Center, University Of Sao Paulo School Of Medicine lesions. Considering the wide distribution of HERV-W 2. Department Of Otorhinolaryngology And sequences within the human genome some studies have Head And Neck Surgery, University Of Sao Paulo School Of determined the origin of the of HERVW transcript in MS Medicine patients through cloning and Sanger sequencing. Here Chronic tonsillar hypertrophy (CTH) is a persistent we aimed to determine the HERV-W expression level of hypertrophy of palatine and pharyngeal tonsils of these patients and the origin of these transcripts using unknown etiology. Respiratory viruses have been Next Generation Sequencing (NGS). Blood samples were detected in high frequencies in tonsils from patients obtained from MS patients, three from patients under with CTH, but their role in CTH pathogenesis is unclear. Natalizumab therapy (monoclonal antibody) and three In this study, Saffold virus (SafV), an emerging human from patients under other therapies. RNA was obtained which has been found mainly in patients using Trizol and before cDNA synthesis it was treated with respiratory or gastrointestinal diseases, was with DNAse. A 730bp amplicon from envelope gene detected by real-time RT-PCR aimed to the conserved was then obtained from each sample and sequenced 5’UTR in patients with CTH. Samples were comprised through Ion Torrent PGM plataform. Reads representing of nasal swabs (NS), nasopharyngeal washes (NW) the transcripts from different sources were then and tissue fragments of palatine (PaT) and pharyngeal mapped to putative chromosomes using a databank (PhT) tonsils obtained from patients (ages: 1-25 containing the most active HERV-W (28 loci) using CLC7 years; mean±SD: 6.18±3.23; median: 5.00) with CTH who underwent tonsillectomy at the University of Sao presented HERV-W expression from at least 4 different Paulo Hospital in Ribeirao Preto, Brazil. Samples were WorkBench. Our workflow indicated that all MS patients September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

34 Oral Presentation collected from November 2011 to March 2013 and show that in immunocompromised patients, such as were previously tested by real-time PCR for a panel of transplanted patients, norovirus infection can lead to common respiratory viruses. A total of 709 samples worsening of symptoms and be confused with clinical from 210 individuals were tested, of which 643 (179 NS, symptoms of graft versus host disease (GVHD). However, 168 NW, 146 PaT and 150 PhT) were from 190 patients routine human caliciviruses (HuCV) screening is not with CTH, and 56 samples (18 NS, 17 NW; 16 PaT and 15 performed. In this prospective study we have monitored PhT) were from a control group (CTRL) of 20 individuals allogeneic stem cell transplant (ASCT) patients for HuCV without CTH. SafV was detected in 45 of 190 (23.68%) infection to investigate the occurrence of HuCV infection, patients with CTH, in at least one sample type (7 NS, 3 to evaluate prolonged viral excretion in feces and long NW, 23 PaT and 23 PhT), and in 8 of 20 (40.0%) CTRL term viral RNA presence in sera. Fecal samples were patients (1 NS, 0 NW; 4 PaT and 4 PhT). The SafV overall collected weekly, and blood samples were obtained every two weeks from ten patients who underwent ASCT, between CTH and CTRL (p = 0.09). In addition, SafV detection frequencies were not significantly different one year. The secretor status was determined by enzyme immunoassayfor a minimum and period the detectionof five months of HuCV and was a maximum performed of betweenspecific detection CTH and frequencies CTRL (p = in0.7335 respiratory and p secretions= 0.1158, by RT-PCR using primers targeting region C of NoV respectivelyand tissue samples for secretions were also and not tissues). significantly Both different in CTH genogroup I and II (GI and GII) and SaV capsid genes. and CTRL SafV was more frequently detected in tonsillar Genomic sequencing and phylogenetic analysis were tissues (15.54% for CTH and 25.81% for CTRL) than also performed for all NoV-positive samples. The results in secretions (2.88% for CTH and 2.86% for CTRL), showed that 6/10 patients (60%) had positive samples suggesting that tonsils could be sites of SafV persistence for NoV, and all of them had a secretor phenotype. SaV and viral shedding. To the best of our knowledge, this were not detected in any of the samples. The main symptoms presented were vomiting and diarrhea. The studies including screening of blood samples of CTH is the first study of SafV infection in tonsils, and further 143 days, and long term presence of viral RNA in serum tissues, sequencing and phylogenetic analysis of these rangedduration from of NoV 29 to excretion 36 days in thefeces patients ranged infected from five with to SafVpatients, strains fluorescent are underway. in situ Financial hybridization support: in tonsillarFAPESP, NoV. Three of the six patients had acute intestinal GVHD. CNPq. All NoV-positive samples were characterized as genotype GI.3. The data highlight the urgent need of the inclusion of HV247 - FIRST REPORT OF NOROVIRUSES HuCV screening in the routine testing performed before OCCURRENCE AMONG ALLOGENEIC STEM CELL transplantation and during follow-up of these patients. TRANSPLANT RECIPIENTS IN BRAZIL: EVIDENCE FOR PROLONGED VIRAL EXCRETION AND LONG- undergoing ASCT in Brazil. FINANCIAL SUPPORT: CNPq, TERM VIRAL RNA PRESENCE IN THE BLOOD FAPEGThis is AND the firstPPGBRPH/UFG report of NoV occurrence in patients Souza, M.1; Lemes, L.N.1; Correa, T. dos S.1; Fiaccadori, F.S.1; Souza, K.M.C.1; da Silva, L.P.2; Arantes, A. de M.2; HV258 - DETECTION AND TYPING OF HUMAN Cardoso, D. das D. de P.1; PAPILLOMAVIRUS BY NESTED MULTIPLEX PCR (NMPCR) REVEALS NEW EPIDEMIOLOGICAL PROFILE 1. LABORATÓRIO DE VIROLOGIA/IPTSP/ UFG - Laboratório de Virologia do Instituto de Patologia Faria, M.G.; Matias, B.F.; Costa, E.P.; Julião, J.A.S.; Tropical e Saúde Pública da Universidade Federal de Goiás, Junior, P.C.F.; Goulart, L.R. Rua 235 s/n st. Universitário, Goiânia - Goiás, 74605-050 UFU - Universidade Federal de Uberlândia, Av. João 2. HAJ/ACCG - Hospital Araújo Jorge da Naves de Ávila, 2121 - Santa Mônica, Uberlândia - MG, Associação de Combate ao Câncer em Goiás, R. 0239, 181 - St 38408-100 Universitário, Goiânia - GO, 74605-070 Epidemiological distribution and genotypic prevalence Human caliciviruses (Norovirus and Sapovirus) are of the human papillomavirus (HPV), globally, remains important acute gastroenteritis agents. Recent studies troubling due to the strong association between HPV

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

35 Oral Presentation infection and neoplasia; present in up to 90% of cases HV262 - USE OF CHIMERIC PROTEINS EXPRESSED IN of cervical cancer. In order to increase the detection and PROKARYOTIC SYSTEM IN THE DEVELOPMENT OF molecular typing of HPV in gynecologic samples, we have A SCREENING METHOD FOR THE EVALUATION OF developed a system nested multiplex polymerase chain ANTI-HTLV-1 ANTIBODIES Santos, D.M. da S.1; do Carmo, A.P.3; Martins, M.L.2; da HPV genotypes, detected by capillary gel electrophoresis. Fonseca, F.G.1; Stancioli, E.F.B.1 Cervicalreaction (NMPCR)secretion forsamples simultaneous were collectedamplification from of 4072 women, randomly chosen at Gynecology Ambulatory 1. UFMG - Universidade Federal de Minas Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, of Clinical Hospital of Federal University of Uberlândia Belo Horizonte - MG, 31270-901 (UFU), for HPV genotyping through NMPCR technique. 2. HEMOMINAS, Rua Grão Pará, 882 - Santa In addition, epidemiological analyses were performed on Efigênia - Belo Horizonte - MG, 30622-020 database of cytological reports (Pap smears) released by 3. IF-ES pathology service of UFU. For genotypic assessment, it primer system to cover all 40 sequences of HPV genotype. retrovirus isolated from human beings and presents Human T-Lymphotropic Virus (HTLV) was the first Towas identify designed genotypes a modified of version high-, ofintermediate- the original MY09/11and low- a wide world distribution. It might be transmitted by breast feeding, sexual contact and blood transfusion. The risk, 40 specific primers were labeled with fluorophores. most important practice to fight this virus consists in typesThe results (multiple were infection) classified (74 as to positive 338 bp) in theand presencenegative against it and, in most of the cases, the carrier stays of amplification for one (single infection) or more HPV asymptomatic.prevention, once Regarding there is no the cure cross-reaction nor efficient treatmentproblems Among the 72 women studied, 45.8% were positive for linked to the currently available diagnostic methods, HPVwhen genotyping. only the β-globin In cytological gene evaluation, (366 bp) was 8.3% amplified. patients it is necessary to create new tests, which would be were observed with cell disorders suggested of HPV infection and 8.3% showed atypical squamous cells of multiepitope protein was tested by immunological tests more efficient and accurate. A recombinant HTLV-1 patients, only 6.1% were compatible with the cytological in a diagnostic test. A recombinant gene containing in order to verify its accuracy and efficiency when used analysis.undetermined A total significance of 44.4% (ASCUS).of the women Among studied the positive were highly immunoreactive epitopes was cloned in Qiagen negative for both genotyping and cytology, while 34.7% pQE30 expression vector and then transformed into showed positive genotype and negative cytology. Among Escherichia coli SG13009 cells. After different induction the patients positive for HPV by NMPCR, 39.4% were detected with single infection, while 60.6% had multiple chromatography columns. The proteins were tested by processes, the proteins were purified using affinity infections. The most prevalent type in simple viral Western blot using infected and non infected patients infection was HPV 52 (38.5%) and multiple infections were HPV 06 (30%). Notably, the discrepancy between showed the best result was used in an Enzyme Linked sera as primary antibodies. The purified protein which genotyping and cytology can be attributed to frequent Immunosorbent Assay (ELISA). In this last test, 80 false-negative smears, which may be due to inadequate different sera were used, being 40 from seronegative sampling or results misinterpretation. On the other hand, donors, 20 from asymptomatic seropositve patients false-positive Pap smears may be related to other cervical and 20 from seropositive patients presenting HAM/ diseases unrelated to HPV. The NMPCR presented in TSP. The results were analyzed using the GraphPad this study is a very promising and effective tool for HPV Prism 5 software. The statistical tests showed that the diagnostics, not only for population screenings, but also means of the HTLV seropositive and seronegative sera for clinical follow-up of patients. FINANCIAL SUPPORT: FAPEMIG, CAPES, CNPQ. the paired samples t-test. These results suggest that this proteindiffered may significantly be used in between clinical serological themselves tests according due to its to capacity of being recognized by infected patients and not by uninfected patients. Individually, both symptomatic

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

36 Oral Presentation and asymptomatic HTLV-1 seropositive patients showed Genetic distance and Shannon entropy values were the seronegative ones according to one-way ANOVA notsequences, related 27,398 to therapy ones with outcome. high quality These were sequences filtered. followedabsorbance by Tukey values test which (p=0.0001). differed This significantly result indicates from were analyzed using median-joining networks and that the protein could be used to identify the presence of Bayesian population structural analysis. These analysis HTLV-1 virus even in situations, such as blood donations, in which the symptoms absence suggests no disease. FINANCIAL SUPPORT: CAPES, FAPEMIG AND CNPQ identified samples with different structures, from high thisconserved result. (oneMutations sub-population) exclusive forto higha type stratified of response ones HV311 - HEPATITIS C VIRUS QUASISPECIES ANALYSIS (6 sub-populations). Networks analysis also confirmed USING ULTRA-DEEP PYROSEQUENCING sequences indicated that this region presents conserved Yamasaki, L.H.T.; Khudyakov, Y.; Vaughan, G.; Raeva, structure,of therapy even were if sequence identified and along physical HVR1. and Amino chemicals acid L.M.G.; Diminitrova, Z.E.; Skums, P.; Rendon, D.S.C.; Jardim, A.C.G.; Bittar, C.; Mello, I.M.V.G.C.; Rahal, P. positions, this conservation seems notable. Potential properties seem flexible. Especially in turns and coils 1. UNESP/IBILCE - Universidade Estadual epitopes positions are concentrated in carboxiterminal Paulista - Instituto de Biociências, Letras e Ciências Exatas, and can vary of number and size between quasispecies Rua Cristóvão Colombo, 2265 Bairro Jardim Nazareth, São These results will contribute to the understanding of José do Rio Preto - SP, 15054-000 HCV quasispecies dynamics and therapy and how a high 2. CDC/HEPATITIS DIVISION - Centers for resolution tool as UPDS is essential to it. Disease Control and Prevention 1600 Clifton Road Atlanta, GA 30329-4027, USA HV340 - EFFECTS OF HTLV-1 INFECTION ON 3. UFU - Universidade Federal de Uberlândia, BONE MARROW CELLS FROM HTLV-1 INFECTED Av. João Naves de Ávila, 2121 - Santa Mônica, Uberlândia - INDIVIDUALS MG, 38408-100 Rodrigues, E.S.1; Favarin, M. do C.1; Macedo, M.D. de1; Hepatitis C is a major public health problem. New Otaguiri, K.K.1; Orellana, M.D.1; Takayanagui, O.M.2; HCV antiviral drugs were released on market on 2010; Slavov, S.N.1; Covas, D.T.1; Kashima, S.1 however, excluding for genotype 1, the most used 1. Regional Blood Center of Ribeirão Preto, therapy used currently is still based on Interferon (IFN) Faculty of Medicine of Ribeirão Preto, University of São Paulo and Ribavirin. Nowadays, genotype 3 is the one with the (Usp); Faculty of Pharmaceutical Sciences of Ribeirão Preto highest rate of treatment failure. Viral genome variability 2. Faculty of Medicine of Ribeirão Preto, Usp, is one of the factors that lead in therapy failure. HCV Brazil presents a high mutability during replication course, The infection of mesenchymal stromal cell (MSC) by implicating in arising of intra-host variants called the human T lymphotropic virus (HTLV-1) in vitro can quasispecies. The hypervariable region 1 (HVR1) from induce alterations in the biological characteristics of envelope protein presents as quasispecies and may be these cells. However, little is known about the effects of related to IFN therapy resistance. Resistant quasispecies this infection on the bone marrow (BM) cells of HTLV-1 may not represent majority of variants population in the infected individuals and the MSC biological functions in host, therefore, in these cases traditional sequencing these patients. Objective: Our objective was to evaluate techniques are unable to detect. For detection of the HTLV-1 infection in BM cells isolated from HTLV-1 minority quasispecies, ultra-deep pyrosequencing asymptomatic carriers (HAC) and symptomatic patients with tropical spastic paraparesis/HTLV-associated even variants with frequency <1% in the population. myelopathy-1 (HAM/TSP). Methods: BM mononuclear Regarding(UPDS) is a this reliable issue, and we efficient determined tool, beingHVR1 ablequasispecies to detect from 14 patients infected with HCV genotype 3 using (CD3, CD4, CD8, CD14, CD19, CD34, CD45, CD73 and the UPDS approach. In total, 64,400 HVR1 sequences cells were isolated and characterized by flow cytometry were obtained from pre-therapy sample. From these cells were separated. In both populations, we evaluated CD105). From these cells, CD4+ T cells and mesenchymal September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

37 Oral Presentation the presence of HTLV-1 by PCR, confocal analysis of the immunoassays kits. Associated risk factors and spatial clustering of HEV seropositivity were also analyzed. Anti-HEV IgG were detected in 50 out of 388 settlers lymphocytesp19 specific proteinin BM from (Gag) HTLV-1 and by infected EIA (p19 individuals protein). (12.9%, 95% CI, 9.5-16.2%), a HEV seropositivity rate whenResults: compared Initially, to we the observed group of annon-infected infiltration individuals. of CD4+ T substantially higher than those previously found across Additionally, the detection of proviral DNA by PCR the Amazon Basin and other regions in Brazil. HEV IgM revealed the presence of integrated provirus in the antibodies were detected in 7/43 (16.3%) anti-IgG cells colonies (CFU-F) was lower in HTLV-1 infected tested CD4+ T cells. The number of fibroblast progenitor determinantpositive samples, of HEV and seropositivity 4 of them had detected a confirmed by multilevel result logisticby immunoblot. regression Increasing analysis age(OR, was 1.033; the only95% significantCI, 1.016- individuals (CFU-F/5 x 105 cells in HAM/TSP (2,11,6), showedHAC (7,52,1), the expression when compared of typical to surface non-infected molecules, subjects and HEV seropositivity was detected in the area. This study differentiation(10,41,1). MSCs potential isolated into from adipocytes HTLV-1 infected and osteocytes patients provides1.050; P data < 0.001). on the No prevalence significant of HEV spatial in a clusteringrural setting of similar to the control MSCs. Moreover, proviral DNA of the Brazilian Amazon. Anti-HEV prevalence observed and p19 viral protein were detected in MSC in the in the present study was considerably higher than those majority of HTLV-1 patients. However, no p19 antigen previously reported in Brazil. Moreover, the detection was detected in MSC supernatant obtained from HTLV- 1 patients Conclusion: These results suggest that MSCs individuals is highly suggestive of the circulation of HEV are potential reservoirs for HTLV-1 infection and can inof this HEV- rural specific population. IgM antibodies FINANCIAL in fourSUPPORT: asymptomatic FAPERJ, contribute to viral dissemination and persistence in the FAPESP, CNPQ host. These results also can elucidate the pathogenesis of HTLV-1 and the related diseases. FINANCIAL SUPPORT: HV353 - IMPLEMENTATION OF NEXT GENERATION CTC, INCTC, FAPESP, FUNDHERP and CNPq. SEQUENCING TECHNIQUES FOR PATHOGEN DISCOVERY IN CEREBROSPINAL FLUID OF PATIENTS HV346 - EVIDENCE OF HEPATITIS E VIRUS WITH ENCEPHALITIS AND MENINGITIS CIRCULATION IN RURAL AMAZONIA Nunes, C.F.; Soares, A.C.; Urbano, P.R.; Gerhardt, D.; Merlone, M.P.1; Vitral, C.L.1; Oliveira, J.M.3; Silva, J.P.1; Romano, C.M. Nunes, M.S.2; Ferreira, M.U.2; Pinto, M.A.3 IMT/USP - Instituto de Medicina Tropical de São Paulo 1. UFF - Universidade Federal Fluminense, da Universidade de São Paulo, Av. Dr. Enéas de Carvalho Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 Aguiar, 470, Jardim América, São Paulo - SP, 05403-000 2. USP - Universidade de São Paulo, Av. Prof. The central nervous system (CNS) may be affected by Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 several agents, including viral bacterial or fungal. CNS 3. FIOCRUZ - Fundação Oswaldo Cruz, Av. infections can trigger severe symptoms and, according to Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 the site of infection, maybe designated as encephalitis or Hepatitis E virus (HEV) is transmitted by the faecal-oral meningitis. Viruses are the most common cause of these route, and represent a common cause of acute hepatitis in diseases, followed by bacteria and fungus. Agents such developing countries. Human cases of HEV infection seem as polyomavirus, Herpesvirus (Simplex, 6 and varicella- to be rare in Brazil, although the virus has been detected tuberculosis and C. neoformans, are responsible for study was to determine the epidemiology of hepatitis mostzoster) of influenzathe CNS infections, A, enterovirus, and have mumps, a high flavivirus, incidence M. in swine livestock and effluents of slaughterhouses. This E in one of the largest agricultural settlements in the worldwide. However, prevalence of different agents Amazon Basin of Brazil. Serum samples collected from varies according to population, individual’s immune 397 individuals aged between 5 and 90 years during a status, age and region of study. In fact, the prevalence population-based cross-sectional survey were tested of these infectious agents is well established in many for anti-HEV antibodies using commercial enzyme countries. However, there is a lack of information

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

38 Oral Presentation regarding the prevalence of these agents in the Brazilian the advent of PCR has greatly increased the sensitivity population. Although there are diagnostic methods of genome detection and, as a consequence, the available for identifying most of the etiologic agents simultaneous detection of multiple viruses has become that cause encephalitis and meningitis (EM), to obtain very frequent, challenging the establishment of disease results in short period is essential for targeting the causality. Chronic tonsillar hypertrophy (CTH) is a very most appropriate treatment of the disease. Here we use frequent ailment and genomes of multiple respiratory next generation technology (Ion Torrent) to investigate viruses are detectable by qPCR in nasopharyngeal putative microbial agents in CNS infections through washes (NW) and tissues from CTH patients without ARI metagenomic approaches in liquor. The advantage of this symptoms. This prompted a search for in situ evidence technique over traditional ones is the capability to detect of productive viral infections in adenoid tissues from not only known agents, but also identify pathogens not patients who shed two or more viruses in respiratory commonly associated to these pathologies in a fast way. secretions. The patients were 179 children with CTH Samples (n=4) from patients with suspected viral EM without ARI symptoms who underwent tonsillectomy is were spiked before extraction with baculovirus and at the Otorhinolaryngology Clinic, University of Sao bovine viral diarrhea viruses as a control for acid nucleic Paulo Hospital in Ribeirao Preto. Remarkably, 47.47% extraction. After a set of centrifugation steps to exclude (85/179) had at least 2 viruses, and 21.78% (39/179) of human cells, genomic DNA/RNA was obtained using patients had 3 or more viruses detected in NW by qPCR. Macherey Nagel commercial kit. Reverse transcription In addition, one quarter of them (45/179; 25.13%) had was performed with random primers using High 3 or more viruses detected directly in the adenoid tissue Capacity (Applied Biosystems) kit. Double-stranded DNA was synthesized using Klenow enzyme Fragment (3’>5 adenoid sections from patients with 2 or more viruses ‘exo-) (NEB), followed by NGS sequencing. Filtering, by qPCR. Selected formalin fixed, paraffin-embedded trimming and assemble of the reads were performed in (IF) for major human respiratory viruses: respiratory CLC genomic workbench software. Our methods appear syncytialdetected invirus NW were(HRSV), stained metapneumovirus by immunofluorescence (HMPV), the samples as internal control were recovered during rhinovirus (HRV), adenovirus (HAdV) and Epstein– analysis.to be efficient In one since sample small investigated, reads of viruses around used 150 to contigs spike Barrparainfluenza virus (EBV). virus Slides (HPIV),were analyzed enterovirus by confocal (HEV), and of Burkolderia (bacterial agent) were assembled. In multiphoton microscopy. Results showed that in most conclusion, we demonstrated that the metagenomic cases (15 patients tested were positive by IF at least methods implemented in the present work are capable for 2 viruses) structural proteins of the same viruses to detect viral and bacterial agents affecting the CNS. co-detected by qPCR in NW were also present in situ by IF in hypertrophic adenoid sections. Importantly, these HV417 - CO-DETECTIONS OF RESPIRATORY VIRUSES BY QPCR IN THE ABSENCE OF SYMPTOMS OF ACUTE ARI symptoms may shed multiple viruses in secretions, RESPIRATORY INFECTION (ARI): NEW INSIGHTS ON duefindings to ongoingstrongly indicateviral activity that children within with a hypertrophic CTH without SOURCES OF VIRUS SHEDDING adenoid. Financial support: FAPESP, CNPq, CAPES Criado, M.F.1; Modena, J.L.P.1; de Paula, F.E.1; de Jesus, B.L.S.1; Pestana, N.F.1; Prates, M.C.1; Silva, M.L.1; HV428 - ANALYSIS OF INFLUENZA A(H1N1)PDM09 Saturno, T.1; Tamashiro, E.2; Valera, F.C.P.2; Lima, VIRAL LOAD IN PATIENTS WITH DIFFERENT W.T.A.2; Arruda, E.1 CLINICAL PRESENTATION Perosa, A.H.; Bellei, N. 1. University of Sao Paulo, School of Medicine, Virology Research Center UNIFESP - Universidade Federal de São Paulo, R. Sena 2. University of Sao Paulo, School of Medicine Madureira, 1500 - Vila Mariana, São Paulo - SP, 04021-001 Viral acute respiratory infections (ARI) are the most frequent illnesses of mankind. Diagnosis of respiratory viruses is useful in medicine and public health, and andThe kineticsthe optimal of viral timing load hasof therapy not been with sufficiently antiviral studied drugs in patients with pandemic 2009 influenza A(H1N1) September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

39 Oral Presentation for critically ill patients is not well established. We Viral and host factors have been described to play a performed viral load analysis in samples from patients role on the different patterns of AIDS progression. The co-circulation of HIV-1 subtype B, F, variant B” pdm09 admitted to Sao Paulo Hospital from July 2009 of B subtype and BF1 recombinants has been fully to(children August and 2013. adults) We withalso confirmedstudied consecutive influenza A(H1N1)samples described in Rio de Janeiro, Brazil. Thus, the aim of this from hospitalized patients which received oseltamivir study was to evaluate the potential association of HIV-1 subtypes circulating in Rio de Janeiro with the distinct was constructed with the target region of M gene from treatment. A standard curve for viral RNA quantification HIV-1 patients under clinical and laboratory follow- to RNaseP to adjust the quantity of virus detected for profiles of disease progression. For this purpose, 246 theinfluenza quality A ofand the viral samples. loads We were studied normalized 153 nasal according swabs according to their progression to AIDS in typical collected from 76 hospitalized patients (mean age ± progressorsup at INI/FIOCRUZ (133; TP), from rapid 1986 progressors to 2011 were (95; classified RP) and standard deviation, 31.3 ± 21.3), 65 ambulatory patients long term non-progressor (18; LTNP). HIV-1 proviral (11.7 ± 14.9) and 12 asymptomatic individuals (36.7 ± 6.1). The mean viral load in the 153 collected samples was sequenced. Neighbor Joining phylogenetic inferences 6.57 (±1.78) log copies/mL. Viral load in symptomatic wereDNA wereperformed amplified in Mega by env-gp120 5 program. nested Chi-square PCR and test then or Fisher\’s exact test were used to compare the groups. (mean ± standard deviation, 6.74 ± 1.69 vs 4.77 ± 1.78 Kaplan Meier method and Cox modeling were performed logpatients copies/mL, was significantly p<0.001). Ten higher hospitalized than asymptomatic patients with to analyze the time until AIDS progression according to severe acute respiratory illness collected during 2013 followed up and samples were collected until clinical asthe follows: HIV subtypes/variants. TP (63.1% B, 24.8% The B”,classification 8.3% F1, 2.2% of the C, HIV-1 0.8% improvementinfluenza season and/or and reduction treated within viral oseltamivir load. Maximum were BF1subtypes and 0.8%according BD); among RP (53.7% the progression B, 24.2% B”, profiles 11.5% were F1, duration of treatment was 30 days. The mean viral 4.2% C, 3.2% D, 2.1% BF1 and 1.1% CRF01_AE) and LTNP load for these patients was 6.64 ± 1.82 log copies/mL. (66.7% B, 27.7% B” and 5.6% F1). Similar distribution of Viral RNA concentration decreased with treatment and HIV-1B and HIV-1B” was observed for the three studied prolonged viral shedding (> 7 days after treatment groups. A trend for a higher frequency of HIV-1F1 was onset) was observed in 70% (7/10) of these patients. observed for the RP group compared to either TP or Slower viral clearance was observed in patients with major comorbidities like chronic liver disease, lupus, differences were made evident in these comparisons. In cardiac disease and leukemia, even under prolonged theLTNP survival groups, analysis, even thoughno differences no statistically in progression significant rate were observed for subtypes B, B” and F1. On the other hand, the group including other less prevalent subtypes treatmenttreatment. management.FINANCIAL These data showed thatSUPPORT: quantification FAPESP (2013/00715-0),of influenza viral CNPQ load would be useful for antiviral of progression to AIDS (HR 1.99 [IC95% 1.16; 3.40] P<0.02).(C, D) and Despite recombinants the fact of being presented a heterogeneous a significant group, risk HV436 - HIV-1 ENV SUBTYPES AND DISEASE probably the presence of HIV-1D strongly contributed to PROGRESSION IN A COHORT OF HIV-1 POSITIVE this association, since this subtype was found here only INDIVIDUALS FROM RIO DE JANEIRO, BRAZIL Leite, T.1; Campos, D.2; Coelho, A.2; Teixeira, S.1; Veloso, described. However, in the multivariate analysis this V. 2; Guimarães, M.1; Morgado, M.G.1 associationin RP group lostand itshas statistical rapid evolution power. profileOur results as previously contrast 1. IOC/FIOCRUZ - Instituto Oswaldo Cruz da with previous ones that described a slowly disease Fundação Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, Rio progression in B” group in comparison with HIV-1 de Janeiro - RJ, 21040-360 subtype B. Taken together, these data try to contribute to 2. INI/FIOCRUZ - Instituto Nacional de the discussion of the possible role of HIV-1 subtypes in Infectologia Evandro Chagas da Fundação Oswaldo Cruz, Av. determining the HIV-1 infection outcomes. Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-360 September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

40 Oral Presentation

HV491 - OPTIMIZATION OF A LOOP-MEDIATED ISOTHERMAL AMPLIFICATION ASSAY FOR NAKED- with Brazilian samples. FINANCIAL SUPPORT: FAPEAM EYE DETECTION OF DENGUE VIRUS INFECTION -order PAIC; toFAPEAM confirm - PPSUS the efficiency REDE; POM of the- FIOCRUZ LAMP protocol Monteiro, D.C.S.; Carvalho, B.K.S.; Nascimento, V.A.; IV100 - EVALUATION OF TETRAVALENT AND Souza, V.C.; Naveca, F.G. CONSERVED SYNTHETIC PEPTIDES VACCINES ILMD/Fiocruz - Instituto Leônidas e Maria Deane - DERIVED FROM DENGUE VIRUS ENVELOPE DOMAIN Fiocruz Amazônia, Rua Terezina, 476 - Adrianópolis, Manaus I AND II - AM, 69057-070 Rocha, R.P.1; Franco, I.R.1; Livonesi, M.C.1; Fumagalli, M.J.1; Rodrigues, N.F.1; da Costa, L.C.F.1; dos Santos, M.C. da S.G.1; Rocha, E.S. de O.2; Kroon, E.G.2; Malaquias, minimumThe development equipped of laboratories, a simplified may system improve for access nucleic to L.C.C.1; Coelho, L.F.L.1 molecularacid amplification, diagnosis of with infectious the possibility diseases, especially of using at 1. UNIFAL - Universidade Federal de Alfenas, resource-poor settings. The Loop-Mediated Isothermal R. Gabriel Monteiro da Silva, 714 - Centro, Alfenas - MG, 37130-000 2. UFMG - Universidade Federal de Minas Amplification (LAMP) is an example of a very sensitive its development, LAMP has been used to detect various Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, pathogenstechnique whichincluding amplifies bacterias, DNA at fungi very highand levels.viruses. Since In Belo Horizonte - MG, 31270-901 the present study, a previously published protocol for Dengue is a major public health problem worldwide, dengue virus (DENV) detection was reevaluated for especially in the tropical and subtropical regions of the best work conditions and naked-eye visualization the world. Primary infection with a single Dengue using a Brazilian DENV isolate. Firstly, a laboratory virus (DENV) serotype causes a mild, self-limiting sample of DENV serotype 4 virus was propagated in febrile illness called dengue fever. However, a subset Aedes albopictus C6/36 cells for ten days at 28°C. of patients experiencing secondary infection with a Subsequently, RNA was extracted from cell supernatant different serotype progress to the severe form of the with a commercial kit and converted to cDNA with disease named dengue hemorrhagic fever (DHF). In this GoScript (Promega). The positivity to DENV infection study, the vaccine potential of a three tetravalent and conserved synthetic peptides derived from Dengue virus described elsewhere. Variables such as temperature envelope domain I (named Pep01) and II (named Pep02 (62°C,was further 63°C, 64°C, confirmed 65°C, by66°C a semi-nestedand 67°C) and PCR incubation protocol and Pep03) was evaluated. A panel of Dengue IgM/IgG time (30, 45, 60, 90 and 120 minutes), as well as positive human serum was used to determine their ability reagents concentrations were evaluated. Moreover, to recognize the synthetic peptides using an indirect the addition of hydroxynaphthol blue (HNB), a reagent ELISA assay. Of a total of 16 dengue IgM/IgG positive that may allow to the visualization of LAMP results serum, only 3 (18.75%) showed IgM against Pep01; 15 by naked-eye, was also tested. All conditions were (93.75%) against Pep02 and 16 (100%) against Pep03. evaluated in triplicate with positive and no-template The presence of IgG against the peptides was evaluated controls. Finally, all reactions were visually analyzed for and14 sera (87.5%) reacted against Pep01; 11 (68.75%) color changes and further subjected to electrophoresis against Pep 02 and 15 (93.75%) against Pep03. Mice immunization experiments showed that these peptides results were achieved when the reaction was conducted were able to induce a humoral response with an absence aton 65°C 2% agaroseover a one-hour gel to confirm period, amplification.with eight units The of best Bst or a low titer of neutralizing activity. The IgM mean titers DNA polymerase; 8mM of MgSO4; 1,4mM of dNTPs; were 37 ± 17 for Pep01 and 300 ± 141 for Pep03. Sera 1M of betaine; 1,6uM/0,2uM/0,8uM of FIP/BIP, F3/B3 from Pep02 immunized animals showed absence of IgM. and Loop primers, respectively. The color change due a The mean total IgG titers of Pep01, Pep02and Pep03 positive reaction was clearly observed when 120uM of were 96 ± 56; 133 ± 46 and 4800 ± 2262, respectively. HNB was included. Further studies, including samples The spleen cells derived from mice immunized with representing all four serotypes, are been conducted in September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

41 Oral Presentation the peptides and infected with a low viral dose of non- adjuvants. The groups given WIV (WIV-MVP, WIV-AddaV, WIV-Gel, WIV-ISA) were vaccinated at 4 weeks of age (only for Pep02 and Pep03), a high expression of IL-10 and were boosted at 7 weeks of age by an intramuscular adapted DENV-1 showed a significant cytotoxic activity route. The 5th group, WIV-IMS, was vaccinated at the IFN-g (P<0.001) compared to DENV-1 derived spleen same time points by the intranasal route. The control cells.(P<0.01) Thus as these well peptides, as reduced and expression specially the of TNF-αPep03, andcan groups included non-vaccinated and challenged pigs induce a humoral response characterized by antibodies (NV/C) and non-vaccinated, non-challenged pigs (NV/ with low neutralizing activities and probably a T cell NC). WIV-MVP (22.1%) and WIV-ISA (21.7%) groups immune response against all DENV serotypes and do lesions consistent with VAERD compared with WIV- notresponse contributed that could to the be immunopathogenesis.beneficial to induce an However, effective AddaVhad significantly (11.3%), higherWIV-Gel percentage (9.3%), WIV-IMS of macroscopic (8.1%) lungand further studies in peptide sequence will be required controls NV/C (6.1%) and NV/NC (0.3%). At the time to induce the production of neutralizing antibodies of challenge, WIV-MVP and WIV-ISA groups showed the against all four DENV serotypes and also to improve greatest geometric mean reciprocal hemagglutination immunogenicity of these peptides. FINANCIAL SUPPORT: inhibition antibody (HI) titers of 640±13 and 1371±12, FAPEMIG; CNPQ; CAPES. respectively, against the homologous vaccine virus. The WIV-AddaV and WIV-Gel groups had mean reciprocal IV183 - THE IMPACT OF ADJUVANT IN WHOLE titers of 149±13 and 184±13, respectively. The WIV-IMS INACTIVATED VIRUS (WIV) VACCINES FOR INFLUENZA and non-vaccinated control groups had HI mean titers A INFECTION IN PIGS WITH VACCINE-ASSOCIATED below the positive cut-off (20). The WIV-IMS did not ENHANCED DISEASE result in VAERD following heterologous challenge, but Souza, K.C.1; Rajao, D.2; Loving, L.C.2; Gauger, C.P.3; also failed to elicit HI antibodies to vaccine antigen. None Vincent, L.A.2 of the groups had cross-reactive HI antibodies against 1. UFRGS - Universidade Federal do Rio the challenge strain (pH1N1). The WIV-MVP and WIV- Grande do Sul, Avenida Paulo Gama, 110 - Farropilhas, Porto Alegre - RS, 90040-060 in serum against homologous and heterologous virus; 2. NATIONAL ANIMAL DISEASE CENTER/ however,ISA groups the had WIV-AddaVsignificantly higherand WIV-Gel levels of IgGgroups antibody also UNITED STATES DEPARTMENT OF AGRICULTURE, had serum IgG antibody against both viruses. These Department of Animal Science 1221 Kildee HallIowa State University Ames, Iowa 50011-3150 immunogenicity and subsequently, a role in VAERD. 3. IASTATE - Iowa State University, Ames, data show that adjuvant plays a significant role in WIV Iowa 50011 IV218 - SELECTION AND CHARACTERIZATION Adjuvants can improve vaccines by modulating and/or FINANCIAL SUPPORT: USDA-ARS, CAPES AND CNPQ. OF MIMOTOPES THROUGH PHAGE DISPLAY FOR magnifying the immune response. Whole inactivated IMMUNOGLOBULIN A DETECTION IN HPV DIAGNOSIS virus (WIV) vaccines can reduce clinical disease against Matias, B.F.; Lima, M.I.S.; Faria, M.G.; Costa, E.P.; Alves, WIV vaccines using oil-in-water adjuvants have been P.T. Pereira, U.P.; Fernandes Jr, P.C.; Goulart, L.R. homologous influenza A virus (IAV) infection; however, associated with enhanced respiratory disease in swine UFU - Universidade Federal de Uberlândia, Av. João when challenged with heterologous IAV of the same Naves de Ávila, 2121 - Santa Mônica, Uberlândia - MG, hemagglutinin subtype. We evaluated the immune 38408-100 response of WIV with different adjuvants in a swine model Screening programs based on Pap smear in most of vaccine-associated enhanced respiratory disease developing countries are not well established and often (VAERD) to test the hypothesis that different types of fail in HPV detection and prevention of cervical cancer adjuvants similarly predispose to VAERD. Pigs were was to develop a new and simple strategy based on IgAdue detection to its lack during of sensitivity HPV infection. and specificity. The strategy Our was aim vaccinated with WIV containing swine δ-cluster H1N2 (human-like,September/October MN08) 2014 IAVVolume formulated 19 – Supplement with 2five - Abstracts/Posters different - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

42 Oral Presentation to identify epitopes or mimotopes by phage display transfection with cloned cDNA of hemagglutinin (HA) that could bind to IgA antibodies in saliva and cervical and neuraminidase segments from wild-type strain secretion samples from patients with HPV infection. We have selected mimetic peptides to the capsid regions strain. However, theses clones are obtained by use of of HPV through a commercial library of random 7-mer plus six segments from influenza A/PR/8/34 (PR8) cervical secretion samples on 15 women that were homologousspecific restriction recombination sites and in in yeast vitro ligation,(HRY) cloning which peptide library (Ph-D.C7C) against purified IgA of sometimes are difficult to perform. In advantage, the and DNA genotyping. After three rounds of selection, 96 fragments containing homologous ends with the vector clonespositive were to high-risk isolated HPV,and sequenced,confirmed bygenerating both Pap 31 smears valid cantechnique be directly is an clonedefficient using and simplein vivo process,recombination. where DNA The sequences. In silico analyses (BLAST, ClustalW2 and I-Tasser programs) revealed 4 sequences that presented the egg supply in pandemic situation, e.g. as occurred in partial alignment to antigenic regions of HPV, such as L1 2009currently (pnd2009). influenza Additionally, vaccine also isthe limited WHO byrecommends capacity of and L2 viral capsid proteins. Clones were submitted to Vero cells as an alternative substrate for recombinant ELISA assay to assess their immunoreactivity against IgA from cervical secretions and saliva of healthy and grow sub optimally in this cell line. Recently, were HPV-infected women. Statistical analysis (ANOVA with reportedinfluenza the vaccine enhanced production, growth of however H1N1 in some Vero virusescell by Bonferroni post-test) showed that clones differed changing an amino acid residue in HA (117N>D). Thus, and also against the control (Irrelevant phage). Our clonessignificantly are potential in both biomarkers positive andfor HPV negative screening samples and to overcome the difficulties of producing recombinant therapeutic monitoring, distinguishing infected patients pandemic influenza vaccine, the objective this work from healthy individuals. These results provide new ofwas the the HA/117N>D development mutation of the (by first HRY) reverse in a chimeric genetics perspectives for HPV detection with a fast and non- virussystem expressing of influenza HA madefrom byisolated HRY andpandemic the insertion (2009) invasive screening strategy. FINANCIAL SUPPORT: capable of improved replication in Vero cells. For this, FAPEMIG; CAPES; CNPQ. the pJG-HW2000-2013 vector was constructed and

IV477 - INFLUENZA: DEVELOPMENT OF A REVERSE GENETICS SYSTEM BY YEAST-BASED HOMOLOGOUS bythe HRY mutation from pJG-HW2000 effect was first to pJG-HW2000-2013. evaluated in prototype The RECOMBINATION AND ITS APPLICATION IN THE pJG-HW2000-2013/PR8influenza PR8 strain. The was PR8 transfected genome wasin HEK subcloned 293T/ IMPROVED VACCINE PRODUCTION IN CULTURE CELL MDCK co-culture and recombinant PR8 (IC-PR8) was Silva Jr, J.V.J.1; Cruz, F. da S.P.1; Bertani, G.R.2; Machado, generated. After, the HA/117N>D mutation was inserted A. de M.V.3; Gil, L.H.V.G.1 by HRY into pJG-HW2000-2013/PR8 and mutant virus 1. CPQAM/FIOCRUZ - Centro de Pesquisas (IC-PR8/HA-117N>D) was generated. Plaque assay Aggeu Magalhães da Fundação Oswaldo Cruz, Av. Professor performed in Vero cell showed replication larger in IC- Moraes Rego, s/n - Campus da UFPE - Cidade Universitária, PR8/HA-117N>D than in IC-PR8. After, the HA segment Recife - PE, 50740-465 2. UFPE - Universidade Federal de Pernambuco, HW2000-2013 and the clone was co-transfected with Av. Prof. Morais Rego, 1235 - Cidade Universitária, Recife - pJG-HW2000-2013/PR8of 2009 influenza isolate andwas clonedchimeric by virusHRY intoIC-PR8/ pJG- PE, 50670-901 HApnd2009 was generated. The mutation HA/117N>D 3. CPqRR/FIOCRUZ MINAS - Centro de was inserted by HRY in IC-PR8/HApnd2009 and mutant Pesquisas René Rachou/ Fundação Oswaldo Cruz, Av. Augusto virus (IC-PR8/HApnd2009-117N>D) was recovered. de Lima, 1715 - Barro Preto, Belo Horizonte - MG, 30190-002 Comparison of the plaque assay and viral growth kinetics between IC-PR8/HApnd2009-117N>D and IC- genetic reverse system and grown in embryonated PR8/HApnd2009 in Vero cell is in progress. FINANCIAL chickenRecombinant eggs. influenzaThe viruses vaccines are generated are produced by cell using co- SUPPORT: CAPES, CNPQ, FIOCRUZ

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

43 Oral Presentation

IV482 - RECONSTRUCTION OF LATENT AND with the ones present in reservoirs , demonstrated their REACTIVATED QUASISPECIES OF SIVMM251 INFECTING RHESUS MONKEYS AFTER TREATMENT potential of deep sequencing for the characterization of WITH THE LATENCY ANTAGONIST INGENOL-B quasispeciesrelation with anddifferent Ingenol-B organs. as potentThese latencyfindings antagonist show the Gonçalves, G. dos S.1; Abreu, C.M.1; Price, S.L.2; Gama, in vivo. L.2; Lewis, M.3; Pianowski, L.F.4; Tanuri, A.1 PIV150 - TWO NOVEL BEGOMOVIRUS SPECIES FROM 1. UFRJ - Universidade Federal do Rio de THE NEW WORLD WITH FEATURES RECALLING OLD Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de WORLD BEGOMOVIRUSES Janeiro - RJ, 21941-901 Godinho, M.T.; Lima, A.T.M.; Xavier, C.A.D.; Zerbini, 2. JHU - The Johns Hopkins University, F.M. Baltimore, MD 21218, Estados Unidos 3. BIOQUAL DFP/UFV - Departamento de Fitopatologia da 4. KYOLAB, Rua Isaura Ap. Oliveira Barbosa Universidade Federal de Viçosa, Campus Universitário, Viçosa Terini n° 231,Jd. Itapuã, Valinhos - SP, 13273-105 - MG, 36570-000 Viral latency is one the major obstacles for obtaining a Begomoviruses (family ) have a circular, cure for HIV infection. Recently, it was proposed a new ssDNA genome encapsidated in twinned icosahedral strategy (“shock-and-kill”) using drugs that reactivate particles. In Brazil, a number of begomoviruses have latent virus in combination with antiretrovirals. It is been described infecting weeds. Here, we describe two important to evaluate the potential of latency antagonist novel begomovirus species infecting Sida acuta plants in reaching viral reservoir, in order to develop an collected from a small area (about 10,000 m2) at Viçosa, state of Minas Gerais in December 2011. Total DNA was that reactivates integrated HIV-1 in J-Lat cells and in extracted from S. acuta samples and the viral genome restingefficient CD4 strategy. T cells Ingenol-B (from humans is a promising and monkeys). PKC agonistRhesus monkeys infected with SIV is an important model to 12 full-length DNA-A component were obtained from study HIV infection. We have analysed the hypervariable fourwas amplifiedsamples, byand RCA, the cloned ICTV-established and sequenced. 89% A totalDNA-A of region V1-V2 of gp120 from SIVmm251 infecting identity threshold was used for taxonomic placement. two rhesus monkeys treated orally with Ingenol-B This analysis indicated that the cloned components throughout 9 weeks, in a dose escalating protocol (1.0 correspond to two novel species, for which the names , 2.5, and 5.0 mg BID), with 7 days cycles on and off Sida golden yellow mosaic virus and Sida yellow spot drugs. Samples from PBMC and plasma from on and virus (SiGYMV and SiYSV, respectively) are proposed. off drug cycles, as well as organs autopsies were used The DNA-A components exhibited a highly divergent 5´ in the study. An amplicon sequencing was performed in half , including part of the intergenic region, the putative Illumina MiSeq sequencer. The data generated for each CP gene and an AV2-like ORF (present only in Old Word sample was submitted to quasispecies reconstruction begomoviruses). The deduced amino acid sequence of using QuRe software, producing different haplotypes the CP had very low identity with other begomoviruses, (subpopulations) and their related percentage. The but the presence of conserved motifs in the CP and Rep gp120 region of reconstructed quasispecies showed a coding regions, characteristic of OW begomoviruses, was detected. Although New World-like begomoviruses subpopulations inside the same sample. The Shannon entropysignificant analysis diversity of thebetween subpopulation samples and diversity even between in each OW-like begomoviruses are found naturally in the NW. sample showed a direct relation with the viral load and FINANCIALhave been foundSUPPORT: in the FAPEMIG, OW, this CAPES is the AND first CNPQ time that in response to Ingenol-B. A compartmentalization of quasispeciesthe diversity in circulating reservoir organs in plasma, was also both observed. fluctuating New subpopulations circulating in plasma emerged during the treatment and the comparison of these subpopulations

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

44 Oral Presentation

PIV160 - COMPLETE GENOME SEQUENCE OF TWO us for the assembly of viral genomes using small RNA NEW VIRUSES ISOLATES ASSOCIATED TO COTTON data sets, the SearchSmallRNA, freely available at http:// BLUE DISEASE BREAKING RESISTANCE IN BRAZIL www.microbiologia.ufrj.br/ssrna/. Genomes sequences Vaslin, M.F.S.1; Fausto, A.K. da S.1; Romanel, E.1; Silva, with 99% and 99,3% of coverage were obtained for T. da F.1; Schrago, C.G.1; Galbieri, R.2; Bélot, J.L.2; Vasli, Acr3 and IMA2, respectively, using CLRDV-PV1 Brazilian M.F.S.1 isolate (HQ827780.2) as reference genome. After that new analysis were performed by the mapping software 1. UFRJ - Universidade Federal do Rio de using the reconstructed genomes as reference genome. Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de Complete putative genome sequences were obtained for Janeiro - RJ, 21941-901 both isolates. RT-PCR assays were performed to validate 2. IMAMT - Instituto Mato-Grossense do the reconstruct genomes and the resulting amplicons Algodão, Av. Rubens de Mendonça, 157. Sala 100, Ed. Mestre Ignácio. Baú, Cuiabá - MT, 78008-000 time the complete genome of these two viruses isolates. Cotton blue disease (CBD) is an important disease that Identitieswere sequenced analysis by showedSanger. Herethat wethe describe new isolates for the share first affects cotton crops in Asia, South America and Africa. high nucleotide and amino acid identities with Cotton In Brazil it is present in almost all the cotton crop areas, leafroll dwarf virus, causal agent of Cotton blue disease. leading to important productivity losses by up to 80% Although, theirs P0 protein are 86.1% identical, indicating in cotton production. The symptoms include leaf rolling, that these new isolates represent a new intense green foliage and a severe to moderate stunting. species. We propose the name Cotton atypical red leaf The disease is transmitted by the Aphis gossypii (Glover) virus (CARLV) to this new virus. FINANCIAL SUPPORT: and associated to Cotton leafroll dwarf virus (CLRDV) CAPES AND FAPERJ from the family Luteoviridae, genus Polerovirus. In order to by pass productivity losses associated, Brazilian cotton PIV210 - SYSTEMIC ACQUIRE RESISTANCE INDUCED BY CELL WALL PEPTIDOGALACTOMANNAN OF THE resistant cotton cultivars. Interesting, since 2006, some FUNGUS CLADOSPORIUM HERBARUM MEDIATES fields are nowce days almost only planted with CBD- VIRUS PROTECTION IN TOBACCO PLANTS groups of plants presenting CBD-like symptoms. This Vaslin, M.F.S.; Montebianco, C.B.; Mattos, B.B.B.; Silva, CBD-resistant cotton fields started to show individual new disease was called atypical vein mosaic disease and T. da F.; Romanel, E.; Bergter, E.B.; Vaslin, M.F.S. infected plants show CBD mild symptoms as leaf rolling UFRJ - Universidade Federal do Rio de Janeiro, Av. at the plant top and small or null internode shortening Pedro Calmon, 550 - Cidade Universitária, Rio de Janeiro - associated with reddish and some withered shape of the RJ, 21941-901 leaves of the central part of the plants. In the subsequent years this phenomenon spread around all the Cerrado Virus protection of susceptible crops is an important and Southeast cotton planting area and it is nowce days goal in the agriculture all over the world. Bioactive an important disease widely distributed in cotton crops agents that can mediate this kind of response areas in Brazil. Sequencing of the POL-CP block of 13 minimizing damages associated with usual vector viruses isolated from symptomatic resistant cotton control by chemical insecticides are each day more receiving importance in plant applied research. Here close to CLRDV was associated to it. However, until we test the ability of a fungal cellular wall glycoprotein nowcollected any, other in the part field of showedits genome that was a virus met known. extremely In from the Cladosporium herbarum to induce systemic order to better characterize the virus associated with acquire resistance against virus infection. C. herbarum this new disease we sequenced the complete genome is the most prominent mold in air spores. It grows over of two distinct viral isolates, Acr3 and IMA2, recovered a wide range of temperatures, and has frequently been from CBD resistant host plants in 2006 and in 2011, reported causing spoilage of meat in cold storage and respectively. Infected plants total small RNAs were associated with the development of respiratory allergic sequenced by Fasteris Co., Switzerland and the libraries disease in humans. Nevertheless, it is also found as a were analyzed at UFRJ using a new software develop by plant pathogen often associated with scab of passion

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

45 Oral Presentation fruit, besides having been reported to cause disease in PIV221 - PROTEIN SYNTHESIS ANALYSIS OF others crops as onion, wheat, oats, peanuts, potatoes, INSECT CELL LINES INFECTED WITH SPODOPTERA tobacco, grapes and coffee. Until now, only one study of FRUGIPERDA MULTIPLE NUCLEOPOLYHEDROVIRUS the interaction of C. herbarum and plants at molecular (SFMNPV) level was reported (Mattos et al., in preparation). Here, Marcio, M.S.; Sihler, W.; Souza, M.L. we checked if the most abundant glycoprotein of the fungus cell wall, the peptidogalactomannan (pGM), may Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica - PqEB s/nº.Brasília - DF, 70770-901 be use for viral protection. Cladosporium herbarum pGM was extracted in sodium phosphate buffer 0.05 M pH Baculovirus have been widely used as successful biopesticides for lepidopteran control. The fall dextrose medium (PDB) for seven days. Suspensions armyworm, Spodoptera frugiperda, is an important 7.0 at 100°C for 2h under reflux, after growth on potato pest in South America damaging several different crops. sprayed on plants of Nicotiana tabacum growing in green A baculovirus pathogenic to this insect, Spodoptera houseof the conditions pGM, in the using concentration a high pressure of 600μg/mL, apparatus. After were frugiperda Multiple Nucleopolyhedrovirus (SfMNV), 24 hours, the plants were infected mechanically with the has a great potential to be used for controlling this pest. Tobacco Mosaic Virus (TMV) with potassium phosphate In the present work kinetics of viral protein synthesis buffer 0.01M pH 7.2. Control plants, sprayed with pGM or water only, showed no sign or symptoms showing which two Spodoptera frugiperda cell lines showed to that the pGM spray by itself do not affect plant wellness. bewas highly carried susceptible out in order to thisto confirm virus (IPLB-SF-21AEprevious results and in The same was observed for plants sprayed with pGM and Sf9). Six different lepidopteran cell lines were assayed: mock inoculated 24 hours later. N. tabacum SR1 cv. plants Bombyx mori (BM-5), Lymantria dispar (IPLB-LD-625Y), sprayed with the pGM before TMV inoculation exhibited Trichoplusia ni (BTI-Tn-5B1-4), Anticarsia gemmatalis milder or no symptoms of the viral disease. Besides (UFL-AG-286) and the two S. frugiperda cells (SF21 that a reduction of approximately 38% of the necrotic per 60mm2 dish were incubated with the SfMNPV mechanically infected 24 hours after pGM spray. These I-19and Sf9).isolate Initially, for 1h adsorption cells seeded time at aand density kept in of TNMFH 1X106 resultslesions showwas observed an important when roleN. tabacum of pGM Xanthiin the inductioncv, where complete medium at 27ºC. At different times post of TMV protection in Nicotiana tabacum. To understand infection (0h, 24h and 72h pi) the medium was replaced the molecular response involved, seven defense-related by phosphate buffered saline pH 6.2. After a 30min genes are being evaluated by qRT-PCR assays. They are the pathogenesis-related genes PR-1a (unknown was added in a 1h pulse. In order to detect the presence ofstarvation radioactively period, labeled a total proteins, of 50 Ci a ofpolyacrylamide [35S]methionine gel and PR-5 (thaumatin-like protein); the phenylpropanoid electrophoresis (SDS–PAGE) was carried out and the gel pathwayfunction), genePR-2 (β1-3PAL (phenylalanineendoglucanase), ammonia-lyase);PR-3 (chitinase) treated for autoradiography. In parallel, morphological and genes involved in plant stress responses and days. Typical cytopathic effects began to be visualized (peroxidase). FINANCIAL SUPPORT: CNPQ, FAPERJ , afteranalysis 48h was p.i. monitored in the S. frugiperdaby light microscopy cell lines. during Although five innate immunity, such as LOX (lipoxygenase) and Prx both cells showed to be very productive, polyhedra formation was even more intense in SF21 cells than CAPES, PROEX AND UFRJ in Sf9 cells. No morphological changes were observed in UFL-AG-286 and BTI-Tn-5B1-4 cells. The LD-625Y and BM-5 cells became highly vacuolated with some visible changes in the cell membrane surface, but none polyhedra production could be observed. Analysis of the kinects of radiolabed proteins showed that the cell protein synthesis was shut off while an intense band of aprox. 30 kD was synthesized in SF21 and Sf9 cells. September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

46 Oral Presentation

This peptide is regarded to be the polyhedrin, the main was secreted, and slightly increased virus titers in vitro. protein component of the occlusion body (polyhedra). No similar band was observed in the other cell lines. This infections of two lepidopteran host species, but the virus was predictable since only these two cell lines showed lethalNo significant concentration differences was 4-fold in lethal lower time in were Trichoplusia observed ni. in During virus infections, the serpin increased cathepsin the susceptibility to both Spodoptera frugiperda cells activity and partially reduced caspase activity. Based on tooccluded SfMNPV virus and particles point out formation. their use These for further results confirmin vitro our observations and previous work, we hypothesize production studies. FINANCIAL SUPPORT: EMBRAPA that acquisition of this serpin gene by HespNPV ancestor may have resulted in increased viral virulence, and PIV295 - CHARACTERIZATION OF A BACULOVIRUS its expression may be advantageous in pest control HOST-ACQUIRED PROTEASE INHIBITOR AND ITS strategies. ABILITY TO AUGMENT VIRAL VIRULENCE Ardisson, A.D.M.P.1; Rohrmann, G.A.2; Bergmann, PIV357 - IDENTIFICATION OF A NEW DCL3 GENE M.R.1; Rollie, J.C.3 IN COTTON GOSSYPIUM RAIMONDII (ULBRICH) D-GENOME AND STUDY OF THE ROLE OF NEW 1. Dept. of Cell Biology, University of Brasília ISOFORMS OF GENE SILENCING RELATED GENES IN 2. Dept. of Microbiology, Oregon State COTTON VIRUS INFECTED PLANTS University 1 1,2 1 3 3. Molecular, Cellular and Developmental Vaslin, M.F.S. ; Romanel, E. ; Moura, M. ; Lei, G. ; Biology Program, Division of Biology, Kansas State University Paterson, A.4 The insect immune system responds innately against 1. UFRJ - Universidade Federal do Rio de many invading pathogens. Defensive cells neutralize Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de Janeiro - RJ, 21941-901 which become melanized through the action of 2. USP - Universidade de São Paulo, Av. Prof. phenoloxidasepathogens by engulfing(PO). POs or trappingare in the them insect into nodulesplasma Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 in an inactive form (proPO) and are activated upon 3. IASTATE - Iowa State University, Ames, Iowa 50011 pathogen invasion by a cascade of serine proteases 4. UGA - University Of Georgia, Athens, GA and serine protease-inhibitors (Serpins). Although 30602, Estados Unidos serpins have been reported in poxviruses, recently the The genus Gossypium is composed by ~50 species in the genome of the baculovirus Hemileuca sp. ranging from 885 Mb per haploid nucleus in D-genome nucleopolyhedrovirusfirst baculovirus serpin (HespNPV). ortholog geneBaculoviruses was described are (G.raimondii) to 2572 Mb in K-genome. Genes involved insect viruses with circular dsDNA infective mainly in plant small RNA biogenesis pathway, keys component of RNA-silencing mechanism, are essential for eukaryote known to manipulate many intracellular host processes development, nutritional and stress responses, suchto moth as apoptosis and butterfly and mitosis, larvae. as While well as baculoviruses host physiology are chromatin regulation and viral defense. Searching for and metabolism, it is not clear whether they can directly Dicer-like (DCL), Argonaute (AGO), RNA-dependent control host innate immune responses. Therefore, RNA polymerase (RDR) and nuclear RNA polymerase in this work, we investigated the functionality of the IV/V (PolIV/V) gene sequence in G. raimondii genome of a prototype baculovirus. We found evidence that the for DCL3, AGO1, AGO4, AGO7, AGO10, RDR1 and Pol IVa. HespNPV serpin and whether it could alter the fitness Beyond(98.3%), these we surprisinglyextra DCL3, AGO1, find duplication AGO4, AGO10 gene and event Pol inhibit both lepidopteran PO activity and a set of serine IVa-2 proteins, G. raimondii genome showed a higher proteinases.HespNpV serpin The genegene is is functional most similar and couldto lepidopteran efficiently number of isoforms of these genes compared to the serpins and presents signatures previously described in others plants species studied here. All these paralogous other insect serpins. When expressed in the prototype baculovirus Autographa californica MNPV, the protein with the unique cotton whole genome duplication event. cotton duplicated genes showed a KS value which fit September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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existence in eudicots. Most of these genes (DLC3, AGO4 was analyzed and compared with other rhabdoviruses andIt is thePol firstIVa) report are involved of an extra-DCL3 in 24-nt andsiRNA Pol productionIVa protein availableCoRSV was in sequencedthe GenBank. by Thethe first CoRSV time, genome and the is sequencebipartite, and secondary steps DNA methylation and chromatin containing negative ssRNA, with 6,522 nucleotides in the remodeling at their target loci. The increase number of RNA1and 5,945 nucleotides in the RNA2. The RNA1 is genes and its isoforms points out a possible role of them in the control the large amount of retrotransposons likely encodes the CoRSV nucleocapsid protein, with a (53%) and repetitive DNA elements present in cotton predictedcomposed molecular by five open weight reading of 49 kDa; frames ORF (ORFs): 2 is likely ORF1 to encode the phosphoprotein with a molecular weight of downregulated as well DCL4 are unregulated in cotton 27 kDa; ORF 3 encodes a 36 kDa protein with homology cultivatespecies. Inplants a previous (from G. study, hirsutum we findspecie) that during the DCL2 Cotton is to viral cell-to-cell movement proteins; ORF 4 encodes leafroll dwarf virus infection. Now, the expression of a 20 kDa protein with unknown function and the ORF5 some of these new genes and isoforms during virus encodes a 60 kDa glycoprotein. Each ORF is separated infection are been assayed by qRT-PCR. Primers for by a conserved tri-modular intergenic spacer. The RNA2 DCL2a, DCL2ab, DCL3a, DCL3b, DCL4; AGO1a, AGo1b, encodes a single protein (ORF6), which has all the AGO1c, AGO2; PollVa1; PollVa2, PollVb were design hallmarks required for assignment as a RNA dependent and are been tested in order to try to understand theirs RNA polymerase. The leader and trailer regions of RNAs 1 possible role during virus:plant interaction. FINANCIAL and 2 are separated from their nearest ORF by truncated SUPORT: FAOERJ, CNPQ AND PIBIC/UFRJ gene junctions containing only the transcription start site or polyadenylation signal, respectively. Phyogenetic PIV378 - THE FIRST COMPLETE GENOME SEQUENCE OF COFFEE RINGSPOT VIRUS; AN EMERGING THREAT as a member of the proposed Dichorhavirus genus, TO COFFEE PRODUCTION AND QUALITY analysis of L protein sequences firmly establishes CoRSV Figueira, A. dos R.1; Ramalho, T.O.1; Sotero, A. de J.1; shares 70% similarity with CoRSV. In this study a wealth Duarte, P. de S.G.1; Wang, R.2; Farman, M.2; Goodin, ofwhich information has an Orchidand resources fleck virus were as generated, type member, serving and M.M.2 as a foundation of future studies for the control of this 1. UFLA - Universidade Federal de Lavras, emerging virus that is an increasing threat to coffee Câmpus Universitário, Lavras - MG, 37200-000 production and quality. FINANCIAL SUPPORT: CNPQ, 2. UKY - University of Kentucky, Lexington, Kentucky 40506 FAPEMIG,PIV383 - CAPES SUBCELLULAR AND CNP&D-EMBRAPA LOCALIZATION CAFÉ. OF COFFEE RINGSPOT VIRUS PROTEINS 1938 in Sao Paulo state, Brazil, and considered a minor Ramalho, T.O.1; Figueira, A.R.1; Sotero, A.J.1; Duarte, diseaseCoffee ringspotfor decades virus thereafter. (CoRSV) was Symptoms first described of coffee in P.S.G.1; Wang, R.2; Farman, M.2; Goodin, M.M.2 ringspot disease include chlorotic or necrotic spots, usually forming concentric rings, which can also be 1. UFLA - Universidade Federal de Lavras, observed in fruits and young branches. CoRSV infected Câmpus Universitário, Lavras - MG, 37200-000 leaves usually fall from infected plants within four to 2. UKY - University of Kentucky, Lexington, Kentucky 40506 loss of photosynthetic capacity and source of shade Coffee (Coffea arabica) is the second most valuable forfive developingweeks after coffeesymptom cherries. appearance, In addition, resulting infection in both traded commodity after petroleum. Although grown of fruits results in their accumulation of compounds commercially in most tropical and subtropical countries, that contribute to depreciate the coffee brew. CoRSV the world supply of coffee is dominated by Brazil, which is transmitted by the false spider mite Brevipalpus produces 35% of the global exports of green coffee . As such, the emergence of Coffee ringspot virus of the family, genus Nucleorhabdovirus, (CoRSV), which is transmitted by the false spider mite basedphoenicis on the and site was of replication. tentatively In classified this study asthe agenome member of Brevipalpus phoenicis, is of critical concern. CoRSV

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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The tymoviruses (family , genus ) are icosahedric, non-enveloped, single-stranded positive family,was first genus described Nucleorhabdovirus. in Sao Paulo stateThe CoRSV in 1938 genome and is consiststentatively of bipartite classified RNAs as a with member negative-sense of Rhabdoviridae polarity. different plant species were reported in Brazil. Most of sense RNA viruses. Only five tymoviruses infecting ORF1, nucleocapsid protein; ORF 2, phosprotein; ORF based on biological, biochemical and serological 3,The viral RNA1 cell-to-cell is composed movement by five protein; Open ReadingORF 4 a Frames:protein properties,these viruses and were no complete identified genomic as distinct RNA tymoviruses sequences of unknown function and ORF 5, glycoprotein. The are available for these viruses, except for the recently RNA2 encodes a single protein, RNA dependent RNA described Tomato Blistering Mosaic Virus (ToBMV). polymerase. Subcellular localization of viral proteins ToBMV was isolated from tomato, and was serologically related to Eggplant mosaic virus (EMV), suggesting silico methods. Therefore, in this study an established a cryptic diversity within Brazilian tymoviruses. strategyis frequently for protein difficult localization to determine was employed, strictly from using in Therefore, we revisited a previously described tobacco- an Agrobacterium-mediated transient expression of viral stock was used mechanically inoculate N. tabacum benthamiana lines that express subcellular markers. infecting EMV to confirm its taxonomic status. Frozen fluorescent protein fusions in transgenic Nicotiana tobacco leaves and the viral RNA was extracted, gel expressed as carboxy-terminal fusions to GFP in ‘TNN’ plantlets. The virus was purified from symptomatic transgenicThe five proteins plants expressingencoded by a the red ORFS nuclear of RNAmarker. 1 were The platform. The paired-end reads were assembled using analysis at confocal microscope showed that GFP-P1 CLCpurified Genomics and sequenced Workbench using version an Illumina6.0.3. The HiSeq assembled 2000 fusion was detected at steady state in both the nucleus contigs were submitted to blastx search against a viral and the cytoplasm. In marked contrast, the GFP-P2 fusion genome database. A contig of 6,256 nucleotides was was localized exclusively in the nucleus. Consistent with found to be similar to tomato blistering mosaic virus. a predicted cell-to-cell movement protein function, the This contig was annotated and tree intact ORFs were GFP-P3 fusion accumulated predominantly at the cell periphery. GFP-P4 accumulated into the nucleus but polyprotein with 1809 amino acids. The ORF2 (1956 could also be found as cytoplasmic aggregates. GFP-P5 nt)identified. encodes The the ORF1 movement (5427 proteinnt) encodes and thethe ORF3replication (573 was targeted to the nuclear envelope and, to a lesser nt) encodes the capsid protein, with 652 and 191 amino extent, at perinuclear membranes. Taken together, the nucleophillic character of the proteins encoded by (UTRs) are 129 and 117 nt long. A phylogenetic analysis ORFs 1, 2, 4 and 5 are consistent with their predicted usingacids, respectively.24 complete The genomes 5′ and 3′available untranslated in GenBank regions roles in formation and viral morphogenesis. This study gives important information and provides is in fact, an isolate of ToBMV, with 88% nucleotide resources to a function prediction and localization of identityconfirmed over that the the entire prior genome. identified Interestingly EMV-tobacco these isolate two CoRSV proteins. ToBMV isolates differ in the ability to infect N. tabacum ‘TNN’ plants. The comparison of the coding region PIV423 - COMPLETE GENOME SEQUENCE OF A of both isolates revealed that most of the nucleotide TOBACCO-INFECTING TOMATO BLISTERING MOSAIC differences among the two genomes are synonymous, VIRUS except for those occurring in the ORF2, which presented Fernandes, J.E.F.1; Melo, F.L.1; Ribeiro, B.M.1; Ribeiro, a high degree of non-synonymous substitution. G.S.2 1. UnB - Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília - DF, 70910-900 2. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica - PqEB s/nº.Brasília - DF, 70770- 901

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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PIV452 - COMPLETE GENOME SEQUENCE OF A NOVEL PIV509 - THE FUNCTIONAL ANALYSIS OF DISTINCT IFLAVIRUS ISOLATED FROM OPSIPHANES INVIRAE TOSPOVIRUS MOVEMENT PROTEINS (NSM) REVEALS Silva, L.A.1; Melo, F.L.1; Tinôco, R.S.2; Fernandes, O.A.3 DIFFERENT BEHAVIOR FROM THE ALFALFA MOSAIC VIRUS (AMV) MODEL SYSTEM 1. UnB - Universidade de Brasília, Campus 1 2 2 2 Universitário Darcy Ribeiro, Brasília - DF, 70910-900 Leastro, M.O. ; Peiró, A. ; Pallás, V. ; Navarro, J.A.S. ; 1 2. FCAV/UNESP - Faculdade de Ciências Resende, R.O. Agrárias e Veterinárias da Universidade Estadual Paulista, 1. UnB - Universidade de Brasília, Campus Via de Acesso Prof.Paulo Donato Castellane s/n, Jaboticabal, Universitário Darcy Ribeiro, Brasília - DF, 70910-900 SP, 14884-900 2. IBMCP/CISIC-UPV - Instituto de Biología 3. PLANT PROTECTION AND RESEARCH Molecular y Celular de Plantas do Consejo Superior de MANAGER OF GROUP AGROPALMA S/A, Alameda Investigaciones Científicas de la Universidad Politécnica de Santos, 466 10. andar Cerqueira Cesar, São Paulo, SP, 01418- Valencia, Ingeniero Fausto Elio, s/n 46022 Valencia 000 Plant viruses have developed a class of proteins responsible for ensuring viral infection and (Lepidoptera: Nymphalidae) and its complete genome dissemination denoted movement proteins (MPs). A new Iflavirus was isolated from Opsiphanes invirae sequenced. O. inviridae, in its larval stage, is considered The MPs interact with the plasmodesma enhancing its a pest, causing defoliation in oil palm, coconut and some size exclusion limit and, in this way, allowing the virus native palms in northern part of South America. This passage. For tospoviruses, it was demonstrated that new virus was isolated from O invirae larvae collected virus movement requires tubules formation driven by in Tailândia, Pará State - Brazil, which showed a brown its non-structural movement protein (NSm). Although, color and discoloration of the posterior and middle parts it is expected that this mechanism would be conserved among the 28 tospovirus species reported so far within ultracentrifugation in a sucrose cushion and the viral of the insect body. The virus particles were purified by RNA was extracted according to TRIzol® Reagent differences in amino acid sequences of viral proteins (Invitrogen) protocol. The viral RNA was sequenced at andthe genus,in their comparison biological features among suchthem as reveals host range. significant Some Macrogen (South Korea) using an Illumina HiSeq 2000 tospovirus species present a narrow spectrum of host platform. The paired-end reads were assembled using plants, e.g BeNMV, while others such as TSWV, TCSV and CLC Genomics Workbench version 6.0.3. The assembled CSNV display a broad host range infecting plants from contigs were submitted to blastx search against a viral a large number of different botanical families. Due to genome database. A contig of 10,083 nucleotides was its main function, the NSm proteins are often assigned stranded RNA genome of positive polarity that possess adaptation. The aim of this study was to evaluate the role afound single to Open be similarReading to Frame Iflavirus, (ORF) which of 9,558 have nucleotides a single- as potential determinants of host specificity and/or (nt), encoding a polyprotein, which is post-translationally and systemic movements using the heterologous Alfalfa processed into viral proteins essential for its replication, mosaicand the virusefficiency AMV of system, four tospovirus that allows MPs the on cell-to-cellfunctional packaging and transmission. The 5’UTR (254 nt) exchangeability of viral movement proteins (MPs) includes an internal ribosome entry site (IRES). The assigned to the “30K family”. Here, differences in the single large ORF encodes both structural (5’terminus) and non-structural (3’ terminus) proteins. The ORF is observed based on the average sizes of the infected areas followed by a 3’UTR (271 nt). The phylogenetic analysis supportedefficiency inby cell-to-cellthe distinct and tospovirus systemic MPs movement tested. wereAlso, we observed that all MPs analyzed were not competent to support the transport of an AMV RNA 3 carrying a CP revealed that this new iflavirus is related to Spodoptera 147765/2012-9) mutant (CP206) defective in virus particles, indicating exigua iflavirus 1. FINANCIAL SUPPORT: CNPQ (PROC. the incapacity of the MPs to transport other complexes different than virions in the AMV context. It was also demonstrated that the MPs of the four tospovirus

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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C-terminal deletion of the MPs revealed that C-terminus to both viruses were used as template to amplify two ofspecies the MP were of BeNMVsimilarly (the efficient only totospovirus form tubules. here However, showing differentFIV and/or regions FeLV ofpositives the gene, and with fifty-nine subsequent of negative sequencing cats a narrow host range) was shown not to be essential for virus movement, in contrast to CSNV, TCSV and TSWV-MPs among all samples. On the second one it was possible which required the entire NSm proteins to guarantee the toand identify analysis. six The single first investigatednucleotide variationregion was points, conserved and features and behaviors among the tospovirus movement with the susceptibility to FIV and/or FeLV infection. proteins.cell-to-cell We movement. suggest that These the results NSm proteinconfirmed plays different a role Onof them,the other the hand, A65S the (A65I) haplotype was significantly analysis showed correlated that in the determination of the viral infection spectrum of host plants based on the differences observed in cell- with the lack of retroviral infection, maybe indicating a to-cell and systemic movement patterns among the protectivethe combination effect. “GGGGCC”Whereas awas polymorphism significantly atcorrelated position tospovirus MPs. FINANCIAL SUPPORT: UNIVERSIDADE 65 have been found in Indochinese Tiger and given the DE BRASÍLIA (PPG BIOLOGIA MOLECULAR), CNPQ, correlation found in this research, more studies about CAPES, FAP-DF, UPV-CISIC, UNIÓN EUROPEA, GOBIERNO the effect on the activity of restriction factors encoded by DE ESPAÑA the A3H gene should be performed. Similarly, research about the effects of the combination “GGGGCC” should VV27 - ANALYSIS OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE APOBEC3H GENE OF effect shown in this work. FINANCIAL SUPPORT: CNPQ, DOMESTIC CATS (FELIS CATUS) AND THEIR FINEPbe carried so that we can confirm a possible protective ASSOCIATION WITH THE SUSCEPTIBILITY TO FELINE IMMUNODEFICIENCY VIURUS AND FELINE VV37 - PARTIAL CHARACTERIZATION OF AVIAN LEUKEMIA VIRUS INFECTIONS INFLUENZA VIRUS (H11N9) IN MIGRATORY BIRDS Costenaro, J.G.; de Castro, F.L.; Junqueira, D.M.; de (ARENARIA INTERPRES) CAPTURED IN AMAZON Medeiros, R.M.; da Silva, T.R.; Knak, M.B.; Almeida, REGION, BRAZIL S.E. de M.; Campos, F.S.; Roehe, P.M.; Franco, A.C. de Araujo, J.1; Júnior, S.M. de A.2; Gaidet, N.5; Hurtado, R.F.1; Walker, D.4; Thomazelli, L.M.1; Ometto, T.1; UFRGS - Universidade Federal do Rio Grande do Sul, 1 3 4 Avenida Paulo Gama, 110 - Farropilhas, Porto Alegre - RS, Seixas, M.M.M. ; Galindo, D.B. ; Webby, R.J. ; Webster, 4 1 90040-060 R.G. ; Durigon, E.L. 1. ICB/USP - Instituto de Ciências Biomédicas Leukemia Virus (FeLV) belong to the Retroviridae da Universidade de São Paulo, Edifício III USP - Administração The Feline Immunodeficiency Virus (FIV) and the Feline - Av. Prof. Lineu Prestes, 2415 - Butantã, São Paulo - SP, immunosuppressive effect in infected domestic cats 05508-900 2. DB/UFRPE - Departamento de Biologia (Felisfamily, catus). are widely Proteins distributed with activity and against induce aretroviruses significant da Universidade Federal Rural de Pernambuco, Rua Dom are conserved between mammals and determined Manoel de Medeiros, s/n, Dois Irmãos, Recife - PE, 52171-900 as restriction factors. Cytidine deaminases of the 3. Agência de Defesa Agropecuária do Estado APOBEC3 gene family are the most studied class of do Pará- ADEPARA, Pa-Brazil restriction factors and the APOBEC3H gene encodes 4. Department of Infectious Diseases, St. Jude two proteins (APOBEC3H and APOBEC3CH) that are Children’s Research Hospital, Memphis, Tn, USA the most responsible for this restriction among cats, 5. CIRAD-ES, Ur Agirs, Montpellier, France through hypermutations in provirus DNA during reverse transcription. Changes on the gene sequence can alter America and Eurasia is known a long time, while their the stability and cellular localization of homologous prevalenceThe prevalence in wild of birds Avian in influenza South America viruses is in largely North proteins to APOBEC3H in mammals. Considering the unknown. Aquatic birds are considered natural reservoir importance of APOBEC3H gene in cats, the investigation of its variability is relevant. Fifty DNA samples of cats country with the highest biodiversity in species of September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersfor - Oral avian Presentation influenza viruses (AIV) and Brazil is the second XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

51 Oral Presentation migratory birds. So, the current study was conducted enveloped, positive sense, single-stranded RNA virus. CoV has been recognized as a major cause of respiratory four samples of migratory birds (Arenaria interpres). disease in several species with important outcomes Samplesto characterize were collectedof the avian in Ilhainfluenza de Canelas, virus detected located in such as the SARS pandemic event. Infectious bronchitis Amazon region, Pará State, in November 2008. Mist virus (IBV), a group 3 coronavirus, causes a costly viral nets were strategically placed near the beach and the disease of chickens that is found worldwide. It can mangrove to capture of birds. In this occasion, a total of cause respiratory disease in chickens of all ages and a 80 samples were collected by orotracheal/cloacal swabs, loss of production and egg quality in mature hens. Some and were put in the VTM medium and immediately strains are nephropathogenic, resulting in renal-induced stored in liquid nitrogen. Blood was collected and sera Proventricular Dilatation Disease (PDD) is a worldwide knownmortality fatal rates disease of up in to birds, 25% mainlyfor susceptible affecting flocks. psittacines PDD viruseswere separated in wild onbirds field. were Real-time used reversewith AIV-M transcriptase TaqMan but also other species. This disease is characterized polymerase chain reaction (RT-PCR) to identify influenza by a gastrointestinal dysfunction with or without positive by molecular test. Then were submitted to viral neurological symptoms. However, bornavirusABV was isolation,Reagents kitinoculated (ABI). Four in embryonatedsamples were Influenzachicken eggs A virus for also found in healthy birds, indicating that additional factors may be required for ABV to cause the clinical disease. To date, 9 different genotypes of ABV including samples,viral replication. three viruses The result were wasisolated, confirmed sequenced per Avian and non-psittacine birds have been detected from birds characterizedInfluenza Virus by Type Haemagglutination-inhibition A Test Kit (Synbiotics). From (HI) these test in Africa, Europe, North America, Japan and Australia. using full panel of HI sera standard. All positive samples All recent studies involved psittacines originating showed similarity with subtype H11N9 groups. Analyze from captivity. No further sampling or diagnose was possible with these birds. According to the available from surveillance of migratory birds have shown great literature, no PDD or ABV cases have been reported in importance,of sequences because confirmed these the birds HI have results. not Ournationality results free-ranging psittacines and few studies were published on the detection of Coronaviruses from parrots. In this Northern and Southern Hemispheres. Therefore, these study we report about the presence of both Coronavirus movementsdefined, and deserve are in special constant attention, migration particularly between after the and ABV infection in apparently healthy, free ranging

cloacal swabes were collected between 2009 and 2010 isolationthe incidence of H11N9 of recent in the outbreaks Arenaria of interpresavian influenza in South in ofpsittacines 7 species inof Brazilpsitacideos for the São first Paulo time. and 142 Mato tracheal Grosso and do America.others continents. To our knowledge, this is the first Sul states, Brazilian Southeast and Midwest. By RT-PCR, 33.8% ABV infections, 15.49% Pancoronavirus infections VV96 - AVIAN CORONAVIRUS AND AVIAN BORNAVIRUS and 6:33% had coinfection ABV-Pancoronavirus. This DETECTION IN FREE-RANGING PSITTACINES work was supported by CAPES/CNPq and FAPESP, grant Lima Neto, D.F.1; Barnabé, A.C.S.1; Caserta, L.1; Martini, number 2011/50919-5. M.C.1; Simas, P.V.M.1; Nagel, N.E.2; Lierz, M.2; Hafez, H.M.2; Felippe, P.A.N.1; Arns, C.W.1 1. UNICAMP - Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz - Barão Geraldo, Campinas - SP, 13083-970 2. FREIE UNIVERSITÄT BERLIN, Kaiserswerther Straße 16-18, 14195 Berlin, Alemanha A key issue in understanding the dynamics of multi- of the pathogen reservoir. Coronaviruses (CoV) are host pathogens in an ecosystem is the Identification September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

52 Oral Presentation

VV113 - PRIMARY AND SECONDARY MUCOSAL IMMUNITY AFTER CHALLENGE WITH BRAZILIAN on vaccinated group A compared to group B. Levels of AVIAN INFECTIOUS BRONCHITIS VARIANT group A. Transcripts for CD4 were significantly lower Okino, C.H.1; Mores, M.A.Z.1; Montassier, H.J.3; Mattos, A compared to unvaccinated/challenged group B. These local and systemic IgG were significantly higher in group G.L.M.1; Brentano, L.1; Coldebella, A.1; Ritterbusch, results indicate an exacerbated local cell-mediated G.A.2; Esteves, P.A.1; Trevisol, I.M.1 and a lower level secondary response to vaccination 1. EMBRAPA SUÍNOS E AVES, Parque andprimary variant immune IBV responsechallenge to for the theIBV fieldevaluated variant interval strain, Estação Biológica - PqEB s/nº, Brasília, DF, 70770-901 post-infection. Otherwise, both local and systemic 2. UFPEL - Universidade Federal de Pelotas, humoral responses were boosted during secondary Capão do Leão - RS, 96160-000 response. FINANCIAL SUPPORT: PROJETO EMBRAPA 3. UNESP - Universidade Estadual Paulista, Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010 03.12.03.012.0.00 Avian infectious bronchitis virus (IBV) is one of the VV172 - ASTROVIRUS DETECTION IN THE BAT most important poultry industry pathogens. IBV is a TADARIDA BRASILIENSIS (GEOFFROY, 1824: gammacoronavirus, characterized for high genomic CHIROPTERA, MOLOSSIDAE) FROM RIO GRANDE DO mutations, with frequent emergence of new variants SUL STATE, BRASIL and incomplete vaccine protection. The aim of this Duppont, P.M.1; Pacheco, S.M.2; Rosa, J.C.A.3; Streck, study was to evaluate humoral and cell-mediated A.F.1; Alves, C.D.B.T.1; da Silva, M.S.1; Budaszewski, immune responses in birds vaccinated with the H120 R.F.1; Weber, M.N.1; Canal, C.W.1 attenuated strain and challenged with Brazilian IBV 1. UFRGS - Universidade Federal do Rio Grande do Sul, Avenida Paulo Gama, 110 - Farropilhas, Porto SPF chickens were equally distributed in three groups Alegre - RS, 90040-060 placedfield isolates in positive genotyped pressure as IBV isolators. variants. At Twenty one-day seven old 2. ISAUVER - Instituto Sauver, Av. Pernambuco, group A birds received full-dose H120 vaccine. At 28 2623 sala 404 Bairro Floresta,Porto Alegre - RS, 90240-005 days of age, all birds from vaccinated group A and from 3. IPVDF/FEPAGRO - Instituto de Pesquisas not vaccinated group B were challenged with 104.0 Veterinárias Desidério Finamor da Fundação Estadual de Pesquisa Agropecuária, Estrada do Conde, 6000, Eldorado do C was mock infected. All birds were euthanized at 5dpi, Sul - RS, 92990-000 tracheasEID50/bird were of removedF3735 Brazilian and evaluated field strain for ciliary of IBV. activity, Group Bats are the second largest group of mammals on the microscopic lesions and viral load to determine degree planet, with about 1,200 species and 174 are found in Brazil. Due to changes and fragmentation of natural immune response genes (innate:TLR3, TLR7, MyD88, of vaccine protection, and for RT-qPCR quantification of habitats, these animals seek shelter alternatives and thus become increasingly exposed to anthropic enviroments. Some bat species are recognized as natural reservoirs wereIFNα; analyzed inflammatory: for anti-IBV IL6; cell-mediated: IgG by ELISA. CD8β, Scores CD3ε, of of several viral families and this feature gives them an lesionsCD4, IFNγ observed and Granzyme by ciliary homolog activity A).and Tears histopathology, and serum important role in the transmission and maintenance of these microorganisms. This work aims to detect the vaccinated group A compared to group B. All immune and levels of viral load, were significantly reduced on presence of astrovirus genome fragments in bat organ samples from Rio Grande do Sul State, Brazil. The bats for the primary immune response in group B. Although related genes tested were significantly up-regulated were caught with mist nets at roost or foraging sites in natural enviroments and entomological network, gloves and long tweezers were used for the urban areas. The onlower tracheal levels samples of mRNA of forbirds TLR7, from MyD88, group A IFNα, (secondary IFN γ, animals were euthanized and sent to the Instituto de response)IL6, CD8β, compared Granzyme to homolog group B, the A and differences CD3 were were found not Pesquisas Veterinárias Desidério Finamor/FEPAGRO for rabies diagnosis. Negative rabies samples were further two birds that resulted as unprotected in the vaccinated significant, what in part may be due to the presence of September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

53 Oral Presentation analysed to detect the presence of astrovirus using RT- PCR. In total, 38 bats samples belonging to thirteen were positive for RVF with infections spread in 7 of 37 species were studied. The samples were submitted to (18.9%)(8/85) of farms. pools. TheSamples higher from rate five of of infection eight municipalities was related RNA extraction followed by RT-PCR. As results, three to the age of 16-30 days. This study is one of the pioneers positive samples (11%) were found in only one species to detect RVF by RT-PCR, as well as adding information (Tadarida brasiliensis). Those positive samples were about the occurrence of RVF in Brazil, providing more collected in urban areas, while the presence of astrovirus representative data about this group of RV. FINANCIAL SUPPORT: FAPESPA; CNPQ. Possibly, the anthropic enviroment may contribute to thewas presence not identified of the in virus, samples since from previous natural studies enviroment. showed VV229 - LONG-TERM CIRCULATION OF INFLUENZA A that bat may be related to human astrovirus, VIRUSES IN SWINE IN BRAZIL but these possibility will be further studied. FINANCIAL Schaefer, R.1; Nelson, M.2; Gava, D.1; Cantão, M.E.2; SUPPORT: CAPES, CNPq, FAPERGS and Propesq/UFRGS. Zanella, J.R.C.1 1. EMBRAPA SUÍNOS E AVES, Parque VV207 - DETECTION OF AVIAN GROUP F ROTAVIRUS Estação Biológica - PqEB s/nº, Brasília, DF, 70770-901 IN FECAL SAMPLES OF BROILER CHICKENS IN PARA 2. FIC/NIH - Fogarty International Center STATE, BRAZIL - National Institutes of Health, 31 Center Drive, MSC 2220 Bezerra, D.A.M.1; Silva, M.J.M.1; Bezerra, D.A.M.1; Silva, Bethesda, MD 20892-2220 USA R.R.2; Soares, L.S.1; Mascarenhas, J.D.P.1 1. IEC - Instituto Evandro Chagas, Rodovia important economic concerns for the swine industry BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 andInfluenza a pandemic A viruses threat (IAVs) for humans. circulating Although in swine Brazil present hosts 2. Laboratório Nacional Agropecuário one of the largest swine populations in the world, there Rotavirus (RV) is a major viral agent of enteric disease has been little evidence prior to 2009 of IAV circulation in humans and animals. They are non-enveloped viruses in Brazilian swine herds. Following the detection of with genome divided into eleven segments of double- pandemic H1N1 (H1N1pdm) IAVs in pigs in Brazil in stranded RNA that encodes 12 proteins. The VP6 protein 2009, surveillance efforts increased. Screening of 1440 is conserved and an excellent target for laboratory nasal swab samples was carried out by RT-qPCR assay. diagnosis as well as being determinant of species/groups Thirty-nine positive samples were submitted to virus isolation. Genetic sequencing was performed by ABI (RVF) infect only birds causing damage to 3130xl and Illumina MiSeq. Five H1N2, four H3N2 and healthof RV these and consequentlyare classified lossesinto eight to the (A-H). poultry The groupmarket. F seven H1N1pdm IAVs, collected from swine in different Presently, there are few data in the literature on the RVF Brazilian states during 2009-2012, were sequenced particularly using molecular methodologies such as the and analyzed. Nucleotide alignments were generated reverse transcriptase-polymerase chain reaction (RT- PCR). In this context, this study aimed to detect RVF in ‘H1p’ (pandemic virus-like), H3, N1p (pandemic virus- broilers in Para state, Brazil, during 2008 to 2011. A total like)for five and data N2, including sets: ‘H1s’ other (human related seasonal human virus-like),and swine of 85 pools of fecal specimens of broilers were collected viruses, collected globally, as background. The four H3N2 in 37 farms located in eight different municipalities in viruses from Brazilian swine are monophyletic (100% the metropolitan region of Belém (Belém, Ananindeua, bootstrap) and closely related to human seasonal viruses Benevides, Castanhal, Santa Isabel do Pará, Inhagapi, Santa Bárbara do Pará e Santo Antônio do Tauá). Viral monophyletic (64% bootstrap) on the H1 tree, and are genome was extracted from stool suspensions and closelyfrom the related late 1990s.to seasonal The H1N2 five H1N2 viruses viruses that circulated are also in humans during 2001-2003. The lower support for for the gene encoding VP6 protein of RVF (RF6F / RF6R). this clade appears to be driven by the early divergence Thesubjected positive to RT-PCR samples using were a specific sequenced primer using pair the designed same of the Brazilian H1 viruses into two distinct sub-clades. primers in RT-PCR. RVF positivity was detected in 9.4% The Brazilian swine IAVs are not monophyletic on the N2

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

54 Oral Presentation phylogeny. Five Brazilian swine viruses of the H1N2 and plasma and serum samples were analyzed from 78 H3N2 subtypes belong to one N2 clade (97% bootstrap) domestic cats that live in an urban area from state of and two H1N2 belong to a second N2 clade (100% Minas Gerais. Plaque reduction neutralizing test was bootstrap). Both clades are closely related to human applied in serum samples to check anti-Orthopoxvirus seasonal H3N2 viruses from the late 1990s. Therefore, neutralizing antibodies. Viral DNA was accessed using Brazilian swine viruses of the H1N2 subtype that phenol, chloroform and isoamyl alcohol method and real contain H1 segments related to human seasonal H1N2 time PCR targeting vgf and ha genes were also applied. viruses have acquired a different N2 of human H3N2 Most animals are female (55.1%) and age range from origin via two different reassortment events. The seven 5 months to 11 years. It was found 13 seropositive viruses of human pandemic H1N1 origin also were not samples (16.7%), with antibodies titers ranging from monophyletic on the H1 or N1 tree, indicating that these 100 to 1600 neutralizing units per ml. Molecular viruses are the result of multiple separate human-to- analysis of seropositive cats showed 7 positives for vgf swine introductions of the H1N1pdm virus, rather than gene and 4 for ha. Poxviruses are ubiquitous among clonal expansion of a single introduced lineage. The co- mammals and the host spectrum is wide. Although in Brazil occurrence of poxviruses are, until now, restricted virus lineages of the H1N1, H1N2, and H3N2 subtypes to rural environment involving dairy cattle and humans, circulation of multiple antigenically diverse influenza investigations conducted in urban areas highlights Brazil’s swine herds, including design of cross-protective the importance to clarify epidemiological chain of this vaccines.introduces FINANCIAL new challenges SUPPORT: for the controlEMBRAPA of influenza (PROCESS in emerging infectious disease. Since smallpox eradication, Nº. 02.11.10600-01) vaccination is discontinued worldwide increasing the number of immunologically unprotected people. VV260 - EVIDENCE OF ORTHOPOXVIRUS These data also reinforce the impact of viral spread to CIRCULATION AMONG URBAN DOMESTIC CATS, urban environments affecting vulnerable populations. MINAS GERAIS, BRAZIL FINANCIAL SUPPORT: CAPES, CNPQ, FAPEMIG, PRPQ Miranda, J.B.; Costa, G.B.; Almeida, G.G.; Figueiredo, UFMG, PPG-MICROBIOLOGIA UFMG. P. de O.; Bonjardim, C.A.; Ferreira, P.C.P.; Abrahão, J.S.; Kroon, E.G.; Trindade, G. de S. VV303 - DEVELOPMENT AND STANDARDIZATION OF A MULTIPLEX RT-PCR FOR THE DETECTION THE UFMG - Universidade Federal de Minas Gerais, PRINCIPAL RESPIRATORY VIRUS IN BIRDS Avenida Presidente Antônio Carlos, 6627 - Pampulha, Belo 1 1 2 2 Horizonte - MG, 31270-901 Sakata, S.T. ; Rosales, C.A.R. ; Reischak, D. ; Orsi, M.A. Cessation of smallpox vaccination occurred 36 years 1. Bolsista Do Projeto Sagres- MAPA/CNPQ ago in reason of its eradication. Because of this, several 2. Laboratório Nacional Agropecuário - outbreaks involving other have been LANAGRO-SP reported worldwide, such as Monkeypox virus which The avian Metapneumovirus (aMPV), the Newcastle is endemic in Africa and Cowpox virus associated to human and animal cases in Europe, specially having cats infectious bronchitis virus (IBV) are the most important as hosts. In Brazil, Vaccinia virus is a causative agent respiratorydisease virus virus (NDV), pathogens Influenza that affect A virus breeding (AIV) hens and of an exanthematous disease, always affecting dairy and broilers, with high importance to the poultry cattle and humans and related to rural environment. farms, both in terms of economic losses, impact in the Furthermore, other mammalian species are known exportation as the possibility of transmission between to be infected, such as equids, monkeys and wild birds species. The aim of the present paper proposes rodents. Taking into account that cats are well known the development, evaluation and standardization of a to transmit Cowpox virus in European urban areas, Multiplex RT-PCR, in order to assess the purity of avian our goal was to investigate Orthopoxvirus circulation vaccines. Reference virus: aMPV subtypes A and B; NDV- among cats in a Brazilian urban environment and its La Sota; AIV subtype H5N2; IBV-M41 were used for implication for epidemiological studies. Whole blood, standardization of the assay and commercial vaccines

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

55 Oral Presentation from different manufactures of biological products genus. Bats and canids are main reservoirs of were screened. For IBV has been applied to RT-PCR with the disease and are responsible by the maintenance of primers directed ORF 1B; for the presence of aMPV, we the rural and urban cycles of rabies. Different species of used an RT-PCR directed to the gene encoding the G herbivores could be affected by rabies in rural cycle but protein subtypes A and B; with respect to the NDV, the cattle are the most important host of rabies at the cycle. region of the gene encoding the F protein of virulent and In the State of Rio Grande do Sul (RS), southern Brazil the avirulent strains was performed, and to virus AIV, a pair urban cycle of rabies was controlled but rural rabies is of oligonucleotides encoding the protein of M1 and M2 still endemic. Recently it has been registered an increase RNA virus matrix gene was used. The pairs of primers of rabies in cattle in the State causing losses at livestock. This work was carried out with the aim to identify target sequence. From reference virus and commercial genetic lineages of RABV circulating in cattle in the RS. used in this work confirmed to be specific for each For this 14 samples of RABV isolated from cattle were products 444pb (aMPV), 310bp (NDV), 244pb (AIV) and submitted to RT-PCR and sequencing of nucleoprotein 179pbvaccines (IBV), was evidencing possible the to differentiation shown the amplificationof sequences gene (N) of virus. For the edition of sequences was targets of each virus concerned. The best results were used CHROMAS and BIOEDIT software and for the phylogenetic analysis was used MEGA 5 software. referring to the annealing temperature of the reaction. After sequence edition, 684 nucleotides of the protein Theobtained multiplex from RT-PCRthe multiplex is a fast RT-PCR and easy identified technique as to“49”, be were aligned and phylogenetically analyzed. In the 14 carried out in any laboratory equipped to perform the samples isolated RABV was clustered with the genetic lineage from haematophagous bat Desmodus rotundus. for further studies of purity of vaccines used in Brazil. To generate the phylogenetic tree genetic sequences of ItPCR. is one This method screening that test can can assist be considered with greater as claritya first stepand RABV samples from cattle isolated in different regions of Brazil (26) and in Uruguai (4) were recovered from viruses, both in quality control of biological products GenBank and included in the work. Based on the (aviancertainty vaccines) the identification and for diagnosis of aMPV, of NDV, these AIV infectious and IBV topology of the phylogenetic tree of studied RABV, three agents in birds. Financial Support: MAPA/CNPq Cesar 1 consists by 4 samples from Uruguai and 18 samples VV316 - SEQUENCING AND PHYLOGENETIC ANALYSIS frommain different clusters (1,regions 2 and of 3) Brazil could (Rio be identified.Grande do Clustersul- 4, OF NUCLEOPROTEIN OF RABIES VIRUS ISOLATED Goias- 1, São Paulo- 5, Rio de Janeiro- 1, Mato Grosso- 2 e FROM CATTLE IN THE STATE OF RIO GRANDE DO SUL Pará- 5). Cluster 2 consists by 15 samples from south and Fernandes, M.E.S.1; Pereira, P.M.C.1; Achkar, S.M.1; southeast regions of the country (Rio de Janeiro- 5 and Oliveira, R.N.1; Ferreira, J.C.1; Rosa, J.C.A.2; Castilho, Rio Grande do sul- 10). The cluster 3 was compound by 7 J.G.1; Almeida, L.L.2; Carnieli, Jr. P.1; Roehe P.M.2,3; samples from midwest and southeast regions (Tocantins- Batista H.B.C.R.1 3, Rio de Janeiro- 1 and São Paulo- 3). Samples from RS 1. INSTITUTO PASTEUR, Av. Paulista, 393 - were included in clusters 1 and 2, with these results Cerqueira César, São Paulo - SP, 01311-000 2. FEPAGRO SAÚDE ANIMAL/IPVDF - from haematophagous bat D. rotundus are current in RS. Instituto de Pesquisas Veterinárias Desidério Finamor, Estrada Anothercould be sample affirm thatof RABV at least isolated two from sublineages cattle in of RS RABV will Municipal do Conde, 6000. Eldorado do Sul - RS, 92990-000 be analyzed to better understand rabies in the region. 3. UFRGS - Universidade Federal do Rio Grande do Sul, Avenida Paulo Gama, 110 - Farropilhas, Porto Alegre - RS, 90040-060 Rabies is a viral infection that reaches the Central Nervous System (CNS), causing fatal encephalitis in all mammals. The etiological agent of this zoonosis is Rabies Virus (RABV), a RNA virus member of Rhabdoviridae family,

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

56 Oral Presentation

VV321 - HERPESVIRUS SIMPLEX TYPE-1 OUTBREAK 3500. Sequences were analyzed with MEGA 6.0, IN MARMOSETS (CALLITHRIX) resulting in 100% of identity with sequences available Ullmann, L.S.1; Kurissio, J.K.1; Linhares, A.G.2; on GenBank KF498959, JQ352184, and JQ780693, Linhares, L.S.A.1; Milanelo, L.2; Araujo Jr, J.P.1 of HHV-1 people is essential due to the lethality of the 1. UNESP - Universidade Estadual Paulista, diseaseconfirming in marmosets. Human Herpesvirus Emphasis 1. should The restrict be given access to Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010 the importance of differential diagnoses mainly with 2. PARQUE ECOLOGICO TIETE, Centro wild animals whereas few epidemiological studies are de Lazer Engenheiro Goulart Rua Guira Acangatara, 70, available. FINANCIAL SUPPORT: FOUNDATION OF THE Engenheiro Goulart, São Paulo - SP. 03719-000 The increasing number of monkeys as pets can bring two different situations: monkeys as hosts to important BIOSCIENCESVV333 - ABC OF INSTITUTE CANINE PARVOVIRUSES (FUNDIBIO). GENOGROUPS human zoonoses and the opposite, man transmitting AND ANTIGENIC TYPES pathogens to the monkeys. One of these pathogens is Streck, A.F.; Borchardt, A.; Daudt, C.; Canal, C.W. human herpesvirus 1 (HHV-1). Different susceptibility LABORATÓRIO DE VIROLOGIA VETERINÁRIA/ levels are observed among the wide variety of UFRGS - Laboratório de Virologia Veterinária da Universidade Neotropical non-human primates and members of the Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9090, Callitrichidae family are considered high susceptible to Agronomia, Porto Alegre - RS, 91540-000 human herpesviruses. We report here an outbreak of HHV-1 in seven healthy marmosets: 3 white-tufted-ear Canine parvovirus type 2 (CPV-2) is a member of the (C. jacchus), 3 black-tufted-ear (C. penicilatta), and one autonomous replicating family that has a hybrid (C. sp.), received at the reception center for animals single-stranded DNA genome which encodes two capsid (RCA), Tietê Ecological Park, a municipal park located in proteins (VP1 and VP2) and two non-structural proteins São Paulo. Marmosets were sheltered near the howley (NS1 and NS2). The CPV-2 emerged from the feline monkeys’ (Alouatta guariba) enclosure. In a period of a parvovirus (FPV) or a closely related virus as a novel month howley monkeys presented clinical signs similar pathogen in the late 1970s and rapidly spread worldwide to herpesvirus with oral lesions that disappeared. in the canine population. Within a few years, the virus During the following 24 days all seven marmosets died underwent a rapid evolution and, new “antigenic” acutely. At necropsy, only one had oral lesions similar to types, termed CPV-2a, CPV-2b and CPV-2c, replaced the hematoma, and all had encephalitis. One week before original CPV-2. Notably, it has been observed that canine the onset on the monkeys, one person with herpes parvoviruses display high evolutionary rates, and for symptoms was seen at the park. The clinical suspicion this reason, nucleotide substitutions are often found. included herpesvirosis and brain tissue was sent for In order to evaluate if the CPV-2a, CPV-2b and CPV-2c diagnosis. As the animals did not show any signs but represent different antigenic types, hypothetical viral encephalitis, rabies was also included on the differential. proteins were generated and analyzed. Additionally, all genomes from CPV-2 were retrieved from GenBank gDNA Tissue Miniprep System kit and a nested PCR with and phylogenetically analyzed. Six distinct genogroups degeneratedDNA purification primers was was performed used to diagnose with ReliaPrepTM herpesvirus were observed. For each genogroup, hypothetical viral proteins were also generated based on the consensus PCR were: DFA (5`-GAYTTYGCNAGYYTNTAYCC-3´), sequences and compared. According to a structural, ILKon the samples.(TCCTGGACAAGCAGCARNYSGCNMTNAA-3´), Degenerated primers used on the first electrostatic and hydrophobical analysis and total polar/ and KG1 (5´-GTCTTGCTCACCAGNTCNACNCCYTT-3´); and on the second PCR: TGV those six genogroups resulted in four main prevalent apolar charges caused by the amino acids modifications, (5´-TGTAACTCGGTGTAYGGNTTYACNGGNGT-3´) and IYG (5´-CACAGAGTCCGTRTCNCCRTADAT-3´). The the VP2 apical structure involved in the virus binding domainstructural represents types. It wasmore observed expressive that electrostaticmodifications and in methodology, ABI 3500 platform with BigDye Terminator hydrophobical changes than the formerly sites used for PCR product was purified and sequenced by Sanger September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

57 Oral Presentation

CPV typing. Therefore, it is suggested that CPV-2 typing need to be revised and that vaccine strains should be Miseq platform. A consensus tree based on nucleotide based on those main prevalent structural types to be sequencespurified products from complete were sequencedgenome was using generated Illumina by more effective. FINANCIAL SUPPORT: CNPQ, FAPERGS, the neighbor-joining method (MEGA5), using maximum CAPES. composite likelihood model and 1,000 bootstrap replicates. The complete genome of canine circovirus VV351 - GENETIC CHARACTERIZATION OF CANINE D11 shared 85.5%-98.0% nucleotide identity with CIRCOVIRUS DETECTED IN STOOL SAMPLES FROM others canine circovirus. The capsid and replicase DOGS IN THE SOUTH REGION OF BRAZIL nucleotide sequences of D11 share 86.7%-98.3% and Cruz, T.F.1; Batista, T.N.2; Baccarin, A.M.2; Gradiz, J.J.2; 82.2%-98.5% identities, respectively, with the known Vieira, E.M.2; Tozato, C.C.1; Kurissio, J.K.1; Araujo Jr, canine circovirus. The capsid and replicase proteins of J.P.1 D11 showed 86.7%-98.3% and 82.2%-98.5% amino 1. UNESP - Universidade Estadual Paulista, acid identity, respectively, with others canine circovirus. Rua Quirino de Andrade, 215, São Paulo - SP, 01049-01 The phylogenetic tree showed that D11 grouped with 2. FURB - Universidade Regional de Blumenau, others canine circovirus. These group forming a distinct R. Iguaçu, 404 - Itoupava Seca, Blumenau - SC, 89030-030 clade with porcine circovirus, wherever avian circovirus are non-enveloped, icosahedral viruses clustered separately. with single-stranded circular genome DNA that belong VV365 - PHYLOGENETIC ANALYSIS OF AICHIVIRUS B to the family . Circoviruses infect birds and IN FECAL SAMPLES FROM CALVES IN BRAZIL mammals, and porcine circoviruses were reported as Rribeiro, J.1; Lorenzetti, E.1; Medeiros, T.N. da S.1; only species in the genus Circovirus that infect mammals. Junior, J.C.R.1; Bronkhorst, D.E.2; Crespo, S.E.I.1; Currently, a specie nonporcine circovirus (canine Favero, L.M.1; Alfieri, A.F.1; Alfieri, A.A.1 circovirus) was detected in samples from dogs. Thus, the objective of this study was to detect canine circovirus in 1. UEL - Universidade Estadual de Londrina, stool samples by quantitative PCR (qPCR) and perform Rodovia Celso Garcia Cid, Km 380 - Campus Universitário, the genetic characterization of the virus. DNA was Londrina - PR, 86057-970 extracted from 45 feces samples collected from dogs 2. UNOPAR - Universidade Norte do Paraná, with hemorrhagic gastroenteritis (n=5), hematochezia Av. Paris, 675 - Jd. Piza, Londrina - Paraná, 86041-140 (n=10), normal stool (n=12), and without information The genus Kobuvirus belongs to the Picornaviridae family, about feces (n=18). The dogs were one months-old to and the virions are non-enveloped, with icosahedral 10 years-old. The animals were from Santa Catarina symmetry and a diameter of 27-30 nm. Currently, the (n=44) and Paraná (n=1) states. The samples were Kobuvirus genus includes three species, designated as tested for canine parvovirus (CPV) and canine circovirus Aichivirus A, Aichivirus B, and Aichivirus C, which can by qPCR. The results determined by qPCR were analyzed cause infections in humans, cattle, and pigs, respectively. qualitatively. Twenty-three samples were positive for The presence of Aichivirus B has been detected CPV and two were positive for canine circovirus. A six worldwide. However, there are few studies about the months-old female dog (D11) from Paraná was co- genetic heterogeneity of these viruses. A total of 30 fecal infected with CPV and canine circovirus. The other samples diarrheic from calves (<30 days) with diarrhea female dog (D10) infected with canine circovirus was were analyzed from a dairy production farm in the state 10 months-old and was from Santa Catarina. The two of Paraná, Brazil. Fecal suspensions were prepared at 10- dogs had hemorrahagic gastroenteritis. The complete 20% (w/v) in PBS buffer and centrifuged at 3,000 x g for 5 min. The RNA extraction was performed with 400 µL of fecal suspensions. The presence of segment double- performedgenome of canineafter RCA. circovirus The wasformation amplified of byfragments rolling- stranded RNA (dsRNA) of rotavirus in the faecal samples circle amplification (RCA). The digestion reaction was was evaluated by polyacrylamide gel electrophoresis circovirus, which has genome DNA of 2,063 kb. The (PAGE) technique. The SN-PCR assay for the bovine larger than 2000 bp confirmed the detection of canine September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

58 Oral Presentation coronavirus was performed to amplify a sequence based to produce artisan cheese using raw milk experimentally on the highly conserved region N gene with a predicted contaminated with VACV and to analyze the viral product of 251 bp. All fecal samples were submitted to viability at different times of the maturation process. RT-PCR assay to detect the 3D region of the Aichivirus The raw milk used for the cheese production was tested B using the primers UNIV-F/R, which target a 216 bp. by PCR for Orthopoxvirus, and it was negative. The Two positive samples to Aichivirus B was performed milk was contaminated with 105 PFU/ml of VACV-GP2. VP1-F/R, which target a 1,159 bp region of the complete Control cheeses were produced separately with raw VP1 capsid gene. Two Aichivirus B region VP1 products milk not contaminated with VACV. Natural yoghurt and commercial rennet were used to produce the cheeses. Genetic Analyser and submitted for nucleotide sequence The control and contaminated cheeses were submitted analysis.were purified, Eleven quantified,out of 30 (36.7%) sequenced fecal insamples an ABI3500 tested to the maturation process during 1, 7, 14, 21, 45 and 60 were positive for Aichivirus B form 3D region, two days. Cheeses production was repeated four times in fecal samples were positive for rotavirus, and none different moments. They were stored in a B.O.D. incubator fecal sample was positive for bovine coronavirus. The at 25ºC. In each previously established maturation day, Aichivirus B had already been described in Brazil and one control and one contaminated cheese were collected also found high infection (38%) in calves with diarrhea. and macerated. For viral isolation, aliquots of macerated The phylogenetic analysis of the VP1 region of Aichivirus cheese were inoculated into BSC-40 cells. VACV viable B showed that the Brazilian strains clustered in the particles were detected in contaminated cheeses until same branch of the genotype A prototype (U-1 strain). 60 days of maturation. These results suggest that artisan Further studies should be performed to characterize cheese produced with milk contaminated with VACV the Aichivirus B strains circulating in all regions of might be a risk for Public Health. FINANCIAL SUPPORT: Brazil. FINANCIAL SUPPORT: FINEP, CAPES, CNPq, and FAPEMIG, CNPQ AND CAPES Fundação Araucária/PR. VV396 - MOLECULAR DETECTION AND VV393 - VIABILITY OF VACCINIA VIRUS IN PHYLOGENETIC RELATIONSHIPS OF AN AMPHIBIAN EXPERIMENTALLY CONTAMINATED CHEESES AT RANAVIRUS ASSOCIATED TO OUTBREAKS OF DIFFERENT TIMES OF THE MATURATION PROCESS MORTALITY IN BRAZILIAN FISH FARM Rehfeld, I.S.; Fraiha, A.L.S.; Matos, A.C.D.; Guedes, Queiroz, S.R. de A.1; de Pádua, S.B.2; Filho, R.N. de M.2; M.I.M.C.; Costa, A.G.; de Souza, M.R.; Lobato, Z.I.P. Araújo, A.P. de3; Maganha, S.R. de L.1; Navarro, J. de O.1; Lima, M.P.1; de Alencar, A.L.F.1; Arruda, E. de P.1; UFMG - Universidade Federal de Minas Gerais, 1 1 1 Avenida Presidente Antônio Carlos, 6627 - Pampulha, Belo Munin, F.S. ; Fernandes, A.M. ; de Sousa, R.L.M. Horizonte - MG, 31270-901 1. FZEA/USP - Faculdade de Zootecnia e Bovine vaccinia (BV), a zoonosis caused by Vaccinia virus Engenharia de Alimentos da Universidade de São Paulo, Av. (VACV), affects dairy cattle and milkers, causing major Duque de Caxias Norte, 225 - Campus da USP, Pirassununga economic impact in the Brazilian dairy production. - SP, 13635-900 2. AQUIVET - Aquivet Saúde Aquática, Rua Previous studies have described the presence of viable Antonio Ernesto Célico, número 359, quadra 8, lote 16, viral particles in milk samples of cows experimentally Residencial Marcia Damha III, São José do Rio Preto - SP, and naturally infected with VACV. Furthermore, others 15061810 studies showed that VACV remains viable in fresh 3. ACQUAPISCIS - Acquapiscis Consultoria e artisan cheese, made with VACV contaminated raw Medicina Veterinária em Aquicultura, Rua Vieira de Morais, milk. However, it is not known if the maturation process 1201, Campo Belo, São Paulo - SP, 04617-014 used during the production of artisan cheese is able to Ranavirus (RV) are emerging pathogens responsible inactivate VACV particles. The artisan cheese produced for epizooties of great ecologic and economic impact in Minas Gerais was declared a Brazilian cultural heritage in 2008, and its sanitary quality is a very important Disease caused by these viruses was detected in Public Health issue. Therefore, the aim of this study was in fishes, amphibians and reptiles around the world. September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

59 Oral Presentation amphibians from Brazil, but until now there is no data VV470 - VIRUCIDAL ACTIVITY OF A PURIFIED COMPOUND ISOLATED FROM PRASIOLA CRISPA AGAINST EQUINE HERPESVIRUS TYPE 1 about RV infection of Brazilian fishes. Currently, tilapia of economically rentable farming activities in Brazil. Due Marinho, R.S.S.1; Ramos, C.J.B.1; Leite, J.P.G.2; Belo, Nile (Oreochromis niloticus) is the fish farm of the most the economic importance of tilapia farming, the present C.A.D.3; Pereira, A.B.3; Teixeira, V.L.1; Paixão, IC.N.P.1; study was aimed to investigate mortality of tilapias from Pinto, A.M.V.1 states of Ceará (CE) and Bahia (BA). For this purpose, 30 moribund and 15 apparently healthy specimens 1. UFF - Universidade Federal Fluminense, Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 were collected during mortality events in tilapia farms 2. FIOCRUZ - Fundação Oswaldo Cruz, Av. installed around Castanhão Dam, Jaguaribara (CE), and Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 from nursery farms in Horizonte (CE) and Paulo Afonso 3. UNIPAMPA - Universidade Federal do (BA). Darkening skin and hemorrhagic foci in the base Pampa, Av. General Osório, 900, Bagé - RS, 96400-100 corneal opacity and bleeding ulcers on the skin with loss Equine herpesvirus type 1 (EHV-1) is a major equine of scalesthe pectoral were observed. fins and perianalConcurrent region, bacterial exophthalmos, infections pathogen. It is etiologic agent for a triad of clinical were also detected. Fragments of liver, pancreas, spleen, signs: abortion, neurological and respiratory disease. It kidney, heart, intestine, stomach, gills, brain and eye is enzootic throughout the world and almost all horses were sampled and stored in buffered formalin solution older than 2 years of age have been exposed. Following 10%. Histopathological analysis revealed impairment of initial exposure, EHV-1 has the ability to develop into an liver tissue at various levels with the presence of vacuolar unapparent, latent infection. It is this ability to reside degeneration associated with necrotizing hepatitis. as a silent and persistent infection in horses which Basophilic inclusions bodies in acinar cells were provides a reservoir of virus for continual transmission. detected in all samples. Some tissue samples showed The terrestrial seaweed Prasiola crispa (Lightfoot) necrosis of the kidney, depletion of hematopoietic tissue, Kützing as collected in ice-free areas, the region near and degeneration of cardiac muscle cells and endothelial the station Polish Arctowski, Admiralty Bay, King George Island (61° 50’ - 62° 15’ S and 57° 30’- 59° 00’ W), in Antarctica. Seaweeds were dried in a dark chamber ofcells. 321bp- Fragments and 625bp-products, of fixed tissues were corresponding submitted toto PCRthe with air circulation at 40°C in dark bags, and stored MCP(polymerase (Major chainCapsid reaction) Protein) for genesequential of Ranavirus. amplification All in a freezer and after a new screening was performed samples tested positive for ranavirus. The amplimer for the removal of waste and other materials getting sequences revealed high percentage of similarity (>96%) dried seaweed. The compound C3F4 was isolated from with other homolog amphibian ranavirus sequences crude extract in dichloromethane in silica gel 70-230 deposited in GenBank. In phylogenetic reconstructions, chromatography column and characterized in Thin- the samples were grouped together in the same clade with other amphibian ranavirus. The results indicate, in C-Nuclear Magnetic Resonance (RMN) spectras. This Layer Chromatography (TLC) and identified by H- and an unprecedented manner, the circulation of frog virus- study aimed to evaluate the in vitro cytotoxic effect and economic implications for tilapia farming from Brazilian Prasiola crispa in EHV-1 replication. The cytotoxic effect like, an amphian ranavirus, in fish farm in Brazil with ofvirucidal C3F4 has properties been measured of C3F4 in compound RK-13 cell purified treated fromwith by FAPESP (Proc. nº 2011/19002-8; 2012/08846-3). different drug concentrations. Cytotoxic concentrations northeast region. This work was financially supported (CC50) values have been determined for acyclovir 1010 ± 2.0 and C3F4 compound C3F4 1884 µM ± 9.0.

on EHV-1 a virucidal assay activity was performed. A virusAs a firstsuspension approach containing to characterize 1x106 thePFU action EHV-1 of C3F4were mixed with C3F4 (50 µM) and acyclovir (200 µM) were kept at room temperature (24 ºC) for 1 h. Meanwhile,

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

60 Oral Presentation control of untreated infected virus was performed in they can emerge silently or aggressively. Wildlife it is the same conditions. Then, treated virus suspension the principle source of these viruses and constitutes a and untreated control were diluted and percentage large and often unknown reservoir of zoonotic diseases of inhibition of EHV-1 infectivity on RK-13 cell was that can be responsible for epidemics or outbreaks determined by plaque assay. The result of virucidal events. The University of Sao Paulo in a partnership activity for dichloromethanic fraction C3F4 was 90% with EcoHealthAlliance-EHA and Columbia University ± 8.5. Acyclovir had slightly virucidal activity on viral particle. For better understanding about mechanisms of of different virus families circulating in animals from the antiviral activity by C3F4 further investigations are wild(NY) and developed urban aareas project with that potential aim the epidemic identification risk. underway. Samples were collected from 758 animals during 2008 to 2013 in Rain Forest region and Amazon Forest in VV473 - EMERGING VIRUSES MONITORING IN BATS, sites close and inside city of Manaus-AM characterized RODENTS AND MARSUPIALS FROM BRAZILIAN RAIN as Pristine, Urban and Intermediate depending of the FOREST REGION AND AMAZON REGION COLLECTED level of conservation. Samples from722 animals (620 BETWEEN 2008 AND 2013 bats, 64 rodents and 38 marsupials) were analyzed in Campos, A.C. de A.1; Anthony, S.J.2; de Araújo, J.3; the Clinical and Molecular Virology Laboratory-USP and Ometto, T.3; Hurtado, R.3; Nardi, M.S.4; Nava, A.5; 216 animals (179 bats and 37 rodents) were analyzed in Solorio, M.R.6; Souza, M.C.P.7; Rostal, M.8; Loh, E.8; duplicate by Columbia University-USA. After the capture Torrelio, C.M.Z.8; Aguirre, A.A.9; Daszak, P.8; Durigon, oral and rectal swabs, feces, urine and blood were E.L.1 collected, directly transferred to liquid nitrogen and 1. Laboratório de Virologia Clínica e Molecular, stored at -70ºC in laboratory. Total nucleic acids were Depto Microbiologia, ICB-II, Universidade de São Paulo And extracted using NucliSens Lysis Buffer (BioMerieux) Ecohealthalliance-EHA and the randomic cDNA synthesis was made with 2. The Center for Infection and Immunity, SuperScript III kit. The presence of thirteen different Columbia University and Ecohealthalliance-EHA viruses and/or families (, , 3. Laboratório de Virologia Clínica e Molecular, Astroviruses Bocaviruses, Coronaviruses, , Depto Microbiologia, ICB-II, Universidade De São Paulo Filoviruses, Flaviviruses, Hantaviruses, Herpes virus, 4. DEPAVE 3, Secretaria do Verde e do Nipah virus, Paramyxoviruses and ) Meio Ambiente da Prefeitura Municipal de São Paulo were analyzed in samples by conventional PCR or Real- and Laboratório de Virologia Clínica e Molecular, Depto Time PCR assays. All amplicons were sequenced by Microbiologia, ICB-II, Universidade De São Paulo 5. Ecohealthalliance-EHA and Depto de sample was positive to Paramyxovirus. Twelve bats were Medicina Veterinária Preventiva e Saúde Animal da Faculdade positiveSanger’s to method Coronaviruses, to results seven confirmation. to Hantaviruses, One rodent one de Medicina Veterinária e Zootecnia da Universidade de São for Paramyxoviruses and ten to Herpes virus. Only one Paulo marsupial was positive to Hantavirus. This data showed 6. Depto de Medicina Veterinária Preventiva e Saúde Animal da Faculdade de Medicina Veterinária e the presence of virus with emergent potential in animals Zootecnia da Universidade de São Paulo and Ecohealthalliance- from Rain Forest and Amazon region. Financial support: EHA EcoHealthAlliance - EHA 7. Laboratório de Virologia Clínica e Molecular, Depto Microbiologia, ICB-II, Universidade de São Paulo and Universidade Santo Amaro 8. Ecohealthalliance-EHA 9. George Mason University. Environmental Science and Policy In the last decades some viruses has been concern among researchers and health professionals because

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation BASIC VIROLOGY -BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

62 Basic Virology: BV

BV7 - EVALUATION OF NITAZOXANIDE ANTIVIRAL BV9 - IN VITRO EVALUATION OF THE ANTIVIRAL ACTIVITY AGAINST ROCIO AND SAINT LOUIS ACTIVITY OF BOTHROPS ALTERNATUS VENOM ON ENCEPHALITIS VIRUSES THE REPLICATION OF RESPIRATORY SYNCYTIAL Barros, H.L.S.1; Polloni, L.C.1; Oliveira, T.F. de M.S.1; VIRUS (RSV) Yokosawa, J.1; Chávez, J.H.2 Polloni, L.C.; Barros, H.L.S.; De Oliveira, F.; Yokosawa, J. 1. UFU - Universidade Federal de Uberlândia, Av. João Naves de Ávila, 2121 - Santa Mônica, Uberlândia - UFU - Universidade Federal de Uberlândia, Av. João MG, 38408-100 Naves de Ávila, 2121 - Santa Mônica, Uberlândia - MG, 2. UFMT - Universidade Federal de Mato 38408-100 Grosso, R. Guadalajara, 1 - Jardim das Américas Cuiabá - Respiratory syncytial virus (RSV), a member of MT, 78060-624 the family, is responsible for high Nitazoxanide (NTZ), a thiazolide anti-infective, presents morbidity and mortality in children throughout the activity against anaerobic bacteria, protozoa, and a range world. This fact runs partly due to the limited treatment options available for this infection, which makes the hepatitis C and B, rotavirus and Japanese encephalitis development of antiviral agents economically feasible a of viruses in cell culture models, including influenza A, virus. Originally developed as a treatment of intestinal priority in healthcare. This study aimed to evaluate the protozoan infections, the antiviral properties of NTZ antiviral activity of crude venom of the snake Bothrops were discovered during the course of its development alternatus against RSV. Through the colorimetric for treating cryptosporidiosis in patients with acquired 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay, the concentration of crude of antiviral activity of NTZ has not been fully elucidated, venom that reduced in 50% the viability (CC50) of Hep- immune deficiency syndrome (AIDS). The mechanism although some studies indicate that NTZ blocks viral 2 cells was determined as 23,1 ng/µL. The evaluation infection via modulation of host cell processes. Rocio of the antiviral activity was also performed by using virus (ROCV) and Saint Louis encephalitis virus (SLEV) the MTT assay. Treating Hep-2 cells with 23,1 ng/µL belong to the Flavivirus genus of the Flaviviridae family. crude venom for 3 h, exposing them with RSV for 1 h, Both viruses may cause important disease, such as and then incubating them for six days showed antiviral human encephalitis. No vaccine or antiviral drug is activity at MOI that ranged from 1,0x10-4 to 9,5x10-4. currently available to prevent or treat such viruses. Thus, the antiviral activity exerted by poison seemed Thus, the aim of this study was to evaluate the in vitro to be dependent on the low amount of virus presented antiviral activity of Nitazoxanide (NTZ) against ROCV to the cells (low MOI). On the other hand, treating cells and SLEV. This evaluation was performed in infected with crude venom after exposing them with RSV (post- VERO cells. The cytotoxicity (CC50) in VERO cells and treatment) did not seem to affect viral activity. These antiviral activity of NTZ against ROCV and SLEV was results may indicate that the antiviral activity of the assayed by the MTT method. In the NTZ CC50% assay, crude venom on RSV in vitro is apparently preventive was obtained the value of 3245,6 µM. Furthermore, in and not therapeutic. Reduced incubating period may the NTZ antiviral effect assay was found the CE50% help elucidate the effect of the venom after limited varying from 630µM to 1254,2 µM. NTZ showed antiviral rounds of viral replication. FINANCIAL SUPPORT: UFU, activity against SLEV, with selectivity indexes ranging FAPEMIG, CNPQ, CAPES, INCT NANO BIOFAR from 2,59 to 5,14, depending on multiplicity of infection (MOI) that was used. No antiviral activity was observed against ROCV. Additional studies should be carried out to investigate in vitro and in vivo NTZ action against other viruses, since antiviral activity against SLEV was demonstrated. FINANCIAL SUPPORT: UNIVERSIDADE FEDERAL DE UBERLÂNDIA

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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BV19 - ASSESSMENT PROINFLAMMATORY CYTOKINES BV20 - FITNESS EVALUATION OF A RECOMBINANT AFTER TREATMENT WITH 17B-ESTRADIOL CELL MURINE NOROVIRUS DURING SERIAL PASSAGES IN LINE (MT-2) CONTINUOUSLY INFECTED WITH HTLV- CELL CULTURE 1 Filho, E.F. de O.; Di Felice, E.; Toffoli, B.; Zonta, W.; Carvalho, L.D.1; Martins, C.P.S.2; Martins, M.L.3; Souza, Mathijs, E.; Mauroy, A.; Thiry, E. J.G.2; Stancioli, E.F.B.2; Fagundes, E.M.S.2; Gomes, R.A.3 Université de Liège - Place du 20 Août 7, 4000 Liège, 1. UESC - Universidade Estadual de Santa Bélgica Cruz, Campus Soane Nazaré de Andrade, Rodovia Jorge Noroviruses are single stranded positive sense RNA Amado, km 16, Bairro Salobrinho Ilhéus-Bahia, 45662-900 viruses which can infect human and different animal 2. UFMG - Universidade Federal de Minas species. Human norovirus (NoV) infections are among Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, the most important causes of gastroenteritis in both Belo Horizonte - MG, 31270-901 children and adults. Infections often occur as outbreaks 3. HEMOMINAS, Rua Grão Pará, 882 - Santa Efigênia - Belo Horizonte - MG, 30622-020 cell culture system as well as a workable animal model, manywhich aspects may be of foodborne. the NoV infection Due to in the human lack areof an still efficient poorly Diseases associated with this infection include Adult understood. The murine norovirus (MuNoV) grows HTLV-1 was the first retrovirus to be isolated in humans. T-cell leukemia (ATL) and myelopathy (TSP / HAM). easily in cell culture in contrast to the Human NoV, and HTLV-1 can be transmitted through infected lymphocytes constitutes an excellent animal model. Recombination present in breast milk, during sex and through blood can dramatically change virulence properties of the transfusions or blood products. Transmission of HTLV- viruses and has been evidenced in silico for different 1 is more effective from men to women and infected human NoV strains isolated from clinical cases. Recently, women are more likely to develop the disease. This after in vitro coinfection of RAW264.7 cells with different pattern is gender-related worldwide and parental MuNoV strains CW1 and Wu20, we obtained a recombinant Wu20/CW1 strain. This recombinant hormonal cause. In this context, this study aims to strain showed reduced plaque size compared to the remains to be clarified. Recent studies point to a possible parental strains. The aim of the study was to observe and lymphocyte culture supernatant permanently infected molecularly characterize the natural genetic evolution evaluate proinflammatory cytokines levels in the human of the recombinant MuNoV strain across in vitro different concentrations (1 uM to 0.0001 uM). Cytokines werewith measured HTLV-1 (MT-2) in the treatedMT-2 cell with culture 17β-estradiol supernatant at after stimulation with PMA and after treatment with adaptreplications. to the Viralcell culture fitness system. is a complex Thus, concept.the recombinant Here we straindefined was this serially fitness replicated as the ability in vitro of a viralin RAW264.7 population cells to (up to 14 passages). Viral plaque sizes of early and late 17β-estradiol at different concentrations (1 uM to 0.0001 and the result points to a change in IL-6, IL-10 and IL-12 progenies were compared with the Image J software. uM), using the Human Inflammatory Cytokine CBA Kit, dependent manner, compared to the untreated control. with the Mann and Whitney non parametric statistical p70 levels in cells treated with 17β-estradiol, in a dose- The greatest change occurred in IL-6 levels compared test.A significant Afterwards, difference viruses from was showndifferent between cell passages them to untreated control (elevation around 5-fold compared were cloned and sequenced. The average plaque size increased from the earlier to the later progenies (from the levels of estradiol in the HTLV-1 carrier may alter the 0.1 mm2 to around 0.5 mm2). Molecular investigations to control cell). These data suggest that fluctuations in are currently performed in order to specify in which genetic region mutations occur and whether or not pro-inflammatory cytokines profile and, consequently, influence the outcome of HTLV-1 pathogenesis. This is the evolution. In addition, two other parameters of in vitro SUPPORT:first study CNPQ,showing FAPEMIG the modulation E FUNDAÇÃO of proinflammatory HEMOMINAS this could explain fitness modifications during in vitro cytokines by HTLV-1 hormone 17β-estradiol. FINANCIAL production and (ii) one step growth kinetics. The data virulence modification will be investigated: (i) virus September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

64 Basic Virology: BV should provide interesting information about genetic The weight and behavior of the animals showed no evolution in the genus Norovirus, especially regarding recombination events and explain how a recombinant no changes in animal behavior or times of death were significant changes. For the LD50>2000 mg/kg group increase in numbers (p<0.001) while urea values strain, first disadvantaged compared to its parental recorded within 48 hours. Platelets showed a significant strains,BV23 could -regain fitness EVALUATION by genetic evolution. OF AN mg/kg R425 concentrations. Although there have been AMINOMETHYLNAPHTHOQUINONE (R425) IN decreased significantly (p<0.001) at the 550 and 175 ACUTE TOXICITY AND ANTI-HSV-1 ACTIVITY IN normal the physiological levels. In histological sections, BALB/C MICE significant changes, the 14-day parameters returned to Garrido, V.; Barros, C.S.; Chagas, C.; Melchiades, major organs (liver, kidney, stomach and duodenum), V.; Ribeiro, C.; Cavalcante, M.; Bittetti, M.; Silva, A.; asthere already was reported reversible with inflammatory other drugs. responses According into the Vargas, M.D.; Neves, A.P.; Teixeira, G.; Paixão, I.C.P.N. OECD 423 guidelines we concluded that R425 has low UFF - Universidade Federal Fluminense, Rua Miguel toxicity in BALB/c mice. Thus, the maintenance of the de Frias, 9, Icaraí, Niterói - RJ, 24220-900 anti-HSV validity tests is mandatory in experimental animals. FINANCIAL SUPPORT: CNPQ, CAPES, FAPERJ, Herpes Simplex-1 virus (HSV-1) is a worldwide UFF (PROPPI) endemic infection and one of the most prevalent pathogenic microorganisms in Brazil. The drugs BV39 - PROSPECTIVE STUDY OF FLAVIVIRUSES IN employed to treat HSV-1 have several side effects SMALL MAMMALS, IN MINAS GERAIS, BRAZIL including resistance, latency, and toxicity. The research Rezende, I.M.de1; Amaral, C.D.2; Sacchetto, L.1; for new effective drugs against these viruses therefore Miranda, J.B.2; Borges, I.A.2; Vieira, F.N.2; Alves, P.A.2; remains crucial. Preliminary in vitro results with an Paglia, A.P.2; Trindade, G. de S.2; Drumond, B.P.1 aminomethylnaphthoquinone (R425), synthesized by The Organic Chemistry Department of UFF have shown 1. UFJF - Universidade Federal de Juiz de Fora, that R425 has low cytotoxicity (CC50= 1722 ±175.9 µM) R. José Lourenço Kelmer - Martelos, Juiz de Fora - MG, 36036- 330 2. UFMG - Universidade Federal de Minas compared with reference drug, acyclovir (CC50= 960 and effective anti-HSV-1 activity (EC50=2.13±0.11 μM), Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, Belo Horizonte - MG, 31270-901 Vero cells infected with HSV-1 KOS strain (MOI-1) and thenμM; EC50= treated 1.00 with μM). different In vitro assaysconcentrations were performed (12.5, 25 in The wildlife is a reservoir of microorganisms that, and 50 µM) of the substance R425 for 24 h. Each drug once transferred to humans, may emerge as public has a degree of systemic toxicity, so that research aims health threats. Most reservoirs animals are mammals, mainly of the orders Rodentia, Chiroptera, Primates and of R425 in BALB/c mice weighing 18-25g and 60-120 Carnivora. The great diversity of rodents, its remarkable daysat finding old. First, out thefor acutethe LD50 toxicity test, level two ofgroups a single of mice dose reproductive potential and their ability to adapt to (n= 5/group) were treated: G1= R425 (2000 mg/kg) different niches are factors that explain, in part, the in 10% DMSO as the vehicle, and G2= the vehicle only emergence and reemergence of some viral diseases. The control. Four groups were also employed for the 14- genus Flavivirus belongs to the group of arboviruses day tests (n= 5/group): G1 and G2 were treated with of greatest global importance, responsible for serious 550 mg/Kg and 175 mg/Kg doses of R425, respectively; diseases such as encephalitis, fever, hemorrhagic fever G3= acyclovir control (50mg/Kg) and G4= 10% DMSO and hepatitis in humans. The members of the genus only. Blood samples were drawn before (D0) and after Flavivirus of major importance to public health are the each experiment (D7, D14) for haematological and Dengue virus (DENV), Yellow fever virus (YFV), West biochemical evaluation (urea, creatinine, uric acid, Nile virus (WNV), Japonese encephalitis virus (JEV) and proteins, albumin, ALT, AST, and alkaline phosphatase), Saint Louis encephalitis virus (SLEV). The aim of this and some organs were removed for histological analysis.

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersstudy - Basic wasVirology: to doBV a prospective study of flaviviruses in XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

65 Basic Virology: BV small mammals in a rural area of Minas Gerais, Brazil. additional dose of bacteria-expressed E1E2 protein. The sera of immunized mice were tested by ELISA and mammals from Laboratório de Vírus/UFMG was used. reacted against the recombinant E1E2 protein as well as TheseTo detect viscera flaviviruses, were collected a collection at Rio of Pomba viscera city, of smallfrom four synthetic peptides (I - IV) covering parts of the E1E2 October 2012 to August 2013 and kept in RNAlater at sequence. In addition, sera of mice of the two immunized -70 °C. Of the 112 samples present in the collection, the groups reacted against viral proteins in Huh7.5 cells liver of 49 animals was initially tested for the presence of DENV, YFV, SLEV, WNV and JEV. The liver was macerated with liquid nitrogen and subsequently, the total RNA producing JFH1 strain HCV in immunofluorescence extraction was performed using Viral RNA Midkit antibodiesassay. Our resultsagainst demonstrate HCV glycoprotein(s). the ability of an artificialFurther (QIAGEN, USA). cDNA synthesis was performed using investigationsantigen, administered are still neededas DNA to vaccine, explore tothe elicit neutralizing specific random primers and then used in a real time PCR (qPCR) capability of these antibodies, and to investigate the assay, which aimed to amplify a fragment of the NS5 cellular immune response elicited by this antigen.

BV56 - EVALUATION OF MITOCHONDRIAL ACTIVITY indeterminateregion of the flaviviruses result and qPCRgenome. assays None will of bethe repeated 49 liver IN CELLS INFECTED BY ADENOVIRUS SEROTYPE 40 andsamples amplicons tested will presented be sequenced. flaviviruses The RNA. serum Some of 109 had Guissoni, A.C.; Parente, J.; Badr, K.; Soares, C.; Cardoso, animals are also going to be tested. All positive samples D. UFG - Universidade Federal de Goiás, Av. Esperança, presence of mammals infected with zoonotic viruses in s/n - Setor Itatiaia, Goiânia - GO, 74001-970 areasare going occupied to be by sequenced humans may to confirm increase the the results. risk of The re- The mitochondria are involved in a variety of metabolic emergence of zoonotic diseases, since these animals may processes including cell ATP production, calcium be close to humans, which may favors the transmission homeostasis, cell proliferation, apoptosis, and synthesis of the infectious agents. FINANCIAL SUPPORT: FAPEMIG, of amino acid, nucleotide and lipid. The distribution, CNPq, CAPES, UFJF, PROPESQ/UFJF. shape and operation of this organelle are regulated by BV46 - HUMORAL IMMUNE RESPONSE ELICITED intrinsic and extrinsic stimuli which may be a virus. BY A DNA BASED IMMUNIZATION ENCODING AN These may induce or inhibit various mitochondrial ARTIFICIAL ANTIGEN CONTAINING SEGMENTS OF E1 AND E2 HCV GLYCOPROTEINS with adenovirus, usually undergoes metabolic changes processes in highly specific manner. When a cell infected Freitas, G.R.O.; Froelich, L.; Oliveira, M.M.; Oliveira, that can trigger apoptosis through mitochondrial T.F.M.S.; Yokosawa, J. pathway. These changes occur not only as a result of viral replication, but also by the interaction between Laboratório de Virologia, UFU - Universidade Federal transcripts and / or viral proteins with factors of cellular de Uberlândia, Av. João Naves de Ávila, 2121 - Santa Mônica, regulation and immune response. Thus, some viral Uberlândia - MG, 38408-100 proteins interact with mitochondrial proteins which Approximately 130 million people are infected chronically regulate cellular responses. In this study we evaluated with hepatitis C virus (HCV). The development of vaccines the activity of the respiratory chain in A549 cells (human has been hampered by the high genetic variability of the lung carcinoma) infected with adenovirus serotype 40 virus as well as by the lack of suitable animal models. In (HAdV-40). The procedure included with the cultivating of A549 cells followed by inoculating of HAdV-40 and antigen containing segments of the HCV glycoproteins observation of cytopathic effect after 30 hours. After E1this and study, E2 thatwe describe have been the reported construction to elicit of neutralizingan artificial this, the infected and control cells were treated with antibodies against HCV. The immunogenicity of the E1E2 MitoTracker ® Green FM, where the probe Mitotracker antigen was assessed in mice by DNA immunization (green) marks all mitochondria, and rosamina (Red) given in three doses. Furthermore, another group of selectively marks just the active mitochondria. The mice was immunized with two doses of DNA and one visualization of these changes was done by A1 Axioscope

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which has many deleted genes, resulting in the absence 579/599 for rosamina and 490/516 for Mitotracker. The imagesfluorescence were microscopegenerated in (Carl AxioCamMR Zeiss) at photographic wavelength system (Carl Zeiss). From the results, it was possible of fibrils. Bioinformatic analysis was also done in order to observe the mitochondria in the cytoplasm as an effectto predict of the the virus mass in ofacanthamoeba the fibrils. In cells addition, displayed it was in verified the viral replication by evaluation of cytopathic performed using a discontinuous glucose gradient, increase in fluorescence intensity zones. Mitochondrial whereagar plates treated with and PAS untreated buffer. A APMV fluctuability were assaysubmitted was theactivity mitochondria was quantified of infected based cells on theHAdV-40 evaluation had higher of the to ultracentrifugation. Electronic microscopy revealed mitochondrialfluorescence intensity activity, of rosamina.suggesting It wasthat observed adenovirus that infection in A549 cells up-regulates electron transport chain. FINANCIAL SUPPORT: FAPEG / CNPQ ofthat treated the enzymatic APMV and treatment M4 was removed quite similar, the fibrils showing and theit was absence corroborated of two proteins, by SDS-Page. one of The approximately protein profile 45 BV61 - FIBRILS OF THE GIANT ACANTHAMOEBA kDa and other of about 80 kDa. These proteins refer POLYPHAGA MIMIVIRUS – INITIAL STUDIES to ORFs R135 and L829 respectively, both related to OF PROBABLE FUNCTIONS OF THIS PECULIAR STRUCTURE to changes in viral replication and cytopathic effect was Rodrigues, R.A.L.1; Silva, L.K. dos S.1; Dornas, F.P.1; fibrils’ synthesis. The absence of the fibrils has not led Boratto, P.V. de M.1; Arantes, T.S.1; La Scola, B.2; Kroon, E.G.1; Abrahão, J.S.1 differentthe same. centrifugation The absence ofconditions. fibrils showed The evaluation no difference of in viral fluctuability but the assay is being done with 1. UFMG - Universidade Federal de Minas Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, temperatures and UV light is underway. The discovery of Belo Horizonte - MG, 31270-901 thefibrils functions in viral ofparticle’s this mimiviruses’ resistance peculiar when exposed structure to high will 2. Université de la Méditerranée, Jardin du bring insights about its biology and may help discussions Pharo - 58, bd Charles Livon -13284 Marseille Cedex 07 about its evolutionary origin and environmental roles. Mimiviridae family comprises giant viruses with huge FINANCIAL SUPPORT: FAPEMIG, CAPES, CNPQ, MAPA, genomic and structural complexity, with Acantamoeba PPG-MICROBIOLOGIA UFMG polyphaga mimivirus (APMV) as its type virus. It BV85 - ANTIVIRAL ACTIVITY OF A POLYSACCHARIDE OF DIMORPHANDRA GARDNERIANA ASSOCIATED WITH MANGIFERIN AGAINST POLIOVIRUS TYPE 1 was firstly isolated in 1992, but only in 2003 it was the Nucleo-Cytoplasmic Large DNA Viruses. During (PV-1) theidentified following as ayears virus, many being singular included characteristics in the group was of Rechenchoski, D.Z.1; Espada, S.F.1; Lopes, N.1; Godoi, described, such as several genes related to translational A.M.1; de Faccin-Galhardi, L.C.1; Nozawa, C.1; Ricardo, apparatus, besides some very distinct morphological N.M.P.S.2; Linhares, R.E.C.1 characteristics, such as two separate portals related to the packaging and delivering of the genome and 1. UEL - Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Km 380 - Campus Universitário, It remains unknown which would be the functions of Londrina - PR, 86057-970 glycoprotein fibrils surrounding almost all viral particle. 2. UFC - Universidade Federal do Ceará, probable roles for this structure. In that purpose, APMV Avenida da Universidade, 2853 - Benfica, Fortaleza - CE, particlesthese fibrils. were Thus, submitted the aim to of a this sequential work is toenzymatic evaluate 60020-181 treatment, with lysozyme and bromelain, to remove the Poliovirus (PV), member of the Picornaviridae family, is a non-enveloped positive single-stranded RNA virus, by transmission electronic microscopy and SDS-Page, causative agent of poliomyelitis, an acute disease of fibrils. Then, it was realized analysis of those particles the central nervous system that may result in paralysis treated and untreated APMV and M4 lineage, the one and death. Currently, poliomyelitis is under control in comparing the eletrophoretic protein profile from September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

67 Basic Virology: BV most part of the world, however, the disease remains BV88 - ANTIHERPETIC ACTIVITY OF NONI (MORINDA endemic in Afghanistan, Nigeria and Pakistan and, CITRIFOLIA) PECTINS therefore, represents a major threat for the world. Rechenchoski, D.Z.1; Lopes, N.1; de Faccin-Galhardi, Considering the importance of viral diseases, the L.C.1; Espada, S.F.1; Godoi, A.M. de1; Bomfim, W.A.1; search for natural products for their control has Ricardo, N.M.P.S.2; Linhares, R.E.C.1; Nozawa, C.1 been encouraged. Dimorphandra gardneriana is a Brazilian native leguminous tree, popularly known 1. UEL - Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Km 380 - Campus Universitário, as faveira or fava-d anta and belongs to the Fabaceae Londrina - PR, 86057-970 family. The plant is widely distributed in the Brazilian 2. UFC - Universidade Federal do Ceará, Cerrado and its fruits are exploited to obtain rutin and Avenida da Universidade, 2853 - Benfica, Fortaleza - CE, 60020-181 constituent obtained from Mangifera indica and has multiplequercetin, pharmacological both bioflavonoids. activities Mangiferin such as is antitumor, the major The natural substances capable of presenting alternative immunomodulatory, antidiabetic, antioxidant, antiviral, mechanisms, unlike synthetic antiviral action, may be antibacterial, anthelminthic, antiallergic and anti- potential candidates for the control of viral infections. These substances have been shown to interact with antiviral activity of a polysaccharide of D. gardneriana many viral targets ranging from adsorption of the virus associatedinflammatory. with This mangiferin study aimedagainst topoliovirus evaluated type the 1 to the host cell to release the new viral particles into the (PV-1). The antiviral action was investigated by plaque extracellular medium, which may result in additional reduction assay (PRA), using different protocols: the mechanisms of action of existing antiviral drugs. In this context and considering the high prevalence of herpes adsorption and virucidal activity. The 50% cytotoxic virus (HSV) infections and the incidence of severe disease concentrationtime-of-addition (CC50) (-2, -1, of 0, the +1, compound,+2, +4, +8), evaluatedinhibition byof in immunocompromised, the search for new antiherpetic compounds, particularly, makes it extremely challenging. The aim of this study is to investigate the antiviral indexMTT assay,(SI) >9.69. was >2000The compound μg/mL and was the tested 50% at inhibitory500, 250, activity of four pectins of noni (Morinda citrifolia), concentration (IC50) 206.25 μg/mL, with selectivity PDNA, PNDB, PDNN and PDNS to HSV-1 in HEp-2 cell of viral inhibition (%IV) of 77.8%, 45.1%, 54.1%, 70.6% cultures. The 50% cytotoxic concentration (CC50) for and125 and65.2%, 62.5 at μg/mL. the time Preliminary, 0h (treatment we showed simultaneously the percent PDNA, PNDB, PDNN and PDNS were 605, 845, 420 e 44 with infection), evaluated 1h, 2h, 4h and 8h thereafter, µg/ml, respectively, by MTT assay. The antiviral action was assayed by plaque reduction assay by the following At the same concentration, the compound inhibited the PV-1respectively, in approximately at the highest 46.8% concentration and 61.5% when (500 added μg/mL). 1h and the virucidal effect. The four compounds showed the protocols: the time-of-addition assay (-2h, 0h, +1h, +2h) and 2h before the infection, respectively. For virucidal 50% inhibitory concentration (IC50) of approximately assay, the compound demonstrated an inhibition around 45 µg/ml. For the PDNS, this concentration was close 26.2% and 30%, while the inhibition of adsorption to the CC50 and, therefore, excluded. For the others, showed a %IV of 65% and 27.5%, at the concentrations were used concomitantly with viral infection, reaching we observed significant antiviral activity when they results suggested that the compound interfered 100% of viral inhibition at the concentration of 100 µg/ inhibitingof 500 μg/mL PV-1 replication. and 250 μg/mL, Therefore, respectively. polysaccharide These ml. In post-infection treatment, only PDNA maintained of D. gardneriana may be a potential candidate for the the activity. No inhibition was observed in the pre- control of PV infection. FINANCIAL SUPPORT: CAPES, infection treatment and virucidal assays for any of the CNPQ, FUNDAÇÃO ARAUCÁRIA, PROPPG/UEL three substances. Thus, the viral inhibition caused by these substances most likely occurs in the initial stages of viral replication. Further studies will be conducted in order to better understanding at what step(s) of HSV-

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1 replication M. citrifolia pectins may act. FINANCIAL the eighth d.a.i and the group that received only VACV- SUPPORT: CAPES/CNPQ/FUNDAÇÃO ARAUCÁRIA/UEL WR the reduction of lymphocytes occurred between the

BV92 - EVALUATION OF THE INFLUENCE EXERCISED treatment with the probiotic was able to reduce letality BY A MICROORGANISM PROBIOTIC DURING VACCINIA andfirst anddelay fourth the d.a.i.reduction The mechanism of lymphocytes through in which infected the VIRUS INFECTION BALB/C MICE animals probably involves a immunomodulation as Andrade, A.C. dos S.P.1; Oliveira, G.P.1; Lima, M.T.1; previously described for other viral infections. However, Calixto, R.S.1; Martins, F. dos S.1; Ferreira, J.M.S.2; this mechanism will be further investigated by other Kroon, E.G.1; Abrahão, J.S.1 experiments. The tested probiotic was able to reduce 1. UFMG - Universidade Federal de Minas the letality of mice and delay the decline of lymphocytes, Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, showing its potential use as a method of controlling Belo Horizonte - MG, 31270-901 infection with VACV. FINANCIAL SUPPORT: FAPEMIG, 2. UFSJ - Universidade Federal de São João del- CAPES, CNPQ, MAPA, PPG-MICROBIOLOGIA UFMG Rei, Praça Frei Orlando, 170, Centro, São João del-Rei, Minas Gerais, 36307-352 BV94 - CLONING AND EXPRESSION OF TRUNCATED HEPATITIS C VIRUS E2 PROTEINS IN BACTERIAL The circulation of Orthopoxvirus in the world and the SYSTEM prevalence of bovine vaccinia in Brazil have generated Moreira-Vieira, F. de F.; Freitas, G.R.O.; Yokosawa, J. establishment of methods for control and prevention of UFU - Universidade Federal de Uberlândia, Av. João poxvirussignificant infections economic is important and social for impacts. society. In Thus, view the of Naves de Ávila, 2121 - Santa Mônica, Uberlândia - MG, this, new strategies to combat these viruses have been 38408-100 studied. Recently, the use of probiotic microorganisms The hepatitis C virus (HCV) belongs to the Flaviviridae has been proposed as a novel therapeutic approach family and to the genus. This virus has an for the control of various viral diseases. Therefore, the enveloped icosahedral capsid, and its genome consists of aim of this study is to understand what are the possible a segment of simple-stranded RNA that has a single open effects caused by probiotics in Balb/c mice infected with read frame (ORF). This ORF is translated to produce a Vaccinia virus Western Reserve (VACV-WR), in relation single protein, which is processed to produce seven to letality and differential leukocyte count. The VACV- structural proteins and three non-structural proteins. There are seven genotypes and several subtypes. The on sucrose cushion and used at a concentration of 106 HCV causes hepatitis C in humans and it is one of the main WR was multiplied and titrated in Vero cells, purified pfu in intranasal infection of Balb/c mice. The probiotic causes of chronic liver disease. It is a blood-borne virus that is transmitted mainly through the reuse of needles by broth De Man, Rogosa & Sharpe (MRS). The animals were injecting drug users and accidents in health care settings. X was obtained from an industrial product and grown in divided into four groups: animals that received (i) only In acute phase, the infection is asymptomatic in 80% of phosphate buffered saline 0.9%, (ii) only the probiotic cases, and chronic infection may envolve to cirrhosis, hepatic carcinoma and cause death. Around the world infection (d.a.i) with VACV-WR. The differential count of more than 350 thousand people die each year due to X (iii) only VACV-WR, (iv) probiotics X for 10 days after leukocytes in whole blood of mice was made. Infected diseases associated with hepatitis C. Early HCV diagnosis isn’t common and treatment is expensive, with many arching back, balanopostitis and weight loss. Uninfected collateral effects and it isn’t always successful, although groups showed clinical signals such as ruffling fur, groups showed no signal of infection. This experiment it can decrease the number of deaths and severe disease. showed that letality in the probiotic-treated group was There is no preventive vaccine for HCV but researchers 50% lower than the group infected with VACV-WR. The around the world are working in search for an effective differential leukocyte count showed that the reduction vaccine. In these studies, the envelope glycoproteins in the proportion of lymphocytes in the group treated E1 and E2 are the main proteins of interest because of with probiotic occurred only between the fourth and their position in the virus surface, turning them into

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

69 Basic Virology: BV targets for the immune system. The objective of this study was to express two truncated proteins containing routes were inoculated only with phosphate buffered selected segments of HCV envelope glycoprotein E2 that intranasal and tail scarification. Control groups for both induces the production of neutralizing antibodies. The gene sequences were cloned into vector pGS-21a for WRsaline 0.9% per animal, (PBS). which The infectionswas multiplied were doneand titratedusing 106 in expression in E. coli of a protein fused with 6xHis-tag. pfu (intranasal) and 105 pfu (scarification) of VACV- Plasmids from selected colonies were sequenced and used in transformation of bacteria BL21-CodonPlus doses.Vero cells In andthis purified experiment, on sucrose the cushion.Germ-free Four animalsgroups, (DE3)-RIPL. The products of transformation were used each one containing five animals, received intranasal for protein expression by induction with IPTG (isopropyl weightinoculated loss. with The VACV-WR infection showed clinicalalso resulted in signals the death such and submitted to electrophoresis in SDS-polyacrylamide ofas perioculartwo animals. alopecy, ruffling Conventional fur, arching mice inoculated of back with and gelβ-D-1-thiogalactopyranoside). and Western Blotting with Bacteria anti-6xHis cells monoclonal were lysed VACV-WR and control groups did not show any clinical antibody. The expression of both truncated proteins was successfully observed. hadsigns. been Other inoculated, four groups a lesionwith five has animals appeared in each 4-6 weredays BV101 - EVALUATION OF INFLUENCE FROM NORMAL submitted to tail scarification. In groups where the virus MICROBIOTA ON VACCINIA VIRUS INFECTION IN groups, all mice survived and did not show any signs of GNOTOBIOTICS AND CONVENTIONALS SWISS MICE morbidity.post scarification All living and animals developed of the into experiments a scab. In thesewere BY TWO DIFFERENT INOCULATION FORMS Lima, M.T.; Andrade, A.C. dos S.P.; Calixto, R.S.; were collected for future assays. The experiments Oliveira, G.P.; Alvim, L.B.; Martins, F. dos S.; Kroon, weresacrificed approved 10 days by afterthe Ethics infection Committee and several of UFMG. samples Our E.G.; Abrahão, J.S. UFMG - Universidade Federal de Minas Gerais, microbiota in pathogenicity and virulence in gnotobiotic study showed that, there is an influence of the normal Avenida Presidente Antônio Carlos, 6627 - Pampulha, Belo and conventional Swiss mice, inoculated with VACV-WR Horizonte - MG, 31270-901 applied intranasally. FINANCIAL SUPPORT: FAPEMIG, CAPES, CNPQ, PPG-MICROBIOLOGIA UFMG Normal microbiota has important role in the control and development of some kinds of autoimmune diseases, BV109 - DEVELOPMENT OF CELL SYSTEM FOR cancers, allergies and also in the formation of immune IN VITRO DETECTION OF NON-NEUTRALIZING ANTIBODIES AGAINST THE DENGUE VIRUS a potential risk factor for the development of animal Gomes, A.V.B.T.1; Rodrigues, N.F.1; Prado, A.A.O.1; diseasesresponse with against high pathogens. frequency Deficientand severity microbiota and also is Carneiro, A.C.A.2; Silva, B.M.1; Malaquias, L.C.C.2; enables ineffective immunization in cases of vaccines Coelho, L.F.L.1 using live attenuated pathogens, such as smallpox vaccines using Vaccinia virus strains. Vaccinia virus 1. UNIFAL - Universidade Federal de Alfenas, is a member of family and is the type virus R. Gabriel Monteiro da Silva, 714 - Centro, Alfenas - MG, of genus Orthopoxvirus. It is currently responsible for 37130-000 zoonotic human infections, affecting workers from 2. UFOP - Universidade Federal de Ouro Preto, Campos Morros do Cruzeiro, s/n - Bauxita, Ouro Preto - MG, dairy cattle, with outbreaks being observed in several 35400-000 of normal microbiota in the development of disease Dengue is a major public health problem worldwide, causedregions by of Vaccinia Brazil. To virus determine infection, the a comparative possible influence study especially in the tropical and subtropical regions of using murine model was performed. Germ-free Swiss the world. Infection with a single Dengue virus (DENV) NIH mice and conventional Swiss mice of 6-7 weeks- serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients (VACV-WR) by two different pathways of inoculation, experiencing secondary infection with a different old were infected with Vaccinia virus Western Reserve September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

70 Basic Virology: BV serotype progresses to the severe forms of the disease, we measured the diameter of 20 viral plaques after 48 h dengue hemorrhagic fever/dengue shock syndrome. of infection with the clinical isolates. We observed that Secondary infection with a different serotype from that CTGV reference isolate CM-01, isolated in RJ in 1999, in primary infection increases the risk of development of had viral plaques with mean diameter of 307.3 µm. On severe complications. In these cases, cross-reactive, non- the other hand, isolates collected in Urupá (URU-07) in neutralizing antibodies can bind to DENV surface and this 2009 and in Jaru, Governador Jorge Teixeira, Espigão D’Oeste, Campo Novo de Rondônia and Ji-Paraná in 2010 presented mean diameters of 249.1 µm, 279.0 µm, 281.0 presentDENV-antibody study we complexes developed are a cell taken system up more able toefficiently identify µm, 241.8 µm, 240.0 µm and 300.5 µm respectively. In theby Fcγ presence receptor of (FcγRIIA)-expressing non-neutralizing antibodies cells. Thus, against in the contrast, isolates from Governador Jorge Teixeira in 2012 (GJT-05) presented plaques with mean diameter of 613.5 µm. To analyze the effect of SG on the formation Kidneythe DENV. cells The (BHK-21). plasmid Since encoding the vector the FcγRIIA has a neomycin receptor of viral plaques, we infected cells with 300 PFU of CTGV resistance,(pTCN- FcγRIIA) we treated was the used cells to with transfect the G418 Baby antibiotic Hamster CM-01 or isolates from RO, in the presence of different concentrations of SG during viral adsorption. After to select the transfected clones. The FcγRIIA expression 01, 60.8% for URU-07 and 64.6% for GJT-05 at 0.1 µg/ was selectedassayed toby beReal used Time in vitro PCR assaysand immunofluoresnce to determine the ml.48h, To we evaluate verified the an replication inhibition ofof 50.2%CTGV isolates for CTGV in CM-the presencestaning. The of non-neutralizing clone that has the DENV high expression antibodies of in FcγRIIA human presence of CDV and ST-246, we infected BSC-40 cells serum. FINANCIAL SUPPORT: FAPEMIG; CNPQ; CAPES. with 300 PFU and added different concentrations of these drugs after adsorption for 48h. Our results showed BV115 - STUDIES ON THE BIOLOGICAL AND GENETIC an inhibition of 53.8% for CTGV, 83.6% for the URU-07 DIVERSITY OF CLINICAL SPECIMENS OF CANTAGALO and 37.9% for GJT-05 at 0.01 µM of ST246. For CDV, VIRUS ISOLATED FROM DAIRY CATTLE IN RONDÔNIA we detected an inhibition of 56.2% for CTGV CM-01, Rezende, B.; Damaso, C. 91.1% for URU-07 and 73.8% for GJT-05 at 30µM of CDV. UFRJ - Universidade Federal do Rio de Janeiro, Av. Therefore, these results suggest that may be interesting Pedro Calmon, 550 - Cidade Universitária, Rio de Janeiro - differences in virus circulating in RO that should be RJ, 21941-901 better investigated. FINANCIAL SUPPORT: CAPES, CNPQ, FAPERJ AND INPETAM Cantagalo virus (CTGV) is a vaccinia virus (VACV) strain isolated in 1999 in Rio de Janeiro state from lesions BV122 - ANTIHERPETIC ACITVITY OF ANNONA in dairy cattle. The disease has caused substantial SQUAMOSA L economic loss in different states of Brazil, and recently Silva, Í.T.S.S.1; Araújo, S.B.2; Fernandes, M.J.B.2; in Rondônia (RO). No antiviral therapy is currently Oliveira, R.A.1; Rebouças, A.S.J.3; Conceição, A.O.1 available for poxvirus-related disease. Therefore, the search for potential antiviral drugs is important. In 1. UESC - Universidade Estadual de Santa addition, the genetic diversity of the different clinical Cruz, Campus Soane Nazaré de Andrade, Rodovia Jorge isolates should be investigated since this feature could Amado, km 16, Bairro Salobrinho Ilhéus-Bahia, 45662-900 contribute to different responses to antiviral drugs. In 2. Instituto Biologico SP, Avenida Conselheiro Rodrigues Alves, 1.252 - Vila Mariana, São Paulo - SP, 04014- this work, we evaluated different clinical isolates from 002 RO collected during outbreaks in dairy cattle after 3. UESB - Universidade Estadual do Sudoeste 2009. The samples were positive for CTGV by PCR and da Bahia, Estr. do Bem-Querer, Km 4, Vitória da Conquista - sequencing of the HA gene. We evaluated some biological BA, 45083-900 features in cell culture and the viral response to three antivirals, i.e. sulfated galactan (SG), cidofovir (CDV) and Annona squamosa L. (Annonaceae) is a tree or shrub cultivated in tropical and subtropical regions of Central virus capacity to spread to neighboring cells. Therefore, America that bears edible fruits known as sugar- ST-246. The size of poxvirus viral plaques reflects the September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

71 Basic Virology: BV apples, ‘ata’, ‘pinha’, or ‘fruta-do-conde’. In vitro and in new effective therapeutics against herpes. In this context, vivo studies revealed the biological activity potential an in vitro study with the diterpene from Canistrocarpus of A. squamosa extracts and special attention has been cervicornis used in this work showed the antiviral done to acetogenins isolated from the seeds, which potential of the diterpene in controlling the replication have antitumoral activity. Despite several biological of HSV-1 and maintaining low cytotoxicity. Hence, the activities reported for Annona species, there is a lack of aim of this work was the isolation of diterpene (4R, 9S, information about antiviral activity of this plant against herpesvirus. Then, for this study, we evaluated the effect of seeds and leaves extract from commercial specimens this14S)-4α-acetoxy-9β,14α-dihidroxydolast-1(15),7-diene end, the air material alga was exhaustively extracted of A. squamosa L. using Vero (ATCC-CL81) cells and withand the CH2Cl2. evaluation The solvent of its antiherpetic was evaporated efficacy under in reducedvivo. To animal herpesviruses. Aqueous, ethanolic, and hexanic pressure, yielding a brownish residue. An aliquot from extracts were obtained by conventional techniques and, the crude extract was dissolved in pyridine and was after solubilization, they were brought to concentrations treated at room temperature with acetic anhydride for from 0.03 mg.mL-1 to 4 mg.mL-1. Cell viability was 65 h. Extraction of the reaction mixture in the usual way established using trypan blue exclusion technique and afforded (addition using H2O Mili-Q® and extraction by cell morphological evaluation. The antiviral assays CHCl3) and was subjected to silica gel chromatography were based on viral titer reduction assay against suid elution by increasing the polarity of pure n-hexane/ethyl herpesvirus type 1 (SuHV-1) and equine herpesvirus acetate mixtures, resulting in 44 fractions. Fractions 26, type 1 (EHV-1) and the results were expressed as inhibition percentage (IP). Leaves and seeds ethanolic spectra data, and new cycles of isolation procedures extracts were less toxic compared to aqueous and 27 and 28 yielded the diterpene, confirmed by 1H NMR hexanic extracts and all of them were suitable to antiviral test in concentrations below 0.5 mg.mL-1. The threewere repeatedgroups of to5 5-month-old obtain a sufficient BALB/c concentration mice were used: for ethanolic extract from seeds showed IP of 82.22 against 1the - diterpene experiments. (15mg/Kg); For the 2 antiherpetic - acyclovir (15mg/Kg); efficacy in vivo,and EHV-1; while hexane extract from seeds showed IP of was clipped and depilated with a chemical depilatory. of A. squamosa L. extracts against animal herpesviruses After3 - DMSO two 1% days, (vehicle). the skin The was right scratched midflank using of each a mousesterile indicating94.24 for SuHV-1.the presence These ofare hydrophobic the first report compounds of activity in seeds with promising antiviral activity. in DMEM with 2% bovine serum was applied to the 27-gauge needle, and 10 μL of HSV-1 KOS (1 X 109 PFU) BV137 - ISOLATION AND THERAPEUTIC EFFICACY IN BALB/C MICE OF DITERPENE ISOLATED FROM beginningscarified area. 1 hour All after mice virus were inoculation. orally gavaged The cutaneous with 200 MARINE ALGA CANISTROCARPUS CERVICORNIS herpesμL solution infection of the was substances scored throughout twice daily the overexperimental 18 days, AGAINST TYPE 1 period. Animals gavaged with diterpene (score = 5,0) Barros, C.; Garrido, V.; Melchiades, V.; Cavalcante, M.; and acyclovir (score = 3,8) had their disease scores Ribeiro, M.; Figueiredo, C.; Souza, K.S.; Pinto, A.M.; reduced after day 10, compared with group 3 (vehicle) Giongo, V.; Teixeira, V.; Paixão, I. (score = 7,0). These results suggest that diterpene may UFF - Universidade Federal Fluminense, Rua Miguel be useful as an oral agent to reduce the severity of HSV-1 de Frias, 9, Icaraí, Niterói - RJ, 24220-900 cutaneous lesions. FINANCIAL SUPPORT: CNPQ, CAPES, FAPERJ, UFF (PROPPI) Herpes Simplex Virus (HSV) infection is a worldwide endemic and is one of the most prevalent infections in Brazil. Although several antiviral substances are available for the treatment of HSV-1, increased viral resistance to usual antiherpetic drugs and the occurrence of severe cases of the disease, mainly in immunocompromised individuals,September/October have 2014 highlighted Volume 19 the – Supplement importance 2 - Abstracts/Posters of finding - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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BV144 - ANTIVIRAL ACTIVITIES OF PHOSPHOLIPASES acting at the entrance into the cell, suggesting its A2 FROM SNAKE VENOM AGAINST DENGUE VIRUS applicability as a tool diagnostic or prototype for drug Caldas, S.; Cecílio, A.B.; Silva, F. de O.; Sanchez, E.F. development. FINANCIAL SUPPORT: Fapemig (BIP- 0056-14, BIP-00168-14, BIP-00139-13), CNPq (Proc. FUNED - Fundação Ezequiel Dias, R. Conde Pereira 482502/2012-6), FUNED. Carneiro, 80 - Gameleira, Belo Horizonte - MG, 30510-010 Introduction - The economic and human burden of BV154 - HSP27 PROTEIN HAS ANTIVIRAL ACTIVITY dengue is substantial in endemic countries. High AGAINST HCV Akinaga, M.M.; Braga, A.C.S.; Carneiro, B.M.; Batista, vaccine demonstrate the need for the development of M.N.; Rahal, P. numbers of cases, absence of specific treatment or drugs against Dengue virus. Recently we have observed UNESP/IBILCE - Universidade Estadual Paulista/ the antiviral activity of phospholipase A2 from Bothrops Instituto de Biociências, Letras e Ciências Exatas, Rua leucurus against Dengue virus. Considering several Cristóvão Colombo, 2265 Bairro Jardim Nazareth, São José studies of antimicrobial activity with animal venoms do Rio Preto - SP, 15054-000 worldwide and the limited number of studies with Several cellular proteins are known to interact with derivatives of snake venom from Brazilian cerrado, the HCV or being necessary to the viral replication this work aimed to assess the anti-Dengue potential of process, such as the family of heat shock proteins phospholipase A2 (pool) and it´s isoforms (K49 and D49) (HSP). The Hsp are usually synthesized in response to cellular stress such as heat shock, nutrient deprivation - First, the maximum non-cytotoxic concentration of and bacterial or viral infections. Among Hsps, Hsp27 phospholipasepurified from B. A2 leucurus and it´s venom. isoforms Material was determined and methods by belongs to a family of small heat shock proteins and has MTT assay in LLC-MK2 cells. Then, the cells were treated shown antiviral activity in hepatitis B virus (HBV) and with the maximum non-cytotoxic concentration of those have observed an increased expression of Hsp27 in Dengue virus-1, 2 and 3 at a multiplicity of infection of human immunodeficiency virus (HIV). Other studies purified molecules and were incubated at 37°C with cells containing an HCV subgenomic replicon compared to the cells without replicon and in patients with hepatocellular carcinoma, high expression of Hsp27 is cells0.05 incompared a 5% CO2 to humidified untreated incubator.controls was After performed 48 hours by of associated with HCV presence. Thus, the present study one-Stepincubation, qRT-PCR. the quantification Subsequently, of viral the RNA antiviral load of treatedassays aimed to evaluate in vitro whether HSP27 has antiviral were performed with both isoforms inactivated by activity for Hepatitis C. For this, human hepatoma Huh7.5 Diethyl Pyrocarbonate (an inhibitor of catalytic activity) cells containing the subgenomic HCV replicon (SGR- to suggest a possible mechanism of action. Results and JFH1 FEO) were transfected with siRNA HSP27 and viral discussion - The maximum non-cytotoxic concentrations replication was assessed 12, 24, 48 and 72 hours after of phospholipase A2 were 40 µg / mL (Pool) and 20 µg/ transfection. At all times HCV replication was greater in cells treated with siRNA Hsp27 (135%, 117%, 115% and reduction of viral RNA in the treated cells, with similar 122% respectively) to cells treated with siRNA control reductionsmL for isoforms. for phospholipase Antiviral assays A2 (Pool) showed and significant isoforms. (100%, 101%, 94% and 98% respectively). These data The trials with inactivated isoforms suggest that the suggest that Hsp27 protein represents a cellular defense catalytic action is not important for antiviral activity. mechanism, playing an antiviral activity in presence of HCV. FINANCIAL SUPPORT: FAPESP to more conclusive results. Moreover, our tests after However, more specific enzyme inhibitors will be tested replication in the treated cells, suggesting an activity at cellviral membrane adsorption level, showed whereas no significant after cell inhibition penetration, of viral no decrease was observed in their viral load. Conclusion - The data show that phospholipases A2 from B. leucurus are able to inhibit the growth of Dengue virus, probably September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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BV170 - REGULATION OF HEAT SHOCK PROTEINS Cebidae, Callithricidae, Saguinus e Alouatta family, and EXPRESSION BY HEPATITIS C VIRUS birds, that can act like secondary hosts. It is transmitted Braga, A.C.S.; Carneiro, B.M.; Batista, M.N.; Akinaga, by blood-sucking arthropods, typically the mosquitoes, M.M.;-Rahal, P. specially the Haemagogus janthinomys. Mayaro is an enveloped virus composed of a single strand of positive- UNESP - Universidade Estadual Paulista, Rua Quirino sense RNA approximately 11.5 kb in length. The genome de Andrade, 215, São Paulo - SP, 01049-010 encodes nonstructural and structural proteins, forming Hepatitis C is a disease caused by hepatitis C virus (HCV), a complex of replication that is required for the starter and it is estimated that about 170 million people are genomic RNA. The nonstructural protein NSP3 have infected with the virus worldwide. During infection HCV an essential function at the replication of the virus and interacts with several cellular proteins to promote viral maturation of proteins, and its formed by tree domains: replication. Studies have shown that many heat shock and C-terminal domain. In order to understand the presence of the virus and some HSPs interact directly replication“macro” domain cycle of orvirus X-domain, we decided N-terminal to clone, express domain withproteins HCV (HSPs) proteins. have This an altered increase expression or decrease profile in inHSPs the and purify the nSP3 protein of Mayaro virus. A DNA expression may assist in understanding the mechanisms fragment equivalent of the coding region of nSP3 was involved in viral replication and provide an alternative in obtained by RT-PCR and was cloned in pET21a vector. viral replication reduction. Thus the present study aimed to evaluate in vitro the expression of heat shock proteins in the presence and absence of HCV. For this human PCR.The plasmidSeveral clones built were was then amplified transferred by Escherichia to Escherichia coli hepatoma Huh7.5 cells were electroporated with the coli(DH5α) (BL21) and theand confirmation he expression of theof thiscloning protein was donewas beby replicon HCV JFH-1 and maintained until approximately detected by SDS-PAGE. We will now take this production 90% of the cells were infected by the virus. These cells were subjected to RNA extraction and cDNA synthesis. characterized. FINANCIAL SUPPORT: CNPQ / FAPESP The differential expression of 84 HSPs and chaperones to a high volume and the protein will be purified and was assessed by qPCR Array comparing uninfected and BV182 - EFFECT OF EXTRACTS SCHINUS infected cells with HCV. The results demonstrate that TEREBINTHIFOLIUS AND PUNICA GRANATUM ON MAYARO VIRUS REPLICATION while four others had reduced expression. These results Ferreira, F.D.1; Salles, S.T.1,3; Meneses, M.D.F.1; mayfive geneshelp in showed understanding increased the expression mechanisms (over involved Log2 2), in Guimarães, T.E.1; Caldas, L.A.4; Meira, G.L.S.1; HCV replication. FINANCIAL SUPPORT: FAPESP Yamamoto, K.I.1,3; Kuster, R.M.2; Campos, R.M.1; da Silva, M.R.S.3 BV177 - CLONING AND EXPRESSION OF THE NONSTRUCTURAL PROTEIN 3 (NSP3) OF MAYARO 1. Laboratório de Interação Vírus Célula, VIRUS Departamento de Virologia, Instituto Microbiologia Prof. Paulo De Góes. Centro de Ciência E Saúde, Universidade Pinhatti, A.K.; Pacca, C.C.; Ribeiro, M.R.; Mota, M.T. de Federal Do Rio De Janeiro. O.; Vedovello,D.; Nogueira, M.L. 2. Núcleo de Pesquisas de Produtos Naturais, FAMERP - Faculdade de Medicina de São José do Rio Centro de Ciências da Saúde, Universidade Federal do Rio de Preto,Av. Brigadeiro Faria Lima, 5416 - Vila São Pedro, São Janeiro José do Rio Preto - SP, 15090-000 3. LAMMP, Departamento de Bioquímica, Instituto de Química, Centro Tecnológico, Universidade The Mayaro virus (MAYV) is an arbovirus of the Federal do Rio de Janeiro. Togaviridae family, genus enzootic in tropical 4. Laboratório de Ultraestrutura Celular South America and endemic in rural areas. This virus Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho. Universidade Federal do Rio de Janeiro. reported in 1957, in the state of Pará. It is maintained in Mayaro virus (MAYV) is an arbovirus that belongs to thecauses sylvatic Mayaro cycle Fever involving and its vertebrates first outbreak like in monkeys Brazil was of the genus Alphavirus, Togaviridae family which causes September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

74 Basic Virology: BV a disease known as Mayaro Fever, with symptoms BV185 - SUSCEPTIBILITY OF BRAZILIAN VACCINIA VIRUS STRAINS TO THE ANTIVIRAL COMPOUND ST- properties of substances extracted from the plants 246 similar to the classic dengue fever. The anti-inflamatory Schinus terebinthifolius (mastic) and Punica granatum Rodrigues, N.F.S.; Pires, M.A.; Almeida, G.M. de F.; (Pomegranate) has already been explored by the Ferreira, P.C.P.; Bonjardim, C.A.; Trindade, G. de S.; Brazilian popular medicine. In this work we tested the Abrahão, J.S.; - Kroon, E.G.; Mota, B.E.F. antiviral effect of these substances against MAYV. The toxicity of these substances in VERO cells was tested by UFMG - Universidade Federal de Minas Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, Belo the incorporation of neutral red after 24h of treatment. Horizonte - MG, 31270-901 The CC50 of each substance was determined. The CC50 of each substance was: 242, 315, 102 and 5000 µg/mL The Vaccinia virus (VACV), which belongs to the Poxviridae family, is the etiological agent of a disease and 590 and 442 µg/mL for Crude Extract and Acetate that affects bovines, humans and other hosts, being ofin acetate,Punica, flavonoidsrespectively. 1 andAfter 2, theoil of determination Schinus, respectively, of non- characterized by the appearance of exanthematous toxic concentrations of these substances, the antiviral lesions at the virus entry site. The transmission occurs effect was tested. Cells were infected for 1 h with Mayaro mainly by direct contact with lesions and it happens virus using a multiplicity of infection of 0,1. Treatment among wild hosts and bovines, and among bovines and of cells was carried out for 24 hours with increasing humans. Recently several outbreaks have been described concentrations. We observed that the substances exhibit in rural properties across the Brazilian territory. In antiviral activity. The IC50 of each was determined by a previous studies, our group observed genotypical and dose response graph, and were 4,3; 4,5; 14 and 830 µg/ phenotypical differences among the viruses that were isolated from these outbreaks. This disease has been respectively, and 12 and 30 µg/mL for Crude Extract considered an important public and animal health andmL µg/mLAcetate in of acetate, Punica, flavonoidsrespectively. 1 andThe 2,selectivity oil of Schinus, index problem, besides compromising local economy. The (ratio CC50/IC50), was determined and the values drug ST-246 (Tecovirimat®) has demonstrated potency were greater than 7 for all substances. We also tested the substances for virucide properties, adsorption However, so far only one isolate of Brazilian VACV (BRZ- and specificity for the treatment of poxvirus infections. impairment and pretreatment. Our results showed more VACV) has been tested for its susceptibility to this drug than 95% virucidal effect for the partitions acetate, (Cantagalo virus, EC50 of 0.0086 µM). Therefore, due to great diversity of BRZ-VACV, it is necessary to evaluate Mastic oil, and acetate of pomegranate did not present further the in vitro susceptibility of these viruses to virucidalflavonoids effect. mastic We and also Crude observed Extract by of pomegranate.transmission different ST-246 concentrations. The susceptibility electron microscopy the damages caused upon the test for eight BRZ-VACV isolates was done by using a virus particles by the virucide substances. Proteomic viral suspension of 150 PFU, which was inoculated into analysis showed differences in the expression of monolayers of BSC-40 cells. The F13L gene in the samples secreted proteins during infection and treatment with to assess the existence of polymorphisms related to drug was amplified by conventional PCR and then sequenced some of the partitions in our substances act directly on resistance. The comet assay was performed to evaluate the virustetrahyamentoflavone particle. FINANCIAL substance. SUPORT: We CAPES, conclude FAPERJ that qualitatively the susceptibility of the isolates. EC50 AND INBEB values for the isolates showed noteworthy variation (from 0.0054 to 0.051 µM), which is within the range of

strains. The CC50 value in BSC-40 cells was higher than 50values µM, describedwhich provides in scientific a selectivity literature index for greater other VACV than 1,000. Sequencing of F13L gene showed that the isolates did not exhibit the previously described polymorphism associated to ST-246 resistance (Gly277Cys). There September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

75 Basic Virology: BV has been drastic reduction in comet formation in ST- an improved pharmacological performance of this 246 concentrations as low as 0.0125 µM for all the compound in antiviral therapies. Until now we evaluated isolates. In conclusion, the results show that EC50 the action of curcumin associated with nanoemulsion (NE-CUR) as photosensitizing drug on PDT system in that ST-246 may be useful in the treatment of human cervical carcinoma cell lines HPV-16 positives (CasKi infectionsvaries significantly by BRZ-VACV. amongst Brazilian VACV isolates and that the nanoemulsions were able to internalize cells BV187 - STUDY OF THE PHOTODYNAMIC THERAPY and SiHa).were Theobserved fluorescence in the microscopyintracellular images environment showed EFFECTS OF CURCUMIN-NANOPARTICLES IN HPV-16 for up 36 hours after incubation in SiHa cells and for POSITIVE CERVICAL CARCINOMA CELLS 48 hours in CasKi cells. The cellular viability assay Matos, R.P.A. de1; Primo, F.L.2; Tedesco, A.C.2; Villa, showed biocompatibility of nanoemulsions, evidenced L.L.3; Calmon, M. de F.1; Rahal, P.1 by the absence of cytotoxicity of empty nanoemulsions. 1. UNESP - Universidade Estadual Paulista, Besides, we observed high phototoxic effect of the Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010 NE-CUR formulation with less than 5% of viable cells 2. USP - Universidade de São Paulo, Av. Prof. after irradiation for both cells lines. Additionally, Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 photodynamic therapy using NE-CUR as photosensitizing 3. INCT-HPV - Instituto Nacional de Ciência drug was able to increase the P53 expression in 2.43 fold e Tecnologia das Doenças do Papilomavirus Humano, R. Dr. change for CasKi cell line. These results show that the Cesário Mota Júnior, 61 - Vila Buarque São Paulo - SP 01221- developed formulation NE-CUR, in combination with 020 photodynamic therapy, has a great therapeutic potential Human Papillomavirus (HPV) are a DNA virus family to be used in the treatment of lesions caused by HPV. FINANCIAL SUPPORT: FAPESP

BV192 - ANALYSIS OF THE ONCOLYTIC POTENTIAL highwith risk more HPVs than are 190 the typesHPV-16 identified, and -18, that and together can be OF AVIAN VP3 PROTEIN areclassified responsible in low andfor more high riskthan HPVs. 70% The cervical most carcinomaimportant cases. The current available treatments are surgery, Costa, C.S.; Knak, M.B.; Costenaro, J.G.; Mees, L.; cryogenics, drugs and antiviral therapies. However, Campos, F.S.; Franco, A.C.; Roehe, P.M. these treatment options have side effects such as pain, UFRGS - Universidade Federal do Rio Grande do Sul, redness, itching, burning and hypersensitivity. So, Avenida Paulo Gama, 110 - Farropilhas, Porto Alegre - RS, due to the large number of people infected by HPV, 90040-060 the need of noninvasive antiviral treatment, with few or no side effects, has been increasing. Thereby, the avian gyrovirus 2 (AGV2). The genome of AGV2 encodes nanotechnology associated with natural compounds threeOur group proteins, has recentlyone of which identified is VP3 a gyrovirus, (AGV2-VP3), named a may be a future treatment. Curcumin is a very versatile protein homologous to apoptin of anemia virus bioactive hydrophobic compound which has anti- (CAV), for which several studies show a pro-apoptotic potential in tumor cells. In view of the similarity anti-neoplastic and chemoprotective properties, and between these two proteins, it became of interest to inflammatory, anti-hyperlipidaemic, anti-angiogenic, also has the ability to decrease the expression of HPV investigate AGV2-VP3 potential to induce apoptosis in oncogenes. Furthermore, curcumin may be used as human tumor cells, since this property might be relevant a photosensitizing agent in Photodynamic Therapy for tumor suppression. Recombinant adenoviral vectors (PDT), as it was shown that several of the effects can be expressing three variants of VP3-AGV2 ( pAd, pAd and potentiated by the combination of curcumin with light. pAd), as well as a control vector pAd expressing the However, curcumin application as an active agent has lacZ gene (pAd), were used for transformation of E. coli been limited by low water solubility and bioavailability. and subsequent cloning. Plasmid DNA was extracted The use of nanoparticles, where the curcumin can and the nucleotide sequences of the recombinant be encapsuled and delivered into cells, could allow

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersadenovirus - Basic Virology: variants BV confirmed. Subsequently, the XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

76 Basic Virology: BV vectors were transfected in 293A cells and cultured until Phytochemical studies employing S. pseudoquina have observation of cytopathic effect, when new subcultures demonstrated the presence of some alkaloids and were performed in attempting to increase viral loads, as determined by titration. The transcription of mRNAs for their inhibitory effects of viral infections. In this study,flavonoids, we evaluated classes of the compounds cytotoxicity which and are the well antiherpes known recombinants adenovirus were inoculated at different activity of three extracts obtained by serial percolation multiplicitiesof VP3-AGV2 of variants infection was in normal confirmed MRC-5 by cells RT-PCR. (lineage The of the stem bark (hexane, ethyl acetate and ethanol cells (lineage of human lung carcinoma); cell viability was isolated from S. pseudoquina. Cytotoxicity was evaluated evaluatedof human by lung MTT fibroblasts) assay (3-bromide and in human (4.5-dimetiltiazol- tumor A549 onextracts), Vero cells and by one using pure MTT biflavone assay, and (strychnobiflavone) antiviral activity 2-yl) -2.5-difeniltetrazolium). Statistical analysis of the was tested against HSV-1 (KOS strain) by viral plaque results evidenced that the three recombinant adenovirus number reduction assay. Results were expressed as expressing variants of AGV2-VP3 inhibit cell growth of 50% cytotoxic concentrations (CC50) and 50% viral both tumor A549 cells and normal MRC-5 cells. However, replication inhibitory concentrations (IC50), respectively, in order to calculate the selectivity index (SI=CC50/ IC50) of each tested sample. Among the tested samples, thatthe inhibitionAGV2-VP3 observed is a potential in A549 candidate cells was forsignificantly a tumor the ethyl acetate extract and the pure compound SPEA2, suppressinghigher than protein. in MRC-5 Future cells. studies These will findings be conducted suggest inhibited HSV-1 replication showing SI values of 14.1 and to examine its potential as an oncolytic protein in more 13.7, respectively. The detected antiherpes activity of S. detail. pseudoquina studied extract seems to be linked to its

BV193 - PRELIMINARY EVALUATION OF ANTIHERPES FINANCIAL SUPPORT: CNPQ, CAPES, FAPEMIG ACTIVITY OF STRYCHNOS PSEUDOQUINA ST. HIL high content of flavonoids, especially strychnobiflavone. Silva, I.T.2; Farias, L.M.3; Xavier, A.A.3; Pádua, R.M.1; BV195 - POLICLONAL ANTIBODIES ANTI-P2, A Leite, J.P.V.3; Simões, C.M.O.2 CAPSID SUBDOMAIN FROM A SAPOVIRUS GI.2 Fagundes, J.M.1; dos Anjos, K.; Lucinda, N.2; Machado, 1. UFMG - Universidade Federal de Minas 2 1 Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, A.M.V. ; Nagata, T. Belo Horizonte - MG, 31270-901 1. UnB - Universidade de Brasília, Campus 2. UFSC – Universidade Federal de Santa Universitário Darcy Ribeiro, Brasília - DF, 70910-900 Catarina, Campus Universitário Reitor João David Ferreira 2. FIOCRUZ MINAS - Fundação Oswaldo Lima - Trindade, Florianópolis - SC, 88040-900 Cruz, Av. Augusto de Lima, 1715 - Barro Preto, Belo Horizonte 3. UFV - Universidade Federal de Viçosa, - MG, 30190-002 Avenida Peter Henry Rolfs, s/n - Campus Universitário, Viçosa - MG, 36570-000 family, it has, at the moment, only one specie member, Herpes Simplex Viruses (HSV) are responsible for many SapporoSapovirus virus. is one Sapovirus of the five genome genus withinis linear, positive- infections of oral, ocular and genital regions, and efforts sense, single-stranded RNA, of approximately 7.3 to 7.5 kb. It has a linked protein at the 5’ terminus (VPg) mainly due to their resistance to the most common and is polyadenylated at 3’ terminus. Sapporo virus is availablehave been treatment made to find (e.g. new acyclovir). drugs for Natural their treatment, products are an inexhaustible source of bioactive molecules, and considerable attention has been given to secondary divided into, not officially, 14 genogroups, whereas GI, batGII, infectiveGIV and GVrespectively. are humans These infective, viruses GIII, are GVI worldwide - GXI are for new antiviral drugs. Strychnos pseudoquina St. Hil. knownporcine infective,because GXII,their GXIII prevalence and GIV areamong mink, doghuman and compounds purified from plants in an attempt to search is a native cinchona-like tree of the Brazilian savanna, caliciviruses, that is very represented by Norovirus, popularly known as “quina” and used in the folk medicine which is the most important virus agent that causes to treat hepatic and stomach diseases, fever, and malaria. acute gastroenteritis. Many epidemiological studies

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

77 Basic Virology: BV are published every month about outbreaks caused by in BALB/c mice infected with a strain of HSV-1 mice. For sapovirus, all of them uses RT-PCR or even RT-qPCR as the experiment three groups were used: G1-1% DMSO detection methods. It is known that in Brazil although - vehicle (n=2); G2-Acyclovir-15 mg/kg (n= 5) and G3- Norovirus is not included into the surveillance system for acute diarrhea it has been found positive samples all over was clipped and depilated with a chemical depilatory. the country; this means that if an effective and cheaper DBD3- 20 mg/kg (n= 5). The right midflank of each mouse method to detect sapovirus was available these viruses inoculated by scratching the skin with a sterile 27-gauge needle.Two days One later, hour 10μL after of inoculation, HSV-1 KOS the(1 X substances 109 PFU) were administered by gavage twice daily for 18 days. The genogroupwould be alsothe subdomain identified. P2 It isfrom known the capsid that Sapovirus protrude animals were monitored and photographed throughout wasGI.2 expressed is worldwide using prevalent,E. coli system. to detectThe sequence this specific of P2 the period to determine the score. The results showed from SapoBR01 was cloned using gateway system to pDEST17 and it was expressed into BL21AI E. coli. The all groups. The disease has natural evolution in G1 (1% protein was found into insoluble fraction; even when low DMSO)the first group signs ofwhile infection most onanimals the 4th from day G2 of inoculation(ACV) and G3 in (DBD3) group started to show signs of healing on the 10th day according to score done. Our results showed mltemperature was used suchto immunize as 15˚C was mice. used A fortotal 24h of of6 induction.injections that concentration of 20 mg/Kg of DBD3 could inhibit The P2 was purified using MagneHis (Promega), 30ug/ the skin lesions caused by HSV-1 without animal death. through subcutaneous back of the animal. After 25 Our conclusion based on in vitro and in vivo data, DBD3 using the purified protein plus aluminum were done seems to be a promising molecule since it has been intraocularly. The serum was separated and then used as adays primary after antibody. the first A injection total of 4 theanimals blood were was immunized collected SUPPORT: CNPQ, CAPES, FAPERJ, UFF (PROPPI) and all the serum collected present positive reaction showing low toxicity and antiviral efficacy. FINANCIAL against the protein expressed. We expect to have human BV203 - GENE EXPRESSION OF INTRACELLULAR feces sample positive to Sapovirus GI.2 to evaluate FACTORS POTENTIALLY INVOLVED IN NATURAL these antibodies against human samples. FINANCIAL RESISTANCE TO HIV IN SERODISCORDANT COUPLES SUPPORT: CNPq. Coltro, V.P.; Santos, I.M.; Pinto, A.R.; da Rosa, R.D. UFSC – Universidade Federal de Santa Catarina, BV202 - TREATMENT WITH MARINE ALGA PRODUCT Campus Universitário Reitor João David Ferreira Lima - AFTER INFECTION WITH HERPES SIMPLEX VIRUS 1 Trindade, Florianópolis - SC, 88040-900 IN BALB/C MICE Garrido, V.; Barros, C.S.; Melchiades, V.; Cavalcante, (HIV) infection is responsible for causing acquired M.; Teixeira, V.L.; Ribeiro, M.; Teixeira, G.; Giongo, V.; Background: Human immunodeficiency virus Paixão, I.C.N.P. transmitted through sexual intercourse, presents UFF - Universidade Federal Fluminense, Rua Miguel compleximmunodeficiency and variable syndrome natural (AIDS). history It and is mainlyshows de Frias, 9, Icaraí, Niterói - RJ, 24220-900 different patterns of disease progression. Currently, The search for new substances against herpes simplex there are many reports of individuals who were exposed virus 1 has advanced in the last decades mainly due to HIV and were not infected, suggesting the existence of to the resistance of existing drugs. Often HSV-1 is not mechanisms that lead to natural resistance to this virus. lethal, but it becomes severe when causes encephalitis in Some of these individuals are seronegative partners of children and immunocompromised patients. Research serodiscordant couples. HIV interacts with many cellular on marine alga shows a high potential bioactive in their host proteins during its replication cycle. Some of these secondary metabolites which could be developed into proteins are required for HIV replication while others new drugs against pathogens. The aim of our study is to exhibit antiviral activity. In this way, HIV replication depends on a delicate balance between cellular co- factors and antiviral restriction factors. This study aims evaluate the efficacy of dolabellanodienetriol product (DBD3)September/October isolated from2014 Volumemarine 19 brown – Supplement alga Dictyota 2 - Abstracts/Posters pfaffii - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

78 Basic Virology: BV to evaluate gene expression of antiviral (APOBEC3G, of infection. Therefore, vaccines against dengue TRIM5alpha, BST-2, SAMHD1, and cFLIP) and proviral should be tetravalent to prevent the occurrence of this phenomenon. In our laboratory, we developed and monocytes from individuals exposed to HIV and a tetravalent vaccine candidate which was made by uninfected,factors (LEDGF/p75 HIV positive and TREX1)and a control in T CD4+ group lymphocytes composed successive insertions of the genes coding for the domain by healthy individuals who have not been exposed III of the E protein from each serotype of dengue virus in to the virus. Methodology: This study was conducted on a cohort of heterosexual and homosexual couples, composed of 9 HIV-exposed seronegative subjects (HESN) multi-wallthe expression carbon vector nanotubes pVAX1. (MWCNTs) In attempt as to a improve nanovector the and 9 HIV-infected patients in HIV (serodiscordant toefficacy deliver of thethis plasmid vaccine intocandidate, cultured in cells.this work For this we usedgoal, couples), and 12 healthy controls (HC) in HIV-negative we made the conjugation of the plasmid DNA with seroconcordant monogamous couples. Serodiscordant MWCNTs using an ultrasonic sonicator bath. Then we couples were recruited at reference centers, and HC were recruited at HEMOSC, both in Florianópolis, SC, Vero cells, just adding this to the culture medium. To analyzeused this the solution expression of plasmid of the DNA+MWCNTs protein encoded to transfect by our vaccine candidate, we compared the level of expression RNABrazil. isolated. Whole Reverseblood samples transcription were collected was performed and CD4 and + between the plasmid mixed with MWCNTs and the geneT lymphocytes expression and was monocytes carried out were through purified qPCR and using total plasmid pure, which was transfected to the cells by the SYBR Green PCR Master Mix, and B2M, RPL13A and UBC method of calcium phosphate transfection. The analysis of the conjugation between plasmid DNA and MWCNTs was made by Raman spectroscopy, transmission electron HIV-exposedas reference seronegative genes. Results: subjects We observed compared significantly to healthy microscopy and agarose gel electrophoresis. Protein controlsincrease andof cFLIP HIV-infected mRNA levels patients, in CD4+ whereas lymphocytes we found noof expression was analyzed by SDS-PAGE, western blotting differences in other genes expression. In monocytes, no plasmid DNA and the MWCNTs conjugated with each data suggest that increase cFLIP levels may play a role other,and immunofluorescence. and the analysis of Theprotein results expression showed thatshowed the insignificant resistance differences to HIV-1 wereinfection. observed. FINANCIAL Conclusions: SUPPORT: Our differences between the plasmid DNA transfected alone CNPq. and when conjugated with MWCNTs. For the next steps we are going to test this vaccine candidate in mice and BV204 - USING CARBON NANOTUBES AS A evaluate the immune response when it is administered NANOVECTOR FOR A TETRAVALENT VACCINE with or without the MWCNTs. FINANCIAL SUPPORT: CANDIDATE AGAINST DENGUE VIRUS FAPEMIG, CAPES, CNPq. Calegari, L.P.; Pessoa, C.R.; Monteiro, J.M. de C.; de Paula, S.O. BV209 - PRODUCTION OF INFECTIOUS RNAS FROM CDNA OF CLONES OF DENGUE VIRUS TYPE 3 (DENV- UFV - Universidade Federal de Viçosa, Avenida Peter 3) AND CHIMERIC CLONES DENV-3/VÍRUS ROCIO Henry Rolfs, s/n - Campus Universitário, Viçosa - MG, 36570- 1 1 1 000 Amarilla, A.A. ; Alfonso, H.L. ; Muller, V. ; dos Santos- Junior, N.N.1; Russo, R.R.1; Soares, A.M.1; Oliveira, A.S.1; Dengue fever is the main disease caused by Arbovirus Rios, W.M.2; Silva, C.L.2; Trabuco, A.C.1; Figueiredo, in the world and it is an issue of big concern in Brazil. L.T.2; Aquino, V.H.1 The virus is represented by four serotypes DENV-1, DENV-2, DENV-3, DENV-4 and the control of them is 1. FCFRP/USP - Faculdade de Ciências based on the elimination of the mosquitos of genus Farmacêuticas de Ribeirão Preto/ Universidade de São Paulo, Aedes, which transmit the virus. Antibodies produced in Avenida do Café, s/nº Ribeirão Preto - SP, 14040-903 2. FMRP/USP - Faculdade de Medicina de Ribeirão Preto/ Universidade de São Paulo, Av. Bandeirantes, infection by a heterologous serotype, a phenomenon 3900, Monte Alegre, Ribeirão Preto - SP, 14049-900 knowna first infection as antibody-dependent may worsen the diseaseenhancement in a secondary (ADE)

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79 Basic Virology: BV

Dengue is an infectious disease caused by dengue virus BV216 - NATURAL VARIANTS OF HUMAN (genus Flavivirus, family Flaviviridae), and transmitted PAPILLOMAVIRUS TYPE 6 EXHIBIT DIFFERENT by the bite of mosquitoes of the Aedes genus, mainly PROMOTER ACTIVITY Aedes aegypti and this disease is an important problem Bonfim, C.M. do1; Sobrinho, J.S.2; Villa, L.L.2; Sichero, of public health worldwide. In the 70s, the emergence L.2; Rahal, P.1 of a new arbovirus caused a major outbreak of encephalitis in Brazil in 1975, in the Ribeira Valley, 1. UNESP/IBILCE - Universidade Estadual Paulista - Instituto de Biociências, Letras e Ciências Exatas, located in the southern of São Paulo state. This new Rua Cristóvão Colombo, 2265 Bairro Jardim Nazareth, São arbovirus was named of Rocio virus (ROCV), which is José do Rio Preto - SP, 15054-000 a member of Japanese encephalitis virus seracomplex. 2. ICESP - Instituto do Câncer do Estado de Recently, an animal model to evaluate the pathogenesis São Paulo, Av. Dr. Arnaldo, 251 - Sumaré, São Paulo - SP, of the disease, showed that the ROCV causes an acute 01255-000 neuroinvasive capacity. However, the mechanisms Recurrent respiratory papillomatosis and anogenital involvedinflammation in neurovirulence in the NCS and and neuroinvasiveness that this virus hasof the a warts are diseases associated with infection by Human ROCV have not been investigated. The objective of this papillomavirus (HPV) type 6. The promoter region of work was the construction of infectious clones of DENV- human papillomavirus, called of long control region 3 and chimeric DENV-3/ROCV. Brazilian DENV-3 (D3BR/ (LCR), contains sequences important for the regulation SL3/2002) and ROCV (SPH34675) isolates were used to of viral replication and transcription of early genes. This region can be used to determine HPV molecular variants of four DNA fragments with ends homologous to whereas nucleotide variability in the LCR can be as high theprepare adjacent the infectious fragments clones. was A strategyused. Complementaryof amplification as 5%. Some transcription factors (TFs) have been shown (cDNAs) representing the entire viral genome to bind to the LCR and modulate viral transcription. The were assembled using a recombination system into aim of this work was to compare the promoter activity of S. cerevisiae cells. The promoter sequence of T7 RNA the HPV-6 variants and search for cellular transcription polymerase was introduced in the viral cDNAs to allow in factors that could explain the differences observed. Were vitro transcription of the viral RNA. Restriction enzyme 6 variants in the pGL3 vector upstream the luciferase amplified and cloned LCR region of 5 different HPV- the assemblies of the cDNAs. In the chimeric clones, the gene. Transient luciferase expression experiments were Edigestion, protein from PCR, D3BR/SL3/2002 and nucleotide sequencingisolated was confirmed replaced by E protein of the ROCV. These cDNAs were used as TRANSFACperformed indatabase C33 cells. was pCMV-β-Gal used to search plasmid for wasputative used template to generate RNAs, which subsequently were as an internal control for transfection efficiency. The introduced in eukaryotic cells. The viral particles have differences in TFs binding among the different variants. been recovered after several passages for all constructs, Then C33 cells were co-transfected with increasing suggesting the infective capacity of RNA generated. This amounts of selected TFs expression plasmids in addition study describes the construction of cDNAs representing to LCR-Luciferase vectors of different molecular variants the entire viral genome from Brazilian isolated of DENV- of HPV-6 to analyze the impact of these proteins upon 3 and chimeric cDNAs between ROCV and DENV-3. These HPV-6 transcription.We observed a 10 fold enhanced constructions could serve as an import tool for studying promoter activity in the HPV-6vc reference variant as different aspects related to pathogenesis of the disease compared to the HPV-6a variant. A transition in nt 16 and could also provide a basis for future vaccines. (G toA) detected in a HPV-6a related variant led to an FINANCIAL SUPPORT: FAPESP and CNPQ. increase in promoter activity similar to the HPV-6vc reference sequence, whereas a transversion in nt 7630 in a HPV-6vc related variant abolished the increased

overlap variable nucleotide positions among molecular variants.activity. Putative Among binding these, only sites ELF1 for FOXA1, superexpression ELF1 and GATA1 was

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

80 Basic Virology: BV shown not to impact upon HPV-6 transcription. CHIP experiments are being performed to analyze the binding between patients and controls was observed for FPR1 used as endogenous control. No significant difference TFs not previously implicated in the regulation of HPV HIV-positive subjects (p<0.001), while the FPR3 was 5.3- earlyof these gene TFs expression, to the LCR. and Inadditional this work, studies were are identified needed fold(p=0.41). higher However, (p<0.0001) FPR2 than had significantlycontrols. Interestingly, lower levels the in since many of these transcriptional factors are mutated increased FPR3 expression in patients was correlated in cancer or are putative cancer biomarkers. with opportunistic diseases (R = 0.31, p <0.001). We hypothesized that the FPR2/FPR3 expression BV219 - MODULATION OF FORMYL PEPTIDE disequilibrium caused by the HIV infection may have RECEPTORS EXPRESSION IN HIV INFECTION contributed to the disruption of the FPR signaling Rodrigues, C.M.1; Almeida, J.F.1; Morais, L.D. da S.1; de pathway, which has led to molecular mechanisms that Sena, A.A.S.1; Borges, A.S.2; Goulart, L.R.1 are still unknown, but with consequences that resulted 1. INGEB/UFU - Instituto de Genética e Bioquímica da Universidade Federal de Uberlândia, Av. Pará, signaling during HIV infection still needs to be thoroughly 1720, Uberlândia - MG, 38400-902 evaluated,in opportunistic and it may infections. represent The a key fine-tuning event in ofthe FPRs HIV 2. ICBIM/UFU - Instituto de Ciências pathogenesis. FINANCIAL SUPPORT: FAPEMIG, CNPQ, Biomédicas da Universidade Federal de Uberlândia, Av. Pará CAPES 1720 - Bloco 2B - Sala 2B221, Uberlândia - MG, 38400-902 BV226 - SYNTHETIC NAPHTHOQUINONES ARE POTENT INHIBITORS OF DENGUE VIRUS REPLICATION activation,Human Immunodeficiency providing opportunities Virus (HIV) infectionfor secondary leads to a severe and rapid depletion of CD4+ T-cells and systemic Amorim, R.1; da Silva, F.C.2; Rocha, D.R.2; Ferreira, V.F. 2; Costa, L.J. da1 process is established with the virus entry through the interactioninfections andof the chronic viral envelope inflammation. proteins (gp120, The infection gp41) 1. UFRJ - Universidade Federal do Rio de with surface cell receptors (CD4 and CCR5) of the host. Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de Janeiro - RJ, 21941-901 has also been described as an alternative co-receptor 2. UFF - Universidade Federal Fluminense, forHowever, HIV, a promiscuous the Formyl Peptide G-protein Receptor coupled 2 receptor (FPR2/ALX) that Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 belongs to the FPR receptor family, which also includes Dengue is a mosquito-borne viral infection caused by FPR1 and FPR3 that are abundantly found in several four serotypes of the dengue virus (DENV 1, 2, 3 and 4), a cells of the human immune system. These receptors member of the Flaviviridae family. It has become a major control several processes that support or prevent the international public health problem in recent years, as there are about 2.5 billion people living in endemic areas. Clinical manifestations of Dengue infection range recentinflammatory demonstration response of according dimer formation to the available among ligand. FPRs Besides the specific HIV-gp120 recognition of FPR2, a Currently, there are no effective vaccines or antiviral their role during HIV infection. We have focused on drugsfrom a against flu-like DENV,illness andto fatal there cases is an of increasinghemorrhagic need fever. to theirduring expression their specific pattern activation in HIV-1 led positive us to investigate subjects, develop and study molecules with potential antiviral in order to assess the involvement of all three FPRs in activity against DENV. The aim of this study was to HIV pathogenesis. Samples of peripheral blood of 47 screen compounds with potential activity against DENV HIV-1 positive patients in chronic phase and 18 healthy from a library of 30 new synthetic naphthoquinones. control subjects were collected at the Clinical Hospital First, we tested Vero cells viability in the presence of Uberlandia. Total RNA samples were extracted and of different concentrations of these molecules using reverse transcription-qPCR (RT-qPCR) assay was neutral red uptake assay. Three molecules (named 118, performed using Taqman probes to quantify FPR1, 143 and 150) were non-toxic at 100 µM concentration, other three (120, 127 and 128) were non-toxic at 50

FPR2/ALXSeptember/October and FPR32014 Volume mRNAs. 19 – Supplement The GAPDH 2 - Abstracts/Posters gene was - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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µM concentration, and four (119, 122, 132 and 146 on previous antiviral and phytochemical studies carried and 152) were non-toxic at 25 µM concentration. Next, out by our group. Cytotoxicity of the extracts was we evaluated the antiviral activity of these molecules evaluated on Vero cells by MTT and sulforhodamine B against DENV-2 in Vero cells at non-toxic concentrations. assays, and the anti-HSV-1 activity (KOS strain) was Viral titers were determined by detection of viral RNA investigated by plaque reduction assay. Results were in the supernatants of infected cells by quantitative real- expressed as 50% cytotoxic concentrations (CC50) and 50% viral replication inhibitory concentrations (IC50), respectively. At 100 µg/mL, crude extracts showed no 120,time PCR127, using 132 aand probe 146) and presenteda pair of primers antiviral specific activity for toxic effects on Vero cells in both cytotoxicity assays, againstDENV-2. DENV-2. From the The eleven molecule tested named molecules, 120 led five to (119, the and inhibited 100% of viral replication. Fractionation highest inhibition of viral replication (95%), followed of crude extracts showed the n-butanol fractions by 132 (86 %), 127 (83%), 119 (79%) and 146 (57%). concentrated toxic constituents. On the other hand, Finally, we determined the EC50 of the molecules 127 aqueous extracts of ES and CP presented CC50 values and 132 as 12.10 µM and 8.21 µM, respectively. These >1,000 µg/mL, along with IC50 values of 19.1 ± 0.1 results indicate that naphtoquinones are a class of and 10.7 ± 1.5 µg/mL, respectively. The preliminary molecules with potential antiviral activity against DENV- results presented in this study encouraged our group to perform further bioguided fractionation and isolation of natural inhibitors, screening against acyclovir-resistant 2.BV228 FINANCIAL - BRAZILIAN SUPPORT: PLANTS CNPQ, CAPES/PROEX, FROM ASTERACEAE FAPERJ. strains and mechanism of action elucidation. These FAMILY WITH PROMISING IN VITRO ANTIHERPES experiments are currently being conducted at our ACTIVITY laboratory. FINANCIAL SUPPORT: CNPq and CAPES. Boff, L.1; Silva, I.T.1; Kratz, J.M.1; Reginatto, F.H.1; Ferraz, A. de B.F.2; Simões, C.M.O.1 BV248 - REVERSE TRANSCRIPTASE INHIBITION EFFECT BY NEW AMANTADINE DERIVATIVES 1. UFSC – Universidade Federal de Santa 1 1 1 Catarina, Campus Universitário Reitor João David Ferreira Franco, G.M. ; Souza, J.G. ; Fagundes, E.M.S. ; de 1 2 1 Lima - Trindade, Florianópolis - SC, 88040-900 Fatima, A. ; Martins, M.L. ; Stancioli, E.F.B. 2. ULBRA - Universidade Luterana do Brasil, 1. UFMG - Universidade Federal de Minas Rua Martinho Lutero - 301 - Universitário, Cachoeira do Sul Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, - RS, 96501-595 Belo Horizonte - MG, 31270-901 Herpes virus type 1 virus (HSV-1) establishes life-long 2. HEMOMINAS, Rua Grão Pará, 882 - Santa latent infections in human hosts, and can be reactivated Efigênia - Belo Horizonte - MG, 30622-020 throughout life. Although most clinical infections do The Human T-lymphotropic virus 1 (HTLV-1) is a not progress to severe cases, immunocompromised retrovirus that infects human and presents high individuals are in special danger and resistant strains prevalence in some regions of the Earth. The HTLV- have emerged, highlighting the need for new antiherpetic 1 is known to cause adult T-cell leukemia/lymphoma drugs. Natural products remain an important source - ATL, HTLV associated myelopathy/tropical spastic of biologically active substances. The contribution of natural products and natural-derived scaffolds in the conditions also important. Its transmission occurs development of antiviral agents is huge, accounting for throughparaparesis cell-to-cell (HAM/TSP), contact, besides being otherthat after inflammatory the entry 57% of all small molecules that reached the market. In the of virus in the cell, the reverse transcriptase (RT) search for antiviral agents, our group has been evaluating performs an essential step to the retrovirus replication: the antiviral activity of Brazilian plants. In this study, we reverse transcription. At this moment, there is no evaluated the cytotoxicity and the in vitro anti-HSV-1 activity of crude extracts and fractions of Eupatorium serratum (ES) and Calea phyllolepis (CP). These two retroviralsspecific treatment used in AIDS to be treatment. used, this The being aim empiricof this study and plants from the Asteraceae family were selected based issymptomatic to evaluate with the corticosteroids, anti-HTLV-1 reverse interferon-α transcriptase and anti-

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

82 Basic Virology: BV activity of three amantadine derivatives, ADA1, ADA2 and BeAn 1999 from C6/36 (Aedes albopictus) cells; after ADA5, moiety (C10H15), for which antiviral properties virus and HIV. Preliminary data are being evaluated viralconfirmation stock had by 1.5 immunofluorescent x 107 plaque forming assay, units was (PFU) filtered per inhad MT2 already and beenC91PL described cells, both to permanently Influenza A, infected Rubella mlthrough as observed a 0.22 inμm Vero membrane cells. An andamount stored of 3 at x 106-70oC. PFUs This of with HTLV-1. The cytotoxicity of the derivatives was OROV, in 200 µl, was inoculated intraperitonially into 24 evaluated by MTT assay in concentrations that ranged females of Swiss albinic mice, 3 weeks old. The animals between 1000µM to 0.0001µM for 24h. A colorimetric were observed for 14 days and showed signals of disease assay in vitro was performed to evaluate inhibitory 5 days post-inoculation. The animals presented loss of activity of the three derivatives, in concentrations that equilibrium, circular march and seizures suggesting ranged between 10µM to 0.0001µM, against reverse they had encephalitis. The fatality rate of the infected transcriptase. Azidovudine (AZT) and lamivudine (3TC) animals was 50%. This experimental model will be activities in the concentration of 10µM were also tested. improved to kill 100% of the animals and will be used in The results showed that both derivatives did not present studies on the pathogenesis of OROV. The experimental toxicity to MT2 and C91PL cells in the tested dilutions. model will also be used to test the protection to OROV In the reverse transcriptase assay ADA1 and ADA2 in infection by previous immunization with a nucleocapsid recombinant protein of this virus. activity 40% and 35% higher than AZT and 17% and 12%the concentrations than 3TC, respectively. of 0.0001μM ADA5 presentedin the concentration inhibitory BV268 - STUDY OF THE EFFECT OF HEPATITIS C VIRUS NS2 PROTEIN IN DNA DAMAGE PATHWAY AZT and 16% than 3TC. Despite further research would Bittar, C.; Campos, G.R.F.; Rahal, P. of 10μM presented inhibitory activity 39% higher than be necessary, these preliminary data demonstrated that UNESP/IBILCE - Universidade Estadual Paulista these new amantadine derivatives could be potential - Instituto de Biociências, Letras e Ciências Exatas, Rua antiviral candidates. FINANCIAL SUPPORT: FAPEMIG, Cristóvão Colombo, 2265 Bairro Jardim Nazareth, São José CAPES, CNPQ do Rio Preto - SP, 15054-000

BV267 - EXPERIMENTAL MODEL OF INFECTION BY Hepatitis c is a worldwide health problem with an OROPOUCHE VIRUS IN MICE estimated incidence of the infection by hcv of 2.2%, corresponding to 130 million people in the world. it is Mamani, P.R.; Souza, W.M.; Badra, S.J.; Figueiredo, estimated that more than 75% of the infected persons L.T.M. develop chronic infection that could lead to cirrhosis CPG/FMRP/USP - Centro de Pesquisa em Virologia and in some cases hepatocellular carcinoma (hcc). the da Faculdade de Medicina de Ribeirão Preto/ Universidade hcc is the third major cause of death by cancer in adults. de São Paulo, Av. Bandeirantes, 3900 - Monte Alegre, Ribeirão Preto - SP, 14049-900 hcv infected patients was an indirect consequence of the Oropouche virus (OROV) belongs to serogroup Simbu of viralat first infection, it was believed however that now the it greatis believed incidence that ofthe hcc viral in the genus Orthobunyavirus (Bunyaviridae). This virus proteins have a direct role in the hepatocarcinogenesis. is transmitted to mammals and birds by mosquitoes although some viral proteins have already shown (Aedes serratus, Culex quinquefasciatus) and midges hepatocarcinogenic potential, there are still many (Culicoides paraensis) that are its primary vectors in questions and a lot to be studied to understand the urban cycle. OROV is the etiological agent of Oropouche role of hepatitis c virus in cancer development. the fever in humans. This is an acute febrile illness that is ns2 protein is usually not studied in the context of endemic in in the Amazon region. OROV is the second hepatocarcinogenesis, however preliminary data arbovirus in number of reported cases in Brazil, only indicates that it has an inhibitory potential of p53 protein supplanted by dengue. We show here our preliminary which has a well known role in tumoral suppression. in results with an experimental model of infection by OROV this work we propose to address the effect of the viral in Swiss albinic mice (Mus musculus). OROV strain protein ns2 in the damage pathway. we used the rt2

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

83 Basic Virology: BV

demonstrating an IC50 value of 0.7263 mM for virus sa biosciences to evaluate the expression of dna damage replication. Caffeine inhibited SGR-JFH1-FEO replication relatedprofiler genespcr array in huh – dna 7.0 damage cells, huh signaling 7.0 cells pathway expressing from around 82 ± 10% and HCVcc replication 79 ± 12% at its maximum safe concentration. Also caffeine reduced 30 the results focusing on genes which presented similar % of viral entry. This inhibition increased two fold when behaviorns2 and huh both 7.0 in cells ns2 infected expressing with cells hcv andjfh-1. virus we analyzed infected particles were exposed to caffeine 1 hour before infection cells. results show 9 genes down regulated. using string of host cells. This data possibly, indicates an interaction v9.1 a network of interaction between these proteins between caffeine and viral glycoproteins. On the other was constructed showing that they relate either directly or indirectly. further analyses are required to understand secretion process. In conclusion, caffeine inhibits HCV what is causing the down regulation of these genes. in replicationhand, there andis no entry/attachment significant influence on ofhost caffeine cells onin vitroviral order to have a more complete understanding of the and has a potential as a new antiviral therapy against effect of ns2 in this pathway we will also investigate HCV alone or in association with conventional drug treatment. FINANCIAL SUPPORT: FAPESP/ CAPES FINANCIAL SUPPORT: FAPESP its profile in hepg2 cells and hepg2 cell expressing ns2. BV278 - EFFECT OF CAFFEINE METABOLITES ON HCV BV277 - CAFFEINE EFFECT ON DIFFERENT STEPS OF REPLICATION HCV REPLICATION CYCLE Batista, M.N.; Andrade, S.T.Q. de; Perissini, L.H.; Batista, M.N.; Carneiro, B.M.; Braga, A.C.S.; Rahal, P. Batista, M.N.; Carneiro, B.M.; Braga, A.C.S.; Rahal, P. UNESP/IBILCE - Universidade Estadual Paulista UNESP/IBILCE - Universidade Estadual Paulista - Instituto de Biociências, Letras e Ciências Exatas, Rua - Instituto de Biociências, Letras e Ciências Exatas, Rua Cristóvão Colombo, 2265 Bairro Jardim Nazareth, São José Cristóvão Colombo, 2265 Bairro Jardim Nazareth, São José do Rio Preto - SP, 15054-000 do Rio Preto - SP, 15054-000

C virus (HCV) infection. It often evolves to a chronic in several liver disorders. It improves abnormal liver diseaseHepatitis and C is thehas liverbeen inflammation considered causedthe major by hepatitis world biochemistry,Caffeine is a phytochemical cirrhosis and related hepatocellular to beneficial carcinoma effects cause of cirrhosis and hepatocellular carcinoma. and our group recently demonstrated this drug capacity Standard treatment using PEG-IFN and ribavirin has to inhibit HCV replication on a dose-response fashion. Caffeine is metabolized to three main compounds: regular treatment has high cost and severe side-effects. theobromine, theophylline and paraxantine. These Therefore,low efficacy new against treatments some HCV are genotypes. needed. Furthermore,Caffeine can molecules differ from caffeine only by loss of a methyl group in different positions. Theobromine lose methyl liver cellular pathways and interfere with cell pathways group at position 1; while theophylline lose the useddirectly by delaythe HCV fibrosis, replication as well cycle. as improve In the thecurrent function study, of methyl group at position 3 and paraxantine lose this the direct relationship between caffeine and viral group at position 7. Theobromine proved to be a good replication cycle was evaluated. To this research, we antitumoral at very low concentrations and to this utilized the subgenomic replicon SGR-JFH1-FEO, the full- purpose it keeps similar effects to those observed to caffeine. Theophylline, showed beyond the antitumoral and Huh-7.5 cell line. Caffeine viability was determined effect, inhibition of ERK and JNK phosphorylation. These onlength Huh-7.5 replicons cells FL-J6/JFH-5′C19Rluc2AUbiby MTT assay. The effect andof caffeine JFH-1; pathways have already been described to be essential to on virus replication cycle was evaluated by luciferase HCV replication process. Thus, in this study we evaluated theophylline and theobromine ability to inhibit HCV and qPCR. We observed in samples treated with caffeine replication. Hepatocellular carcinoma cell lineage aassay, dose-dependent western blotting, reduction indirect on immunofluorescence virus replication (Huh-7.5) were cultured and stably transfected with of subgenomic and full-length replication systems. subgenomic replicon (SGR-JFH1-FEO). Initially the cells Caffeine inhibited viral replication on different steps, expressing the SGR-JFH1-FEO were transferred to 96

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

84 Basic Virology: BV wells plates and after 24 hours different theophylline/ or vaccine to cure or prevent DENV infection. Therefore, theobromine concentration were added: 1 µM to 5 new approaches are needed to control Dengue virus mM on 10-fold dilution series. Cells were incubated infection and disease. Tizoxanide (TIZ) is the active for 24 h, 48 h and 72 h and tested for drug cytotoxicity compound of Nitazoxanide (NTZ), a thiazolide licensed by MTT assay. Viral RNA expression was evaluated by for the treatment of parasitic gastroenteritis. In this luciferase reporter assay. Caffeine metabolites showed study, the anti-DENV-2 activity of TIZ was evaluated in more toxicity on tumoral liver cell line (Huh7.5), what cultured cells. DENV-infected Vero cells were treated is compatible with literature, and IC50 of theophylline with TIZ at different concentrations. The replication of was higher than caffeine, 1.2 mM (caffeine 0.726 DENV in the control and TIZ-treated cells was examined mM). When analyzed on 1 mM, caffeine inhibits HCV by virus titration. TIZ was also administered at different replication around 60 %, while theophylline inhibits HCV time points of DENV infection to determine the stage at replication around 45 %. It was not possible to analyze theobromine effect at same concentrations of caffeine inhibited the replication of DENV in cell culture in a dose- or theophylline. Theobromine has low saturation point dependentwhich TIZ affectedmanner with DENV 50% replication. inhibitory TIZ concentration significantly and was tested at following concentrations: 1 – 10 µM. value of 3.07 ug/ml, a non-toxic concentration in Vero At analyzed concentrations of theobromine, there was cells (50% cytotoxic concentration = 7.58 ug/ml). The no effect on HCV replication. In conclusion, theophylline selective index calculated was 2.5. The viral yields when inhibited HCV replication around 45 % at maximum safe the cells were treated with TIZ after the adsorption concentration and theobromine did not affect the virus period decreased by 90% at 5 ug/ml, compared to the replication at the tested concentrations. Therefore, we control. TIZ showed little (up to 20%) or none antiviral concluded that caffeine has a more potent effect before effect in other stages of viral replication (virucidal; 1, metabolism and this should be considered on a possible 12, 24 h pre-treatment, adsoption, penetration, virus utilization of caffeine as therapy. FINANCIAL SUPPORT: release).Our results corroborate with those found in Shi FAPESP et al (2014) study which indicated that NTZ has anti- Japanese encephalitis virus activity, acting at the early- BV285 - TIZOXANIDE INHIBITS THE REPLICATION mid stage of viral replication. These results suggest OF DENGUE VIRUS-2 IN VERO CELLS the potential application of NTZ/TIZ in the treatment Yamamoto, K.A.1; Salles, T.S.1; de Meneses, M.D.F.2; Campos, R.M.2; Brown, D.T.3; Vancini, R.3; da Silva, are being analyzed in order to elucidate the mechanism M.R.S.2; Ferreira, D.F.2 of flavivirusaction of infection.TIZ. FINANCIAL At the moment,SUPPORT: further CNPQ, analyses CAPES, 1. IQ/UFRJ - Instituto de Química da FAPERJ, INBEB, FARMOQUIMICA S.A. Universidade Federal do Rio de Janeiro, Avenida Athos da BV293 - ACTION OF BACCHARIS DRACUNCULIFOLIA Silveira Ramos (antiga Av. 6), 149 Bloco A - 7° andar, Cidade D.C. EXTRACT AGAINST HERPESVIRUS TYPE I Universitária, Rio de Janeiro - RJ, 21941-909 2. IMPG/UFRJ - Instituto de Microbiologia Cecílio, A.B.1; Dutra, A.G.S.2; Silva, F. de O.1; Caldas, S.1; Paulo de Góes da Universidade Federal do Rio de Janeiro, Filho, A.A. da S.1 Av. Carlos Chagas Filho, 373 - Cidade Universitária, Rio de 1. FUNED - Fundação Ezequiel Dias, R. Conde Janeiro, Ilha do Fundão Rio de Janeiro - RJ, 21.941-590 Pereira Carneiro, 80 - Gameleira, Belo Horizonte - MG, 3. NC State Biochemistry - Department of 30510-010 Molecular and Structural Biochemistry, 120 Broughton Drive, 2. PUC- Pontifícia Universidade Católica Raleigh, NC 27607, Estados Unidos Introduction: The Herpesvirus type 1 (HHV-1) has an Dengue virus (DENV), a mosquito-borne virus (family overall prevalence of about 90% in the human population. Flaviviridae, genus Flavivirus), is a leading cause of This prevalence can be explained by the establishment illness in the tropics and subtropics. More than one- of a persistent infection in the host. The infection has third of the world’s population is in areas at risk for become a public health problem worldwide, where infection, and there is no available antiviral treatment besides the usual manifestations of the disease including

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

85 Basic Virology: BV latency may arise numerous complications, such as eye Rhinovirus, the causative agent of the common cold, infections. Plant extracts are commonly used in research is responsible for enormous damages to the world against diseases and secondary metabolites provide economy and health. Lactoferrin (Lf), a glycoprotein defense against pathogens and may have antiviral activity. present in mucosal secretions of mammals, is widely The Baccharis dracunculifolia, known as alecrim-do- studied for its antiviral activity. The mechanisms of Lf campo, belongs to Asteraceae family and is considered antiviral activity already described include intracellular the most important source of green propolis. According biochemical processes, interaction with cell surface and to the literature, the plant is used in folk medicine and competition for virus receptors. Our aim is to study the has antioxidant, antimicrobial and cytotoxic activity. interaction of lactoferrin with HeLa H1 cells and how it Therefore, the evaluation of the antiviral activity of B. interferes with the HRV14 infection cycle, investigating dracunculifolia against HHV-1 indicates an alternative the possible interaction of Lf with molecules present for treatment and prevention of this disease. Material at the cell surface, such as glycosaminoglycans. We and Methods: Cytotoxicity assays for B. dracunculifolia observed the effect, to the cells, of Lf and NaClO3 using extract were performed and tested in interval of 48 MTT cytotoxicity assay, and used confocal microscopy to hours. Through the tetrazolium salt colorimetric assay - MTT cytotoxicity was determined and the 50% EC50 effective concentration was determined by sigmoidal ofinvestigate Lf and sulfation Lf ability inhibition to bind nonspecifically of cells HS with to heparanNaClO3. Time-lapsesulfate (HS), microscopyperforming, forexperiments this, the fluorescent showed that labeling bLf 48 hours. From this analysis the antiviral assay without causes morphological changes to the cellular surface. adsorptionregression. was The performed, EC50 determined testing the was concentrations 195,3μg/mL inof Structures similar to transitional blebs are observed immediately after bLf addition. After 30 minutes, bLf used in the experiment of 48 hours. The supernatant is observed bound to the cell membrane, while after was285 tocollected, 75 μg/mL subjected of the crude to DNA extract. extraction A MOI and= 0.1 Real- was 60 minutes a perinuclear distribution of bLf could be Time PCR. Results: The antiviral activity was detected observed. Cells treated with sodium chlorate for sulfation inhibition presented less bLf binding as compared to indicate that the antiviral activity is effective in an assay control cells, suggesting a role for heparan sulfate during withoutusing the previous concentrations adsorption of 285 of athe 165 virus. μg/mL. Conclusions: Our tests bLf binding to the cell. Our data show that bLf binds to The crude extract of B. dracunculifolia is an important HeLa H1 cells surface and is slowly internalized (60 min) approach in order to study a promising compound to moving to the perinuclear region of the cell. Sulfation treat the infection of Herpesvirus. FINANCIAL SUPPORT: inhibition suggests that the initial interaction of bLf FAPEMIG, FUNDAÇÃO EZEQUIEL DIAS – FUNED AND is dependent on negative molecules, such as heparan PONTIFÍCIA UNIVERSIDADE CATÓLICA DE MINAS sulfate. The bLf binding to glycosaminoglycans may be GERAIS - PUC / MG. slow internalization of the molecule suggests that it may BV297 - THE ROLE OF BOVINE LACTOFERRIN/ interferethe key for with an unspecific intermediate antiviral to late mechanism stages of of the bLf. viral The HEPARAN SULPHATE INTERACTION ON HUMAN infection cycle. FINANCIAL SUPPORT: FAPERJ, CNPq, RHINOVIRUS 14 INFECTION FINEP, CAPES. Denani, C.B.2; Santos, R.2; Carvalho, C.A.M.1; Roxo, T. 2; Real-Hohn, A.1; Barros, C.A.2; Silva, J.L.1; Oliveira, A.C.1; Gomes, A.M.O.1; Gonçalves, R.B.2 1. UFRJ - Universidade Federal do Rio de Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de Janeiro - RJ, 21941-901 2. UNIRIO - Universidade Federal do Estado do Rio de Janeiro, Avenida Pasteur, 296 - Urca, Rio de Janeiro - RJ, 22290-240

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BV313 - INHIBITORY ACTIVITY OF SEVERAL FUNGI of the extracts with remarkable antiviral activity were AND PLANT EXTRACTS AGAINST DENGUE VIRUS obtained from cultures of endophytic fungi, which are TYPE-2 Barbosa, E. de C.1; Francisco, F.L. de M.1; Mota, G.C.1; extracts presented interesting effective concentration in the process of taxonomic identification. Eight fungal Alves, T.M. de A.1; Carlos, L.Z.1; Rosa, L.H.2; Calzavara- 50 (EC50) values, at doses between 3.1 µg/mL and Silva, C.E.1; Kroon, E.G.2; de Oliveira, J.G.1 12.5 µg/mL, with no cytotoxicity at 100 µg/mL. The molecular and cellular mechanisms of antiviral activity 1. CPqRR/FIOCRUZ MINAS - Centro de of those extracts are under investigation by our group. Pesquisas René Rachou/ Fundação Oswaldo Cruz, Av. Augusto FINANCIAL SUPPORT BY FIOCRUZ, CPQRR, CNPQ AND de Lima, 1715 - Barro Preto, Belo Horizonte - MG, 30190-002 FAPEMIG 2. UFMG - Universidade Federal de Minas Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, BV325 - HEPATITIS B VIRUS SURFACE ANTIGEN Belo Horizonte - MG, 31270-901 (HBSAG) QUANTIFICATION IN PATIENTS WITH Dengue virus (DENV) is an important human pathogen, CHRONIC KIDNEY DISEASE: RELATION TO VIRAL which causes a wide spectrum of clinical illnesses REPLICATION AND HISTOLOGICAL FINDINGS ranging from a silent or mild febrile infection, self-limited Lima, A.S.N.2; de Castro, I.R.D.2; Bernardino, J. de S.T.2; dengue fever to a severe dengue hemorrhagic fever and Alves, R.2; de Carvalho, I.M.V.G.1; Feldner, A.C. de C.A.3; dengue shock syndrome. In fact, Dengue fever is the Silva, A.E.B.3; Carvalho-Filho, J.R.3; Ferraz, M.L.C.G.3 most prevalent arthropod-born viral diseases in the 1. Instituto Butantan, Av. Vital Brasil, 1500, Butantã, São Paulo - SP, 05503-900 vaccine available for dengue. Indeed, the development of 2. Universidade Federal de São Paulo, anworld. antiviral Currently, drug thereis considered is neither a reasonablespecific treatment alternative nor Laboratório de Hepatologia Molecular Aplicada, for dengue treatment. In this study, a total of 2940 Departamento de Gastroenterologia fungi and plant extracts obtained from the Fiocruz 3. Universidade Federal de São Paulo, collection of plant and fungi extracts (COLAB) were Departamento de Gastroenterologia screened for antiviral activity against Dengue virus Patients with chronic kidney disease (CKD) on type 2 (DENV-2). Extracts of different anatomical parts hemodialysis are at higher risk of HBV infection and at of a wide range of plants and fungi isolates were tested increased risk of developing liver cirrhosis, especially in vitro using BHK-21 cells in the presence of DENV. after undergoing kidney transplantation (KT). the inhibition of the cytopathic effect (CPE) caused by differentiate between HBV inactive carriers and patients DENVThe antiviral and also effect by ofthe those 3-(4,5-dimethylthiazol-2-yl)- extracts was verified by withQuantification HBeAg-negative of HBsAg chronic serum hepatitis levels (qHBsAg) B, as well can as help in 2,5-diphenyltetrazolium bromide (MTT) colorimetric identifying patients with a lower probability of response assay. In addition, the potency of the extracts (EC50) to antiviral therapy with interferon. The aim of this that showed antiviral activity was further examined. study was to evaluate potential correlations between For that, BHK-21 cells were treated simultaneously with different concentrations of those extracts (ranging in patients with CKD. Three groups of chronic HBV from 100 to 0.78 µg/mL) in the presence of DENV-2. carriersqHBsAg, were serum included: HBV DNA Group levels 1 and – Normal histological renal functionfindings A total of 114 out of the 2940 extracts tested showed (controls); Group 2 – CKD patients under hemodialysis; antiviral activity. Thirty-two out of the those 114 and Group 3 – KT recipients. Serum qHBsAg and HBV extracts were obtained from plants belonging to 17 distinct families: Malpighiaceae (6), Erythroxylaceae HBsAg Quantitative and the Abbott RealTime HBV (1), Melastomataceae (2), Myrtaceae (1), Rubiaceae assays,DNA levels respectively. were quantifiedHBV genotyping using was the performed ARCHITECT by (1), Fabaceae (1), Clusiaceae (1), Combretaceae (1), sequencing and phylogenetic analysis. Liver histology Leguminosae (2), Lythraceae (2), Asteraceae (4), was assessed using the METAVIR scoring system. A total Amaryllidaceae (3), Sapindaceae (3), Ochnaceae (1), of 104 patients was included as follows: Group 1: n=50, Begoniaceae (1), Annonaceae (1), Primulaceae (1). Most

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2: n=21; and 3: n=33. Mean age was 42.0±12.2 years, one of the main cases of blindness in underdeveloped countries. Although there is little information about the was observed in 46% of the patients. Median serum ALT early stages of infection, it is known that transmission levelwith was 72% 1.4 males. times Significant the upper liverlimit fibrosisof normal. (F2/F3/F4) Fifty-two occurs by direct contact with skin or mucosa with some patients (50%) were genotyped, with genotypes D and A type of abrasion, when in contact of person presenting being the most prevalent (56% and 33%, respectively). virus secretion. The most used compound for the HBV viral load was obtained for 92 patients (88%), tratament of such infections is acyclovir. However, the with higher levels found in Group 3 (median of 8.90 emergence of HSV-1 strains resistant to antiviral drugs as log IU/mL; p<0.001). qHBsAg was available for 61 acyclovir, makes the search for new molecules, especially subjects (59%), with a mean of 4.0±0.7 log IU/mL. Mean with different mechanisms of action, a constant urgency. qHBsAg levels were similar among groups (4.02±0.71, The aim of this study was to evaluate the possible 4.34±0.41, and 3.80±0.90 log IU/mL for Groups 1, 2, and 3; p=0.231). HBeAg-positive patients exhibited monoterpene fraction extracted from algae Plocamium higher levels of qHBsAg as compared to HBeAg-negative brasiliensesantiviral effect, on ofVero synthetic cells infectedsubstance with β lapachone HSV-1 virus, and subjects (median 4.41 vs. 3.49 log IU/mL; p=0.020). checking the cytotoxicity and the virucidal effect. Results Comparable levels of qHBsAg were observed in subjects obtained in plaque reduction assay showed that synthetic log IU/mL; F2/F3/F4: 3.94±0.78 log IU/mL; p=0.644). fraction obtained from Plocamium brasilienses at a with mild and significant liver fibrosis (F0/F1: 4.04±0.80 substances β lapachone AN6 and 39-42 monoterpene and HBV viral load (rs=-0.061; p=0.658). This analysis No significant correlation was seen between the qHBsAg concentration of 50μM were able to significantly reduce whileviral plaquethe fraction forming 39-42 units. Plocamium The β brasilienses lapachone wasshowed not confirms the association between KT and higher HBV cytotoxic at any concentration used (50μM and 500 μM) butviral are load. higher qHBsAg in HBe-positive levels seem patients.to be influenced Up to now, neither the correlationby renal function between status, qHBsAg nor and by HBV liver DNA fibrosis levels stage,could forhigh control cytotoxic cells. effect The at concentrationsvirucidal analysis above demonstrated 100μM, but at 50μM concentration values approached the one found 2012/11081-9 AND CAPES 42 of Plocamium brasiliensis were able to prevent not be confirmed in CKD subjects. FINANCIAL SUPPORT: viralthat βpenetration lapachone into substance Vero cells. AN6 Other and fractionexperiments 39- BV331 - EFFECT OF Β LAPACHONE AND PLOCAMIUM are underway to determine in which step of the viral BRASILIENSES MONOTERPENE ON HERPES VIRUS replicative cycle the tested substances are interfering. SIMPLES TYPE 1 (HSV-1) FINANCIAL SUPPORT: CAPES; CNPQ; FAPERJ; PROPI- Souza, K.F.C.S.1; Carvalho, D.G.1; Brito, V. de L.3; Lima, UFF G.M.3; Silva, T.C.3; Garcia, D.G.2; Martins, D. de L.1; Teixeira, V.L.1; Paixão, I.C.N. de P.1; Burth, P.1 BV352 - ANTIVIRAL ACTIVITY OF SYNTHETIC PEPTIDES AGAINST HSV-1 AND AICHI VIRUS 1. UFF - Universidade Federal Fluminense, Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 Silva, P.A.; Boas, L.V.; Migliolo, L.; Mendes, G.; de Lima, 2. UFRJ - Universidade Federal do Rio de L.M.P.; Franco, O.L. Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de UCB - Universidade Católica de Brasília, W5 Norte, Janeiro - RJ, 21941-901 Brasília - DF, 70790-160 3. IEPIC - Instituto de Educação Professor Viral infections affect all living organisms. For control Ismael Coutinho, Travessa Manoel Continentino, 32, São Domingos, Niterói - RJ, 24210-150 and prevention of such infections agents, public health and vaccination programs have been developed and Infection with herpes simplex type-1 virus (HSV-1) can the antiviral appear as a valuable alternative for virus cause various diseases such as mucocutaneous infections, treatment. In this context, antiviral peptides have been encephalitis in immunocompromised patients and characterized with different mechanisms of action neonates, as well as genital infections and keratitis, being

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersthat - Basic are Virology: specific BV for each viral type. However, in recent XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

88 Basic Virology: BV years, studies on antimicrobial peptides have reported Cytomegalovirus pp28 tegument protein, encoded by the an additional antiviral against both enveloped and non- UL99 gene, is essential for viral replication and functions enveloped viruses, making them promise candidates for antiviral drugs.OBJECTIVES: The present study aimed to the cytoplasm of infected cells. In this work we aim to evaluate the antiviral activity of synthetics peptides, Pa- determineduring the the final function acquisition of the of RhCMV137 the viral envelopeprotein, the in MAP 1 and variants, clavanin MO, Cn-AMP1, and LL-37 RhCMV pp28 homologue. In order to generate a RhCMV against human herpes virus 1 and Aichi virus.MATERIALS AND METHODS: The peptides were synthetized by the cloned into the PGS284 shuttle vector, which will be used forlacking homologous the RhCMV137 recombination gene, its flankingand allelic regions exchange were using C-18 reversed-phase HPLC, and molecular masses in bacteria, creating a RhCMV with substitution of wereF-MOC determined stepwise solid-phase by using mass method, spectrometry further purified MALDI- by the RhCMV137 for the kanamycin resistance gene. In addition, we aim to perform structural and biophysical in Vero cells was evaluated by MTT method and antiviral studies of pp28 and RhCMV137 proteins, to better activityToF/ToF was Ultraflex determined III. The by cytotoxic reduction effect of the of virusthe peptide titers. understand their biochemical roles and to study their RESULTS: The peptides clavanin MO, Pa-MAP 1.8 and Pa- structure–function relationship. For this, the RhCMV137 MAP18br showed a higher cytotoxicity at concentrations and UL99 genes were cloned into the pET28a expression over 12 µM. Moreover antiviral assays, showed that the vector and experiments of protein expression are peptide Pa-MAP 1 caused 90% of inhibition of HSV-1, currently underway. The studies of the RhCMV137 in a virucidal manner, with a SI higher than 4,8, and 0% protein will be important for determination of its of Aichi virus. LL-37 showed 90 % of inhibition of Aichi function as compared with the pp28 protein, and for virus replication with a SI of 3,4. Other peptides did not validation of the RhCMV as an animal model for CMV showed any antivirus effects. CONCLUSIONS: This study studies. In addition, the RhCMV137 defective mutant could also be tested as a vaccine against CMV in Rhesus for Aichi virus treatment. In conclusion, the data suggest monkeys. FINANCIAL SUPPORT:FAPESP, CAPES. thatwas thePa-MAP first report1 could of be an utilized antiviral as peptidea potent with candidate potential for virus infections control. BV392 - EVALUATION OF APOPTOSIS INDUCED BY OROPOUCHE VIRUS NSS PROTEIN BV362 - STRUCTUTRE AND FUNCTION OF THE Oliveira, A.S.; Acrani, G.O.; Criado, M.F.; Silva, M.L.; RHESUS CYTOMEGALOVIRUS RH137 PROTEIN Arruda, E. Santos, E.S.; Sperança, M.A.; da Silva, M.C.C. FMRP/USP - Faculdade de Medicina de Ribeirão UFABC - Universidade Federal do ABC, Rua Catequese, Preto/ Universidade de São Paulo, Av. Bandeirantes, 3900 - 242 - Jardim, Santo André - SP, 09090-400 Monte Alegre, Ribeirão Preto - SP, 14049-900 (CMV) are ubiquitous members of Oropouche virus (OROV) is the second most common sub-family, associated with serious arboviral disease in Brazil and a major cause of febrile diseases in immunocomprised hosts. Due to the strict infections in South and Central America. OROV belongs to the Bunyaviridae family, genus Orthobunyavirus. pathogenicity in vivo are performed in animals, using OROV genome is a tri-segmented single-stranded specie-specificity, the studies of CMV biology and negative sense RNA, composed of large (L), medium (Macaca mulatta), susceptible to infection with Rhesus (M) and small (S) segments. The L segment encodes CMVthe CMV (RhCMV), specific are of considered the target specie.the ideal Rhesus research macaques model the polymerase; the M segment encodes a poly-protein for these studies, becuase their close relationship to whose cleavage products are the surface glycoproteins humans. Studies of the functional characterization of (Gn and Gc) and nonstructural protein (NSm). The S viral proteins are important for the understanding of segment encodes the nucleocapsid structural protein viral replication and pathogenesis in vivo. We have (N) and the non-structural protein NSs. It has been previously demonstrated using, a HCMV Bacterial previously shown that the non-structural proteins (NS) of other Bunyaviruses are involved in metabolic

ArtificialSeptember/October Chromosome 2014 Volume (BAC) 19 – system, Supplement that 2 - theAbstracts/Posters Human - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

89 Basic Virology: BV processes such as apoptosis and synthesis of type I IFN. order to study acute and persistent HRSV infection. We have previously demonstrated that OROV induces Neonate BALB/c mice (10 day-old), were infected apoptosis of HeLa cells and this requires the synthesis intranasally with 104 PFU of HRSV A long strain (ATCC). of viral proteins. The pathogenesis of OROV infection On days 3, 7, 14, 30, 60 and 90 post infection (pi), is not completely understood, but apoptosis seems to animals were euthanized with ketamine and xylazine play a central role. Based on that, this study was done to and tissues from the respiratory tract (nostrils/trachea evaluate the occurrence of apoptosis induced by OROV and lungs), liver, spleen and mesenteric lymph nodes infection, and by NSs transfection in HeLa, Glioblastoma were harvested for quantitation of viral RNA by real- and HCE (human corneal epithelium) cell lineages. Semi- time RT-PCR, and by plaque assay. qPCR showed that HRSV RNA was detected both in lungs and lymph nodes containing coverslips and maintained respectively with on day 7 pi, but remained detectable in lymph nodes MEM,confluent DMEM monolayers and SHEM, were and seeded then ininfected the 24-well with platesOROV on days 30, 60 and 90 pi. HRSV RNA was detectable in or transfected with plasmid containing the NSs gene nostrils/trachea on day 90 pi. Remarkably, viable HRSV (pcDNA3.2- NSs), or with empty vector (pcDNA3.2) was recovered in mesenteric lymph nodes on days 7, as a negative control. Subsequently, the plates were 30 and 60 pi. This study successfully established an incubated at 37°C, 5% of CO2 for 24 or 36 hours. Then experimental model for HRSV intranasal infection, with additional important evidence that the virus remains viable in mice for prolonged periods of time. FINANCIAL antibodymonolayers direct were to fixedOROV withand paraformaldehydeTUNEL assay for DNA and SUPPORT: FAPESP, CNPq, CAPES subjected to immunofluorescence (IF) with polyclonal transfection by the NSs protein, and also intense BV404 - HCMV INDUCED CHEMOTERAPIC progressivefragmentation. labeling The of IF fragmented confirmed DNA the infectioncharacteristic and RESISTENCE IN MRC-5 AND U251 CELLS of apoptosis was noted at the times of 24 and 36 h pi. Santos, C.J.; Souza, A.C.S.; Silva, M.C.C. These results demonstrate that OROV NSs protein is pro- UFABC - Universidade Federal do ABC, Rua Catequese, apoptotic for different tumor cell lines, and may become 242 - Jardim, Santo André - SP, 09090-400 an important experimental tool for that purpose. Human Cytomegalovirus (HCMV) is a ubiquitous virus BV395 - HUMAN RESPIRATORY SYNCYTIAL VIRUS that leads to lifelong persistent infection and can cause (HRSV) EXPERIMENTAL INFECTION IN NEONATE serious diseases in immunocompromised individuals. BALB/C MICE In the past years accumulating evidences suggest an de Jesus, B.L.S.; Criado, M.F.; de Andrade, M.A.A.M.; de association between HCMV infection and cancer. The Sousa, E.; Arruda, E. virus has been detected in many types of cancers, especially in glioblastoma multiforme (GBM), the most FMRP/USP - Faculdade de Medicina de Ribeirão prevalent and malignant tumor of the central nervous Preto/ Universidade de São Paulo, Av. Bandeirantes, 3900 - Monte Alegre, Ribeirão Preto - SP, 14049-900 HCMV can transform cells, previously studies suggest HRSV is the single most important viral cause of thatsystem. it might Although increase there tumor is no directmalignancy confirmation through thatthe lower respiratory tract infections and re-infections action of viral proteins that disrupt cellular pathways in children worldwide. Passive immunoprophylaxis involved in the cell cycle, apoptosis, angiogenesis, cell reduces hospitalizations, but vaccines or effective invasion and cellular immune response. Interestingly, therapies are not available. The neonatal mouse model other studies also demonstrated that the virus is capable of HRSV infection, which most closely mimics human of induce degradation of nuclear complexes, leading infants, offers a possibility to study pathogenesis of to a increase of cell resistance to chemoterapy. In this HRSV-induced airway disease, mechanisms of HRSV study, we analyzed the effect of HCMV infection in cell counteraction of the host immunity, and for preclinical resistance to the chemotherapic agent Temozolamide testing of interventions. We have successfully established an experimental HRSV neonate BALB/c mice model in and human glioblastoma cell line (U251) were treated (TMZ) by MTT assay. Human lung fibroblasts (MRC-5) September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

90 Basic Virology: BV with TMZ (100 µm) in presence and absence of virus at Transcriptase Assay Kit. Furthermore, the cytotoxicity of multiplicity of infection 1 and 10, respectively (M.O.I. both extracts were evaluated on human MT2 cells. The 1 and 10). The results demonstrated that at 120 hours IC50 (the concentration of a drug that is required for 50% post treatment TMZ reduced the amount of cells in inhibition) and the CC50 (the concentration of a drug both cell lines, as compared to untreated cells. The that is required for 50% of the cytotoxic effect) values viral presence itself also reduces the cellular amount were estimated by using Sigma Plot program. Our results in both cell lines. The presence of HCMV in treated cells show that both the hexane and the dichloromethane- increases cell survival in approximately 50% in MRC-5 methanolic extracts of P. crispa reduced the HIV-1 RT and 40% in U251 cells when comparing with no infected catalytic activity in a dose-dependent manner. The IC50 cells. The data reinforce a possible association between values obtained for both hexane fractions, F14 and F15, HCMV infection and tumor resistance to chemotherapic were similar (50ug/mL), while de the IC50 for the F16 treatment. Further experiments are been conducted fraction was 70ug/mL. The dicloromethane fraction in other glioblastoma tumor cell lines. In addition F11 showed an IC50 value of 25ug/mL. The CC50 values expressing individual HCMV proteins were obtained for both hexane fractions, F14 and F16, as well constructed in order to test the effect of single proteins in as the dicloromethane F11 fractions were similar, 0,8mg/ drug resistance. FINANCIAL SUPPORT: FAPESP/CAPES. mL, while the hexane F15 fraction was considered to be toxic for the MT2 cells. Conclusion: the hexane and BV405 - THE ANTI-HIV-1 REVERSE TRANSCRIPTASE the dichloromethane fractions of the algae P. crispa ACTIVITY OF THE GREEN ALGAE PRASIOLA CRISPA reduced the HIV-1 reverse transcriptase activity with Amorim, L. dos S.C.1,3; Ribeiro, M. de S.R.2; Brito, low cytotoxic effect indicating a promising role for these C.J.B.2; Stephens, P.R.S.1,3; Oliveira, R.G. de O.1; Lyra, algae extracts in the AIDS-therapy research. FINANCIAL J.S.P.O.2,3; Teixeira, V.L.1; Paixão, I.C.N. de P.1; Pereira, SUPPORT: Capes, CNPq, FOPESQ-UFF and FAPERJ H. de S.P.1 BV408 - THE ANTI-HIV ACTIVITY AND LOW 1. UFF - Universidade Federal Fluminense, CYTOTOXICITY OF OXOQUINOLINE DERIVATIVES ON Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 MT-2 CELL 2. UFRJ - Universidade Federal do Rio de 1,3 1 2,3 Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de Amorim, L. dos S.C. ; Stephens, P.R.S. ; Lyra, J.S.P.O. ; Janeiro - RJ, 21941-901 Pereira, H. de S.P.1; Ribeiro, M. de S.R.1; Neurauter, A.L. 3. FIOCRUZ - Fundação Oswaldo Cruz, Av. de A.3; de Souza, M.C.B.V.1; Sangrillo, F.S.S.1; Cunha, Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 A.C.C.1; Ferreira, V.F.F.1; Paixão, I.C.N. de P.1 1. UFF - Universidade Federal Fluminense, been established as the etiological agent of the AIDS, Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 anSince appreciable the human number immunodeficiency of drugs have virusbeen developed (HIV) has 2. UFRJ - Universidade Federal do Rio de and approved for the treatment of the HIV-infected Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de patients. Viral enzymes and particularly the Reverse Janeiro - RJ, 21941-901 Transcriptase (RT), which is an essential catalytic 3. FIOCRUZ - Fundação Oswaldo Cruz, Av. activity in the retrovirus replication cycle, have been Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 chosen as the target for drug development. Despite of According to UNAIDS 2013, there were more than 34 million people living with HIV worldwide. Brazil has a nucleoside and non-nucleoside reverse transcriptase population of approximately 198 million inhabitants inhibitors,the remarkable virus pharmacological mutations are induced profiles by of antiretroviral the classical and (from 1980 to 2013) more than 480.000 AIDS cases therapy and drug-resistance is usually reported. In were diagnosed, with more than 35.000 new cases per this work the anti-HIV-1 RT activity of the hexane and year. Currently, there are no effective HIV/AIDS vaccine dichloromethane-methanolic fractions of the extracts or cure, although the introductions of antiretroviral of the green algae Prasiola crispa were investigated by using the Molecular Probe EnzChek® Reverse individuals with access to treatment. However, the drugs significantly improve the prognosis of infected September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

91 Basic Virology: BV emergence of drug-resistant viral strains is on the increasing. Therefore numerous studies have been at 0, 4, 8, 12, and 24 h post infection (pi). Coverslips wereat 4°C then for 1stained h, and by fixed IF withand 4%analyzed paraformaldehyde by confocal some low-toxicity and low-cost anti-HIV substances. microscopy. Primary antibodies used for IF were Thedeveloped, aim of suchthis asstudy preventive was to strategiesevaluate the in order cytotoxicity to find directed to plasma membrane (transferrin receptor and reverse transcriptase (RT) activity of oxoquinoline TrF), endoplasmatic reticulum (ER, calnexin alpha - derivatives on MT-2 cells infected with HIV-1. The cells in 96-multiwell plates were treated with various concentrations of the compounds for 72 h. Then, the AtCNX2), early trans-Golgitimes pi HRSV network proteins (giantin), appeared earlyto concentrate and late solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl nearendossomes the nucleus, (SNX2 starting and CD63, heavy respectively) accumulation and at 4 HRSV. h pi, tetrazolium bromide (MTT, Sigma)was added to in a region suggestive of ER and Golgi. Thereof, HRSV evaluate cell viability. The 50% cytotoxic concentration proteins homogeneously disperse over the cytoplasm, (CC50) was calculated by linear regression analysis of the dose response curves and measurement of ELISA granules, and at 24 pi they tend to be localized at the p24 antigen in supernatants from MT-2 cell lines treated celllosing periphery, the early patternconsistent of concentration with virus inbudding well-defined from with oxoquinoline derivatives and infected by HIV-1. The anti-HIV-1 RT activity of oxoquinoline derivatives were localization of HRSV structural proteins with Golgi or investigated by using the Molecular Probe EnzChek® ERthe all plasma through membrane. the 24-h period. There wasImportantly, no significant there was co- Reverse Transcriptase Assay Kit. The oxoquinoline striking disorganization and dispersion of giantin and derivatives studied by our group have important effects on HIV replication, as we have observed more than 60% of RT viral inhibition. Cytotoxicity levels lower than studyCNX2 points over time, to important indicating targets loss of cell integrity damage of in ER HRSV and 30% were observed on substances. Further pre-clinical infectedGolgi, likely cells, due underscoring to HRSV inflicted the need cell for damage. experiments This studies are needed to better evaluate these substances to assess the mechanisms for cell damage, including ER as potential candidates for microbicides or sistemic stress. FINANCIAL SUPPORT: CAPES and FAPESP drugs. FINANCIAL SUPPORT: FIOCRUZ, CNPq, FAPERJ, CAPES, FOPESQ-UFF-PROPPI. BV422 - BIOINFORMATICS ANALYSIS OF THE PRO- APOPTOTIC NSS PROTEIN OF OROPOCHEVIRUS BV419 - INTRACELLULAR LOCALIZATION PATTERN Souza, M.M.; Oliveira, A.S.; Acrani, G.O.; Arruda, E. DURING HUMAN RESPIRATORY SYNCYTIAL VIRUS USP - Universidade de São Paulo, Av. Prof. Almeida (HRSV) INFECTION IN A549 CELLS Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 Cardoso, R.S.; Criado, M.F.; Silva, M.L.; da Silva, L.L.P.; Neto, E.A. Oropouche virus (OROV), of the family Bunyaviridae, is the second most common cause of arboviralfebrile USP - Universidade de São Paulo, Av. Prof. Almeida illness in Brazil, but its pathogenesis is practically Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 unknown. OROV causes apoptosis of infected cells, both Human Respiratory Syncytial Virus (HRSV) is the single in vitro and in vivo. Previous results have shown that the most important respiratory viral pathogen for children non-structural protein NSs of OROV induces apoptosis worldwide. Nearly 95% of kids have been infected by in eukaryotic cells. A bioinformatics analysis of NSs was HRSV before 5 years of age. Despite its importance, little is done in order to predict structural and physico-chemical known about cell biology of HRSV infection in respiratory parameters of the protein, which can help to guide experiments, and to understand how its relationship analysis of A549 cells infected with HRSV A (strain long) to apoptosis happen. The physicochemical parameters atcells. different We performed time points indirect post infection, immunofluorescence using antibodies (IF) were analyzed by the Protparam protocols: molecular for HRSV and for protein markers of cellular organelles. weight: 10.6 kDa; theoretical pI: 10.7;amino acid composition: 4 cysteine residues, 4 negatively charged well plates were infected with HRSV (MOI=1), absorbed Sub-confluent monolayers of A549 cells grown in 24- September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersand - Basic 12 Virology:positively BV charged residues; extinction coefficient: XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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1.829 assuming all Cys residues are reduced; estimated and insects, organisms which are quite different for the half-life: 30 hours for mamilian cells; instability index: presence of cholesterol. In both, infection is productive 66.49,indicating an unstable protein; hydropathicity and leads to the formation of equally infectious viral (GRAVY): -0.232,indicating NSs to be hydrophilic). particles. This work aims to evaluate the thermostability Secondary structure prectiction by Jpred showed seven of the envelope of MAYV particles originated from mammalian and mosquito cells in order to evaluate the structural characteristics of these virus particles. To molecularbeta strands. homology BLASTn searchmodeling. has However,not indentified 3 structurally proteins with sufficient similarity to be helpful as template for hamster kidney) and C6/36 (Aedes albopictus larvae) killer toxin (which matches 42% of NSs sequence achieve that, virus particles purified from BHK-21 (baby withsimilar 23% proteins identity), could and be two identified: human Williopsismakriimethylesterases (matching 36% of NSs sequence with 36% identity).Ab showcells werethat particles labeled with obtained the fluorescent from BHK-21 probe and laurdan C6/36 initio modelling was done by QUARK,validated using cellsand analyzed have similar by fluorescence membrane spectroscopy.organization, despiteThe results the PROCHECK, and visualized by PyMol. After validation,the difference in their lipid compositions, suggesting that cholesterol may not be the only determining factor for with 3 and other with 4 beta strands, and showed the maintenance of the organizaton of the virus envelope. modeling confirmed presence of two beta sheets,one The effect of high temperature on the virus morphology was analyzed by light scattering and viral infectivity was Thesedisulfide analyses bonds help in coil guiding regions experiments nearthe C-terminus. to structural A measured by plaque assay. Both particles were able to targetssearch byof NSs Dali in identified host cell similaritymetabolic pathways,in with streptavidin. order retain infectivity even when exposed to temperatures to make advances in the understanding of its function. of 45 and 60 °C, suggesting a high thermostability for FINANCIAL SUPPORT: CNPQ AND FAPESP MAYV derived from BHK-21 or C6/36 cells. FINANCIAL SUPPORT: FAPERJ, CNPQ, CAPES, FINEP, UFRJ, INBEB BV445 - STUDY OF THE ORGANIZATION AND STABILITY OF THE LIPID ENVELOPES OF MAYARO BV450 - EVALUATION OF THE ANTIVIRAL POTENTIAL VIRUS PARTICLES PURIFIED FROM VERTEBRATE OF PLANT EXTRACTS ANNONA MURICATA, AND INVERTEBRATE CELLS CAESALPINIA ECHINATA AND SPONDIAS MOMBIN OF Ferreira, V.N. dos S.; Carvalho, C.A.M.; Ferreira, D.F. NORTHEAST BRAZIL AGAINST DENGUE-2 VIRUS IN Silva, J.L.; Gomes, A.M. de O. C6/36 AND VERO CELLS Farias, K.J.S.; Machado, P.R.L.; de Almeida Jr, R.F.; UFRJ - Universidade Federal do Rio de Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de Janeiro - Lima, T.L.C.; Nascimento, Y.M.; de Araújo, J.M.G. RJ, 21941-901 UFRN - Universidade Federal do Rio Grande do Norte, Mayaro virus (MAYV) is an enveloped alphavirus Campus Universitário Lagoa Nova, Natal - RN, 59078-970 that belongs to the Togaviridae family. MAYV has a The dengue virus is the most prevalent arthropod-borne genome composed of a positive-sense ssRNA molecule virus causing morbidity and mortality in tropical and measuring nearly 12 kb. The replication cycle of the subtropical regions of the planet. As it is a disease with virus involves vertebrates and invertebrates. Cholesterol no vaccine or effective treatment, it is important to study is an important lipid component in animal cells and the antiviral potential of medicinal plants to reduce the is primarily responsible for dynamic and structural patient’s viremia, one of the factors that may cause the maintenance of cell membranes, and forms, along with severe form of the disease. This study examined the sphingomyelin, organized regions of membrane known antiviral potential of three plant extracts from Brazilian as “lipid rafts”. Studies have suggested the involvement Northeast: Caesalpinia echinata, Annona muricata and of cholesterol and lipid rafts at different moments Spondias mombin, against dengue-2 virus (DENV-2) in of the replication cycle of several enveloped viruses. C6/36 and Vero cell cultures. The stock solutions were However, it is interesting to note that arboviruses have 68 mg/mL for C. echinata, 100 mg/mL for A. muricata an alternate cycle that involves infection of mammals and 100 mg/mL for S. mombin. In the cytotoxicity assay,

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

93 Basic Virology: BV different concentrations of each extract were prepared DENV-4). DENV infection can be asymptomatic or a from stock solutions and tested in Vero and C6/36 cells to self-limited, acute febrile disease ranging in severity. It determine the ideal concentration to experiments. Those is estimate that there are 390 million dengue infections cells were seeded in 96-well plates and after incubation each year, of which 96 million manifest apparently. In periods of 1, 6, 12, 24, 48, 72 and 96 hours of contact 2013 were reported 4819 cases in the State of Alagoas, with the extracts, cell viability was evaluated by MTT technique and the method of exclusion of Trypan blue against the virus exists, the sole measure of control is 0.4%. Therefore, the concentration which was taken to limitingBrazil. At the the Aedes present mosquito time, vectors. no specific Therefore, intervention there is a be used in the assay of antiviral activity was 0.68 mg/mL, great need to develop new compounds for the treatment 1 mg/ml and 1mg/mL for C. echinata, A. muricata and of patients infected with DENV. Propolis is a resinous S. mombin, respectively. In the assay of antiviral activity mixture collected by honey bees from plant sources. The monolayers of Vero and C6/36 cells seeded onto 24-well composition of the propolis depends on the season, the plates were infected with DENV-2 at a multiplicity of vegetation, and the area of collection. The red propolis infection (MOI) of 1. At 1h post-infection the inoculum is founded in Brazil northeast and presents a diverse was removed. Vero and C6/36 cells were incubated in biological activities such as antimicrobial, antioxidant and antitumoral. The aim of this work was to investigate antiviral activity of the red propolis etanolic extract experiments:the presence of(i) defined1 h after concentrations infection, and of theat 24-hour extracts against dengue virus type-2 (DENV-2). Concentrations intervalsas described for 7above days; at (ii) defined 1 hour time after intervals, infection in and different at 12- of red propolis, ranging from 200 to 1,56 µg/mL, were hour intervals for 7 days. Infected cell supernatants were evaluated for cytotoxicity by MTT assay in VERO E6 cells. collected at 24, 48, 72, 96, 120, 144, and 168 hours post- The effect of red propolis over DENV-2 (NGC strain) was evaluated by replication inhibition, which was measured of DENV-2 RNA copies by Real-Time Quantitative Reverse by MTT assay using two different methodological Transcriptase-Polymeraseinfection, cleared by centrifugation Chain forReaction the quantification (qRT-PCR) strategies: pre-treatment and pos-treatment. The and plaque assays (PFU). Our results demonstrated that results were expressed as 50% cytotoxicity (CC50) and 50% effective (EC50) concentrations, respectively, in Vero cell cultures at 24-hour and 12-hour intervals in order to calculate the selectivity indices (SI=CC50/ whenA. muricata compared induced to auntreated significant cells, reduction but not in viralin C6/36 yield EC50). The etanolic extract of red propolis showed cells; contrary to the other two extracts, which did not a CC50=164,22 µg/mL. In the pre-treatment 10% of show a reduction in viral yield. Based on these data, we viral replication was inhibited by 25 µg/mL of red conclude that A. muricata has an inhibitory effect against propolis. In the post-treatment, the concentration that DENV-2 virus in Vero cells culture. inhibit 50% of viral replication (EC50) was 38,67 µg/ mL, and the selectivity index (SI) was 4,24. The results BV460 - EVALUATION OF POTENTIAL ANTIVIRAL showed that red propolis have a promising antiviral ACTIVITY OF RED PROPOLIS AGAINST DENGUE activity in the post-treatment. More studies should be VIRUS carried out to investigate their mechanism of action and the action against others serotipes of dengue vírus. Veras, R.M. de A.; Machado, D.; Rocha, M.A.L. Goes, FINANCIAL SUPPORT: The authors are grateful to the T.; Sales, C. de B.P.M.; Queiroz, A.; Moura, F. de B.P.; CAPES (Fellowship VDMM), FAPEAL (PPSUS: 60030 Moreira, M.S.A.; Borges, A.A.; Muller, V.D.M. 000740/2013, Pronem 20110722-006-0018-0010), UFAL - Universidade Federal de Alagoas, Av. Lourival MCT, FINEP, CNPQ (479822/2013-1 e 404344/2012-7), Melo Mota, Ciudad Universitaria - AL, 57072-900 INCT-INOFAR/CNPq (573.564/2008-6),Ministerio da Saude/CNPQ/SESAU AlCAPES Dengue fever, the most important arthropod-borne viral disease, is caused by dengue viruses belonging to the into four antigenically related serotypes (DENV-1 to Flavivirus genus, Flaviviridae family, and are classified September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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BV463 - EVALUATION OF ANTIVIRAL ACTIVITY OF Further experiments are been carried out to completely COPAIBA OIL AGAINST DENGUE VIRUS TYPE 2 evaluate the antiviral activity and to investigate which Melo, D.M.; Veras, R.M. de A.; Rocha, M.A.L.; Goes, T.; compounds are involved in the detected anti dengue Muller, V.D.M.; Borges, A.A. activity. FINANCIAL SUPPORT: CNPq/ FAPEAL/ CAPES UFAL - Universidade Federal de Alagoas, Av. Lourival BV464 - VPG AMINO ACID SEQUENCE CONSERVATION Melo Mota, Ciudad Universitaria - AL, 57072-900 AMONG DIFFERENT HUMAN RHINOVIRUS SPECIES Dengue viruses (DENVs) cause the most common Watanabe, A.S.A.; Moreira, L.; de Oliveira, P.S.L.; arthropod-borne viral disease. Repeated reemergence Granato, C.; Bellei, N. of dengue in sudden explosive epidemics often cause UNIFESP - Universidade Federal de São Paulo, R. Sena public alarm and seriously stress healthcare systems. It Madureira, 1500 - Vila Mariana, São Paulo - SP, 04021-001 is estimate that there are 390 million dengue infections each year, of which 96 million manifests apparently. Human rhinovirus (HRV) infections are the most The dengue severity is divided into dengue without frequent cause of common cold and accounting for warning signs, dengue with warning signs, and severe approximately 50% of all upper respiratory tract dengue. Dengue virus belongs to the Flaviviridae family, in 3 distinct species: HRV-A, B and C, with A and C infections described. Currently the HRVs are classified related serotypes (DENV-1 to DENV-4). Currently there species clinically associated with more severe disease. genus Flavivirus and is classified into four antigenically Several studies have demonstrated that host factors, substantial vector control efforts have not stopped as age, immune status and severe clinical outcome itsare rapid no licensed emergence vaccines and global or specific spread. therapeutics, Copaiba oil, and is extracted from the trunks of Copaifera trees species. understand of HRV molecular epidemiology raised new concernsare not associated about the mechanisms with HRV specific involved species. in pathogenesis A better analgesic, antimicrobial, antifungal, antileishmanial, of the different species. In this scenario the importance antiproliferative,It has been reported antimutagenic, for anti-inflammatory and others. However, activity, of viral physiology is evident. The HRV replication antiviral properties have not been studied yet. The aim mechanism is initiated through a small genome binding of this work was to investigate the antiviral activity of protein called VPg (Viral Protein of the genome). HRV copaiba oil against dengue virus type-2 (DENV-2). To replication is initiated by VPg uridylylation. The VPg evaluate the cytotoxicity by MTT assay in VERO E6 cells, serves as a primer for initiation of intermediate RNA the monolayer was treated with concentrations ranging negative strand synthesis, which will subsequently be from 1,000 to 1.56 µg/mL. The effect of copaiba oil transformed into a genomic positive RNA strand. Several over DENV-2 (NGC strain) was evaluated by replication studies have demonstrated that VPg plays a critical role inhibition, which was measured by MTT assay using in HRV replication. Structural evaluation of VPg, and its two different methodological strategies: pre-treatment role in the different species replication could clarify the and post-treatment. The results were expressed as 50% infections dynamics and clinical diversity presented by cytotoxicity (CC50) and 50% effective concentrations amino acid sequence conservation among the different (EC50), in order to calculate the selectivity indices patients. The first aim of the pilot study was assessed the (SI=CC50/EC50). The copaiba oil did not show CC50 to HRV species, and the second objective was to perform VERO E6 cell monolayers up to a concentration of 1,000 the prediction of the VPg structure. Two hundred and µg/mL. When the monolayer was treated with copaiba fourteen VPg sequences were obtained from GeneBank, oil after the viral infection (post-treatment), there was a with the following distribution: HRV A – 131 sequences, high inhibition of viral replication, and the EC50 obtained HRV B – 49 sequences and HRV C – 34 sequences. The was 19.4, and the selectivity index was >51.5. When the analysis of sequences conservation was performed cells monolayer was treated with 25 µg/mL before viral using the Jalview 2.8.1 software. Molecular modeling of infection (pre-treatment), it was possible to inhibit 43% the HRV VPg structure was performed using the Yasara of viral infection. These results showed that the copaíba software. The VPg peptide was composed of 20 amino oil have a promising antiviral effect against DENV-2. acid sequence, among different HRV species sequence September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

95 Basic Virology: BV conservation ( 80%) was: HRV A – 85%, HRV B – 65% also sustains the need for laboratory tests cheap, simple and HRV C – 55%. We could successfully predict the and quick to be adopted routinely in women. Such tests ⎕ structure of two species, A and B, and the molecule aim to reduce STDs and increase the correct and early structure was very similar between both species. Unlike diagnosis of infection thus avoiding complications in the expected the amino acid sequence was high conserved course of illness and treatment. FINANCIAL SUPPORT: only in HRV A species. In HRV C species the sequence LACEN/AL - Laboratório Central de Saúde Pública de conservation was relatively low. The differences in amino Alagoas UNCISAL - Universidade de ciências da saúde de alagoas PROBIC – UNCISAL - Programa Institucional de species replication rates. Further studies can elucidate theacid VPg sequences role in HRV may pathogenesis. reflect a possible difference in the BolsasBV488 de - ANTI-HIV-1 Iniciação Científica ACTIVITY OF NEW DITERPENES BV483 - RETROSPECTIVE STUDY OF SEXUALLY DERIVED FROM THE BROWN SEAWEED DICTYOTA TRANSMITTED DISEASES (STDS) IN PREGNANT AND PFAFFII NON-PREGNANT WOMEN IN PEOPLE ATTENDED BY Oliveira, I.B.1; Pardo-Vargas, A.3; Cirne-Santos, C.C.2; LACEN MACEIÓ-AL Castellanos, L.3; Teixeira, V.L.1; Paixão, I.C.N.P.1 Antão, K.L.1; de Sá, J.P.O.1; Pinheiro, T.M.L.2; de Lima, 1. UFF - Universidade Federal Fluminense, 2 1 1 M.C. ; Pinheiro, M. da S. ; Maria, F.H. de O.S. Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 1. UNCISAL - Universidade Estadual de 2. FIOCRUZ - Fundação Oswaldo Cruz, Av. Ciências da Saúde de Alagoas, Rua Doutor Jorge de Lima, Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 113, Trapiche da Barra, Maceió - AL, 57010-300 3. UNAL - Universidade Nacional da Colômbia, 2. LACEN Avenida Carrera 30 # 45, Bogotá, Cundinamarca 111321, Colômbia Our basic strategy for controlling and prevention of Sexually Transmitted Diseases (STDs) will be through constant information and educational activities that Human immunodeficiency virus type 1 (HIV-1) is the focus on: risk perception, changes in sexual behavior (AIDS), a clinical syndrome which causes profound etiologic agent of Acquired Immunodeficiency Syndrome and promotion and adoption of preventive measures to inside a cell, integrates its genetic material into the DNA immunosuppression. HIV infects CD4+ cells and, once pregnant and non-pregnant women with characteristics suchemphasize as: early condoms sexual use. behavior, Our aim different was to drawsocio-economic a profile in number of copies of infective viral particles, making the of the host. Thus, it gets efficient replication, with a large and cultural aspects and age groups, who spontaneously organism susceptible to the acquisition of opportunistic sought treatment at Central Laboratory (Lacen/AL) for infections, leading to the development of AIDS. Since the routine examinations as pre-natal vaginal discharge introduction of the highly active anti-retroviral therapy and pap smear, followed by referral to the preventive (HAART), the life expectancy of HIV-infected individuals examination or treatment and depending on the case The has increased, even in cases in which the virus remains total sample of cases was 5326 women, from which 3885 active. However, the discovery and development of non-pregnant and 1441 pregnant. Ages ranging from new drugs candidates is required, as current drugs do 11 to 61 and the data were collected from the medical not completely eradicate HIV from infected tissues and records in LACEN-AL from January 2004 to December the long-term use of these drugs is restricted by the 2005. The correlation between the data obtained from emergence of drug-resistant viruses, metabolic disorders microorganisms associated with STD prevalence in and toxicity. Marine natural products have demonstrated pregnant and non-pregnant women were: Candida sp, pharmacological activities against a great number of Gardnerella vaginalis, Monilia, Staphylococcus aureus, pathogens, including HIV-1. In the latest years, natural Streptococcus agalactiae, Condylomata acuminata compounds capable of inhibiting somehow HIV-1 have (HPV). The results show that the rate of pregnant women been target of study. The aim of this study is to show the with STDs is very high and it supports the importance of potential anti-HIV-1 activity of 3 dolabellane diterpenes having a thorough prenatal checkup regarding STDs. It

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersderived - Basic Virology: from the BV brown seaweed Dictyota pfaffii. Were XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

96 Basic Virology: BV used MT-2 cells, which were maintained at 37 °C, 5% of site-directed mutagenesis in the TSWV NP sequence, CO2 and used for cytotoxicity and antiviral assays. For the antiviral assay were used the viral isolated from HIV- multimerization, which supports a “head-to-tail” 1 IIIB. Cytotoxicity and antiviral assays was performed by interactionidentified regions model. involved However, in RNAthe tospovirusbinding and RNPs protein is MTT method. Results of cytotoxicity assay showed that uncharacterized at the molecular level and the lack the compounds were not cytotoxic and MT-2 viability of structural information about NPs prevents a more detailed description of those interactions. Recently, the NPs tridimensional structure from viruses of the 3,4.was Thehigher compound than 90% also for were concentrations less toxic than up the to positive250μM, related viral families Arenaviridae, resulting in a CC50 between 1345μM ± 2 and 1456μM ± and Bunyaviridae have been elucidated providing structural basis for understanding the tospovirus control, neviparine (325μM ± 1,4). The antiviral activity NP behavior. Whereas no structural models are replicationwas tested atin concentrations 83%, 69% and between 52% of 6,25μMcases, resulting and 25μM. in available for Tospoviruses NP, several studies made At 6,25μM, the compounds 1, 2 and 4 inhibited the viral for Orthobunyaviruses NP could be used as reference 0,7, respectively. Thus, these results suggest that such because of their kinship. NP proteins have distinct size diterpenesEC50 values could of 2,9μM be considered ± 0,2, 4,1μM potential ± 0,4 newand agents6,16μM for ± and quite distinct folds. Nevertheless there are several HIV-1 therapy. Therefore, further studies on the precise similarities in the architectural principles by which both mechanism of activity and on the in vivo antiviral activity proteins form NP-NP multimers and NP–RNA complexes. are needed. FINANCIAL SUPPORT: FAPERJ, CAPES, Using Homology Modeling technique we determined CNPQ, PROPPI-UFF that GRSV NP structure possesses two interacting N- and C-arms and a globular positively charged groove domain BV494 - STRUCTURAL INSIGHTS INTO TOSPOVIRUS into which RNA is deeply encompassed. Tospovirus NUCLEOPROTEIN NP proteins share a high level of amino acid identity, Paula, D.F.; Lima, R.N.; Lucas, F.; Resende, R.O. indicating that GRSV NP structure could be used as UnB - Universidade de Brasília, Campus Universitário a model for all members of the genus. The results Darcy Ribeiro, Brasília - DF, 70910-900 tridimensional structure for Tospovirus NP protein. reported here provide for the first time new insights into between viral and host proteins that alter the routine of BV495 - EXPRESSION PROFILE OF FOUR CELLULAR Efficient viral infection requires appropriate interactions the cell to promote the virus infection. Viral proteins GENES DURING 36 HOURS OF HUMAN ADENOVIRUS can also interact to form oligomers, such as some viral TYPE 5 INFECTION proteins of tospoviruses. The genus Tospovirus belongs Giehl, I.C.; Rigotto, C.; Staggemeier, R.; Jesus, L.F. de; to the family Bunyaviridae. All members of this genus Henzel, A.; Spilki, F.R. have three segmented single stranded RNA genome. The L segment encodes the viral RNA-dependent RNA FEEVALE - Universidade Feelave, Av. Dr. Maurício polymerase (RdRp). The M segment encodes two Cardoso, 510 | Bairro Hamburgo Velho, Novo Hamburgo - RS, glycoproteins (Gn/Gc) and the nonstructural protein 93510-250 NSm responsible for the viral movement. The S segment During an infection, human adenoviruses, such as encodes the nonstructural RNA silencing suppressor serotype 5 (HAdV-5), encode several proteins that can protein (NSs) and the nucleocapsid protein (NP). perturb cellular mechanisms that regulate cell cycle Multiple copies of the NP form oligomers that interact progression and apoptosis, as well as mRNA production with the viral genomic and antigenomic RNAs to build and translation. It is known that a small number of ribonucleoprotein complexes (RNPs) that are functional viral gene products can induce massive reprogramming templates for RNA replication and transcription carried of cellular gene expression. In order to understand out by the RdRp. Moreover, the NP interacts with what might be the impacts of adenoviruses on cells of NSm for cell-to-cell transport of the RNPs. Previously, different organisms, studies evaluating the effect of studies based on yeast two-hybrid system (2YHS) and infectious viral particles on expression of cellular genes

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

97 Basic Virology: BV are frequent. Therefore, the aim of this study was to This protein plays an essential role in viral infectivity evaluate the expression of four cellular genes involved and progression to AIDS. It has been reported that Nef in immune response, cell proliferation and cell cycle interacts with different cellular partners to perform progression, in several time points during the infectious several functions in the viral replicative cycle, but the cycle of HAdV-5. HAdV-5 was inoculated onto human function related to increased viral infectivity in primary lung carcinoma cell line (A549), at 1 m.o.i (multiplicity lymphocytes and macrophages has not been described. of infection). Negative control, mock inoculated cells, Nef may mediate downregulation of surface expression was performed in order to normalize the results. At of CD4 membrane molecules and may prevent apoptosis different time intervals upon infection (1h, 2h, 4h, 6h, in T cells infected with HIV-1. It has been reported that 12h, 18h, 24h, 30h and 36h), total RNA was extracted the cellular protein Alix/AIP1 plays a central role in by a commercial kit (PureLink®), followed by cDNA directing the ESCRT machinery and this is essential for synthesis (SuperScript® III). Quantitative real time the budding of certain enveloped viruses such as HIV-1. PCR (qPCR) was performed using primers that targeted We are investigating the role of the interaction between the following genes: TGFBR1, CCNDBP1, DHFR and Nef and cellular protein Alix/AIP1. Nef interaction with Smurf2. 18S gene was used as the endogenous control. Alix/AIP1 have been previously mapped to the amino The comparative threshold cycle (Ct) method was used acid residues YPLTF present at the positions 135-139 to quantify changes in gene expression in infected on the C-terminal of the Nef protein from the HIV NL4- group when compared to negative control. Values were 3 isolate. The objective of this study is to elucidate expressed as fold differences (FD) between test and the interaction between these two proteins has an control treatments. In order to quantify the HAdV-5 present in the infected group, qPCR was performed using siRNA knockdown assays were performed in Hek293T primers designed to amplify the hexon protein gene of cellinfluence cultures. on The the increaseknockdown in viralwas carried infectivity. out from For this, the HAdV, namely VTB2. Our preliminary results showed transfection of siRNA against Alix/AIP1. The expression two major peaks with increased expression of the four genes, one appearing early in infection (4h) – FD values antibodies against Alix showing a 96% of knockdown between 1.80 and 3.15 - and the other standing out at afterof Alix 24h was when monitored it was usedby Western 50mM blottingsiRNA. So with we specificstarted 30h – FD values between 2.96 and 4.34. In other periods, it was observed an increased, decreased or unchanged after 24h of knockdown. Interestingly, infectivity assays gene expression, with no pattern followed by the four oftesting viruses transfection produced ofin plasmidsthese conditions NL 4.3 and showed NL 4.3 an ΔNef Alix genes. VTB2 gene was detected only in infected group, dependence to increase viral infectivity using NL 4.3 and in all time points from 1h to 30h, with values ranging this increase was not observed when was used the NL from 2.01x104 to 2.74x109 genome copies/5µL, in an ascending pattern over time. These results represent of NL 4.3 in knockdown cells of 60% was observed as the erratic behavior of cell gene expression during 4.3 ΔNef. In these experiments an infectivity reduction viral infection, alternating between overexpression and these results indicate that the Alix/AIP1 protein has a normoexpression of the studied molecules. FINANCIAL rolewell inas increasedthe reduction infectivity observed of HIV-1in NL in4.3 the ΔNef presence virus. So, of SUPPORT: FEEVALE, CAPES, FAPERGS, CNPq. Nef protein.

BV496 - THE ROLE OF INTERACTION BETWEEN VIRAL NEF AND ALIX/AIP1 IN INFECTIVITY OF HIV-1 da Silva, G.P.D.; Costa, L.J.; Mendonça, L.M. UFRJ - Universidade Federal do Rio de Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de Janeiro - RJ, 21941-901 Nef is an accessory protein expressed early during the replication cycle of the primate lentiviruses HIV and SIV.

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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BV502 - EVALUATION OF ANTIVIRAL ACTIVITY OF BV508 - DISCOVERY OF PERILLYL ALCOHOL EXTRACTS OF BACTERIA ISOLATED FROM SOILS AND PERILLIC ACID ANTI-HSV-1 ACTIVITY AND MOUNDS AGAINST HUMAN HERPES VIRUS TYPE 1, MECHANISM OF ACTION KOS STRAIN Ribeiro, C.P.1; de Amorim, L.M. da F.1; Santos, T.F.Q.1; Padilla, M.A.1; Kohn, L.K.1; de Moraes, A.P.1; Morandi, da Silva, V.A.G.G.1; Bloom, D.C.2; Paixão, I.C.N.de P.1 B.C.2; Garboggini, F.F.2; Martini, M.C.1; Arns, C.W.1 1. UFF - Universidade Federal Fluminense, 1. UNICAMP - Universidade Estadual de Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 Campinas, Cidade Universitária Zeferino Vaz - Barão 2. UFL - Universidade da Flórida, Gainesville, Geraldo, Campinas - SP, 13083-970 FL 32611, Estados Unidos 2. CPQBA Infection by herpes virus simplex type-1 (HSV-1) causes Human herpes virus type 1 (HHV-1) belongs to the several diseases, ranging from cutaneous, oral and family and represents serious threats for genital infections to fatal encephalitis. Acyclovir is the public health and increasing in the severity of ilnesses reference compound used in antiviral therapy. With the in immunocompromised patients. In addition, there are emergence of strains of HSV resistant to the current mutant strains resistant to available drugs. Since the antiviral drugs, new antiviral agents, especially those with different modes of action, are urgently needed. of HHV-1, the use of natural products as antivirals can In this study, we examined the mechanism of action of bedifficulties an alternative. associated The aim with of preventionthis study was and to treatment evaluate perillyl alcohol (POH) and perillic acid (PA) in inhibiting in vitro 93 extracts of bacteria isolated from soils in vitro replication of HSV-1 KOS strain, on Vero cells, mounds for antiviral activity against HHV-1. Isolated by plaque assay. The cytotoxicity of the compounds was microorganisms were incubated in culture medium for measured by MTT assay. Our results demonstrate that four weeks at 30°C and these inoculums were extracted these compounds have high anti-HSV-1 activities in a by liquid-liquid extraction with ethyl acetate. Antiviral concentration range that was not cytotoxic to Vero cells. activity was measured by virus titration technique and Interestingly, despite the high anti-HSV-1 activity in Vero the percentage of viral inhibition (PI) was calculated for cells, the compounds helped the virus to reactivate from virus treated with samples. The extracts were considered Trigemal ganglia neurons culture. DNA fragmentation active when PI was higher than 97%. From all extracts indicating that probably they are not causing apoptosis equivalent to 5% of them and six showed toxicity at the inassay Vero confirmed cells (infected the low or cytotoxicity not). However, of these the compounds,compounds concentrationtested, five of tested them were(50µg/mL). considered The extracts active, whichCDPA10, is did not show virucidal activity, and therefore do not CDPA22, CDPA26 and CDPI92 presented 99% of PI. inactivate the viral particle. In addition, PCR analyses These extracts were selected for an evaluation to identify showed that these substances are not inhibiting viral the selectivity index (SI) and were considered promising genome replication, suggesting that the high anti-HSV-1 the extracts that showed IS value greater than 3.0. Of all, activity is not due to inhibition of viral replication. Our CDPA10, CDPA22 and CDPA26 presented the best values western blot results suggest that any of these compounds of SI with 17.10, 11.37 and 3.84 respectively. Finally, are affecting the total HSV-1 protein production. Since these extracts were evaluated for their mechanism of action through the addition of the extract and/or viral and protein production, we performed a virion egress dilution at cell monolayer at different times. As a result, assay,we did which not find revealed any inhibition that POH in viral is probably genome preventingreplication the virus of getting out of the cells. Work is currently inactivators with 99% of activity; and CDPA26 was active underway to determine if these compounds block capsid asthe an CDPA10 inhibitor and of replication CDPA22 were with classified99% of action. as viruses These assembly or virion morphogenesis. Perillyl alcohol and initial results of this work were promising; however additional studies are necessary to evaluate the chemical and in vivo assays toward development of new strategies compounds responsible for the activity. FINANCIAL inperillic anti-HSV-1 acid have therapy. a promising FINANCIAL profile SUPPORT:for further CAPES/in vitro

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV SUPPORT: CNPq, FAEPEX and FAPESP FAPERJ/CNPQ/PROEX/ FOPESQ-UFF-PROPPI XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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BV512 - PATTERN OF BACTERIAL MICROBIOME IN the total reads in the outcome cures). Interestingly, THE RESPIRATORY TRACT OF INFLUENZA PATIENTS Correa, R.A.1; Borges, L.G. dos A.2; Giongo, A.3; Correa, four times more in the outcome cures than in the unclassified sequences at phylum level were observed R.A.1; Gregianini, T.S.4; Franco, A.C.1; Roehe, P.M.1; outcome deaths. A higher number of Porphyromonas Campos, F.S.1; Veiga, A.B.G.2 and Staphylococcus reads were observed in the outcome deaths. On the other hand, a higher number 1. UFRGS - Universidade Federal do Rio Grande do Sul, Avenida Paulo Gama, 110 - Farropilhas, Porto the outcome cures. Some genera such as Haemophilus, Alegre - RS, 90040-060 Prevotellaof Pseudomonas and Streptococcus and Rhodococcus presented were high identified number in 2. UFCSPA - Universidade Federal de Ciências of reads in at least half of the samples analyzed. We da Saúde de Porto Alegre, R. Sarmento Leite, 245 - Centro Histórico, Porto Alegre - RS, 90050-170 have observed that different bacteria genera could 3. PUCRS - Pontifícia Universidade Católica be presented in the virus infection depending on the do Rio Grande do Sul, Av. Ipiranga, 6681 - Partenon, Porto outcome. More analyses need to be performed in order Alegre - RS, 90619-900 to identify patterns in the community diversity for both 4 IPB/LACEM - Instituto de Pesquisas Biológicas/ death and cure outcomes aiming to correlate those Laboratório Central de Saúde Pública do Estado, Av. Ipiranga, 5.400, Jardim Botânico, Porto Alegre - RS, 90610-000 bacteria to the influenza infection and host immunity. clinically more aggressive, mainly due to an imbalance inPandemic host immune strains activity of Influenza against the A virusvirus, appearwhile in topost- be pandemic periods the mortality is lower and probably intervention. Host immunological response and factors there are many flu patients cured without medical outcomes among infected patients in a population. associated with virus strains may define different A virus can be a result of an associative network betweenStudies havevirus-bacteria shown that toward mortality the host. rate Thus, by influenza the aim of the present study was to show a pattern of bacterial diversity in the microbiome of upper respiratory tract of

Ainfluenza H1N1 2009 patients virus with in differentthe post infectionpandemic outcomes. period were Ten selectednasopharingeal in a double samples blind, of patientsrandomized infected study by approvedinfluenza by the Research Ethical Committee of UFCSPA. Samples were divided in two groups (cure and death) based on the patient prognostic. In order to identify the microbiome, DNA was extracted and amplicons from the V4 hyper-variable region 16S rRNA gene were generated and sequenced on an Ion Torrent PGM, using a 314 chip. A total of 703,585 usable reads were generated and analyzed using Prinseq and MG-RAST. The most prevalent phyla in the samples were Bacteroidetes (40% of the total reads in the outcome deaths and 24.5% of the total reads in the outcome cures) and Firmicutes (36.7% of the total reads in the outcome deaths and 29.5% of

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV ENVIRONMENTAL VIROLOGY: EV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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EV22 - DETECTION AND MOLECULAR EV40 - EVALUATION OF VIROLOGICAL PARAMETERS CHARACTERIZATION OF AICHIVIRUS FROM DURING MIMIVIRUS PRODUCTION WASTEWATER DIRECTLY DISCHARGED INTO Silva, L.C.F.1; Boratto, P.V.M.1; Dornas, F.P.1; Campos, URUGUAY RIVER, URUGUAY R.K.1; Kroon, E.G.1; La Scola, B.2; Abrahão, J.S.1 Tort, L.F.L.1; Burutarán, L.1; Victoria, M.1; Lizasoain, 1. UFMG - Universidade Federal de Minas 1 1 2 2 A. ; García, M. ; Miagostovich, M. ; Leite, J.P.G. ; Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, 1 Colina, R. Belo Horizonte - MG, 31270-901 1. Laboratory of Molecular Virology, Sede Salto, 2. Aix-Marseille Université, 58 Boulevard Cenur Del Noroeste, Universidad de La República, Uruguay Charles Livon, 13284 Marseille, França 2. Laboratory of Comparative and Acanthamoeba polyphaga mimivirus (APMV) was Environmental Virology, Oswaldo Cruz Institute (FIOCRZ), Ministry Of Health an English hospital during a pneumonia outbreak Aichivirus (AiV) belongs to the genera Kobuvirus, inside andfirst belongs isolated to few Mimiviridae years ago fromfamily. a APMV water prompted cooler, in Picornaviridae family, and there are three AiV genotypes capable of infecting human: A, B and C. AiV is a human of various genes never-before-seen in viruses and enteric virus with a global prevalence between 0,9 to theirthe creation roles in of virus-host an open fieldinteractions. of study In about recent function years, 4,1% in gastroenteritis sporadic cases, and, these virus several giant viruses have been isolated from different water. The aim of this study was to assess the viral community has experienced a remarkable advancement contaminationhave also been of detected AiV in insewage environmental directly discharged superficial inenvironments the comprehension and specimens. of the Althoughmimivirus the replication scientific into Uruguay River, and to characterize AiV genotypes cycle in the last years, few studies have been devoted circulating in Uruguay. For this purpose, sewage to the investigation of the methodological features and samples (n=96) were collected biweekly from March conditions for mimivirus production. This work aimed 2011 to February 2012 in four Uruguayan cities: Bella investigate the conditions for cultivation of mimiviruses Unión, Salto, Paysandú and Fray Bentos. Each sample isolates to obtain informations about the production was concentrated by ultracentrifugation method. A of infectious particles, total viral particles and viral Nested RT-PCR directed to 3CD region of AiV genome, DNA. Our results suggest that low viral doses are more was performed. Positive samples were sequenced and phylogenetic analysis were performed in order to yielding up to 5000 TCID50 for each inoculated TCID50. determine the genotypes present in the samples. A wide Besidesefficient methodological for the production information, of infectious these data particles, also dissemination of AiV was observed in the sewage samples analyzed with a 57.3% of positivity, being detected with infectious particles (in TCID50) that are produced during a high prevalence in the four sampled cities. Beside a mimivirusreveal, for cultivation the first time, in laboratory the ratio conditions. between Then, total andcan clear seasonality was not observed in the present study, a higher positivity was observed in the coolest seasons FINANCIAL SUPPORT: CAPES, CNPQ, FAPEMIG; MAPA. (autumn and winter). The phylogenetic analysis showed help prompt mimivirological studies in different fields. that all the positive samples belong to genotype B. This and showed the potential risk of infection with this virus ofstudy the representlocal populations the first detectionthrough recreational of AiV in our activities country, or consumption of water from the Uruguay River, in which untreated sewage is directly discharged.

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Environmental Virology: EV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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EV41 - ACANTHAMOEBA POLYPHAGA MIMIVIRUS INHIBITS TRANSCRIPTION OF AMOEBAL SUBTILISIN- eukaryotic cell. LIKE SERINE PROTEINASE MRNA AND CIRCUNVENTS examples of fight for supremacy involving a virus and a CELL ENCYSTMENT EV51 - COMPARISON OF DIFFERENT METHODS FOR ISOLATION OF GIANT VIRUSES Boratto, P.V.M.1; Almeida, G.M.F.1; Botelho, L.1; Lima, A.1; Costa, A.O.1; Santos, D.A.1; La Scola, B.2; Kroon, Arantes, T.S.; Andrade, K.R.; Silva, L.C.F.; Dornas, E.G.1; Abrahão, J.S.1 F.P.; Silva, L.K. dos S.; Rodrigues, R.A.L.; Kroon, E.G.; Abrahão, J.S. 1. UFMG - Universidade Federal de Minas Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, UFMG - Universidade Federal de Minas Gerais, Belo Horizonte - MG, 31270-901 Avenida Presidente Antônio Carlos, 6627 - Pampulha, Belo 2. Aix-Marseille Université, 58 Boulevard Horizonte - MG, 31270-901 Charles Livon, 13284 Marseille, França The giant viruses are an important and unique group Acanthamoeba is a genus of free-living amoebas distributed worldwide known as agents of public the year 2003. The Acanthameba polyphaga mimivirus health concern for causing disease in humans. On (AMPV),of viruses so whose named first was representative isolated from waswater discovered collected in natural environments, they can act as hosts for many a cooling tower for air conditioning in the British city microorganisms, including members of the family Mimiviridae, one of the largest and most complex group community because of its unique size, bigger than some of viruses ever described. Many studies have investigated bacterialof Bradford. and The also AMPV by tocaused provide interest genes in neverthe scientific before the interactions between these protozoa and some of its observed in other viruses. After the discovery of the harbored microorganisms (mostly pathogenic bacteria) AMPV, many other giant viruses have been isolated from but few have explored the mechanisms involving their different samples and localizations through various interaction with giant viruses. In the present study, we protocols of isolation. This study aims to compare two explored the behavior presented by Acanthamoeba spp. mimiviruses isolation techniques: the agar PAS isolation and Mimivirus (APMV) in response to different conditions technique and the direct inoculation of samples in of incubation and interaction. The investigated scenarios Acanthamoeba castellanii monolayers growth in PYG were: (1) Acanthamoeba trophozoites infection, (2) liquid medium. For these analyses, were initially used Acanthamoeba mature cysts infection, (3) trophozoites 10 oyster samples previously enriched in middle rice infection shortly after being subcultured in encystment during 30 days. After the enrichment, the samples were medium and (4) trophozoites infection shortly before being subcultured in encystment medium. The isolation in cultivation of A. castellanii was performed regulation of some genes involved with encystation in usingsubjected 100µl the of tests sample, in order that to werecheck subjected the efficiency. to three The Acanthamoeba in response to APMV infection were also passages lasting three days each for the visualization investigated. The results obtained suggest that amoeba of cytopathic effect. For the agar PAS isolation test the encystment is a crucial phenomenon for avoiding viral infection success, and therefore it is the target of of A. castellanii. After three days, 10 µl of each sample an arm wrestling between the virus and amoeba. We wereenriched collected sample and were inoculated first inoculated on an in agar a fresh PAS culture plate demonstrate that once amoeba encystment is triggered, containing a monolayer of A. castellanii. The plate was observed during three days for viewing lysis plaques APMV and, remarkably, APMV is able to interfere with the on agar. After theconductingthe test it was possible to expressiontrophozoites of the become serine significantly proteinase mRNA less permissive related to the to observe that the technique of isolation in culture of A. amoebal encystment. This work suggests that amoebas castellanii presented a positivity of 40% of de isolation can use encystment as a form to protect themselves after the third pass. Already the agar PAS teste showed an from viral infections, while APMV can interfere with isolation rate of 50%. Through the results obtained was the encystment process by regulating an amoebal gene. observed that the agar PAS technique was more effective These results may represent one of the most ancient than isolation technique on cultivation of A. castellanii. September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Environmental Virology: EV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

103 Environmental Virology: EV

In addition, a sample which showed no cytopathic for Real-Time PCR assays, targeting the conserved effect after three passage in culture of A. castellanii was helicase gene of mimivirus, and the 18s ribosomal gene positive in agar PAS, demonstrating greater sensitivity of Acanthamoeba. Phylogenetic analyses were also of the technique. The agar PAS technique allowed the performed to investigate the relationship of these new isolation in a short time using a smaller sample volume. samples with Mimiviridae family. Our results showed a The agar PAS isolation system has proved to be an higher positivity of mimiviruses in respiratory isolation area (36%) compared to other settings: reception the isolation of giant viruses in less time compared to (15%), stairs (10%), elevators (4%), hallways (8%) and isolationefficient, sensitivein culture and of A.less castellanii fastidious and method can be allowing widely nursery (10,5%). In addition, a correlation was observed used. FINANCIAL SUPPORT: FAPEMIG, CAPES, CNPQ, between the presence of amoebae and mimiviruses: 26 MAPA, PPG-MICROBIOLOGIA UFMG out of 29 positive samples for the helicase gene were also positive for the 18s gene. The phylogenetic analysis EV52 - EVALUATION OF THE DISTRIBUTION showed a close relationship among our samples to other OF MIMIVIRUS IN A HOSPITAL ENVIRONMENT mimiviruses. In conclusion, this study showed that the AND CORRELATION WITH THE PRESENCE OF respiratory isolation area presents a greater positivity ACANTHAMOEBA to mimiviruses, indicating a possible risk factor for the Silva, L.K. dos S.; Andrade, K.R.; Rodrigues, R.A.L.; occurrence of pneumonia. Moreover, the presence of Boratto, P.V.M.; Arantes, T.S.; Silva, L.C.F.; Dornas, F.P.; mimiviruses appears to be directly related to the presence Kroon, E.G.; Clemente, W.T.; Abrahão, J.S. of Acanthamoeba. More studies are needed to clarify this UFMG - Universidade Federal de Minas Gerais, relationship and the role of mimiviruses as pathogens Avenida Presidente Antônio Carlos, 6627 - Pampulha, Belo of the respiratory system. FINANCIAL SUPPORT: CNPQ, Horizonte - MG, 31270-901 CAPES, FAPEMIG, MAPA, PPG-MICROBIOLOGIA UFMG. In 1992, during a pneumonia outbreak, a giant virus EV60 - AUTOMATED COMPOSTING ALLOWS THE was isolated from a cooling tower of a Hospital in DISINFECTION OF DIFFERENT ADENOVIRUS IN Bradford, England and was called Acanthamoeba SWINE MANURE polyphaga mimivirus (APMV). APMV infects amoebae Henzel, A.1; Soliman, M.C.1; Ziber, G.1; Staggemeier, of Acanthamoeba genus and is the prototype of the R.1; Rigotto, C.; Sá, M.2; Pujol, S.2; Aita, C.2; Spilki, F.R.1 Mimiviridae family, which is clustered with the Nucleo- Cytoplasmic Large DNA Viruses (NCLDVs). Pneumonia 1. FEEVALE - Universidade Feelave, Av. Dr. Maurício Cardoso, 510 | Bairro Hamburgo Velho, Novo is a major cause of death related to infection around Hamburgo - RS, 93510-250 the world; however, between 20 to 50% of cases have 2. UFSM - Universidade Federal de Santa unknown etiology. Therefore, identifying new causative Maria, Avenida Roraima, 1000 - Camobi, Santa Maria - RS, agents of pneumonia is a public health concern. 97105-900 Some studies have pointed out the APMV as potential respiratory tract pathogens and probable causative The automated composting is a method for stabilization agents of pneumonia in humans. In order to better of organic wastes. This technique is considered effective understand the role of mimiviruses as etiological agents for elimination of pathogens in manure swine. Among of this disease and its association with the presence the potential pathogenic microorganisms present of amoebae, we evaluate the distribution of these in manure are adenovirus (AdV), members of the viruses in different settings of Hospital das Clínicas/ family, consisting of double-stranded DNA UFMG. Altogether, 153 samples were collected from genome, are often found. However, little is known about reception, stairs, elevators, hallways, nursery and respiratory isolation areas. The samples were collected the efficiency of this technique in removing viruses. using swabs and after were eluted in PBS. After this two automated composting system in elimination of The aim of this work was to evaluate the efficiency of procedure, total DNA was extracted from the samples different AdV species (canine, CAV; avian, AvAdV; bovine, by phenol-chloroform method and used as template BAV; human, HAdV and swine, poAdV) in liquid swine

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Environmental Virology: EV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

104 Environmental Virology: EV manure (LSM). For this, two automated composting act as reservoirs from which contaminant viruses can units were developed. First (treatment 1), consisted in be resuspended and return to the water column by a mix of LSM, wood shavings and phosphoric acid; and the second (treatment 2), only LSM and wood shavings human adenoviruses (HAdV) have been found to be the were used. The frequency of application of the manure mostseveral prevalent natural enteric or artificial virus phenomena.in biosolids. AmongIn the city them, of in the two piles of composting and its revolving occurred Florianópolis, Santa Catarina (SC), Brazil, located in the every seven days and the temperature of the piles was protected area of the Peri Lagoon Municipal Park. The measured daily throughout the process (156 days). Sangradouro River (SR) receives the Peri Lagoon water Throughout this period, samples were collected of raw during the rainy episodes and this water is drained to LSM, of the compost (LSM more shavings) before and the ocean located in the neighborhood. This river also after its revolving. In both treatments was done viral receives wastewater and sewage from residences. This analysis. The samples were diluted with Minimum study aimed to quantify HAdV either in surface waters Essential Medium (MEM) for the extraction of viral or sediment, as well as to evaluate the integrity and DNA and the adenoviral species were characterized infectivity of HAdV in these samples collected along six points of the SR. A sum of 96 samples were collected by a differential step of high resolution melting. From during the summer and winter seasons of 2013. Two thethrough raw LSM the added amplification the two bypiles real-time of composting PCR followed during liters of surface water samples were concentrated by the process were detected in all samples BAV (28/28). negatively charged membranes and 20g of sediment Other viral species such as HAdV and poAdV in 7.14% (01/14) in treatment 1 and CAV and poAdV 7.14% glycine buffer followed by polyethylene glycol (PEG) (1/14) in treatment 2. During the process of composting precipitation.samples were The concentrated nucleic acids andwere clarifiedextracted usingfrom (with the addition of manure every 7 days), adenovirus samples before and after DNAse treatment in order to loads remained constant in both treatments before and evaluate genome copies of HAdV (by qPCR) derived from after its revolving, showing that the time between the intact or undamaged viruses respectively. The samples positive for genome copies were used to infect A549 cell removal. On maturity phase of the compost, when it monolayers for plaque assay (PFU) for viral infectivity wasapplications no LSM was, of the there manure was wasno detection not sufficient of adenovirus for viral determination. For surface waters, the incidence of HAdV was 17/24 (70.8%) ranging from 105 to 108 however, was continued adenovirus being detected gc/L contained 70.8% undamaged HAdV particles (UP), untilin treatment the end 1 ofdemonstrating composting efficiencyin treatment of the 2. process;Further from which 14/17 (82.4%) contained infectious HAdV analysis will be conducted to measures of temperature, ranging from 103 to 104 PFU/L during the summer. In pH, dry matter and moisture to understand this result. winter, this incidence was 15/24 (62.5%) ranging from FINANCIAL SUPPORT: CNPQ, FAPERGS, PROJETO MAIS 105 to 108 gc/L contained 58.3% UP, and no infectious ÁGUA, FEEVALE viruses were detected by PFU. For sediment samples, the incidence was 9/24 (37.5%) ranging from 109 to 1010 EV78 - PRESENCE OF HUMAN ADENOVIRUSES IN gc/Kg contained 37.5% UP, from which 6/9 (66.7%) SURFACE WATER AND SEDIMENTS IN SANGRADOURO contain infectious HAdV ranging from 103 to 104 PFU/ RIVER, SANTA CATARINA, BRAZIL Kg during the summer. The results of winter collection Elmahdy, E.M.; Schissi; C.D.; Fongaro, G.; Nascimento, revealed 9/24 (37.5%) ranging from 108 to 109 gc/Kg M.A.; Barardi, C.R.M. contained 37.5% UP and no infectious samples by PFU. UFSC – Universidade Federal de Santa Catarina, We could conclude that HAdV genomes can be more Campus Universitário Reitor João David Ferreira Lima - concentrated in sediment but its infectivity can be lost Trindade, Florianópolis - SC, 88040-900 due to physical and chemical composition of the samples. FINANCIAL SUPPORT: CNPq/TWAS and CNPq Universal Human enteric viruses are a common microbial 472804/2013-8. which can reach the aquatic environment through the discharge of untreated sewage. Aquatic sediments can

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Environmental Virology: EV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

105 Environmental Virology: EV

EV89 - SEDIMENTATION AND SURVIVAL EVALUATION particles. Solid-liquid separation and survival studies OF PATHOGENS IN SWINE EFLLUENT AND SLUDGE FOR SAFE REUSE PURPOSES viable methods that allow studying the disinfection of of these prevalent pathogens in effluents and sludge are Fongaro, G.1; Kunz, A.2; Schissi, C.D.1; Magri, M.E.1; these matrices. This can predict the safe reuse of these Zaguini, J.1; Barardi, C.R.M.1 secondary-products as biofertilizers in agriculture or back to the swine facilities. FINANCIAL SUPPORT: CNPQ 1. UFSC – Universidade Federal de Santa 472804/2013-8. Catarina, Campus Universitário Reitor João David Ferreira Lima - Trindade, Florianópolis - SC, 88040-900 EV108 - ADENOVIRUS AND ENTEROVIRUS IN 2. EMBRAPA, Rodovia SP 340 - Km 127,5 - SURFACE WATERS IN THE WATERSHED SINOS RIVER, Tanquinho, Jaguariúna - SP, 13820-000 RS, BRAZIL Rigotto, C.; Dalla Vecchia, A.; Staggemeier, R.; Soliman, food and water, characterizing high contents of solids, M.C.; Souza, F.G. de; Giehl, I.; Henzel, A.; Rigotto, C.; organicThe swine matters, effluent phosphorus is composed and by nitrogen.urine, feces, Solid-liquid digested Spilki, F.R. separation and aerobic treatment are routinely used FEEVALE - Universidade Feelave, Av. Dr. Maurício to reduce the suspended solid concentration in the Cardoso, 510 | Bairro Hamburgo Velho, Novo Hamburgo - RS, 93510-250 aerobic reactor (AR) before settling tank, were used to evaluateliquid fraction. the survival In this study,rates oftreated the following swine effluent pathogens after Adenovirus (AdV) and enterovirus (EV), among other viral agents are responsible for different pathologies, mainly associated with gastroenteritis. These non- artificially seeded: somatic coliphage phiX-174 enveloped viruses are eliminated through the feces of (phi-X), Human Adenovirus (HAdV-2) and Salmonella symptomatic or asymptomatic individuals released untilTyphimurium. 120 h, in triplicate. Imhoff cones The pathogens(v. 1L) were survival filled within liquid the in high concentrations in water bodies and remain for andeffluents solid andfraction the settling were determined experiments after were 0, performed0.08, 0.16, long periods due to its high resistance to environmental 0.33, 0.75, 2.5, 5, 10, 24, 48, 72 and 120 h. In the sludge conditions. The Sinos River watershed serves 1.5 million (solid-fraction) this parameter was measured after 24, people (94% urban) living with serious problem caused 48, 72 and 120 h of settling time. The enumeration of by intense contamination of its waters. The watershed is geographically divided into three stretches: upper performed by integrated cell culture assay–preceded by (source, low population density and small farms), reversethe HAdV, transcription phi-X and S.(ICC-RT-qPCR), Thyfimurium weredouble respectively layer agar medium (increased urban density) and lower (mouth, strong population density and industrial concentration). The present study evaluated the occurrence of AdV method and by ISO 6579 (2002). In effluent, the survival and EV in water samples by qPCR in catchment points fraction)rate was: the 12.2% survival for S. rate Thyfimurium, after 120h 64%was 1.6% for HAdV-2 for S. for public supply in Sinos River, main watercourse and 76% for phi-X-174 after 120h; In sludge (solid- of watershed. Five-hundred mL of raw water were The settling rate of the three pathogens was also collected monthly in sterile bottles in eight catchments evaluatedThyfimurium, by measuring64% for HAdV-2 their reductionand 75% forin thephi-X-174. liquid- of water treatment plants (WTP) along the river for 24 months. Initially the samples were submitted to virus concentration by adsorption-elution method and constantfraction. S.in Thyfimurium the other periods was significantly evaluated; HAdV-2reduced atfrom 20 sequence nucleic acids were extracted with a commercial effluent at 45 min and 5h post sedimentation remaining kit (RTP DNA/RNA Virus Mini Kit). For EV detection an additional step (cDNA) was performed using the kit High correlatedmin, 2.5 and with 24h, theand phi-Xsedimentation at 10, 20 min,of solid 2.5, 5particles. and 10h. Capacity cDNA Reverse Transcription. For both viruses, The sedimentation of the HAdV and phi-X was positivity primers targeting conserved regions of the genome were 72h of settling in the secondary sludge. This means used for qPCR reaction. All 178 samples were negative thatHAdV-2 the and pathogens S. Typhimurum are present increased mainly significantly in the solidafter for EV. In the upper part of the river high rates of AdV September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Environmental Virology: EV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

106 Environmental Virology: EV were observed, with prevalence and average loads of: These agents are transmitted by fecal-oral route, human adenovirus (HAdV, 62% and 5,94x103 GC/L); being associated with a number of diseases, especially canine adenovirus (CAV-1 an 2, 66% e 1,04x106 GC/L); gastroenteritis, either in human beings or animals. (AvAdV,16%) and bovine adenovirus Despite the health impacts caused by outbreaks of these (BAV,4%). In the middle section viruses were observed viruses, the investigation of their presence in drinking in the upper stretch, plus the presence of porcine adenovirus PAdV (4%). In the lower stretch lower rates mandatory in many countries, including Brazil. Studies in of HAdV (48%) and CAV (39%) were observed when Brazilwater, aiming sewage the effluent detection and of recreational enteric viruses waters in mineral is not compared to other stretches. There was a gradual decline water have not yet been carried out. In this context, this in the detection rates for HAdV, CAV and AvAdV along study aimed to detect the presence of enteric viruses; the three sections of extension, which may be associated human adenovirus (HAdV), human enterovirus (EV) with the presence of inhibitors in the samples and the and human rotavirus genogrupe A (GARV), in mineral increasing of urbanization, since the lower stretch of water and discuss the possible sources of transmission. the watershed suffers intense anthropogenic pressure Samples of 1.5 L and 500 mL were analysed for virus by and represents great human demand by the water PCR and total and fecal coliforms by AQUACULT® kit. The use. Continuous monitoring of viral contamination is most prevalent virus was Adenovirus, 32.5% followed necessary to reduce negative impacts on public health. by rotavirus 25% and enterovirus 17.5%. HAdV was also FINANCIAL SUPPORT: CNPQ, FAPERGS, PROJETO MAIS present in 100% of mineral water containers (20 L) with ÁGUA, FEEVALE viral loads ranging from 1.34x103 to 9.94x104 gc/L. For bacteriology, total and fecal coliforms were absent in EV127 - ENTERIC VIRUSES IN BOTTLED MINERAL all samples and only three samples out of 60 analysed WATER COMMERCIALIZED IN BRAZIL presented contamination by thermotolerant bacteria. Rigotto, C.; dos Santos, V.R.; Staggemeier, R.; Dalla It is concluded that, following the example of what Vecchia, A.; Henzel, A.; Rigotto, C.; Spilki, F.R. has been considered in relation to the public supply of FEEVALE - Universidade Feelave, Av. Dr. Maurício drinking water, stricter measures for microbiological Cardoso, 510 | Bairro Hamburgo Velho, Novo Hamburgo - RS, control should also be applied to mineral water, so that 93510-250 this becomes indeed a safer alternative. FINANCIAL SUPPORT: CNPQ, FAPERGS, PROJETO MAIS ÁGUA, Brazil is the 6th largely consumer of mineral bottled FEEVALE water worldwide, stemming from increasing living standards and the general perception of population EV134 - VIRAL RECOVERY RATES OF HADV, EV that bottle mineral water would be the healthier and AND RV EVALUATED IN DIFFERENT STAGES OF safer option for consumption, when compared to other CONCENTRATION BY ADSORPTION-ELUTION sources. Although several studies suggest that bottled PROCESS IN NEGATIVELY CHARGED MEMBRANES water may have the same or worse bacteriological and Giehl, I.C.; Dalla Vecchia, A.; Rigotto, C.; Souza, F.G. de; chemical quality as tap water. The presence of microbial Soliman, M.C.; Staggemeier, R.; Giehl, I.C.; Henzel, A.; contaminants depends on source characteristics, Spilki, F.R. methods, asepsis of the packaging material and product storage. This scenario is being aggravated by the fact FEEVALE - Universidade Feelave, Av. Dr. Maurício Cardoso, 510 | Bairro Hamburgo Velho, Novo Hamburgo - RS, consumer in reusable containers of 20 L. At present, 93510-250 thethat 64%Brazilian of mineral resolution water ANVISAin Brazil isRDC distributed N.54 (June to final of Viral particles are often highly diluted in water and 2000) regulates the microbial quality of natural mineral water requiring the detection of fecal coliforms and are applied combined with molecular techniques to thermotolerans bacteria. Among the enteric viruses three achievedifficult a to higher detect, analytical thus virus sensitivity concentration virus detection methods of the most studied, as environmental contaminants from environmental samples. Currently, the virus are the adenoviruses, enteroviruses and rotaviruses. concentration method by adsorption-elution using

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Environmental Virology: EV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

107 Environmental Virology: EV negatively charged membrane is the most widely EV146 - MORPHOLOGICAL AND QUANTITATIVE used technique to concentrate viruses in different ANALYSIS OF MARINE VIRUSES IN ARRAIAL DO CABO environmental matrices, however there are few studies AND GUANABARA BAY, RJ, BRAZIL aiming the assessment of quantitative virus recovery Pedrosa-Macena, L.G.1; Paula, J.E.F.B.1; Santana- from different types of enteric viruses under the same Pereira, P.1; Amorim, L.1; Ferreira, D.F.2; Silva, R.2; conditions. The aim of this study was to evaluate the Damaso, C.R.A.2; Giongo, V.1; Paixão, I.C.N.P.1 volume samples (500 mL) and determine the recovery 1. UFF - Universidade Federal Fluminense, Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 rateefficiency in different of this method stages forof virusthe processconcentration using inhuman small 2. UFRJ - Universidade Federal do Rio de adenovirus (HAdV), enterovirus (EV) and rotavirus (RV) Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de as prototype viruses. Inoculums containing previously Janeiro - RJ, 21941-901 known viral titers of these viruses were individually and separately spiked into 500 mL of previously autoclaved Viruses are the most abundant biological agents in MilliQ water. One milliliter from each step of the aquatic environments. Responsible for most of the concentration method was withdraw at the beginning of genetic diversity and interference in marine ecological the process and viral genomes were extracted using RTP processes, viruses distributed in limnic and marine DNA/RNA Virus Mini Kit and the viral nucleic acids were ecosystems, such as oceans, lakes, coral reefs, glaciers, analyzed by real-time quantitative PCR with Platinum Sybr Green qPCR SuperMix-UDG, Invitrogen kit. The structure and composition of microbial communities mangroves, among others. Viral activities influence the results showed higher rates of viral recovery before study aimed to analyze marine viruses (cyanophages) morphologicallyand biogeochemical and flows determine of matter their and energy.abundance, This 77%concentrating at the end (step of 1)the in concentration relation to the when final stepcombined (step spatially and seasonally in regions of Arraial do Cabo and with5), except other for viruses HAdV inwith the highest sample. recovery EV and efficiencyRV showed of Guanabara Bay located in the state of Rio de Janeiro. Thus better recovery rates in the early stages ranging from techniques were performed as Transmission Electron 30% to 74% when evaluated separately, compared to Microscopy (contrasted with uranyl acetate 1%) and rates of 14% to 39% when evaluated combined. Results Flow Cytometry, which was used in three different obtained for HAdV when associated to other viruses in concentrations of SYBR Green I dye in the proportion of 5x10 -3, 5x10 -4 and 5x10 -5 from the stock solution. It EV and RV genome suggests a possible competition of was evidenced phages of Order (Families HAdVthe sample when andin the the presence reduction of otherin recovery viruses. efficiency Overall, thefor Myoviridae, Podoviridae and ) in Praia dos data showed wide variation in recovery rates (4% RV to Anjos (Arraial do Cabo), the Aterro da Praia Grande and 77% HAdV) using RNA and DNA genome viruses at the cytometry results indicated that the summer (3.61 ± Enseada de Jurujuba (Guanabara Bay). The seasonal flow study showed better recovery rates before concentration 2.74 x 10 8.part.mL-1) and spring (2.85 ± 1.8 x 10 8.part. andfinal loss stage in the of concentration,intermediate stages, as previous suggesting studies. that virus This mL-1) had higher viral abundances. Spatially at Arraial concentration protocol could be replaced or excluded do Cabo the sampling area with highest abundance was Praia dos Anjos (4.44 ± 2.62 x 10 8.part.mL-1) future comparative analyzes with environmental followed by Praia do Forte (3.23 ± 1.87 x 10 8.part.mL- without harming the efficiency of viral recovery, however, 1), otherwise the Guanabara Bay the highest abundance presence of inhibitors. FINANCIAL SUPPORT: CNPQ, was represented in the Aterro da Praia Grande (3.33 FAPERGS,matrices arePROJETO needed MAIS to ÁGUA, assess FEEVALE the efficiency in the ± 1.17 x 10 8.part.mL-1) followed by the Enseada de Jurujuba (2.57 ± 1.37 x 10 8.part.mL-1). Being found the presence and abundance of marine viruses in the study areas, especially in the period with higher solar incidence (Spring and Summer), showing a bacterial viability of maintenance for viral replication. Thus it concluded that September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Environmental Virology: EV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

108 Environmental Virology: EV this environment is promising for future analyzes of / mL). These data support the characterization of these role of viruses, organisms and microorganisms involved environments such oligotrophic, which are those with in the dynamics of the marine ecosystem. FINANCIAL a low supply of nutrients. Therefore it was possible to SUPPORT: CNPQ, FAPERJ, PROPPI AGIR determine the presence of viral particles associated with Siderastrea stellata and the water column in both EV164 - VIRAL ABUNDANCE AND MARINE sites. The relationship between nutrient availability, ENVIRONMENT ON ENDEMIC SIDERASTREA content of Chl-a and bacterial abundance supported STELLATA CORAL REEF IN ARRAIAL DO CABO AND the feasibility of viral replication. Bacteriophages are BÚZIOS/RJ therefore regulators of bacterial abundance, as well as Santana-Pereira, P.; Giongo, V.; Pedrosa-Macena, L.G.; responsible for nutrient cycling. FINANCIAL SUPPORT: Paula, J.E.F.B.; Voguel, V.R.; Nepomuceno, A.; Crapez, CNPQ, FAPERJ, PROPPI AGIR M.A.; Paixão, I.C.N.P. EV200 - PASSION AT SECOND SIGHT: AN UPDATE OF UFF - Universidade Federal Fluminense, Rua Miguel GENOME SEQUENCING AND ANALYSIS OF SAMBA de Frias, 9, Icaraí, Niterói - RJ, 24220-900 VIRUS Coral reefs are known to be holobiont and integrated Assis, F.L.1; La Scola, B.2; Kroon, E.G.1; Abrahão, J.S.1 assemblage of the coral animal, its symbiotic algae, protists, fungi, a diverse consortium of Bacteria and 1. UFMG - Universidade Federal de Minas Archaeas and even viruses. The symbiosis can be Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, understood through de evaluation of corals and your Belo Horizonte - MG, 31270-901 2. Université de la Méditerranée, Jardin du symbionts, the breakdown of which can result in disease Pharo - 58, bd Charles Livon -13284 Marseille Cedex 07 and mortality. Little is known, however, about the consequences of viruses that infect them. Studies have The Samba virus (SBV) was isolated in Brazilian Amazon shown that the virus highly dynamic aquatic ecosystem forest in 2011. At that time, a previous genomic analysis components have a central role in these environments was performed using a partially sequenced molecule. acting as antimicrobial agents in the control of the Nowadays, using the Illumina Deep Sequencing quantity and diversity of coral symbionts such as Technology, we obtained the complete genome sequence bacteria and phytoplankton. Viruses collaborate for the of SBV. For these proposes, the DNA of sucrose-gradient redistribution of micro and macro nutrients throughout the water column and in food webs. In this work, total were assembled using the CLC workbench 6 software. purified SBV was extracted, sequenced and the raw data and dissolved inorganic nutrients in the water column nitrate, nitrite, phosphate and ammonium were pairs (bp) with a C-G content of 28%. This new molecule isThe ~32kb final genome shorter of SBVthan is athe molecule previous of 1.181.380SBV genome base trophic conditions of the environment and its limitations. announcement, but still longer than Achantamoeba Spatiallyquantified higher and analyzed concentrations by grouping of nutrients data, estimating were found the in Búzios (Bz) and seasonally during the summer and mimivirus. The new genome of SBV was predicted poliphaga mimivirus (APMV), the first discovered autumn. The EFM technique was used to count and determination of the abundance of bacteria and virus report), ranging in size from 113nt to 8834nt, with a to encode to 971 ORFs, (33 more ORFs than the first particles, samples of seawater and coral mucus were mean size of 1068pb. The ORF’s density was 0.821 genes subjected to serial dilutions and labeled with 3 dyes (AO, per kb (1216 bases per gene), with a coding percentage SYBR Green I and DAPI). The highest bacterial abundances of 87.9%. Its arsenal was comprised by 142 metabolic- were found during the winter in the coral mucus samples associated cellular protein and up to 180 general in Bz (15.1 x 107 cels. / mL) and in seawater in Arraial metabolic processes, besides some regulatory proteins. do Cabo (AC) (7.23 x 107 cels. / mL). The abundance Among the predicted ORFs, 45% was comprised by of viral particles was found higher in summer in Bz in hypothetical proteins, and 85% showed best hit against coral mucus samples (2.70 x 107 part. / mL) and during autumn in AC samples from seawater (3.24 x 107 part. sequences and six tRNA sequences being three cognates APMV. We identified four aminoacil-tRNA sintetase September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Environmental Virology: EV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

109 Environmental Virology: EV to leucine, besides one to histidine, cysteine and precisely infer the real risk of these pathogens to cause tryptophan. Curiously, the C-G% of the tRNA sequences a disease. The methods applied were the plaque assay was 48.6%, greater than that found in the entire genome. (PA) and the integrated-cell-culture real-time PCR All the predicted ORFs matched orthologous into NVCOG (ICC-RT-qPCR). Firstly, a bioaccumulation experiment database (average similarity of 93,67%), meaning that was conducted to determine the optimal intake period new ORFans were not predicted. A total of 58 clusters of virus by the oysters. They were allocated in an the SBV genome, which was similar to that detected in a known amount of HAdV-2. The times of 3h, 5h, 8h, APMVconsisting and ofHirudovirus 179 paralogous sangsue. proteins The reciprocal were identified best hit in 10h,aquarium, 12h and and 24h the were water evaluated, was artificially and the seededperiod of with 8h of bioaccumulation was selected. Secondly, samples of oysters were kept at 4oC to mimic the market storage analysis identified 917 bona fide orthologous proteins temperature during commercialization, so the viral Moumouvirusbetween SBV and(group APMV. B) and The 485 same ones analysis with Megavirus identified stability could be measured. The animals remained alive chiliensis339 bona (group fide C). orthologous The phylogeny proteins based shared on DNApol with sequence grouped the SBV into mimivius clade A. This reduction in the infectious viral load was observed. updated analysis has a prominent relevance once it might Finally,until 96 the hours, viral anddepuration during thisrate period,was measured no significant up to be used as a guide to future genomic studies. Its broad 120h using a depuration tank supplied with an UV light. protein arsenal, the great number of uncharacterized A tank without UV light was used as a negative control. putative proteins and the current discussions regarding After 24h of depuration in the tank with UV, and 48h in virus evolution encourages us to go deeper into genomic the tank without UV, no infectious viruses were detected analysis of giant virus. FINANCIAL SUPPORT: CNPQ, by plaque assay. The results of the ICC-RT-qPCR are being FAPEMIG, CAPES, MAPA, PRPQ limit when compared with plaque assay, it is possible that EV208 - BIOACCUMULATION, STABILITY AND infectiousfinalized, but viruses once could this technique be detected has for a highera longer detection period. DEPURATION OF HUMAN ADENOVIRUS IN OYSTERS This work shows that the depuration tank was effective CRASSOSTEA GIGAS to inactivate HAdV-2 and that UV disinfection speeds the Pilotto, M.R.; Garcia, L.A.T.; Barardi, C.R.M. depuration. It also provides information to complement UFSC – Universidade Federal de Santa Catarina, the current legislation regarding depuration and viral Campus Universitário Reitor João David Ferreira Lima - inactivation. FINANCIAL SUPPORT: CNPq Universal Trindade, Florianópolis - SC, 88040-900 2012-1013 471755/2011-7 The Brazilian government implemented, in 2012, EV243 - INFLUENCE OF TIME AND TEMPERATURE OF the National Programme for Hygienic and Sanitary STORAGE ON HUMAN ROTAVIRUSES VACCINE STRAIN Control of Bivalve Mollusks (PNCMB). The PNCMB sets STABILITY IN WATER MATRICES minimum requirements to ensure the quality of bivalve Damazo, N.A.; Moresco, V.; Barardi, C.R.M. mollusks, as well as supervise the attendance of these requirements, based only on bacterial standards. Some UFSC – Universidade Federal de Santa Catarina, areas of mollusks production need to depurate their Campus Universitário Reitor João David Ferreira Lima - animals before selling them, but it does not give any Trindade, Florianópolis - SC, 88040-900 Human rotaviruses (RVA) are involved with many includes viral detection. The aim of this study was diarrhea outbreaks, most in children under 5 years tospecification evaluate the about stability the depurationand depuration method, of Human neither old and mainly due to the poor sanitary conditions. Adenovirus type 2 (HAdV-2), considered one of the most Nowadays, two vaccines are available and their positive resistant and prevalent viruses in the environment, in impacts on public health improvement were already oysters Crassostrea gigas. The focus of the work was to showed. However, there are no studies evaluating use only in vitro cell culture methodologies to determine the impact of these vaccine strains in the aquatic the infectivity of the virus, once these techniques can environment. The goal of this study was to evaluate the

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110 Environmental Virology: EV

EV251 - DISTRIBUTION AND MORPHOLOGIC of the RVA vaccine strain (RotaTeq) in environmental DIVERSITY OF VIRIOPLANKTON IN ARRAIAL DO waters.influence The of RVA the was time propagated and temperature in MA104 on cells the stabilityand the CABO, CABO FRIO UPWELLING REGION, RIO DE titre of the viral stock was measured by plaque assay JANEIRO STATE, BRAZIL (PFU) as previously standardized (2,2x107 PFU mL-1). Pedrosa-Macena, L.G.1; Paula, J.E.F.B.1; Pereira, P.S.1; A quantity of 3mL was seeded in 17mL of three different Vogel, V.A.R.1; Nepomuceno, A.M.1; Crapez, M.A.1; water matrices: (SWL) surface water lagoon (water Ferreira, D.F.2; Amorim, L.M.F.1; Giongo, V.A.1; Paixão, supply); (SDW) surface drinking water (unchlorinated); I.C.P.1 and (BWL) backish water lagoon (recreational area). Aliquots of 1mL were stored at the temperatures of 22ºC 1. UFF - Universidade Federal Fluminense, Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 and 4ºC to mimic respectively environmental and lab 2. UFRJ - Universidade Federal do Rio de storage temperatures. The RVA stability was evaluated Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de by PFU at time zero, and at 5, 10, 30, 60, 90 and 120 Janeiro - RJ, 21941-901 days of storage. The viral titre reduction was expressed as log reduction values (Log10 = Nt/N0). In all cases, Viruses has been described as the most abundant the higher Log10 reduction values were observed in biological components and dynamic in different the water samples stored at room temperature (22ºC). aquatic ecosystems, being its greatest reservoir the For the water matrix SWL, the samples showed a Log10 oceans, where the concentration ranges from 107 to reduction of 2.99 and 1.40 at 22ºC and 4ºC respectively 109 mL-1. However, few virus ecology studies have and for the water matrix SDW the Log10 values were addressed the assessment of virioplankton community 2.75 and 0.78 respectively for 22ºC and 4ºC both at in Brazilian tropical aquatic ecosystems. These is due the end of 120 days of storage. Samples belonged to in large part, by the majority of studies carried out in BWL stored at 22ºC showed a complete inactivation the area of environmental virology, are focused on just of the RVA after 90 days of storage (up to 4 Log10 the aspects of public health and disease-causing agents reductions). These samples stored at 4ºC also showed and not the ecological aspects. We carried out a study the higher values of log reduction in this temperature with the aim of assessing virioplankton distribution (2.44) after 120 days. The presence of other different and morphological diversity and determining how microorganisms, as well as the distinct physicochemical their variation in the marine waters of Arraial do Cabo, properties of the samples SWL, SDW and BWL can explain the difference on the Log10 reduction values activities. Subsurface seawater samples were collected influenced by upwelling events and anthropogenic observed. This can be due to predation and the water from Anjos Bay (AC581) and Forno Bay (AC570), more composition that can affect the virus survival and/or to promote the virus aggregation to solid particles in the others sampling sites (AC571, AC582 and AC561) were influenced by anthropogenic activities and the three water. Further experiments will involve the study of the located in more external portion of AC region, therefore stability of the RVA genome by RT-qPCR in order to check upwelling occur and with lesser input of anthropogenic receiving high influence of oceanic environment where on the genome persistence. FINANCIAL SUPPORT: CNPQ activities. Viral abundances were highest in site AC581 UNIVERSALthe influence 471755/2011-7; of the temperature CAPES and time of storage also (4.44 ± 2.62 x 10 8.part.mL-1) and AC570 (3.23 ± 1.87 x 10 8.part.mL-1), which were located in the inner

andbay 2.51 and x more 10 8.part.mL-1, influenced respectively). by anthropogenic The lowest and viral less influenced by upwelling (means of 3.33 x 10 8.part.mL-1 located in site AC571 (1.32 ± 0.4 x 10 8.part.mL-1). Sites AC582,counts wereAC561, found which in mostpresented influenced little wastewater oceanic seawater input, showed viral abundance mean of 1.4 ± 0.5 x 10 8.part. mL-1 and 1.53 ± 0.28 x 10 8.part.mL-1 respectively. The September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Environmental Virology: EV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

111 Environmental Virology: EV highest virioplankton abundances was during summer, (83.3%; 20/24), and Pampa (75%; 18/24). Viral load ranged from 1.47 to 7.95 x 10 8.part.mL-1, followed ranged from 1.76 x 103 to 5.11 x 107 genome copies/L. by spring with minimum of 1.24 x10 8.part.mL-1 and The results suggest intense anthropic contamination of maximum of 5.7 x 10 8.part.mL-1, coinciding with surface water in the region. FINANCIAL SUPPORT: CAPES, the seasons in which upwelling phenomenum occurs. FAPERGS, CNPQ, PROJETO MAIS ÁGUA, UNIVERSIDADE Most morphotypes have tails, belonging to Myoviridae, FEEVALE. Podoviridae and Siphoviridae. The majority of tailed phages in Arraial do Cabo region were found at AC581, EV287 - ASSESSMENT OF VIRIOPLANKTON ABUNDANCE AND ECOLOGICAL FACTORS IN this study showed that phage represented an important GUANABARA BAY, RIO DE JANEIRO STATE, BRAZIL fractionat the most of virioplankton antropogenic community influenced in area. Arraial In additiondo Cabo Santana-Pereira, P.1; Barbosa, J.E.F.1; Pedrosa, L.G.M.1; coastal region. FINANCIAL SUPPORT: CNPQ, FAPERJ, Vogel, V.A.R.1; Nepomuceno, A.M.1; Crapez, M.A.1; PROPPI AGIR. Ferreira, D.F.2; Amorim, L.M.F.1; Giongo, V.A.1; Paixão, I.C.P.1 EV254 - ENTERIC VIRUS IN SURFACE WATERS FROM 1. UFF - Universidade Federal Fluminense, URBAN AREAS IN RIO DOS SINOS WATERSHED, RS, Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 BRAZIL 2. UFRJ - Universidade Federal do Rio de Staggemeier, R.; Heck, T.M. da S.; Andriguetti, N.B.; Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de Soliman, M.C.; Souza, F.G. de; Rocha, S.; Faria, N.A.; Janeiro - RJ, 21941-901 Henzel, A.; Rigotto, C.; Spilki, F.R.; Almeida, S.E. de M. Little is known about the ecology of viruses living in FEEVALE - Universidade Feelave, Av. Dr. Maurício seawater columns over tropical estuarine ecosystems, Cardoso, 510 | Bairro Hamburgo Velho, Novo Hamburgo - RS, 93510-250 controlling their distribution. The aim of this study The increased population density in urban areas is often especially the influence of environmental factors the distribution and abundance of viruses and their The four stream targets of this work are situated in relationshipsis to assess for with the hosts first and time environmental an attempt tovariables elucidate in thisreflected highly in urbanized a higher contamination and industrialized of water region, resources. within the eutrophic estuary of Guanabara Bay, Rio de Janeiro, the municipalities EstânciaVelha, Campo Bom, Novo Brazil. Virus abundance ranged from 1.09 to 4.51 x 108 Hamburgo, form the Rio dos Sinos watershed, southern Brazil. These streams are recipients of large amounts brazilian aquatic system examined so far. The highest of urban and industrial sewage (mostly without any virusparticles•mL-1, abundance being found among in thisthe highest study observedwas in GB537 in any treatments). The aim of this study was the detection (average 3.33 x 108.part.mL-1) and GB 566 (2.57 x 108. part.mL-1), which were located in the inner bay and genomes in water samples collected from the streams EstânciaVelha/Portãoand quantification of(EstânciaVelha human adenovirus and Portão (HAdV) cities), Schmidt (Campo Bom city), Pampa and Luiz Rau (Novo oceanicless influenced seawater by located marine in seawater. sites GB557 In the(average other 1.39hand, x Hamburgo city). In total, 102 water samples (500 108.part.mL-1)the lowest viral and counts GB546 were (average found 1.45 in mostx 108.part.mL- influence ml each) from 17 different sampling points, at were 1). Interestingly, virus and bacterial abundances were collected bimonthy from September/2012 to to July/13. strongly correlated (r= 0.70; P <0.01) as well when virus After, concentration by adsorption / elution, extraction abundance were plotted against chlorophyll-a (r= 0.85; of viral DNA was performed. The virus detection was P <0.001). Further correlation analysis showed that performed by qPCR using primers with the potential alignment of highly conserved regions of the genome models when plotted against phosphate (r= 0.75; P of the virus. According to the results 83.33% (85/102) <0.001);virioplankton ammonia abundance (r= 0.78; also P revealed<0.001) andhigh temperature fits to linear were positive for the presence of HAdV: Schmidt (91.7%; (r= 0.59; P <0.05) and showed an inverse correlation to 22/24), EstânciaVelha/Portão (83.3%; 25/30), Luiz Rau salinity (r= -0.45; p> 0.05) and conductivity (r= -0.39;

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112 Environmental Virology: EV

P > 0.05). All investigated phages were tailed phages of (N=57) were reanalyzed for 5’end ORF2 region by the Myoviridae and Podoviridae families, both belonging others nested (for GI) and semi nested (for GII) in order to the Caudovirales order, which represent the most to obtain amplicon for genotyping. After the samples abundant in nature, and account for 96% of the phages described so far. Understanding the correlation among molecular characterization in an automated sequencer. virus abundance and other biological and physical- Thewere obtainedpurified usingsequences a commercial were edited,kit and submittedaligned and to chemical measurements consist in an important compared to others available in gene bank (NCBI) and in the site NoV genotyping tool. Of 57 positive samples abundance and its hosts in tropical estuaries submitted by TaqMan® real time PCR and/or semi nested RT-PCR, toapproach pollution. to elucidate FINANCIAL the main SUPPORT: factors influencingCNPQ, FAPERJ, viral 53 were retested for 5’end ORF2, since four samples PROPI-UFF. a new analyze, so, in 47.2% (25/53) the NoV genome EV328 - PARTIAL CHARACTERIZATION OF wasshowed detected, insufficient of these quantity 12% (3/25) of material belonging which to GI,allowed 24% NOROVIRUS GI AND GII IN WATER AND SEWAGE (6/25) to GII and 64% (16/25) for both. The most frequent SAMPLES FROM BELÉM CITY, PARÁ STATE, BRAZIL GI and GII genotypes were GI.8 (n=8) and GII.4 (n=12), Teixeira, D.M.1; Spada, P.K. de P.1; Bandeira, R. da S.1; respectively, but others genotypes were also observed Soares, L. da S.1; Gurjão, T.C.M.1; Morais, L.L.C. de S.1; with lower incidence as GII.6 (n=3), GII.9 (n=2), GII.12 Sousa, M.S. de2; Mascarenhas, J.D.P.1; Fumian, T.M.3; (n=1), GII.14 (n=1), GI.1 (n=1) and GI.4 (n=2). Due to the Gabbay, Y.B.1 low quality of sequences obtained, eight samples could 1. IEC/SVS/MS - Seção de Virologia do Instituto Evandro Chagas, Rodovia BR-316 km 7 s/n, Levilândia, that in the less rainy period (July to November) the NoV Ananindeua - Pará, 67030-000 positivitynot be genotyped increased for and, GI and in the three months for GII. of highest It was verified rainfall 2. NÚCLEO DE MEDICINA TROPICAL/ (December to June) it decreased. The results obtained UFPA - Núcleo de Medicina Tropical da Universidade Federal in the present study indicate the circulation of NoV GI do Pará, AV. Generalíssimo Deodoro, 92, Belém - Pará, 66055- and GII in aquatic environments in Belem, revealing 240 the degradation that these water bodies have suffered, 3. Laboratório de Virologia Comparada e as a result of poverty or lack of sanitation in our city, Ambiental/FIOCRUZ - Laboratório de Virologia Comparada allowing input of pathogens in these ecosystems, along e Ambiental da Fundação Oswaldo Cruz Pavilhão Hélio e Peggy Pereira, Av. Brasil, 4365 - Manguinhos, Rio de Janeiro - CEP: 21040-900 inwith Brazil. its effluents. FINANCIAL According SUPPORT: to our FUNDAÇÃO knowledge AMAZÔNIA it was the Enteric viruses excreted in feces from infected first detection of GI.1 and GI.8 in environmental samples individuals dispersed in aquatic environments by CNPQ; INSTITUTO EVANDRO CHAGAS/SECRETARIA DE sewage discharge. Among these viruses, the norovirus VIGILÂNCIAPARAENSE EM DE SAÚDE/MINISTÉRIO AMPARO À PESQUISA DA SAÚDE. (FAPESPA); (NoV) is actually considered the main cause of gastroenteritis outbreaks worldwide, resulting from EV347 - SEARCHING FOR CIRCO-LIKE VIRUS- the ingestion of contaminated food and water as well as BRAZIL (CLV-BR) IN CEREBROSPINAL FLUID AND is also associated with hospitalizations. This research ENVIRONMENTAL SAMPLES aimed partially characterize the NoV (GI/GII) in Nagasse-Sugahara, T.K.1; Castrignano, S.B.1; Garrafa, different water matrices and in untreated sewage from P. 2; Dergovics, F.L.1; Mehnert, D.U.2; Barrela, K.M.2; Belém city. Environmental samples (2 liters) collected in Monezi, T.A.2; Barbosa, M.R.3; Garcia, S.C.3; Bonanno, V.M. dos S.3; Kisielius, J.J.1 1. IAL - Instituto Adolfo Lutz, Av. Dr. Arnaldo, seven points were concentrated on filtering membranes extracted by silica method and initially submitted to 355 - Cerqueira César, São Paulo - SP, 01246-000 to obtain a final volume of 2 mL. The nucleic acid was semi nested RT-PCR (RNA-dependent RNA polymerase) 2. ICB/USP - Instituto de Ciências Biomédicas and TaqMan® real time PCR (ORF1/ORF2 junction). da Universidade de São Paulo, Edifício III USP - Administração The positive samples for both molecular methods September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Environmental Virology: EV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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- Av. Prof. Lineu Prestes, 2415 - Butantã, São Paulo - SP, EV350 - DETECTION OF PORCINE CIRCOVIRUS TYPE 05508-900 2 (PCV2) AND GYROVIRUS TYPE 2 (AGV2) IN SWINE 3. CETESB - Companhia Ambiental do Estado SLURRY de São Paulo, Av. Prof. Frederico Hermann Jr.,345, Av. Prof. Correa, R.A.1; Teixeira, T.F.2; Cibulski, S.P.2; Santos, Frederico Hermann Júnior, 345 - Alto de Pinheiros São Paulo H.F.2; Soliman ,M.4; Staggemeier, R.4; Henzel, A.4; - SP, 05459-010 Rigotto, C.4; Spilki, F.R.4; Roehe, P.M.3 Using a metagenomic approach, our group recently 1. UFPEL - Universidade Federal de Pelotas, discovered two new viruses, which we named circo-like Capão do Leão - RS, 96160-000 virus Brazil (CLV-BR) strains hs1 and hs2, in a sample 2. FEPAGRO - Fundação Estadual de Pesquisa of human feces collected in 2003, as published in Virus Agropecuária, R. Gonçalves Dias, 570, Porto Alegre - RS, Research in 2013. The viruses contain a circular DNA 90130-060 genome approximately 2500 nucleotides long encoding a 3. UFRGS - Universidade Federal do Rio replication initiator protein (Rep) and were characterized Grande do Sul, Avenida Paulo Gama, 110 - Farropilhas, Porto by molecular biology and electron microscopy techniques, Alegre - RS, 90040-060 as well as bioinformatics analysis. In the phylogenetic 4. FEEVALE - Universidade Feelave, Av. Dr. tree, the viruses did not cluster with members of the Maurício Cardoso, 510 | Bairro Hamburgo Velho, Novo already established viral families but with some circo- Hamburgo - RS, 93510-250 Environmental contamination by swine manure may and bat feces. Objective. To investigate the presence of like viruses identified in reclaimed water and rodent CLV-BR viruses in various types of samples. Material therefore important to analyze and detect potential viralrepresent contaminants a significant in liquid risk waste to of human pigs. In health. the present It is samples (55 individual samples) sent to IAL for viral study, pig slurry (PS) were tested for the presence of and methods. We tested 11 pools of cerebrospinal fluid detection; 5 pools of wastewater samples and 5 pools the following viruses: chicken anemia virus (CAV), of reclaimed water samples collected between January avian gyrovirus type 2 (AGV2), porcine cirovirus type 2005 and November 2006 (177 samples of each type) 2 (PCV2), porcine bocavirus type 1 (PBoV1), Torque and provided by ICB-USP; 2 samples of wastewater teno sus virus 1 and 2 (TTSuV1, TTSuV2). The DNA was (São Lourenço, SP, and Taboão da Serra, SP) and 2 raw extracted with a commercial nucleic acid extraction. water samples (Guarapiranga dam, SP, and São Lourenço Different, previously described quantitative polymerase spring, SP) collected in December 2013 and provided by chain reactions (qPCR) were employed to examine 213 CETESB. All samples were analyzed by PCR targeting the samples of slurry, of which 28 (13,1%) were found Rep gene of both CLV-BRs. Results. Among the samples to contain PCV2 genome fragments. Nineteen (8,9%) tested, the target sequence was found in one pool of contained AGV2 genome fragments. Only 6 samples wastewater samples from ICB-USP (pool-2). This result (2,8%) were contaminated with both PCV2 and AGV2. CAV, PBoV1, TTSuV1 and TTSuV2 genomes were Discussion. Metagenomic studies have contributed to not detected in the samples examined. These results was confirmed by sequencing and BLAST analysis. the detection of large numbers of new viruses. After the indicate that AGV2 and PCV2 can be detected in swine discovery of a new virus, further studies are required manure. Further experiments will be conducted in order to evaluate different kinds of samples where the virus to examine whether such samples might also contain infectious virus. determine whether the virus can be associated with might be detected, to identify its specific host and to EV413 - HUMAN AND MURINE NOROVIRUS RECOVERY samples collected in 2005 supports the hypothesis that FROM TOMATOES SAMPLES disease. The finding of CLV-BR in a pool of wastewater (a) this virus continued to circulate in the state of São Correa, A. de A.1; Melgaço, F.G.2; Correa, A.3; Luz, I.2; Ganime, A.C.2; Miagostovich, M.P.2 also have come from human waste. Paulo two years after first being detected and (b) it may 1. UFF - Universidade Federal Fluminense, Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Environmental Virology: EV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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2. Laboratory of Comparative and methodology for detecting NoV in a broad range of food Environmental Virology, Oswaldo Cruz Institute, Fiocruz samples, the results observed in this work provides a new 3. Laboratory of Virology, Fluminense Federal perspective for the study of salad vegetables as a vehicle University for viral transmission in outbreaks of gastroenteritis Norovirus (NoV) is the most important agent of food associated to NoV. FINANCIAL SUPPORT: MCTI/CNPQ/ borne outbreaks of acute gastroenteritis and their ANVISA NO. 23/2012 . EV530 - SHELLFISH DEPURATION TANKS FOR identification in foods is difficult due to the food matrix INACTIVATION OF RECOMBINANT HUMAN ADENOVIRUS AND MURINE NOROVIRUS IN (skimmedcomplexity. milk) This studyand polyethylene evaluated the glycol efficiency (PEG), of twofor ARTIFICIALLY SEEDED SEAWATER Humanviral concentration NoV GII.4 (NoV methods, GII.4) and organic Murine flocculation Norovirus Garcia, L.A.T.; Nascimento, M.A.; Barardi, C.R.M. samples (cherry, grape and common tomatoes). For UFSC – Universidade Federal de Santa Catarina, (MNV-1) recovery from three tomatoes classifications Campus Universitário Reitor João David Ferreira Lima - buffer was used as elution buffer; for PEG precipitation; Trindade, Florianópolis - SC, 88040-900 Glycineorganic 0.05M/Tris-HCl flocculation, Glycine 0.1M with 0.05M/Tris-HCl 3% beef extract 0.1M buffer, according to the International Organization for pathogens from mollusks tissues. The seawater Standardization (ISO/TS 15216-1:2013), was applied. All disinfectionShellfish depuration during the is adepuration process that process aims isto important eliminate the samples were tested in the whole form; grape tomato and ultraviolet (UV) light treatment is the most used method worldwide. Viral models are usually employed as obtained was treated or not treated with 1,3% cationic surrogates of fastidious viruses in viability studies. The samples were also tested sliced. The final concentrate detergent cetyltrimetylammonium bromide (CTAB) aim of this study was to determine methods based on chloroform/butanol (1:1 vol/vol), for PEG precipitation, seeded in seawater and apply these assays to assess for organic flocculation, and with equal volume of in order to remove inhibitory substances from the virus theGFP-fluorescence inactivation ableof rAdV-GFP to detect recombinantand murine adenovirus norovirus extract . The viral RNA was extracted by QIAamp viral RNA mini kit® (Qiagen) and the cDNA produced, using tanks with and without UV light treatment. Kinetics ofin rAdV seawater GFP-expression in recirculation was previously shellfish depurationmeasured by real time PCR using primers and probes previously by UV-spectrophotometer. Flow cytometry (FC) and random primers. The viral quantification was performed virus titre and detection limit of each method. Traditional described. For the flocculation method, cherry tomato GII.4 (5.0%) and MNV-1 (0.6%). Whole/sliced grape methodfluorescence of plaque microscopy assay was (FM) done were in used order to to determine compare andsamples common showed tomatoes low recoverysamples efficiencypresented forrecovery NoV seawater was evaluated with and without UV treatment. respectively) and for MNV-1 (2.26%, 4.61%, and 4.05%, Theits efficiency time post-infection with FC and established FM. Disinfection for the detection of seeded of respectively).efficiencies for The NoV samples GII.4 (16.9%,treated with 47.7%, CTAB, and showed 33.4%, lower sensitivity, when compared with FM, which was When the PEG method was applied, cherry tomato similarinfected toGFP-fluorescent plaque assay. cellsSeawater rAdV wasdisinfection 24h. FC showedresults better recovery efficiency than the untreated samples. samples showed a 0.06% and 2.1% recovery for NoV showed that rAdV-GFP declined 99.99% after 24h and GII.4 and MNV, respectively. For the grape tomatoes 48h with and without UV treatment respectively. MNV whole/sliced and common tomatoes, the respective was completely inactivated after 24h in both treatments. As conclusion, the depuration tanks were effective to NoV GII.4 and 11.2%, 13.8% and 8.8% for MNV. The inactivate rAdV-GFP and MNV and the UV disinfection recovery efficiencies were 6.8%, 51.6% and 2.5% for treatment accelerated the process. FINANCIAL SUPPORT: can be the method of choice for recovery those viruses CAPES fromresults tomatoes obtained samples. suggest Since that there the organic is no standardized flocculation

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Environmental Virology: EV HUMAN VIROLOGY -HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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HV4 - LOW - RISK HPVS PREVALENCE IN CERVICAL HV6 - PREVALENCE OF HPV 18 DNA IN CERVICAL SAMPLES OBTAINED FROM WOMEN WITH SAMPLES OBTAINED FROM WOMEN WITH ALTERED ABNORMALITIES IN THE UTERINE CERVIX BY USING CITOLOGY LBC METHODOLOGY Gomes, A.C.C.; Vago, A.R.; Lopes, L.V.A.; Ebani, E.C.; Calastri, L.P.; Vago, A.R.; Lopes, L.V. de A.; Fonseca, Andrade, L.O. L.P.; Toppa, N.H. UFMG - Universidade Federal de Minas Gerais, UFMG - Universidade Federal de Minas Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, Belo Avenida Presidente Antônio Carlos, 6627 - Pampulha, Belo Horizonte - MG, 31270-901 Horizonte - MG, 31270-901 Aims: Numerous epidemiologic and laboratory studies Aims: Mounting evidence from both laboratory and epidemiological studies, indicate that sexually a persistent HPV infection and the development of transmitted HPV infections may be the leading cause of cervicalhave confirmed pre-malignant a strong and causal malignant association lesions. between Several cervical cancer (CC) world-wide. The presence of these studies also demonstrated HPV DNA in more than 95% of cervical neoplasia (CIN) and cancer (CC). High-risk Pap smear, however, a normal Pap smear does not prove species, namely types 16 and, to a lesser extent, types theHPV absence types is ofmost HPV often infections. first detected According by an to abnormal the HPV 18, 45, 56, 31, 33, 35, 51, 52, 58, are types that are most phylogenetic tree, the so-called low-risk (LR) HPVs, frequently found in pre-malignant and malignant lesions, namely types 6, 11, and rarely 42, 43 and 44, are most while low-risk types (namely types 6, 11, and rarely 42, commonly associated with benign or condylomatous 43 and 44) are most commonly associated with benign lesions on the anogenital tract. This study was aimed to investigate the prevalence of the LR-HPVs 6 and 11 types these lesions. This study was aimed to investigate the in a women-group from the Brazilian population which prevalenceor condylomatous of HPV18-oncogenic lesions are rarely type among identified a women- within presented cervical low-grade squamous intraepithelial group from the Brazilian population which presented lesions, especially ASCUS and CIN1. Methods: By using a cervical cell abnormalities. Methods: By using a Hemi- conventional PCR protocol and primers directed to HPV nested PCR protocol and primers directed to HPV E6-E7 E6-E7 genes, we examined the prevalence of HPVs 6/11 early-transcribed genes, we examined the prevalence in DNA samples isolated from 190 LBC (Liquid-based of HPV18-DNA in 190 LBC (Liquid-based Cytology) Cytology) cervical samples, obtained from patients cervical samples obtained from patients from Belo from Belo Horizonte, Minas Gerais state, which were Horizonte, Minas Gerais state, which were diagnosed diagnosed as ASCUS (67), CIN1(116) and CIN2/CIN3(07). by Cytopathology analysis as ASCUS (67), CIN1(116) Results: Among the analyzed samples, the general and CIN2/CIN3(07). Results: Almost ninety percent prevalence of HPVs 6/11A was of 9% (18/190), with of samples were positive to HPV-DNA (169/190) by 7% (05/67) of positivity in ASCUS and 11% (13/116) using a Nested- PCR protocol and primer-sets directed in CIN1 cases. These LR HPVs were not found in any of to the HPV L1 conserved gene sequence. Among the all the HSIL samples. Conclusion: Our results pointed out analyzed samples, the general prevalence of HPV18-DNA a relatively high incidence of LR-HPVs 6/11 among the was of 16% (31/190), with 15% (10/67) of positivity analyzed women population who presented low-grade in ASCUS, 17% (20/116) in CIN1 cases and in 14% cervical intraepithelial neoplasia. By considering that (1/07) of CIN2/CIN3 samples. Conclusion: Our results 32% of women with CIN1 will exhibit lesion persistence pointed out a relatively high incidence of HPV 18 type and that, 10% of them will progress to CIN3, these data infection among the analyzed women population, who emphasize the importance of vaccination (especially the were mainly diagnosed as exhibiting low-grade cervical quadrivalent one, against HPVs 6, 11, 16 and 18 types) lesions. These patients would require a strict clinical in preventing the establishment of pre-invasive cervical accomplishment and/or treatment, in order to prevent lesions. a subsequent cervical lesion progression.

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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HV10 - PREVALENCE OF HPV 45 DNA IN CERVICAL HV29 - ANIMAL MODEL OF ARTHRITIS MAYARO SAMPLES OBTAINED FROM WOMEN WITH CITOLOGY VIRUS-INDUCED ABNORMALITIES Santos, F.M.; Oliveira, M.D.; Dias, R.S.; Monteiro, J.M. Vago, A.R.; de Freitas, G.V.; Lopes, L.V. de A.; Andrade, de C.; de Paula, S.O. L.O.; Silva, M.X. UFV - Universidade Federal de Viçosa, Avenida Peter UFMG - Universidade Federal de Minas Gerais, Henry Rolfs, s/n - Campus Universitário, Viçosa - MG, 36570- Avenida Presidente Antônio Carlos, 6627 - Pampulha, Belo 000 Horizonte - MG, 31270-901 The Mayaro (MAYV) virus belongs to the genus Cervical cancer is one of the most common causes of Alphavirus and the family Togaviridae, and is considered cancer-related death in women and is associated with an arbovirus of epidemiological importance for the persistent infections with a high-risk (HR) subset of countries of South America, once that is responsible human papillomavirus (HPV). According to the HPV for causing acute febrile illness, often followed by phylogenetic tree, the HR HPVs, namely types 16 and, polyarthritis. In Brazil the MAYV is endemic in the to a lesser extent, types 18, 45, 56, 31, 33, 35, 51, 52, Amazon region, but the risk of emergency in other 58, are primarily associated with CIN. Very recently, the regions of the country as well as in other continents has preliminary results about immunogenicity and safety of a novel 9-valent HPV L1 Virus-like Particle (VLP) vaccine animals infected outside the endemic areas. Thus, due (Merck) were presented. This vaccine is directed against tobeen the demonstrated potential of become by the identification a public health of travelersproblem and the L1VLPs from types 6/11, 16, 18, 31, 33, 35, 45, 52 the impact that the alphaviral disease has, not only in the and 58. However, there is a scarcity of data concerning human life quality, but also on the economy, the objective the HPV 45 prevalence among the worldwide women, of this work is to develop an animal model of disease including the Brazilian ones. Aims: The purpose of this MAYV-induced for understanding the mechanisms by work was to investigate the HR-HPV 45 prevalence which arthritis is caused and the determination of the among women from Belo Horizonte city, MG state, primary sites of infection. We utilized Balb/c mice as Brazil. Methods: By using a conventional PCR protocol experimental models, which were divided into three and primers directed to HPV E6-E7 genes, we examined groups. Being an infected in the right rear footpad and the HPV45-DNA prevalence in 190 LBC (Liquid-based the other subcutaneously in the thorax below the right Cytology) cervical samples. These cases included 67 ASCUS, 116 CIN1 and 7 CIN2/CIN3. Results: HPV45 route, and the third was the negative control, which showed to be the second most prevalent type in each wasforelimb, inoculated to assess only whetherPBS. The there animals is influencewere evaluated of the cervical lesion group (ASCUS, CIN1 and CIN2/3), when for weight loss and clinical signs at 24-hour intervals compared with other HR-HPV tested. Among the all for a period of 21 days. In addition, the animals were analyzed samples, the general prevalence of HPV45-DNA subjected to physical tests that assess muscle strength. was of 16% (31/190), with 16% (11/67) of positivity in Observed that the group infected in the right forelimb ASCUS and 17% (20/116) in CIN1 cases. This HR-type lost weight and showed various clinical signs such as was absent among the high-grade lesions. Conclusion: Our results pointed out a relatively high incidence of infected in the rear footpad did not lose weight and HPV 45 type infection among the analyzed women, who alsoruffled did fur, not abnormal show any march, of these curved clinical body. signs. The However animals were mainly diagnosed as exhibiting low-grade cervical by strength test performed, we found that both infected lesions. By considering that most of women submitted to the cytology cervical screening are diagnosed as to control. These results show that infected groups had CIN1, our data emphasize the relevance of the HPV45 lossgroups of strengthshowed significantly caused by arthralgia, lower mean and time also compared that the type inclusion in newly designed vaccines to effectively route of infection is an important factor in determining prevent this HR-type dissemination among the sexually the clinical signs to be developed by the animals. active women.

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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HV35 - EVALUATION OF ANOGENITAL HUMAN PAPILLOMAVIRUS INFECTION IN ASYMPTOMATIC epidemiology of HPV infection in our state. MEN FROM RIO DE JANEIRO, BRAZIL as to the identification of risk factors associated with the HV42 - SULFATED POLYSACCHARIDE OF Menezes, W. da R.1; Afonso, L.A.1; Dobao, E.2; Gouvea, ADENANTHERAPAVONINAINHIBITS HERPES T.D. 3; Carestiato, F.N.1; Cavalcanti, S.M.B.1 SIMPLEX AND POLIOVIRUSIN HEP-2 CELLS 1. Lab Diagn Virologico, UFF Godoi, A.M.2; Lopes, N.2; Rechenchoski, D.Z.2; Faccin- 2. Setor De Dematologia Da Santa Casa De Galhardi, L.C.2; Nozawa, C.2; Ricardo, N.M.P.S.1; Misericordia Linhares, R.E.C.2 3. Setor de DST da UFF 1. UFC - Universidade Federal do Ceará, Currently, the genital tract infection by human Avenida da Universidade, 2853 - Benfica, Fortaleza - CE, papillomavirus (HPV) is one of the most prevalent sexually 60020-181 transmitted diseases in the world. However, there are still 2. UEL - Universidade Estadual de Londrina, gaps in the knowledge regarding the etiology of penile Rodovia Celso Garcia Cid, Km 380 - Campus Universitário, cancer, and the natural history of HPV infection in men Londrina - PR, 86057-970 is not yet fully elucidated. This study aimed to determine The herpes simplex 1 (HSV-1), member of Herpesviridae the prevalence of HPV infection in penile swab samples, family, is an enveloped double-stranded DNA virus that derived from a clinically asymptomatic male population. affects various age group, causing lesions in the oro-facial For this purpose, 261 samples were collected between region. The drug of choice for the disease treatment is the January 2011 and July 2013 in different institutions in the acyclovir(ACV), but its continued use can select resistant city of Rio de Janeiro, including a hospital, a dermatology strains. Poliovirus (PV), member of Picornaviridae clinic, a university and a metallurgical company. We family, is a non-enveloped positive single-stranded RNA also analyzed the epidemiological variables of these virus, known as the causative agent of poliomyelitis, a subjects through the application of a socio-demographic disease that affects the central nervous system, causing questionnaire to aid in the investigation of possible risk its treatment. Several studies report the use of natural productsflaccid paralysis. with antiviralThere is noactivity, effective including chemotherapy sulfated for andfactors. Restriction The viral Fragment identification Length was Polymorphism made through (when the polysaccharides. This work evaluated the antiviral effect needed)generic andtechniques. type-specific An overall Polymerase prevalence Chain Reaction,of HPV of a sulfated polysaccharide of Adenanthera pavonina infection was observed in 16.47 % (43 individuals). (SPLSAp), in HEp-2 cells, against HSV-1 and PV-1. The The most prevalent HPV type was HPV 6 (34.88%), 50% cytotoxiticy of the polysaccharide (CC50) was followed by HPV 16 (23.25%), HPV 11 (16.27%), HPV carried out by MTT method and the antiviral activity by 45 (9.30%) and HPV 58 (2,32 %). Hence, infection was plaque reduction assay (PRA), using different protocols associated with low-risk oncogenic types in 53.66% of of treatment. The SPLSAp CC50 was determined as the studied individuals, and high-risk oncogenic types 500µg/ml.A 50% inhibitory concentration (IC50)of were detected in 46.34 % of them. The age of the subjects 15µg/ml with a selectivity index (SI) of 33.3 were found studied ranged between 18 and 65 years with a mean for HSV-1, and for PV-1, an IC50 of 1.18 µg/ml and SI of age of 26.30 years. Among the epidemiological variables, 423.73. The inhibition of HSV-1 was demonstrated by the time-of-addition and time-of-removal tests, with the of men who have sex with men, for the group who have highest percentage of viral inhibition (%IV) of 100% and keptstatistical anal significantintercourse results during were sexual found relationship, for the group and 99.3%, respectively, at the concentration 200 µg/ml. A for the group that using condom regularly (p<0.1). We maximum inhibition of viral protein synthesis was also described a lower prevalence of HPV infection among asymptomatic male population than those reported the highest concentration(200µg/ml). The HSV-1DNA in some recently published studies. We believe that synthesisdemonstrated was bytotally the immunofluorescence(IF)inhibited by SPLSAp at 25µg/mlassay at the results may contribute to a clearer view about the as demonstrated by PCR.The combination index (CI)of circulation of HPV in the general male population, as well September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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SPLSAp with ACV showed an antagonistic effect to the severe cases and deaths. Previous studies of our group reference drug. The maximum %IV for PV-1 was found at demonstrated de co-circulation of DENV-1 and DENV-2 the time 0 hour of infection and the SPLSAp also showed and data from City Hall also indicated the introduction virucidal effect of 74.1% at the highest concentration of DENV-4 in JF. Given the hyperendemicity of DENV tested (100 µg/ml). By IF assay the SPLSAp inhibited and the lack of data regarding seroprevalence of dengue 100% of PV-1 at 100 µg/ml, in a dose-dependent in healthy population in JF, the aim of this work was to response. These results suggested that the maximum perform a serological study to check the presence of anti- inhibitory effect of SPLSAp in the replication of HSV-1 DENV antibodies in healthy persons living in JF, Minas occurred after the step of viral penetration and for PV-1 Gerais. Whole blood samples were collected from 340 in the initial stages.Besides, the SPLSAp low toxicity individuals from September to October/2013 and from and high selectivity make the compound a potential February to May/2014. A rapid immunochromatography candidate for further studies in the control of these test (Dengue Duo Cassete PanBio kit) to detect IgM and infections. IgG antibodies to DENV in human serum samples was used. From 340 samples, 16.17% (55 samples) were HV44 - SEROPREVALENCE OF DENGUE IGM AND positive: 19 samples were IgM positive and IgG negative, IGG ANTIBODIES AGAINST DENGUE VIRUS AMONG 12 samples were IgM negative and IgG positive and 24 HEALTHY INDIVIDUALS IN JUIZ DE FORA, MINAS, samples were IgM positive and IgG positive. This data BRAZIL indicated a high seroprevalence in individuals living Siqueira, T.R.1; Mendonça, L.A.1; Veiga, R.2; Fernandes, in Juiz de Fora, besides the occurrence of primary and G.C. Coelho, L.F.L.3; Drumond, B.P.1 possible secondary infections in individuals in this 1. UFJF - Universidade Federal de Juiz de Fora, city. Further experiments will be performed in order R. José Lourenço Kelmer - Martelos, Juiz de Fora - MG, 36036- to check the presence of neutralizing antibodies and 330 the presence of antibodies that may facilitate DENV 2. LAWALL - Laboratório de Análises Clínicas infection in a heterotypic infection through ADE like em Juíz de Fora, Av. dos Andradas, 341 - Centro, Juiz de Fora mechanism. Information about the DENV circulation as - MG, 36036-000 well population immune status are important factors for 3. UNIFAL - Universidade Federal de Alfenas, dengue control. R. Gabriel Monteiro da Silva, 714 - Centro, Alfenas - MG, 37130-000 HV49 - PREVALENCE OF HUMAN PAPILLOMAVIRUS INFECTION AND PHYLOGENETIC ANALYSIS OF HPV- Dengue virus (DENV) infection is the most important 16 E6 VARIANTS AMONG INFECTED WOMEN FROM arboviral disease in the world. Dengue viruses are NORTHERN BRAZIL 1 2 1 serotypes designated DENV-1 to DENV-4. DENV infection Silvestre, R.V.D. ; Lopes, B.P.T. ; Sousa Jr, E.C. ; 2 2 1 mayclassified result into in foura range antigenically of disease,ranging and genetically from distinct acute, Passetti, F. ; Ferreira, C.G.M. ; Mello, W.A. febrile illness to severe, life-threatening hemorrhagic 1. IEC - Instituto Evandro Chagas, Rodovia disease. Each serotype can cause human disease and BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 mortality. Infection with one DENV serotype is believed to 2. INCA - Instituto Nacional do Câncer, Praça induce protective immunity against homotypic infection. Cruz Vermelha, 23, Centro, Rio de Janeiro - RJ, 20230-130 However, immunity to one DENV serotype may be related The main cause of cervical cancer in the world is to more severe disease in heterotypic infection through high risk human papillomavirus infection (mainly a process believed to be associated with antibody- represented by HPV-16 and HPV-18), that are associated dependent enhancement (ADE) of infection. Juiz de Fora to the development of malignant transformation of the (JF) is a medium size city and considered the regional epithelium. HPV prevalence exhibits a wide geographical capital and the main reference for health care of macro variability and HPV-16 variants have been related to region Zona da Mata, in Minas Gerais, Brazil. JF has been an increased risk of developing cervical intraepithelial facing dengue epidemics since 2010, with reports of lesion. The aim of this study was to describe DNA-

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HPV prevalence and HPV-16 variants among a women St. Louis encephalitis virus (SLEV), a member of the population from Northern Brazil. Methods: One hundred Flaviviridae family, Flavivirus genus, is a causative agent and forty three women, during routine cervical cancer of encephalitis in the Americas. In Brazil, sporadic cases of SLEV infection have been reported since 1953, but the inquiry and were screened through a molecular HPV test.screening, HPV-16 at Jurutivariants Project, were fulfilleddetermined an epidemiological by sequencing 2007, concomitant to a Dengue virus (DENV), serotype 3 the HPV-16 E6 open reading frame. Results: Forty two first outbreak of SLEV in Brazil was identified only in samples were considered HPV positive (29.4%). None that SLEV circulation in Brazil is largely unknown, and of those had abnormal cytology results. HPV prevalence thereoutbreak. may This be finding,epidemiological along with implications other reports, of indicates the co- varied between different age groups (Z(U) = 14.62; p = <0.0001) and high-risk HPVs were more frequent among younger ages. The most prevalent type was HPV- sequencecirculation of ofa SLEV SLEV, strain DENV isolated and from other a flaviviruseshuman patient in inBrazil. Brazil, Here, strain we describeBeH 355964. the firstPhylogenetic complete analysis genome as European (87.5%). Conclusions: HPV prevalence in was performed to determine BeH 355964 genotype our16 (14%) population and it variantswas higher were than classified, described predominantly, by others using the full-length genome and the envelope (E) gene and the most prevalent HPV types were high-risk HPVs. The European HPV-16 variant was the most prevalent 355964 as within genotype V. Although the number of among HPV-16 positive samples. Our study reinforces singlesequences, gene sequences separately. available Both analyses is greater classified (such as BeHthe the fact that women with normal cytology and a positive E gene), but the complete genome phylogenetic shell molecular test for high-risk HPVs should be submitted offers best supports and provides further information to continuous follow up, in order to verify persistence about the virus. of infection, promoting an early diagnosis of cervical cancer and/or its precursors. HV57 - PROTEOME CHANGES IN A549 CELLS INFECTED WITH HADV-40 HV53 - FIRST COMPLETE GENOME SEQUENCE OF Guissoni, A.C.; Parente, A.F.; Badr, K.; Souza, K.; SAINT LOUIS ENCEPHALITIS VIRUS (SLEV) ISOLATED Soares, C.; Cardoso, D. FROM A HUMAN HOST IN BRAZIL UFG - Universidade Federal de Goiás, Av. Esperança, 1,2 3 4 Vedovello, D. ; Drumond, B.P. ; Rafael, E. ; Ullmann, s/n - Setor Itatiaia, Goiânia - GO, 74001-970 L.S.2; Favaro, E.A.1; Terzian, A.C.B.1; Figueiredo, L.T.M.5; Teixeira, M.M.4; Araújo Jr, J.P.2; Rahal, P.6; Nogueira, M.L.1 to the host cells. Determination of these changes can beThe observed viral infection from cell-virus may cause relationship a lot of modificationswhich is not 1. FAMERP - Faculdade de Medicina de São important only in the direction of viral biology, but also José do Rio Preto,Av. Brigadeiro Faria Lima, 5416 - Vila São at the cellular, which support to elucidate of various Pedro, São José do Rio Preto - SP, 15090-000 phenomena, including the discovery of new targets for 2. UNESP - Universidade Estadual Paulista, antiviral therapy. In the context of viral infection, which Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010 considered one of the fundamental possible aspects that 3. UFJF - Universidade Federal de Juiz de Fora, lead to protein changes in functional levels for many cells R. José Lourenço Kelmer - Martelos, Juiz de Fora - MG, 36036- 330 such as, cell signaling pathways, protein degradation, 4. UFMG - Universidade Federal de Minas apoptosis, and cytoskeletal rearrangement. These Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, proteins are extremely important since it responsible Belo Horizonte - MG, 31270-901 for many of biological functions of the cell. Thus, the 5. USP - Universidade de São Paulo, Av. Prof. viruses are evolved to reverse cellular proteins for their Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 6. UNAERP - Universidade de Ribeirão Preto, proper release. For the study of these protein changes Av. Costábile Romano, 2201, Ribeirania. Ribeirão Preto - SP, thatbenefit, induced which by includes viruses, maintenance, proteomics analysis, replication which and 14096-000 has been widely used to study these changes, was our

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

121 Human Virology: HV methodology tool that used in this study. In this context, Gerais state often presents high indices of dengue. The we infected A549 cells (human lung carcinoma) with city of Juiz de Fora, MG, has been facing dengue epidemics HAdV-40 to observe the cellular proteins differentially since 2009/2010, with the record of severe cases and which expressed after viral infection (treated) versus to deaths. This work aimed to investigate the circulation of uninfected cells (control). After this, the proteins were dengue virus (DENV) in clinical samples from patients with clinical symptoms of dengue, in 2012 and 2013, method in Nanodrop, followed by digestion. The using molecular and serological approaches. Samples of extracted bypeptides osmotic were lysis separated and quantified on the by LCthe system Bradford in serum from patients were used for total RNA extraction, two dimensions, nanoUPLAC (Waters) coupled to a mass and the total RNA was used for detection of DENV by RT- spectrometer Q-TOF (Syanpt, Waters). The ionization PCR. Clinical samples from patients who had fever for up of the peptides was performed on nano-eletronspray to six days were also tested for Dengue virus NS1 protein. Furthermore, analysis of patient records was performed in which we could check the day the fever began, the 321source differentially in positive expressedmode (nanoESI+) proteins and were examined observed, by 202MS, hematological data (hematocrit, total leukocyte count, fromaiming the to treated identification and 119 andfrom quantification control. The differentially of proteins. and platelet count) and serologic response (IgM and/ or IgG). A total of 166 serum samples were analyzed following functional categories: regulation of cellular for molecular detection, being six DENV positive (one immunity,expressed proteinsmechanisms of the of treated signal were transduction, classified intocellular the metabolism, protein degradation, cell organization, cell samples were DENV-2 positive). Sixty clinical samples cycle, and DNA processing. These data suggest that, the wereserum tested sample for wasNS1 DENV-1and 11 positive(18.33%) and were five positive. serum infection of A549 cells with HAdV-40 caused changes in From the analysis of patient records 39 (29.32%) were the cellular machinery and prioritizing pathways that IgM positive, nine (6.77%) were IgG positive and 15 (11.28%) were IgM and IgG positive. In addition, from 109 patients hematological data were available. Among promoteHV58 efficient - MOLECULAR viral progeny. AND SEROLOGICAL these, 18 (16.51%) patients had less than the reference INVESTIGATION OF DENGUE INFECTION, IN JUIZ DE value, 32 (29.35%) developed leukocyte hematocrit FORA, MINAS GERAIS below 3.500/mm3 and 25 (22.94%) had platelet counts Botelho, J.1; Mendonça, L.A.1; Siqueira, T.R.1; below the reference value. The results indicated the co- Sacchetto, L.P.1; Rezende, I.M.1; Fernandes, G.C.1; circulation of serotypes 1 and 2 in Juiz de Fora and the Kroon, E.G.2; Drumond, B.P.1 combination of molecular and serological tests allowed 1. UFJF - Universidade Federal de Juiz de Fora, R. José Lourenço Kelmer - Martelos, Juiz de Fora - MG, 36036- of the disease and 22 in the convalescent phase. The 330 knowledgethe identification of viral oftypes 50 circulating patients in in thethe municipality acute phase 2. UFMG - Universidade Federal de Minas and the diagnosis of dengue in patients using different Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, techniques constitute valuable information to control Belo Horizonte - MG, 31270-901 dengue. Financial support: FAPEMIG, CNPq, CAPES, UFJF, Dengue is the most important arboviral disease PROPESQ/UFJF. caused by Dengue virus (DENV), family Flaviridae, genus Flavivirus. DENV comprises four genetically and antigenically distinct serotypes, termed DENV-1 through DENV-4. Infection by any DENV serotype can cause a wide range of clinical symptoms that vary from classic dengue to life-threatening syndromes characterized by hemorrhage and capillary leakage, dengue hemorrhagic fever and dengue shock syndrome. Brazil has the highest incidence of dengue cases in the Americas and Minas

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HV59 - MOLECULAR CHARACTERIZATION OF in patients on hemodialysis can be related to host HEPATITIS C VIRUS IN END-STAGE RENAL DISEASE immunological features and geographical patterns. PATIENTS UNDER HEMODIALYSIS HV64 - DEVELOPMENT OF REAL TIME PCR de Carvalho, I.M.V.G.1; Alves, R.2; Bernardino, J. de PLATFORM FOR VIRAL MENINGOENCEPHALITIS S.T.2; Feldner, A.C. de C.A.2; Ferraz, M.L.C.G.2; Silva, DIAGNOSIS AND MOLECULAR EPIDEMIOLOGY OF A.E.B.2; Filho, J.R.C.2 MENINGOENCEPHALITIS AT CHILDREN HOSPITAL IN 1. Instituto Butantan, Av. Vital Brasil, 1500, MINAS GERAIS STATE, BRAZIL Butantã, São Paulo - SP, 05503-900 Oliveira, D.1; Candiani, T.2; Almeida, G.1; Luiz, A.P.F.1; 2. Departamento de Gastroenterologia/ Alvarenga, P.1; Castro, F.1; Abrahão, J.1; Coimbra, R.1; UNIFESP - Universidade Federal de São Paulo, R. Sena Kroon, E.1 Madureira, 1500 - Vila Mariana, São Paulo - SP, 04021-0 1. UFMG - Universidade Federal de Minas Several new direct-acting antiviral agents are in Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, development or already approved for the treatment of Belo Horizonte - MG, 31270-901 chronic hepatitis C virus (HCV) infection. The effectiveness 2. FHEMIG - Fundação Hospitalar do Estado of many of these drugs is still dependent on whether de Minas Gerais, Alameda Vereador Álvaro Celso, 100, Santa resistance-associated variants pre-exist before therapy. Efigênia, Belo Horizonte - MG, 30150-260 Certain clinical conditions can change immunological Meningitis is a worldwide disease, which main etiologic characteristics of the host and modify genetic features of agents are viruses, bacteria and fungus. Viruses are the viral populations. The aim of this study was to perform main causes of central nervous system (CNS) infection a molecular characterization of HCV isolates from end- around the world. In Brazil, there are 11,500 cases stage renal disease (ESRD) patients under hemodialysis / year putative reported cases of viral meningitis. (HD). Nested PCR and Sanger sequencing were used to obtain genetic information on the NS5B region from etiological agent. Human Herpesvirus 1 (HHV-1), Human a cohort of 73 treatment-naïve subjects with chronic HerpesvirusHowever, for 2most (HHV-2), cases Humanthere is Herpesvirus no identification 3 (HHV-3), of the HCV infection was selected (31 ESRD-HD and 42 with viruses from the Enterovirus genus (ENTV) and viruses normal renal function). Genomic sequences from the Los from the Flavivirus genus(FLAV) are the main etiological Alamos databank were used for comparative analysis. agents of viral meningitis. The objective of this work Bioinformatics methodologies such as phylogenetic was to develop a real-time PCR platform for HHV 1, HHV reconstructions and informational entropy were used 2, HHV 3, ENTV and FLAV diagnosis in cerebrospinal to analyze datasets separately by geographical location, HCV genotype and renal function status. ESRD-HD meningitis and apply that platform on CFSs collected of patients presented HCV genotypes 1a (n=14), 1b (n=12), hospitalizedfluid (CSF) ofchildren patients from with Hospital clinical InfantilJoão suspect ofPaulo viral II 2a (n=1), 2b (n=2) and 3a (n=2). Control subjects were (HIJPII). Primers were designed (or based on literature), infected with genotypes 1a (n=9), 1b (n=21), 2b (n=4) targeting conserved regions in the genome of these virus and 3a (n=8). Each genotype exhibited typical branching (HHV 1, HHV2 and HHV 3)or genus (ENTV and FLAV). patterns, already described. Separation of HCV subtype The viral isolates HHV 1 EK, HHV 2 (ATCC VR 590), HHV 3 1a into two distinct clades was observed in most HC05, Enterovirus C (Sabin) and Yellow fever virus(17D Brazilian sequences, in contrast to subtype 1b isolates. strain) were used during the initial primer tests. Then The entropy analysis of genomic sequences from the 57 samples of CSFs(10ul -140 ul) from patients with ESRD-HD group reveled two amino acid peak positions meningoencephalitis of probably viral etiology were which were absent in most control sequences. These collected from 2010 to 2013 at HIJPII for tested in our amino acid positions are closely related to a particular platform.The reactions used in this platform showed epitope for cytotoxic T lymphocytes and T helper cells recognition (CCDLDPQARVAI; positions 2663–2673 HHV -3, 110% ENTV and 92% FLAV.When an analytical sensitivityhigh efficiency114% test was performed, for HHV-1 our and data HHV-2, demonstrated 113% for in the H77 reference sequence). The identification of September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV specific mutant epitopes within the NS5B HCV protein XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

123 Human Virology: HV high sensitivity, with detection limit up to 1 PFU/µl for neurological manifestation as confusion and behavior HHV 1, ENTV and FLAV. When the samples were tested alterations. With the aim of characterizing the etiological our platform was able to identify the viral etiologic agent in 71.9% of cases (41 cases). Among these positive were sent to our laboratory. The samples were submitted samples 29(50.87%) were positive for ENTV, 6 for HHV-3, toagent, RNA cerebrospinal extraction (Viral fluid (CSF)RNA samplesQIAamp from Extraction the patient Kit, 5 for FLAV (4 DENV-2 and 1 co-infection DENV-2/DENV- Qiagen, USA) followed by reverse transcription using 3) (8.7%) and 1 positive for HHV-1/2 (1.7%). ENTV was random primers (MMLV, Promega, USA). The cDNA most prevalent agent what is consistent with others was used as the template in a real time PCR designed works in the literature. The detection of DENVs in 8.7% of to amplify the NS5 region of virus of genus Flavivirus. cases is an important result because these are emerging neurotropic agents. Finally this platform could be useful for the rapid diagnosis of viral meningoencephalitis. (CSF).Specific To amplification identify the inmember the real oftime genus PCR Flavivirus with a melting that temperature of 79,5ᴼ C was observed for the sample is important for therapy, severe cases surveillance and viralThe identificationemerging agents of viral surveillance. agent of neurologic infection thewas etiological the etiological agent.The agent optimalof such infection, alignment a ofspecific the C-prM PCR regionfor Dengue using virusClustalW was and performed MEGA version confirming 4 showed DENV high as HV65 - DENGUE VIRUS 3 GENOTYPE I INFECTION identity among the nucleotide sequences of the sample WITH NEUROLOGICAL MANIFESTATIONS, BRAZIL and other DENV-3sequences deposited in GenBank. Oliveira, D.1; Machado, G.3; Almeida, G.1; Abrahão, J.1; Phylogenetic trees of the C-prM region were constructed Campos, M.2; Kroon, E.1 and showed that the DNA fragment sequence clustered 1. UFMG - Universidade Federal de Minas with other sequences of DENV-3genotype I from Brazil, Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, Asia and Central . Neurological manifestations of DENV Belo Horizonte - MG, 31270-901 are possibly underreported in Brazil, this report serves 2. FIOCRUZ MINAS - Fundação Oswaldo as an alert to the importance of DENV diagnosis in CNS Cruz, Av. Augusto de Lima, 1715 - Barro Preto, Belo Horizonte infections. The number of cases and the impact in public - MG, 30190-002 health maybe more important than suspected because 3. Hospital Risoleta Tolentino Neves, Rua das Gabirobas, 1 - Vila Clóris, Belo Horizonte - MG, 31744-012 viral infection in Brazil. the viral pathogen is not identified in most cases of CNS Dengue virus(DENV) is the most important mosquito- HV66 - GENOTYPING AND PHYLOGENY OF DENGUE borne disease of humans. Worldwide approximately VIRUS 1 AND DENGUE VIRUS 4 IN DIVINÓPOLIS/MG, 2,5 billion of people live in areas with risk of dengue BRAZIL, 2013 transmission and estimated 50 million dengue infections 1 1 1 occur annually. In Brazil more than 200.000 cases were Dutra, K.R. ; Corrêa, V.M. ; Lopes, D.O. ; Drumond, B.P.2; Nogueira, L.M.3; Ferreira, J.M.S.1; Santos, L.L.1 habitants).The distinct DENV (1,2,3 and 4) might be 1. Laboratório de Biologia Molecular da usuallyreported associated in the first 45to daysa systemic of 2013 and (105,5 dynamic cases/100.000 disease. Universidade Federal de São João Del-Rei (Campus Centro Among the plethora of clinical symptoms caused by DENV, Oeste Dona Lindu) 2. Laboratório de Virologia da Universidade or neglected. In this work we describe a case report Federal de Juiz de Fora ofneurological a patient manifestationspresenting neurological has been manifestations undernotified 3. Laboratório de Pesquisa em Virologia, likely associated to DENV-3.This case occurred in Belo Departamento de Doenças Dermatológicas, Infecciosas e Horizonte, Minas Gerais State, Brazil. In October 2012, Parasitárias, Faculdade de Medicina de São José do Rio Preto our lab was contacted by a local Hospital to perform (FAMERP) the diagnosis of a 21 old patient that presented dengue Dengue is a disease transmitted to humans through the symptoms and also neurological symptoms. The patient bite of infected female mosquitoes of the genus Aedes. stayed in hospital for 27 days presented wide range of The Dengue virus (DENV) is an enveloped virus belonging

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

124 Human Virology: HV the family Flaviviridae with four distinct serotypes: HV75 - DETECTION OF NOROVIRUS RECOMBINANT DENV1, DENV2, DENV3, DENV4. Currently, these four STRAINS ISOLATED FROM ACUTE GASTROENTERITIS serotypes are circulating in almost all states of Brazil and CASES IN BRAZIL an increase in genetic variability between each serotype Fumian, T.M.; Alves, R.; Assis, M.; Simões, M.; Andrade, has been described. In Minas Gerais, the number of cases J.; Leite, J.P.G.; Miagostovich, M.P. has been grown, therefore the genotyping will provide information on dengue virus distribution in human FIOCRUZ MINAS - Fundação Oswaldo Cruz, Av. Augusto de Lima, 1715 - Barro Preto, Belo Horizonte - MG, populations over time and place. Using phylogeny studies 30190-002 is possible to identify mutations and recombination events accumulated over the years. The most common Norovirus (NoV), a major cause of acute gastroenteritis gene used in studies of molecular epidemiology of DENV (AGE) outbreaks worldwide, are constantly evolving. This is E gene, due to the importance of its product in the binding and entry of the virus into the cell, especially in these viruses spread and remain in human population. ability is reflected in the speed and efficiency with which the humoral response of the human host. Mutations in The present study aimed to investigate the presence of this gene may be related to the increased virulence of viral recombination events – one of the main driving forces strains. In this study, 100 samples of whole blood from that shapes NoV evolution – among different genotypes patients with suspected dengue, from Divinópolis/MG , in Brazil. Fecal samples were tested using primers in the year 2013 were analyzed. Viral RNA was extracted targeting region B (ORF1) and region D (ORF2) of NoV from the serum with QIAamp ® Viral RNA Kit ( QIAGEN ®, USA) and viral detection was performed by reverse positive results with both methodologies were subjected genome by using specific primers. Samples yielding transcription (RT - PCR). The serotyping was performed to investigate the possibility of a recombination event, by sequencing the viral fragment of 511 pairs obtained by RT - PCR. The sequences obtained were compared Mon 431/432 and G2SKR. Amplicons obtained were and ORF1/2 junction region was amplified with primers to reference sequences deposited in GenBank using the BLASTN software. Of the 100 samples analyzed, 26 were Reaction Kit® and an ABI Prism 3130xl DNA sequencer. purified and sequenced using the Big Dye Terminator positive for DENV. 12 samples were sequenced, ten were positive for DENV 1 and two positive for DENV 4. One was made according to the Noronet (http://www.rivm. nl/mpf/norovirus/typingtool)Assignment of the strain to specificand the NoV phylogenetic genotypes for each serotype to amplify the envelope protein gene. analysis was performed using MEGA version 5.05. In Phylogeneticsecond RT - PCRanalyzes was areperformed being performed with specific by MEGA primers 6.0 total, we analyzed 37 samples, and found recombination software. These results explain the epidemic occurred in events in 19 (51%) of these. We detected eight different the city, since DENV1 do not circulate in the municipality combinations of NoV recombinant genotypes, being: GII. for almost 10 years, and there was no report of circulation Pe/GII.4 (n=12); GII.P7/GII.6 (n=7); GII.P7/GII.14 (n=1); of DENV4 in Divinópolis history before 2013. The results GII.Pe/GII.17 (n=2); GII.P13/GII.17 (n=1); GII.P21/GII.3 (n=1); GII.Pg/GII.12 (n=5); GII.P16/GII.3 (n=2). These of DENV in Brazil and its relations with DENV isolated fromof this other work willcontinents, improve besidesthe evolution the determination analyze and flow of results were confirmed by phylogenetic and Simplot circulating genotypes in Divinópolis. This study can be breakpoint located near the ORF1/2 overlap. Our study analysis with references strains, and identified the applied to surveillance programs of health services and revealed a high circulation of NoV recombinant strains in preventive interventions. This study will also provide Brazil, revealing that this type of event is not uncommon the screening of molecular markers, associating them among divergent genotypes. In addition, we highlight with the pathogenicity and immunogenicity of DENV. the importance of this mechanism for successfully NoV Financial support: FAPEMIG maintenance and spread in human population. To our

concerning NoV recombination from cases of AGE in Brazil.knowledge, this is the first description of a large study

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HV76 - MOLECULAR INVESTIGATION OF DENGUE isolates in this study and other isolates from Brazil VIRUS INFECTION IN GOIANIA, GOIAS, BRAZIL, 2012– and Latin America was observed. Therefore, molecular 2013 monitoring is important to establish a complete data base Oliveira, A.C.R.; Guimarães, V.N.; Cunha, M. dos P.; for surveillance studies in hyperendemic population, Souza, M.B. de L.D.; Cardoso, D. das D. de P.; Almeida, collaborating with investigations of the virus dynamics T.N.V.; Fiaccadori, F.S. and the prospective pattern of future epidemics. IPTSP/UFG - Instituto de Patologia Tropical e Saúde HV79 - HANTAVIRUSES VERSUS DENGUE DISEASE: Pública/ Universidade Federal de Goias, Rua 235 - s/n, Setor RETROSPECTIVE DIAGNOSIS OF A HANTAVIRUSES Universitário, Goiânia - Goiás, 74605050 CASE CLINICALLY MISTAKEN WITH DENGUE DISEASE Dengue is an important public health problem da Costa, V.G.; Silva, A.C.M.; Floriano, V.G.; Moreli, M.L. worldwide and it is spread for tropical and subtropical UFG - Universidade Federal de Goiás, Av. Esperança, countries. Dengue virus (DENV) is an arbovirus s/n - Setor Itatiaia, Goiânia - GO, 74001-970 as DENV-1-4. Since the introduction of DENV-1 in Brazil, theclassified epidemiological in four serologically scenario related is characterized viruses designated by the this context, we can include diseases caused by dengue Viral diseases result in nonspecific clinical picture. In co-circulation of four DENV, resulting in successive virus (Family Flaviviridae) and hantavirus (Family epidemics with alternating prevalence among serotypes. The pathogenesis of this infection has been associated picture, or a more severe disease, and it is constantly Bunyaviridae). The dengue causes a flu-like clinical with the occurrence of a cross-serotype immune associated with epidemics in urban areas. Conversely, the hantaviruses is primarily a rural disease, it is of serotypes circulating as well as the characterization of transmitted to man by inhalation of aerosols waste from theresponse genomic in secondary variants infections.have been Thus,considered the identification important infected wild rodents. The cases of hantaviruses occur perspectives on disease control, since the introduction of sporadically, and, as well as dengue fever, there is seasonal new variants can be a risk factor for severe dengue. In this distribution. In view of this, our objective was to report context, the aim of this study was perform a molecular a case of hantavirus disease that went unnoticed in investigation of DENV infection in Goiania, Goias, during municipality of Jataí-Goiás. Thus, the present study was approved by the ethics committee of Federal University of serotypes/genotypes of the virus. 278 samples were of Goiás (n°348/2010). Blood samples of 202 patients collectedthe epidemic from period patients 2012- with 2013, symptoms and the of identificationdengue fever suspected dengue were screened, retrospectively, which in basic health centers, which were submitted to RNA were collected between the years of 2012 and 2013. extraction followed by a hemi-nested RT-PCR for the Thus, the samples were stored in the serum bank of C-prM genome region to detect the viral RNA and to the medical center municipal health from Jataí. For the identify the serotype. The molecular characterization processing of the samples was used an in house ELISA was performed for all RNA positive samples by using a recombinant N protein of Araraquara virus. nucleotide sequencing of the junction region E/NS1 Thus, was found a case with positive anti-hantavirus IgM and phylogenetic analysis. It was observed a positivity antibody. The patient was a 44-year-old Brazilian male rate of 20.5% (57/278). From the 57 DENV samples, 18 with fever and myalgia presented. Sample collection (31.6%) were DENV-1 and 39 (68.4%) DENV-4, which occurred on March 18, 2012. For this case the optical density was 0.92, which was obtained in triplicate, while that demonstrated this co-circulation with a DENV-4 the cut-off value was 0.12. The detection of anti-dengue predominanceis in agreement in withepidemiological findings from surveillance Brazil and of 2013. Goias IgM antibodies by ELISA was negative. Regarding to laboratory data obtained, during hospital admission, was found abnormal, only mild thrombocytopenia thePhylogenetic occurrence analysis of genetic classified variants the DENV-1with differences samples asin (115x103/mm3), but two days after was observed genotype V, with two different clades, which may reflect elevated hematocrit (64%), thrombocytopenia as genotype II, a phylogenetic proximity between the (89x103/mm3) and leukocytosis, however without left evolutionary history. For DENV-4 samples, classified September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

126 Human Virology: HV shift (segmented neutrophils=11218/mm3). Finally, in asthmatic and non-asthmatic pediatric population after supportive symptomatic treatment the patient from Goiania-Goias, as well as to evaluate the possible progressed to the stage of convalescence. The record role of these infections in asthma exacerbations. The study enrolled four to fourteen-old children from four diagnosis of hantavirus, since that laboratory tests healthcare centers, divided into three groups according of this case shows the difficulty in the laboratory to the clinical symptoms and the physician’s evaluation: central public laboratories (LACEN). However, this may stable asthma, exacerbated asthma and just ARI. delayrequire diagnosis a qualified or diagnosticdiscourage referenced that samples system, are sent as the to Between August/2012 through August/2013, 225 nasal diagnostic centers, mainly because of hantaviruses to samples were collected (aspirates – 193, nasal swabs – be a severe acute disease. In conclusion, this case of 32). For the molecular screening, a Multiplex Nested-PCR hantavirus disease, retrospectively diagnosed, serves as a warning and indicates that the number of cases of targeting the nucleoprotein gene for FLU (FLUA, B and C)protocol and the was F gene performed, for RSV (RSVA using and specific B). It set was of observed primers country. a global detection rate of 5.77% (13/255), being FLUA disease can be been significantly underestimated in the HV81 - INFLUENZA VIRUS AND RESPIRATORY and four (4/13 – 30.76%) positive samples respectively. SYNCYTIAL VIRUS INFECTIONS AMONG Mostand RSVA of the the positive most prevalent, samples withbelonged five (5/13 to the – group38.46%) of EXACERBATED ASTHMATIC CHILDREN FROM exacerbated asthmatic children (7/92 – 7.6%) and no GOIANIA-GOIAS coinfection cases were detected. The results indicated the Castro, Í. de A.1; Costa, L.D.C.2; Oliveira, A.C.R.1; de circulation of viral pathogens among asthmatic children, Souza, K.M.C.1; Cardoso, D. das D. de P.1; Souza, M.B. especially among asthma exacerbation cases, although de L.D.1; Fiaccadori, F.S.1 the comparison between the population groups was 1. IPTSP/UFG - Instituto de Patologia Tropical e Saúde Pública/ Universidade Federal de Goias, Rua 235 - broader assays including other frequent respiratory s/n, Setor Universitário, Goiânia - Goiás, 74605050 virusesnot statistically will improve significant. the knowledge Hence, further of viral analysis ARIs in with this 2. FM/UFG - Faculdade de Medicina da Universidade Federal de Goias, Faculdade de Medicina, 235 treatment guidelines. c/ 1a. s/n, S. Universitário,Goiânia - Goiás, 74605-020 specific population, and help the establishment of new HV91 - VIRUS SURVEILLANCE IN PATIENTS WITH Acute respiratory infections (ARIs) are among the most CLINICAL DIAGNOSIS OF DENGUE FROM A MEDIUM- SIZED CITY OF RONDONIA STATE, BRAZIL childhood worldwide. Many aetiological agents are Pereira, W.V.E.G.; Gusmão, A.F.; Guioti, F.; Ferreira, relatedsignificant with causes ARIs, ofalthough morbidity more and than mortality 80% of in these early A.C.; Mondini, A. Virus (FLU) and Respiratory Syncytial Virus (RSV). The FCFAR/UNESP - Faculdade de Ciências Farmacêuticas symptomsinfections arerange caused from bydry viruses, cough, mainlywheeze, by runny Influenza nose da Universidade Estadual de São Paulo, Rodovia Araraquara and fever to severe episodes of pneumonia. Asthma - Jaú Km 1, Araraquara - SP, 14801-902 is a chronic condition that affects a large number of The infection by any of the four serotypes of the dengue children and its emergence has been associated with an virus (DENV 1-4) can be asymptomatic or produce interaction of genetic predisposition and environmental a disease that ranges from a mild febrile illness to life factors. Furthermore, episodes of asthma exacerbation, threatening hemorrhagic manifestations. In Brazil, the clinical condition with rising prevalence over the majority of dengue disease diagnostics are obtained by past few years, may be related to respiratory viral serology and clinical and epidemiological criteria. Virus infections. Considering ARIs as a key factor of asthma exacerbations, there are few studies in Brazil reaching reaction or the detection of the nonstructural protein the pediatric population. In this context, this study aimed 1isolation, are usually identification performed ofin DENVreference by polymeraselaboratories chainfor a to investigate the occurrence of FLU and RSV infections limited number of cases. No further tests to detect other

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

127 Human Virology: HV arthropod borne viruses (arboviruses) are performed identify the phylogeny pattern of the virus circulating in febrile patients. In our study, we are testing samples in the epidemic period of 2013, using the complete with clinical diagnostic of dengue from a medium sized sequence of envelope gene (E gene). DENV-1 positive city from the west part of Brazil for DENV 1-4 and samples were subsequently subjected to RNA extraction alphaviruses. We collected blood samples from febrile by the “primer walking” method for the sequencing of the patientsother flaviviruses, that had clinical along with diagnostic orthobunyaviruses of dengue from and entireand RT-PCR envelope amplification gene (1485bp). using AllDENV-1 nucleotide specific sequences primers August 2013 to January 2014 in Ji-Paraná (Rondonia), of Goiás were aligned with other sequences available which is allegedly an area with the circulation of at the GenBank, representing all DENV-1 genotypes, several arboviruses. Viral RNA was extracted and a using the ClustalW algorithm and edited using Jalview to detect members of the Flavivirus, Alphavirus and was inferred with jModelTest program according the Multiplex-Nested-RT-PCR with genus-specific primers Akaikesoftware. information The evolution criterion model that(AIC) best and fits a theNeighbor- dataset to identify DENV 1-4, yellow fever virus, mayaro virus, Joining method available in the software MEGA6 was oropoucheOrthobunyavirus virus and genera seven and other species-specific viruses was performed. primers used in order to analyze the phylogenetic relationship. We were able to collect 103 blood samples for the study. The analyzes demonstrated that all isolates of DENV-1 So far, we have tested 12 samples and we could not detect Goiás belong to genotype V. The set of isolates form two DENV or any other arboviruses. However, 89% of the clades within the genotype V, revealing the co-circulation samples still remain untested. It is undeniably important to provide differential diagnostic for febrile illnesses, not sequences isolated in Rio de Janeiro and Espírito Santo only for a better management of the patients but also to -of 2010, two distinct while thelineages. second The is firstformed clade by is isolatescomposed from of understand the epidemiology of the viruses that may be Americas countries like Venezuela - 2006 and Puerto circulating in a certain location and the control measures Rico - 2010, and together other Brazilian states such as that should be taken to control their spread. Roraima - 2009, Pernambuco - 2010 and Rio de Janeiro - 2011. Furthermore, these results indicate that despite HV93 - CO-CIRCULATION OF TWO DIFFERENT an endemic viral serotype/genotype in a particular LINEAGES OF DENGUE VIRUS TYPE 1 IN WEST geographic region over a prolonged period, this does CENTRAL REGION OF BRAZIL not mean that only one variant are associated with Cunha, M. dos P.; Guimarães, V.N.; Souza, M.B. de L.D.; this condition, revealing the role of co-circulation and Cardoso, D. das D. de P.; Castro, Í. de A.; de Souza, replacement lineages as responsible by hyperendemicity K.M.C.; Fiaccadori, F.S. IPTSP/UFG - Instituto de Patologia Tropical e Saúde of co-circulation of two lineages of DENV-1 detected in dengue in urban centers isolates. This is the first report Pública/ Universidade Federal de Goias, Rua 235 - s/n, Setor West Central region of Brazil. Universitário, Goiânia - Goiás, 74605050 HV99 - MONITORING OF CYTOMEGALOVIRUS Dengue virus (DENV) is the most important arboviral INFECTION IN RENAL TRANSPLANT RECIPIENTS pathogen in the world and has been spread throughout Costa, A.P.F.1; Souza, L.M.S.1; Joventino, K.M. de S.1; the tropical and subtropical regions. It is a positive single Santos, W.K. da S.1; Pereira, M.G.1; Quintana, V.H.A.2; strand RNA virus belonging to genus Flavivirus, family Araújo, J.M.G.1; Freitas, J.C. de O.C.1; Farias, K.J.S.1; Machado, P.R.L.1 inFlaviviridae, the years 80, classified and after a inlow four or silent antigenically co-circulation distinct with 1. UFRN - Universidade Federal do Rio Grande otherserotypes serotypes, DENV-1-4. DENV-1 In Brazil, re-emerged DENV-1 in was 2008 first becoming detected do Norte, Campus Universitário Lagoa Nova, Natal - RN, predominant in later years. Nevertheless, the population 59078-970 structure of the viruses transmitted in this region is not 2. USP - Universidade de São Paulo, Av. Prof. Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 well understood. In this context, the phylogeny analysis of DENV-1 strains from Goiás was performed in order to

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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The cytomegalovirus (CMV) is responsible for high HV102 - LACK OF ASSOCIATION OF TNF-ALPHA morbid-mortality in solid organs transplanted patients AND IL-6 GENES METHYLATION WITH CLINICAL and it is associated with the increase in the predisposition OUTCOME IN DENGUE INFECTED PATIENTS to acute and chronic rejection to allograft. The objective Morais, S.M. de S.1; Gomes, A.V.B.T.1; Filho, S.L. de M.1; of this study was to carry out the monitoring of an Ferreira, J.M.S.2; Malaquias, L.C.C.1; Coelho, L.F.L.1 infection by CMV through the polymerase chain reaction (PCR) in mononuclear peripheral blood cells (PBMC) 1. UNIFAL - Universidade Federal de Alfenas, R. Gabriel Monteiro da Silva, 714 - Centro, Alfenas - MG, samples and serum from patients who underwent renal 37130-000 transplant in the Onofre Lopes University Hospital 2. UFSJ - Universidade Federal de São João del- (HUOL), Natal, Rio Grande do Norte. In the prospective Rei, Praça Frei Orlando, 170, Centro, São João del-Rei, Minas study 45 patients who underwent the renal transplant at Gerais, 36307-352 HUOL between February 2013 and January 2014 were included. Peripheral blood was collected on the 2nd, 4th, Dengue virus (DV) is an enveloped virus, positive 8th, 12th and 24th weeks after the transplant to obtain single-stranded RNA, transmitted to humans through the PBMC and serum. The DNA extraction was carried the bite of infected female Aedes aegypti mosquito. out according to the QIAamp® DNA kit protocol and the There are two main clinical manifestations caused by molecular detection of CMV according to the nested- DV infection named Dengue Fever (DF) and Dengue PCR. It was observed in the pre-transplant that 93.3% Hemorrhagic Fever (DHF). DNA methylation is the best (42/45) presented IgG anti-CMV antibodies, being that great importance in silencing and gene regulation. In characterized epigenetic modification in DNA exerting and 6.6% IgG-/IgM-. In relation to the CMV molecular eukaryotes, it is found predominantly in the carbon 5 detection89% of the in receptors serum and were PBMC IgG+/IgM-, samples, 4.4% 62.2% IgG+/IgM+ (28/45) position of cytosine followed by a guanine in the CpG presented positivity in PBMC, and 24.4% (11/45) positive when it occurs in regions rich in CpG dinucleotides dinucleotide. This modification is biologically relevant of the monitoring collections. Detection of CMV DNA in known as CpG islands, there are common in promoter serumresult both correlates in the PBMCwith retarded and in the graft serum, function in at least(27.3%), five regions of certain genes. This work aims to analyze the fever (45.5%) and gastrointestinal disease (54.5%). In relation to the monitoring of the infection by CMV, it IL-6 in symptomatic and asymptomatic dengue patients. Thesemethylation two cytokines status in were the promoter involved inregion DHF pathogenesisof TNF-α and collection presented positive detection. On the 4th week and the promoter methylation was investigated using thisobserved positivity that 2.7%increased of the to patients 10.5%, onincluded the 8th in week,the first to 16.6%, on the 12th week, there was a reduction to 11.4% extracted from 23 symptomatic and 18 asymptomatic methylation specific PCR (MS-PCR). The DNA was and on the 24th week, none of the patients presented positivity in the serum. In PMBC, the positivity found was treatment. The converted DNA was used to amplify the dengue patients and submitted to sodium bisulfite of 16.2% on the 2nd week, with an increase to 26.3% methylated and unmethylated regions using two sets on the 4th week, 36.1% on the 8th week and 45.7% on of different primers for each gene in MS-PCR reaction. the 12th week, with a reduction in the detection on the 24th week to 36.3%. Finally, the results demonstrated in 65,22% of symptomatic patients and in 66.67% of Promoter methylation of the TNF-α gene was detected an increased seroprevalence of IgG anti-CMV in the asymptomatic patients and, for the IL-6 gene, in 30.43 group studied, in relation to the molecular detection, an and 61.11% of symptomatic and asymptomatic dengue increased positivity was found in PBMC or serum, with the association between the detection of the viraemia in and IL-6 was not statistically different in symptomatic patients, respectively. Promoter methylation of TNF-α serum and the clinical manifestations associated with compared with asymptomatic cases (p values of 0.8267 the infection. Financial support: FAPERN.

IL-6for TNF- genes α and 0.6536clinical foroutcome IL-6). Ourin dengue findings infection. indicate no association between methylation status of TNF- α and

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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HV104 - CMV GLYCOPROTEIN B GENOTYPES HV107 - VISUAL INSPECTION OF THE CERVIX: OF CHILDREN WITH CONGENITAL INFECTION PRECISE, EFFECTIVE AND INEXPENSIVE METHOD HOSPITALIZED IN THE INTENSIVE CARE UNIT USEFUL IN THE CANCER PREVENTION IN PUBLIC Cardoso, E.S. de C.; Gadelha, S.R.; Marin, L.J. HEALTH UNITS FROM REGIONS WITH HIGH PREVALENCE OF HPV UESC - Universidade Estadual de Santa Cruz, Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Barreto, D.M.V.S.; Almeida, S.M.V.S.; Mariano, A.P.M.; Bairro Salobrinho Ilhéus-Bahia, 45662-900 Gomes, L.G. da S.; Costa, G.B.; Marin, L.J.; Gadelha, S.R. The cytomegalovirus (CMV) is the most common agent UESC - Universidade Estadual de Santa Cruz, Campus of congenital infection in the world. It is not clear why Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, some newborns with congenital CMV infection are Bairro Salobrinho Ilhéus-Bahia, 45662-900 symptomatic or not. One of the major glycoprotein The human papillomavirus (HPV) is responsible for of the virus is the glycoprotein B (gB). This protein is one of the most prevalent sexually transmitted diseases polymorphic and highly immunogenic. Most of the wild and is the major cause of death from cervical cancer viral strains are grouped in four major gB genotypes. worldwide. Development of simple and inexpensive Recognizing the vital role of this glycoprotein in virus- approaches to screening early lesions of cervical cancer host interaction, different genotypes can be associated in low-resource regions is essential and should be with virulence and different clinical outcomes. In fact, investigated. The aim of this study was to evaluate the it has been suggested that these genotypes could have use of the technique of visual inspection of the cervix for screening of cervical lesions and consequent prevention viral replication. In this study, it has been performed aspecific neonatal tissue screening tropism orfor some detection mechanism of symptomatic to facilitate for the visual inspection was the presence of white congenital CMV infection. It has been included all lesionsof cervical in thecancer. transformation The definition zone of anear positive the squamoustest result newborns under three week who were admitted to columnar junction (JEC), 1 minute after direct application the Intensive Care Unit of the Hospital Manoel Novaes, of a 5% solution of acetic acid. This technique increases in Itabuna, Bahia. Urine samples were collected in screening for cervical lesions without additional costs. collectors bags, and subsequently, saliva samples also For cervical cytology smears were prepared and stained were collected with sterile swabs. Samples of saliva and urine collected were subjected to PCR without prior System. The virus was detected by nested PCR. It was DNA extraction. It was used a positive and negative usedby Pap the smear primers and MY09 classified and accordingMY11, followed to the by Bethesda primer controls in all PCR reactions. Children with congenital infection will be assessed and monitored by a medical data of 195 women from public health units in southern team until the second year of life. If necessary, they will BahiaGP5+ andregion GP6+. were In analyzed. his study, Theclinical prevalence and epidemiological of HPV in the be treated. The genotypes of CMV glycoprotein B will be region was 47.7% by nested PCR and cytology detected 35.4% of intraepithelial lesions and atypical. The test will be carried out a real-time PCR. By the time, it was collecteddetermined samples by RFLP. of 17 For children. quantification There was of noviral positivity load, it positive predictive value, 85% and 83%, respectively. The in any samples. This fact can be explained because the performancevisual inspection was calculated showed highusing specificitythe standard and method high small number of children included in the study until this of detecting the nested PCR. In fact, it was observed that moment. Financial support: UESC the techniques (cytology, molecular detection and visual inspection) are complementary and it would be ideal that these methods could be performed together, in order to perform a better screening. However, in the basic health units and in areas with less resource, this is not feasible. The visual inspection method proved been effective in detecting lesions caused by HPV, an inexpensive tool and precise, demonstrating that this approach can be September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

130 Human Virology: HV included in a routine gynecological visit. In addition, this Acre. C. quinquefasciatus, the culicidae found in higher method can be applied in large-scale screening and may abundance in Cuiabá, is considered a secondary vector be an alternative to cytology exam (in our study, visual for OROV transmission. Culicoides paraensis, considered inspection showed better performance when compared the main vector for OROV, was not captured during the to cervical cytology). Moreover, its application provides a simple and safely early detection of subclinical infections from cities of Pará affected by Cuiabá-Santarém highway and premalignant lesions, reducing cases of invasive andstudy. in Serology primates to in OROV the wasPantanal already of identifiedMato Grosso in humans do Sul cancer and metastasis. Financial suport: PR OROV in Mato Grosso. *Financial support: CAPES/CNPq. HV110 - MOLECULAR DETECTION OF OROPOUCHE recently, corroborating the molecular identification of VIRUS IN MATO GROSSO, BRAZIL HV112 - SEROPREVALENCE OF DENGUE AMONG THE Cardoso, B.F.1; Serra, O.P.1; Heinen, L.B. da S.; Zuchi, ASSYMPTOMATIC INDIVIDUALS IN ALFENAS, MINAS, N.; dos Santos, M.A.M.2; Slhessarenko, R.D.1 BRAZIL Prado, A.A.O.; Gomes, A.V.B.T.; Rodrigues, N.F.; de 1. UFMT - Universidade Federal de Mato Grosso, R. Guadalajara, 1 - Jardim das Américas Cuiabá - MT Lima, M.M.; Malaquias, L.C.C.; Coelho, L.F.L. 78060-624 UNIFAL - Universidade Federal de Alfenas, R. Gabriel 2. MT-Laboratório Monteiro da Silva, 714 - Centro, Alfenas - MG, 37130-000 Oropouche fever is the second most frequent arbovirus Dengue is an arboviral disease transmitted to humans through the bite of infected female mosquitoes of region. Oropouche virus (OROV) is an Orthobunyavirus the genus Aedes. Among the most common clinical foundin notifications in neighboring in Brazil, states reported of Mato mainly Grosso in the (MT). Amazon In manifestations caused by Dengue virus (DV) infection are Central and South America genotypes I, II and III of OROV the Dengue Fever and Dengue Hemorrhagic Fever. The have been reported. This study aimed to investigate the disease is now endemic in 112 countries in tropical and circulation of orthobunyaviruses from Simbu serogroup subtropical regions and Brazil is one of the most affected in MT. To achieve that, viral RNA extracted from the regions in the world accounting for approximately 60% serum of 529 patients with acute febrile illness obtained between 2011-2012 in 20 cities of MT and total RNA from covering systematically the population exposed to DV 145/319 pools of Culex quinquefasciatus and 57/102 (non-infected,of notifications symptomatic in the Americas. and asymptomatic) The lack of hinders studies of Culex spp. mosquitoes captured in 2013 from 200 the knowledge of predisposing factors which may censitary sectors in the urban area of Cuiabá, MT were contribute to the development or maintenance of the reverse transcribed to cDNA using a primer designed to the S segment of Orthobunyavirus genus. Nested RT-PCR the seroprevalence of IgM/IgG anti-DV in the population for Simbu serogroup (300 pb) was followed by nucleotide ofdisease Alfenas, in a southern specific population. Minas Gerais. This Three study hundred aims to serum verify sequencing and phylogenetic analysis. 4/529 (0.75%) samples were collected to verify the presence of IgM/ human samples from Cuiabá, Várzea Grande and Nova IgG anti-DV in using Panbio Dengue Duo Cassette (Alere Mutum and 3/202 (1.48%) mosquitoes pools, two of C. Australia) . The results showed that from 300 samples, quinquefasciatus and one of Culex spp. negative for other 239 (79.67%) were negative; 37 (12.33%) were positive and 24 (8%) were indeterminate (the presence of a the serogroup Simbu. The nucleotide sequences showed weak band in the IgM or IgG result). Among the samples homology11 flaviviruses with andstrains five alphavirusesfrom Belém-PA were and positive Manaus- for that were positive, 24 (64,86%) are IgM positive, 4 AM belonging to genotype I of OROV. The preliminary (10.81%) are IgG positive and 9 (24,32%) are positive phylogenetic analysis revealed that the samples belong for IgM and IgG. The Panbio Dengue Duo Cassette can to subgenotype IA, similar to strains from Pará obtained detect the high levels of IgG characteristic of secondary from humans, sloths and Ochlerotatus serratus and C. infections and IgM levels associated with primary quinquefasciatus mosquitoes and to subgenotype IB, similar to human isolates from Pará, Maranhão and the positive samples 24 (64,86%) were derived from dengue with high sensitivity and specificity. So, among September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

131 Human Virology: HV primary infection and 13 (35,12%) were derived from were unique to this isolate were not observed in the secundary infection. This data showed a high prevalence other isolates from the same genotype/lineage. Among of anti-dengue antibodies in the population of Alfenas, these aa substitution one is conservative at position 222 MG and also the circulation of the virus in the region. (located in domain II of the E protein) and one is non- conservative at position 306 (located in domain III of the HV116 - MOLECULAR CHARACTERIZATION OF A E protein). To check the virulence of the isolate, Swiss DENGUE VIRUS 1 ISOLATED IN ALFENAS, MINAS mice were infected intraperitonally with 1 x 104 pfu or GERAIS, BRAZIL inoculated with PBS. After 7 days of infection the liver of Franco, I.R.1; Rocha, R.P.1; Fumagalli, M.J.1; da Silveira, N.J.F.1; Elias, T.C.1; Fagundes, L.G.1; Nogueira, M.L.2; 3 1 1 Drummond, B.P. ; Malaquias, L.C.C. ; Coelho, L.F.L. ofinfected edema, animals hemorrhage showed and the necrosis. presence It ofis alsoinflammatory observed 1. UNIFAL - Universidade Federal de Alfenas, theinfiltrate presence around of steatosis the central when veins compared and also to focal uninfected points R. Gabriel Monteiro da Silva, 714 - Centro, Alfenas - MG, animals, suggesting the presence of a viral replication 37130-000 2. FAMERP - Faculdade de Medicina de São molecular characterization and analysis of a virulence José do Rio Preto,Av. Brigadeiro Faria Lima, 5416 - Vila São in the liver of these animals. This is the first report of Pedro, São José do Rio Preto - SP, 15090-000 potential of a DENV-1 strain isolated from South of Minas 3. UFJF - Universidade Federal de Juiz de Fora, Gerais, Brazil. R. José Lourenço Kelmer - Martelos, Juiz de Fora - MG, 36036- HV117 - INVESTIGATION OF DENGUE VIRUS 330 INFECTION IN GOIANIA, GOIAS, BY MOLECULAR Dengue is a major public health problem worldwide, METHODS AND SEROLOGICAL especially in the tropical and subtropical areas with Oliveira, T.S.; Guimarães, V.N.; Cunha, M. dos P.; e around 2.5 billion people living in areas at risk regions Souza, M.B. de L.D.; Cardoso, D. das D. de P.; Carneiro, of the world. The disease is caused by a positive single R. dos S.; Fiaccadori, F.S. strand RNA virus that belongs to genus Flavivirus, family Flaviviridae. There are four serologically related viruses IPTSP/UFG - Instituto de Patologia Tropical e Saúde designated as DENV-1, 2, 3 and 4. Infection with one of Pública/ Universidade Federal de Goias, Rua 235 - s/n, Setor these serotypes causes a mild, self-limiting febrile illness Universitário, Goiânia - Goiás, 74605050 called dengue fever. However, a reduced number of Dengue is a major challenge to public health in Brazil patients could develop the severe forms of disease called and worldwide. Dengue virus (DENV) is an arbovirus of dengue hemorrhagic fever and dengue shock syndrome. In this study, we performed a molecular characterization four antigenically distinct serotypes DENV-1-4. Infection of envelope gene from a DENV-1 strain isolated from a withthe family one of Flaviviridae, these serotypes genus may Flavivirus, causes a diseaseclassified whose into dengue hemorrhagic fever patient from Alfenas, South of spectrum ranges from clinically asymptomatic or a Minas Gerais (BR/Alfenas/2012). The phylogeography mild and self limited febrile illness called dengue fever analysis using nucleotide sequences from the DENV-1 to severe clinical forms. In Brazil, the co-circulation of E gene shows that these samples clustered within the four serotypes has led to successive epidemics and Goias genotype V, lineage L1 together with other Brazilian state, usually present high number of dengue cases. In DENV-1 isolates and also with the Venezuelan isolates. 2013 were related alarming statistics, with an increase Thereby, it was observed that the introduction of this of 401% in the number of reported cases and 25% in lineage probably occurred from Rio de Janeiro state, the number of deaths. The Epidemiological Surveillance Southeast Brazil and after, the virus has spread to Minas System plays an important role in the continuous Gerais state where it had a local evolution evidenced monitoring infections, nevertheless, it has been shown by the distance between the BR/Alfenas/2012 and the other related isolates. Fifteen conservative and/or non- of new cases. To increase the detection of DENV in early conservative aminoacids substitutions were observed in infections,the relevance the of NS1 laboratory research diagnosis was introduced in the confirmation in Public the deduced polyprotein sequence. Two aa substitutions Health Laboratories. However, the time of infection has

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alphaviruses in Cuiabá, MT.Culicidae specimens were of choice. Therefore, the present study evaluated the captured in 3 locations from 200 censitary sectors from occurrencea decisive influence of DENV on infection the results in regardingGoiania, Goias the method in the epidemic period of 2012-2013 among the population in pools (1-10 mosquitoes) according to sex, species, with clinical suspected of dengue infection (up to seven dataCuiabá and in collection2013, identified point.Minced with dichotomy pools were key, subjected allocated days) using a combination of serological and molecular to total RNA extraction, duplex-RT-PCR followed by methods. Serum samples were collected from 278 symptomatic patients who were attended in basic health centers between October/2012 and May/2013. The encephalitismultiplex-semi-nested-RT-PCR (SLEV), dengue 1 (DENV-1) to five alphavirusesand mayaro samples were submitted to the research of serological and 11 flaviviruses.Positive samples for Saint Louis markers NS1, IgM and IgG using a commercial kit subjected to nucleotide sequencing.A region of the SLEV (DuoTest-Bioeasy), as well as viral RNA detection by (MAYV) viruses were amplified by single RT-PCR and RT-PCR for the C-prM region. The positivity for infection was 43.9% (122/278), with rates of 30.5%, 13.6% and Theenvelope minimum gene wasinfectious amplified rate for (MIR) phylogenetic was calculated analysis. 20.5% for NS1, IgM and RNA markers, respectively. In forPositive positive pools forspecies.Between MAYV were confirmed11.090 bymosquitoes, RT-qPCR. 24.5% of DENV infection cases, NS1 was the only marker detected, demonstrating its advantage mainly favoring pools, 1/137(MIR=1.04) of Aedes aegypti positive for early diagnosis with higher rate of positivity between 4 DENV-14.556 females and 1/67 classified (MIR=2,6) in 13 Culex species sp. constituted for SLEV. 593For and 5 days. The IgM was detected singly in 16.4% with MAYV, 10/67(MIR=26,2) Culex sp., 7/162(MIR=3.7) Cx. quinquefasciatus, 4/137(MIR=4.1) Ae. aegypti and For the RNA, the highest rate was observed between 1 andstatistical 3 days. significance The combination for the of sixth these and three seventh markers, days. RT-qPCR. For DENV-4, 55/137(MIR=57,1) Ae. aegytpi, notably constitute a powerful tool in the diagnosis 34/54(MIR=162.7)5/54(MIR=24) Cx. Cx. comp. comp. pipiens pipiens, were 68/162(MIR=36) confirmed by DENV. Monitoring of dengue epidemics by laboratory Cx. quinquefasciatus, 46/67(MIR=120,4) Culex spp., 1/6(MIR=142.8) Limatus sp., 2/4(MIR=500) system, it is relevant to establish a complete data base to Psorophora spp., 3/7(MIR=375) Ps. varipes albigenu, beconfirmation, used for surveillance with an active studies. and effective notification 1/1(MIR=1000) were positive.In previous studies, SLEV,

HV120 - MOLECULAR INVESTIGATION OF NATURAL in patients from the urban area of Cuiabá during a large INFECTION BY FLAVIVIRUSES AND ALPHAVIRUSES IN outbreakMAYV and of the dengue.The four serotypes SLEV genotypeof DENV wereV-A, previoulsy identified ADULT MOSQUITOES CAPTURED IN CUIABÁ, MATO GROSSO, BRASIL Culex sp. female.Culex and Aedes species presented Serra, O.P.1; Cardoso, B.F.1; dos Santos, F.A.L.1; Heinen, logreported of 3.52-4.08 in humans copies/µL from Cuiabá, of MAYV.Experimentaly, was identified in a L.B. da S.1; Zuchi, N.1; Ribeiro, A.L.M.2; Rodrigues, these species have been demonstrated as competent J.S.V.1; Myiazaki, R.D.1; Slhessarenko, R.D.1 vectors for the virus, but their participation in the 1. UFMT - Universidade Federal de Mato epidemiological cycle of MAYV transmission is unclear. Grosso, R. Guadalajara, 1 - Jardim das Américas Cuiabá - MT DENV-4, responsible for the majority of the human 78060-624 2. HUJM - Hospital Universitário Júlio Müller, species, possibly due to hematophagy in humans.Cuiabá Av. Fernando Corrêa da Costa, nº 2367, Bairro Boa Esperança, presentscases in MT a varietyduring 2012-2013,of culicidae was species, identified associated in several to Cuiabá - MT, 78060-900 environmental conditions favorable to the occurrence Arboviruses belonging to Flavivirus and Alphavirus of arboviral outbreaks, emphasizing the importance of genus are considered an important public health entomological and virological surveillance. FINANCIAL issue in Brazil, mainly in tropical areas.The aim of the SUPPORT: CAPES, FAPEMAT, UFMT study was to identify the frequency of hematophagous mosquitoesSeptember/October naturally 2014 Volume infected 19 – Supplement by flaviviruses 2 - Abstracts/Posters and - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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HV121 - IMPROVEMENT OF NOROVIRUS GII/4 HV123 - DIFFERENCES IN THE INCIDENCE OF HPV SUBTYPING PROTOCOL TYPES AT A NORTHERN POPULATION IN BRAZIL, Oliveira, L.M.1; Fumian, T.M.2; Miagostovich, M.P.2; BASED IN THE WORLDWIDE PROFILE Andrade, J.S.R.2; Colombo, A.C.F.1; Paixão, S.1; Nagata, Silva, A.K.1; Silvestre, R.V.D.1; da Paixão, C.G.S.1; T. 1 Ferreira, L.S.S.1; Lima, S.F.2; Junior, L,B.D.2; Mello, W.A.1 1. UnB - Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília - DF, 70910-900 1. IEC - Instituto Evandro Chagas, Rodovia 2. FIOCRUZ - Fundação Oswaldo Cruz, Av. BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 2. Laboratório Paulo C. Azevedo, Avenida Norovirus (NoV) are a major cause of acute gastroenteritis Comandante Brás de Aguiar, 99 - Batista Campos, Belém - (AGE), responsible for almost 50% of AGE outbreaks PA, 66035-000 worldwide, affecting all ages. Despite the high genetic The Human papillomavirus (HPV) is a sexually transmitted diversity, the genogroup II, genotype 4 NoV (GII.4), are infectious agent, commonly found worldwide. It causes the main agent of these outbreaks. Every two years asymptomatic infections in mucous membranes that oft a new GII.4 variant arises by the capsid protein (VP1) are eliminated naturally about two years, but can cause amino acids changes as a new pandemic strain, resulting important clinically diseases depending of the viral type, in a high antigenic variability of this NoV group. It had with persistence or progression of infection, such as high been common to determine the subtype (or strain) by grade lesions and cancer. Among the diseases caused by sequencing the part of VP1 gene, however, recent study HPV, cervical cancer represents one of biggest challenge of NoV taxonomy suggested the use the whole VP1 to public health. Taking into account assessments gene sequences for GII genotyping and GII.4 subtyping conducted by the National Cancer Institute (INCA), in analysis. In this study new primer set were designed by 2014, it was estimated that cancer of the cervix already multiple alignment of NoV GII.4 sequences for complete ranks third overall in Brazil, with the highest rates found VP1 gene recovery by RT-PCR (GII-4 Subtyping 5019 For: 5´-GGC AAG AGC CAA TGT TCA G-3´ and GII-4 in the metropolitan area of Belém/Pará. The gross rate Subtyping 6821 Rev: 5´-TGT TAT TTT CAA AAT CAA YYT casesin North is about region 35/100000 as 23.57/100000 women, becoming women. Specificallythe second TTT GGT T-3´). Six NoV positive stool samples previously cancer in incidence in Pará capital. For prophylactic use, two vaccines have been developed trying to cover the major circulating types, with the intention of decrease confirmed by RT-qPCR were used for RT-PCR with the the incidence of genital HPV-related disease. Brazil was primer set described above and five samples amplified adopted quadrivalent vaccine that combat subtypes 6, successfully. The amplicons were gel-purified and multiplesequenced alignment using the sameand phylogenetic primer set. All tree five NoVconstructed samples genital warts and the other two are causally implicated bywere MEGA6 classified with as representative NoV GII.4, Sydney sequences 2012 variant, of each usingGII.4 in11, 70% 16 and of 18cervical of which cancers. the first The two aim are of related this study to 90% is toof subtypes. The possible presence of recombination was identify which HPV types are circulating in a routine investigated using RDP4, and our results showed that no gynecology treatment in Belém city, Pará. Was used 84 recombination was found among the samples analyzed. samples collected from women aged 20-50, submitted The use of new primer set was successful to determine to the Pap test, previously detected as HPV positive by the subtype of NoV GII.4. FINANCIAL SUPORT: CAPES Hybrid Capture second generation. These samples were

region of L1 in viral genome. Then, they were subjected totested the test by PCR“Linear with Array biotinylated HPV” that specific is able primersto identify for 37 a

products with oligonucleotide probes and detection different HPV types from hybridization of the amplified different types of high risk HPV with prevalence of 59 by colorimetric determination. The study identified 10 September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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(18.7%), 31 (16.7%), 16 (12.5%) and 58 (10.4%). We production of an antiserum and the development of a real time RT-PCR for diagnosis of infections by CHIKV in prevalence of HPV 55 variants (14.58%) and 81 (10.4%). humans and mosquitoes. FINANCIAL SUPPORT: CNPQ- Finally,also identified we conclude 15 different that the types occurrence of low riskof HPV HPV, in with our 150815/2014-0 population shows a trend different from the worldwide prevalence types included in quadrivalent vaccine. With HV128 - ADENOVIRUS INFECTION IN ALLOGENEIC these founds we suggest the strengthening of networks STEM CELL RECIPIENTS: EVALUATION OF VIRAL tests of patients by cytology and, if possible associated to EXCRETION AND VIREMIA a molecular HPV test, to reduce the incidence of cervical Santos, H.C.P.1; Fiaccadori, F.S.1; Cardoso, D. das D. cancer in our quite different epidemiological scenario. de P.1; Arantes, A. de M.2; Silva, L.P.2; Almeida, T.N.V.1; FINANCIAL SUPPORT: MS / SVS / IEC Souza, M.1 1. IPTSP/UFG - Instituto de Patologia Tropical HV124 - PRELIMINARY WORK WITH CHIKUNGUNYA e Saúde Pública/ Universidade Federal de Goias, Rua 235 - VIRUS IN A BIOSAFETY LEVEL 3 (BSL-3) LABORATORY s/n, Setor Universitário, Goiânia - Goiás, 74605050 1 2 3 de Figueiredo, M.L.G. ; Amarilla, A. ; Romeiro, F. ; 2. Unidade de Transplante de Medula Óssea do Badra, S.J.3; Aquino, V.H.2; Figueiredo, L.T.M.3 Hospital Araújo Jorge/ ACCGO 1. Evandro Chagas Institute, Brazilian Ministry The human adenoviruses (HAdV) infect people of all ages of Health worldwide, causing a wide range of clinical syndromes, 2. School of Pharmaceutical Sciences of The depending on the viral type. In immunocompromised University of São Paulo hosts, mainly those undergoing allogeneic stem cell 3. Virology Research Center, School of Medicine transplant (ASCT), persistent and severe infection are of Ribeirao Preto of The University Of Sao Paulo common, resulting in a bad prognosis. HAdV diagnosis Chikungunya (CHIKV) is an Alphavirus (Togaviridae) is not included in routine laboratory exams of ASCT that has reemerged in tropical and subtropical regions of patients in public hospitals in Brazil. Therefore, studies Asia and Africa. The main vectors of this virus are Aedes involving HAdV infection in this group of patients are still aegypti and Aedes albopictus. Last year, CHIKV has been scarce. Thus, the objective of this study was to monitor introduced in the Americas, in the Caribbean. However, an ASCT recipients (admitted to Hospital Araújo Jorge in autoctonous case was found in North America. Therefore, Goiânia, Goiás) for HAdV occurrence and also correlate probably, this virus will reach Brazil and will produce viral positivity with clinical symptoms and patients’ large outbreaks of acute febrile illness with arthritis. prognosis. For this, one fecal and one total blood sample Thus, it is important to develop diagnostic methods for were obtained from each patient prior to the ASCT and, this virus in our country. We describe here a preliminary after that, samples were obtained weekly. Patients were work with the prototype of CHIKV (S27-African strain), monitored for a minimum of one month and a maximum isolated in 1953. In a biosafety level 3 laboratory, the of 19 months. Up until now, 98 serum samples and 62 virus has been rehydrated from a 30 years old ampoule fecal samples, obtained from 19 ASCT recipients, have (ATCC, V548-001-522), inoculated into C6/36 (Aedes been tested for HAdV. Serum and fecal samples were albopictus) cells and also intracerebrally in baby mice. screened by Nested-PCR, using primers targeting a partial Five days after infection, despite not showing cithopatic region of the hexon gene. Fecal samples were further effect, the infection of CHIKV in C6/36 cells was screened by a commercial enzyme immunoassay (EIA) (RIDASCREEN Adenovirus, R-Biopharm). All samples time RT-PCR. Forty eight to 72 hours after infection, 70% were negative by the EIA. However, ten stool samples ofconfirmed the infected based mice on immunofluorescentdeveloped encephalitis test and after real- from four patients were positive for HAdV by Nested- PCR, and ten serum samples from seven patients, were infected cells and brains of mice with encephalitis were also positive. In total, ten patients (53%) had at least one collected5 days all and animals stored were at -70 dead. oC as Cell virus culture seeds. fluidsThese ofseeds the positive sample (serum or fecal) for HAdV. Five HAdV- will be used in further experiments that will include the

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posterspositive - Human Virology: patients HV presented diarrhea, and five patients XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

135 Human Virology: HV presented graft versus host disease. In one patient, HV131 - EVALUATION OF SERA OF MICE IMMUNIZED HAdV-positivity in fecal samples preceded viral positivity WITH A CHIMERIC ANTIGEN CONTAINING SEGMENTS in the serum. So far, three positive samples, from three OF E1 AND E2 ENVELOPE PROTEINS OF HEPATITIS C distinct patients, were sequenced. Two of those samples VIRUS were characterized as HAdV F serotype 40 and one as Oliveira, M.M.1; Freitas, G.R.O.2; Froelich, L.2; HAdV C type 57. This data highlights the importance of Yokosawa, J.1 the inclusion of HAdV testing in the routine laboratory exams of ASCT patients. Preliminary results also suggest 1. Laboratório de Virologia, Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia that fecal screening for HAdV could be potentially used (UFU), Uberlândia, MG. to predict HAdV viremia, and thus contribute to an early 2. Programa de Pós-Graduação em Imunologia clinical intervention and prevention of disseminated e Parasitologia Aplicadas, ICBM, UFU, Uberlândia, MG. infection. FINANCIAL SUPPORT: FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE GOIÁS (FAPEG); Hepatitis C is a disease that affects the liver and is a major PROGRAMA DE PÓS GRADUAÇÃO EM BIOLOGIA DA cause of chronic liver disease worldwide, progressing RELAÇÃO PARASITO-HOSPEDEIRO (PPGBRPH) frequently to cirrhosis and liver cancer. Approximately 3% of the world population is infected chronically with HV129 - DEVELOPMENT OF A REAL TIME-PCR hepatitis C virus (HCV). The transmission occurs through BASED IN SYBR GREEN FOR RAPID DETECTION OF contact with contaminated blood and until now there is CHIKUNGUNYA VIRUS Romeiro, M.F.; de Souza, W.M.; Figueiredo, M.L.G.; of a vaccine is due to the HCV genetic diversity, and the Tolardo, A.L.; Figueiredo, L.T.M. existenceno effective of only vaccine. one Thesystem difficulty for virus in replication the development in cell culture, based on a single genotype (2a). Conserved FMRP/USP - Faculdade de Medicina de Ribeirão segments of HCV E1 and E2 glycoproteins that had been Preto/ Universidade de São Paulo, Av. Bandeirantes, 3900 - reported to elicit neutralizing antibodies were selected Monte Alegre, Ribeirão Preto - SP, 14049-900 to construct the chimeric antigen GST-E1E2, which was Chikungunya virus (CHIKV- S27-African strain) is a expressed in bacterial system and used to immunize mice. mosquito-borne Alphavirus and one of the prevalent re- Sera obtained from these mice showed to be reactive, emerging arbovirus in tropical and subtropical regions by ELISA, against synthetic peptides corresponding of Asia and Africa. The main vectors of this virus are Aedes aegypti and Aedes albopictus. Last year, CHIKV this reactivity, in this study, we transfected CHO-K1 cells has been introduced in the Americas, in the Caribbean. withto HCV the segments plasmid presentEFJFH c-NS2, in the which GST-E1E2. encoded To confirm for the However, an autoctonous case was found in North HCV proteins core, E1, E2, p7, and NS2. Zeocin-resistant America. Therefore, probably, this virus will reach Brazil clones were selected and expression of the HCV core and will produce large outbreaks of acute febrile illness with arthritis. We show here a real-time RT-PCR based assay (IFA) by using an anti-core monoclonal antibody. on SYBR green I, using primers that amplify part of the protein was confirmed by indirect immunofluorescence gene of NS1 of CHIKV. This new real-time RT-PCR was four also showed reactivity by IFA against pEFJFH validated using a CHIKV in Vero cells and in suckling c-NS2-transfectedOut of five sera that cells. showed In conclusion, to be reactive these by results ELISA, baby mouse brain, both passaged in a biosafety level 3 laboratory. This RT-PCR, used as a routine, could become humoral response in mice. FINANCIAL SUPPORT: CNPQ, a useful tool for rapid diagnosis of infections by CHIKV FAPEMIG,indicated PROPP-UFU, the capacity CAPES, of GST-E1E2 UFU. to elicit specific in Brazilian patients and mosquitoes, allowing manage its disease, and monitor the spreading of CHIKV through Brazil.

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136 Human Virology: HV

HV139 - CIRCULATION OF DIFFERENT SEROTYPES clades formed in DENV-1 phylogenetic reconstructions, OF DENGUE VIRUS IN SAO JOSE DO RIO PRETO, SAO indicating two possible lineages. For DENV-2 and DENV- PAULO 4, one clade grouped all SJRP samples, indicating a single Colombo, T.E.1,2; Vedovello, D.1; Biselli, J.1; Drumond, possible lineage. This data shows that the phylodinamics B.3; Cabrera, E.1; Nogueira, M.L.1 of dengue circulation can be much more complex than expected even in a small city, with circulation not only 1. FAMERP - Faculdade de Medicina de São of different serotypes but also different strains as we José do Rio Preto,Av. Brigadeiro Faria Lima, 5416 - Vila São already showed before for DENV-3. These data reinforce Pedro, São José do Rio Preto - SP, 15090-000 the importance and need for surveillance programs 2. UNESP - Universidade Estadual Paulista, to detect and trace the evolution of these viruses. Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010 3. UFJF - Universidade Federal de Juiz de Fora, R. José Lourenço Kelmer - Martelos, Juiz de Fora - MG, 36036- CAPES FINANCIAL SUPPORT: PRONEX; INCT-DENGUE; FAPESP; 330 HV141 - GASTROENTERIC VIRUS SHEDDING BY The four serotypes of dengue virus (DENV 1–4) ASSYMPTOMATIC DAY-CARE CHILDREN FROM (family Flaviviridae, genus Flavivirus) are antigenically GOIÂNIA, GOIÁS and genetically distinct. The emergence of epidemic Corrêa, T. dos S.; Souza, M.; Santos, H.C.P.; Fiaccadori, dengue in the Americas, as well as worldwide, has been F.S.; Souza, K.M.; Abdelhaleem, K.R.; Almeida, T.N.V.; characterized by a rise to hyperendemicity. In the present Cardoso, D. das D. de P. work we looked the DENV transmission in Sao Jose do Rio Preto from 2011 to 2014. Were used serum samples IPTSP/UFG - Instituto de Patologia Tropical e Saúde Pública/ Universidade Federal de Goias, Rua 235 - s/n, Setor Universitário, Goiânia - Goiás, 74605050 Theof suspected viral surveillance and confirmed was DENVperformed patients with provided multiplex by Gastroenteric viruses, such as rotavirus A (RVA), human RT-PCRthe Public using Health Flavivirus Authority generic to profile primers DENV based circulation. on non- adenovirus (HAdV), human calicivirus (norovirus and structural protein (NS5), followed by nested assays with sapovirus), human astrovirus (HAstV), are important acute gastroenteritis agents. These agents affect follows: 65,41% (522/798) DENV-4, 22.56% (180/798) mainly children, elderly, and immunocompromised DENV-1,species-specific 11,28% primers. (90/798) The DENV-2 DENV and serotypes 0.75% (6/798) were as individuals. Outbreaks of AGE are common in semi- coinfection, showing a complex pattern of serotypes closed environments, such as hospitals, schools and day- circulation. Statistical analysis of the mean ages (years) care centers; however, asymptomatic viral excretion has of DENV-1 (38,12 ± 18,82), DENV-2 (38,06 ± 18,78), been reported by children and immunocompromised DENV-4 (36,57 ± 16,69) and coinfection (29,66 ± 18,94) individuals. The main objective of this study was to screen fecal samples (previously tested and negative = 0,4470, F = 0,8876) between means. The correlation for norovirus and sapovirus genogroups I and II by RT- betweencases showed of the that gender there was of noDENV significant patients difference according (P to infecting serotype also showed that there was no for RVA, HAdV and HAstV detection. Sample collection tookPCR) placeobtained in a from day-care children center less in than Goiânia, five years Goiás, of from age, 2 (P = 0,5634) and DENV-4 (P = 0,7781). Up to now, 9 October 2009 to September 2011. For RVA and HAdV DENV-1,significant 15 difference,DENV-2, 33 DENV-1DENV-4 have (P = been 0,4868), subjected DENV- to screening, all samples were tested by a commercial sequencing of the envelope gene, and 8 DENV-1, 7 DENV- enzyme immunoassay (EIA) (RIDASCREEN Rotavirus/ 2, 7 DENV-4 had their genome completely sequenced. Adenovirus – R-Biopharm); samples were further The phylogenetic analyses of serotypes 1, 2 and 4 tested by polyacrylamide gel electrophoresis (PAGE) for RVA-genome detection. For HAstVs screening, an RT- with genotypes that circulating in Brazil (genotypes PCR reaction was performed, using primers targeting V,show Asian that American the samples and identifiedAmerican, in for this types study 1, grouped2 and 4 the ORF2 region. All positive samples were submitted respectively). Looking inside the genotypes two distinct to molecular assays for characterization. From all 42

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

137 Human Virology: HV children, 10 (23.81%), between two and three years old, were positive for one of the viral agents. From those, two (4.7%) were positive for RVA, by both methodologies, sampleswith species-specific for DENV in SJRP primers from in 2008 serum to 2012 from and samples 373 and characterized by RT-PCR (targeting the VP7 and (68.56%)from dengue were suspected positive for patients. DENV-1. We 20 amplified DENV-1 have 544 VP4 genes) as G2/Pnon-typable. Three (7.14%) were been subjected to sequencing of the envelope gene positive for HAdV and characterized, by PCR/NestedPCR and/or full genome sequencing and were used for followed by genomic sequencing of a partial region of phylogenetic reconstruction. Our analyses showed that the hexon, as species F (serotypes 40/41). Five (11.9%) the all samples belong to Genotype V and formed two were positive for HAstV, and characterized as serotype 1. Data reveal the occurrence of asymptomatic viral as L1 and L6. Comparing the two lineages, the following excretion by the children in the day-care environment aminowell defined acid groups(aa) substitutions divided into twoare lineagesobserved: identified L338S constituting a potential risk for viral dissemination and (leucine to serine, aa position 338), K394R (lysine to the occurrence of outbreaks. FINANCIAL SUPPORT: arginine, aa position 394), L428V (leucine to valine, aa UNIVERSIDADE FEDERAL DE GOIÁS position 428) and I436V (isoleucine to valine, aa position 436). The I457R (isoleucine to arginine, aa position 457) HV142 - IDENTIFICATION AND BIOLOGICAL was recognized only one sample (BR/SJRP/287/2011). CHARACTERIZATION OF DENGUE SEROTYPE 1 Additionally, epitope analysis of samples showed that ISOLATES (DENV-1) DETECTED IN SÃO JOSÉ DO RIO the two lineages are recognized by different receptors of PRETO, SÃO PAULO T and B lymphocytes indicating that exhibit differences Pinheiro, T.M.1; Biselli, J.M.1,2; Colombo, T.E.1,2; in immune response. Our preliminary data show the Drumond, B.P.3; Ullmann, L.S.2; Araújo Jr, J.P.2; Batista, existence of differences between the samples, evidencing I.C.A.4; Silva, C.E.C.4; Vedovello, D.1,2; Nogueira, M.L.1 the importance of understanding the biological DENV 1. FAMERP - Faculdade de Medicina de São characteristics in vitro and in vivo correlating to the José do Rio Preto,Av. Brigadeiro Faria Lima, 5416 - Vila São likely epidemiological consequences. Pedro, São José do Rio Preto - SP, 15090-000 HV148 - CHARACTERIZATION OF AN ATYPICAL 2. UNESP - Universidade Estadual Paulista, Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010 DENV-2 STRAIN ISOLATED IN BRAZIL 3. UFJF - Universidade Federal de Juiz de Fora, Salvador, F.S.1; Amorim, J.H.2; Ferreira, L.C. de S.2; R. José Lourenço Kelmer - Martelos, Juiz de Fora - MG, 36036- Romano, C.M.1 330 1. IMTSP - Instituto de Medicina Tropical de 4. FIOCRUZ - Fundação Oswaldo Cruz, Av. São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 470, Jardim Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 América, São Paulo - SP, 05403-000 Dengue is an infectious viral disease transmitted by 2. ICB/USP - Instituto de Ciências Biomédicas the bite of infected Aedes mosquitoes (Aedes aegypti da Universidade de São Paulo, Edifício III USP - Administração and Aedes abopictus). It is considered a serious public - Av. Prof. Lineu Prestes, 2415 - Butantã, São Paulo - SP, health issue in tropical and subtropical regions where 05508-900 the vector is best adapted. Dengue virus (Flaviviridae family, Flavivirus genus) has four antigenically distinct main arboviruses that infect man worldwide. From a serotypes (DENV 1-4) subdivided into genotypes and patientDengue presentingvirus is a flavivirus, dengue withoutand is responsible warning signs for the in lineages with different degrees of virulence. Therefore, Brazil it was isolated a strain of Dengue Virus (named the aim of this study was to perform an investigation to JHA) with capacity of neurovirulence in mice using 100 correlate the molecular and biological characteristics of NS1 fragment of this sample characterized it as DENV A viral surveillance was performed with Multiplex RT- 2.PFU To Amplificationstudy this virus and in Sanger more sequencingdetail, we performed of a small samples of DENV-1 identified in São José do Rio Preto. PCR using Flavivirus generic primers based on non- the complete sequencing of viral genome using NGS structural protein (NS5), followed by Nested assays technology in Ion Torrent platform. Viral RNA was

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

138 Human Virology: HV reverse transcribed and three fragments of 4kb with 2. UFMG - Universidade Federal de Minas Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, Belo Horizonte - MG, 31270-901 overlap were amplified using the SuperScript III high- kit (Invitrogen). Reads were assembled and analyzed in Bunyaviridae family viruses are considered important fidelity one-step reverse transcription-PCR (RT-PCR) CLC genomic Workbench. Consensus complete genome emerging and reemerging infectious agents, capable of was aligned to 208 globally sampled DENV genomes causing different clinical features in humans, including and genotyping was performed by maximum likelihood fever, encephalitis and hemorrhage. Little is known reconstructions. A second dataset comprising only about the interaction of some of these viruses with the envelope region of DENV2 was built to estimate genetic human immune system, justifying the need for studies on distance among sequences using PAUP. Recombinant analysis was done in RDP software. We also visually This study aimed to quantify the expression of a set of 5 inspected JHA genome by comparing it to well-known genesthe influence related ofto Orthobunyavirushuman innate immunity and innate (TLR3, immunity. TLR7, neurovirulent DENV2 strains. Phylogenetic analysis of TLR9, IFNg and IFNb) after infection by the Apeu virus complete genomes showed that JHA strain belongs to (APEUV). Peripheral blood mononuclear cells (PBMC) American genotype of DENV2, but it is more divergent collected from 5 healthy individuals were exposed to than expected from viruses of the same genotype. While APEUV in two distinct conditions: PBMC infected with the intra- and inter-genotype average genetic distance m.o.i.=1 and m.o.i.=3; and uninfected controls (mock was around 2.5% and 10% respectively, JHA was almost infected PBMC; uninfected PBMC and fresh PBMC (T0 6% divergent from American genotype viruses. No which were not submitted to any process of incubation). evidence of recombination was found that could explain After infection and 4 hours of incubation, total mRNA such divergence. Although this strain was quite divergent was extracted from cells and used to obtain a cDNA pool, from general American viruses, JHA was almost identical for qPCR assays based on TaqMan system. The group to a single strain isolated in Trinidad Tobago in 1953 T0 was used as “normalizer group”. Expression of TLR9 mRNA was increased in the infected group (m.o.i. 1 and to neurovirulence, the genome was then compared m.o.i. 3). IFNb messenger expression was even higher in (Trin53). To identify specific sites that would be related to others (neurovirulent and non-neurovirulent these groups (15-fold higher than in T0 group). There American). Several non-synonymous changes were observed throughout the genome in comparison to TLR7 and IFNg genes in all tested groups, as well as any American viruses consensus, but two sites found in was no significant changes in the expression of TLR3, envelope gene were suggestive of neurovirulence ability mock and uninfected groups. These results indicate that (E126 and E390) since they differ from other American TLR9significant and IFNb changes genes in are expression related to of changes all tested in the genes human on but are similar to neurovirulent viruses. Others already immune response in the early stages of APEUV infection reported the importance of these sites for neurovirulence and further studies with these and other genes are being in mouse model. Ongoing neurovirulence reversion conducted aiming to elucidate the molecular processes experiments using cell culture and mice should better involved in the infection of human cells by APEUV. clarify the particular characteristics of JHA genome neurovirulence. HV153 - IDENTIFICATION OF G AND P GENOTYPES OF GROUP A ROTAVIRUS AMONG CHILDREN IN RIO HV149 - EVALUATION OF THE EXPRESSION LEVELS BRANCO, ACRE, BRAZIL OF GENES INVOLVED WITH INNATE IMMUNITY IN Neves, M.A.O.1; Menezes, E. de F.C.2; Silva, M.C. de M.2; HUMAN CELLS INFECTED BY APEU VIRUS Silva, L.D.2; Loureiro, E.C.B.2; Gabbay, Y.B.2; Rodrigues, 1 2 1 Ferreira, J.G.G. ; Villani, F.N.A. ; Oliveira, J.G. ; Ferreira I.C.2; Silva, R.U.2; Soares, L. da S.2; Mascarenhas, J.D.P.2 P.C.P.2; Calzavara Silva, C.E.1 1. UEPA - Universidade do Estado do Pará, 1. CPqRR/FIOCRUZ MINAS - Centro de Rua do Una, nº 156, Belém - Pará, 66.050-540 Pesquisas René Rachou/ Fundação Oswaldo Cruz, Av. Augusto 2. IEC - Instituto Evandro Chagas, Rodovia de Lima, 1715 - Barro Preto, Belo Horizonte - MG, 30190-002 BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000

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In developing countries, group A rotavirus (RVA) cause HV157 - RELATIONSHIP OF HPV GENOTYPES death in more than 197,000 children each year and is FROM CERVICAL AND ANAL SITES AMONG HIV recognized as a major viral agent of acute gastroenteritis SEROPOSITIVE WOMEN in infants and young children. Its structure is composed Volpini, L.P.B.; Boldrini, N.A.T.; Miranda, A.E.; Freitas, by 12 proteins, two structural proteins are located at L.B.; Spano, L.C. outer capsid of the virion; the VP7 (Glycoprotein) and the VP4 (Protease-sensitive) are widely studied once that UFES - Universidade Federal do Espírito Santo, Av. Fernando Ferrari, 514, Goiabeiras, Vitória - ES, 29075-910 induce immunological response and are involved in viral neutralization. To date, 27 G and 37 P genotypes have The human papillomaviruses (HPV) have 198 described been reported by sequence analysis. This study aimed genotypes and approximately 40 have tropism for the to identify G and P genotypes of RVA in fecal samples of anogenital region. Infection with oncogenic or high-risk children from Rio Branco, Acre. Material and Methods: HPV types (HR-HPV) in the anus or cervix sites can lead to invasive squamous cell carcinoma and coinfection from children with and without diarrhea were subjected with HIV can increase the risk of cancer in both sites. toFor enzyme-linked RVA identification, immunosorbent 488 stool assay samples (RIDASCREEN). recovered Furthermore, the HPV cross-infection between cervical The viral RNA of positive samples was extracted and and anus is common, both acting as a reservoir to subjected to reverse-transcription polymerase chain another. The aim of this study is to describe HPV types reaction (RT-PCR). The primers used were Beg9/End9 found in cervical and anal specimens from HIV-positive and 4con3/4con2 for the G and P type, respectively. women attending the Reference Center for STD/AIDS, Vitória-ES, from September 2013 to June 2014. Cervical and anal samples were obtained with cytobrush from For RVA genotyping the product of the first round of 73 HIV-seropositive women, aged between 18 and amplification (RT-PCR) was subjected to a second round 65 years. Viral DNA was extracted using the QIAGEN Gof (G1,amplification G2, G3, G4, (seminested G9, and G12) PCR) and with P-types the same (P[4], primer P[6], QIAamp® DNA Mini Kit. HPV was screened with the P[8],used inand RT-PCR P[9]). in Results: combination RVA positivitywith specific was primers observed for sets of PGMY09/11 primers and genotyped by Reverse in 9.6% (47/488) of the samples. The predominant G Line Blot (RLB) with probes for 32 genotypes. The types detected in 2012 in Rio Branco were: G2 40.4% results showed HPV detected in 74% (54/73) of women (19/47), G3 23.4% (11/47), G12 21.3% (10/47) and in cervical and/or anal sites, being 45.2% (33/73) G1 4.2% (2/47), and with regard to the P types: P[4] and 65.8% (48/73) at each site, respectively. In 50% 40.4% (19/47), P[8] 29.8% (14/47) and P[6] 21.3% (27/54) of the positive cases, HPV was present at both (10/47). Five samples were not typed for VP7 gene and sites. Twenty six different genotypes were found and four for VP4. Genotype G2P[4] was the most prevalent the most frequently detected in the anal and cervical combination accounting for 40.4% (19/47), mostly in sites were, respectively, HPV-16> 6> 51 and 53 and January with 58% (11/19) of the cases. Conclusion: HPV-45> 31> 16, 52, 53 and 69. Infection with the same genotype at both sites was present in 48.1% (13/27), circulating in the post-vaccine period in Rio Branco, and 69.2% (9/13) of them were HR-HPV. Mixed infection theThis capital study ofallowed the Acre the State, identification Brazil, and of showedRVA genotypes similar occurred in 57.4% (31/54). These preliminary data results if compared with other studies conducted in show an important and partial correlation between HPV genotypes detected in both sites and suggest a common G2P[4] has been observed after the introduction of the source of infection or even, spread between these Rotarixnorthern vaccine. Brazil Furthermore, where a significant the epidemiological predominance basis of anatomical sites. Detection and typing of HPV in HIV- of the cyclic phenomenon of rotavirus genotypes is not positive women are important for monitoring of anal fully elucidated, and this represent challenges with and cervical lesions. The persistence of HPV infection regard to the effectiveness of rotavirus vaccines against and the presence of lesions will be analyzed, which will the variability of genotypes detected. FINANCIAL generate information that will clarify the natural history SUPPORT: INSTITUTO EVANDRO CHAGAS of HPV infection and persistence in this vulnerable group of women. FINANCIAL SUPPORT: UFES, FAPES September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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HV158 - STANDARDIZATION OF THE REAL TIME Four patients with HHV-6 (33.3%) had acute GVHD and POLYMERASE CHAIN REACTION IN DETECTION overlap. HHV-7 occurred in 31/57 (54.3%) in a median AND MONITORING OF EARLY BETAHERPESVIRUS of 15 days after transplantation (range 0-98 days). In REACTIVATION IN HEMATOPOIETIC STEM CELLS 3/31 patients with HHV-7 (9.7%) died in less than 100 TRANSPLANT PATIENTS days after transplantation and their main cause of death Bonon, S.H.A.; Oliveira, R.S.; Dellariva, T.C.; Lima, was acute GVHD and bacterial infection. Eight patients R.G.; Costa, S.C.B. with HHV-7 (25.8%) had acute GVHD and overlap. Active betaherpesvirus infection monitoring using sensitive University of Campinas/Faculty of Medical Sciences/ techniques are very important to use preemptive Laboratory of Virus therapy. Supported by FAPESP. of infectious agents are being deployed in routine, HV162 - IDENTIFICATION AND MONITORING OF toMolecular minimize methods the consequencesfor the detection and and avoid quantification possible INFLUENZA VIRUS A AND B, IN POPULATION MACEIÓ clinical manifestations of herpesviruses, providing Antão, K.L.; de Sá, J.P.O.; de Lima, M.C.; Pinheiro, early treatment with appropriate antiviral, especially T.M.L.; Pinheiro, M. da S.; de Barros, L.H.R.; Maria, CMV. Real Time Polymerase Chain Reaction (qPCR) in F.H. de O.S. plasma is one of the options to be used in monitoring of UNCISAL - Universidade Estadual de Ciências da patients. Also, it is necessary to develop such techniques Saúde de Alagoas, Rua Doutor Jorge de Lima, 113, Trapiche “in house” to minimize costs with commercial kits. The da Barra, Maceió - AL, 57010-300 objective of this study is to standardize an qPCR “in house” using Taqman technology for the detection and syncytialViral agents, virus such(RSV) asare influenza responsible viruses for outbreaks A and of B, and HHV-7) in HSCT patients. An nested PCR and CMV acuteparainfluenza respiratory 1, 2infection and 3, adenovirus(ARI), causing and a respiratory high rate antigenemiaquantification was of also betaherpesvirus performed and DNA the (CMV, comparison HHV-6 of morbidity and mortality, especially in children and between these techniques was evaluated. Plasma the elderly. This work aimed to study these viruses samples from 57 patients were prospectively obtained as etiologic agents of ARIs in patients of all ages. Also aimed to assess the association between seasonality the transplant. Nested PCR for betaherpesvirus and and respiratory virus. The samples of nasopharyngeal CMVweekly antigenemia from the day were of transplantused in all untilsamples. day +100The qPCRafter secretion (NPS) were used for patients under 5 years of technique is being developed and standardized. The age and the combined swab (nasal and oral) for patients results will be compared to healthy group matched for above 5 years of age. These samples were collected at sex and age and with cardiac transplant patients group. the Center Sentinel Health Unit Dr. Ib Gatto Hawk, from Fifty seven patients were analised by Nested-PCR and lower and / or upper respiratory tract by up to three CMV antigenemia. Active CMV infection was detected by days. ARI and / or in the period November 2012 to N-PCR in 37/57 (64.9%) with a median of 34 days (range November 2013 a total of 12 months of observation. The 2-91 days) and CMV antigenemia in 24/57 (42.1%), analyzes of 488 samples of male (203) and female (285) median 42 days (range 0-91 days) after transplantation. sexes were performed by IIF in virology sector LACEN- Five patients (13.5%) with active CMV infection died in less than 100 days after transplantation (median 50 days, of monoclonal antibodies (MAbs). Of the 488 samples, range 46-100 days) and two of them (40%) had CMV AL. With the indirect immunofluorescence using a panel disease. Fourteen with active CMV infection (24.6%) had acute GVHD and overlap. Five patients (8.8%) had CMV RSV.42.4% It is (207) observed showed that IFA 25.8% positive (126) for of influenza the cases A were and disease in the gastrointestinal tract (GIT), biopsy proven. B virus, parainfluenza types 1, 2 and 3, adenovirus and Active HHV-6 in plasma was diagnosed in 12/57 patients (21%) for a median of 24 days after transplantation (range positive for influenza A; 10.0% (49) for adenovirus; 4.1% 0-84 days). A patient with HHV-6 positive (8.3%) died by (20) for RSV; 3.5% (17) for parainfluenza 3; 2.5% (12) CMV disease in less than 100 days after transplantation. for parainfluenza virus 2; 1.8% (9) for parainfluenza 1; September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersand - Human 0.6% Virology: (3) influenza HV B. The monthly distribution of XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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for each virus. This fact makes clear the need to maintain continuouspositive cases surveillance shows a distinctvirology, epidemiologicalin order to mitigate profile the /were ASGUS), cytologically whereas classified 50% (1/2) as atypical, for high-risk squamous HPV or impact of these viruses during periods of peak activity. glandular cells of undetermined significance (ASCUS Suporte Financeiro: LACEN/AL - Laboratório Central low-grade (including HPV suggestive alterations and de Saúde Pública de Alagoas UNCISAL - Universidade cervicalcompared intraepithelial to CH2; five presentedneoplasia intraepithelialgrade I), 80% lesions (4/5) de ciências da saúde de alagoas PROBIC/UNCISAL - of these was positive for high-risk, and a single sample

ProgramaHV163 - Institucional PARADIGM de SHIFTBolsas de IN Iniciação DIAGNOSIS Científica OF weclassified note that as invasivea large percentage squamous (17%)cell carcinoma of HPV detection revealed PATIENTS ATTEND FOR GYNECOLOGIC ROUTINE ina high-risk molecular oncogenic tests that in areCH2. not According reported to in the cytological findings, EXAM IN BELÉM METROPOLITAN REGION, PARÁ Silva, A.K.1; Paixão, C.G.S da1; Silva, A.K.1; Ferreira, occur by the subjectivity of the Pap smear, thus showing L.S.S.1; Lima, S,F.2; Junior, L.B.D.2; Mello, W.A.1; necessarytests being to classified implement as aone molecular of several tool biases to complement that may Silvestre, R.V.D.1 routine gynecologic examinations, thus increasing the 1. IEC - Instituto Evandro Chagas, Rodovia capacity to predict changes arising from HPV infection. BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 FINANCIAL SUPPORT: MS / SVS / IEC 2. Laboratório Paulo C. Azevedo, Avenida HV165 - DIVERSITY OF HADV IN HUMAN STOOLS Comandante Brás de Aguiar, 99 - Batista Campos, Belém - PA, 66035-000 Vicentini, F.1; Gomes, Y.M.1; Rodrigues, M.C.1; Mello, I.O.1; Leite, J.P.G.2; Miagostovich, M.P.2; Spano, L.C.1 precursor lesions of cervical cancer, new technological 1. UFES - Universidade Federal do Espírito advancesIn order are to being improve implemented. the efficiency In screening of diagnosis programs of Santo, Av. Fernando Ferrari, 514, Goiabeiras, Vitória - ES, for cervical cancer is utilized for decades the Pap test, 29075-910 which is the primary method for diagnosis, however, 2. FIOCRUZ - Fundação Oswaldo Cruz, Av. depends on several factors, getting too subject to results Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 bias. Currently new microscopic and molecular tools Adenoviruses (HAdV) have long been associated with have united to complement these results with greater diarrhea, but to establish a causal relationship with accuracy. In this study, we investigated the relationship between cytological and molecular diagnostics for feces of other types not enteric. In order to characterize Human Papillomavirus (HPV), about high and low thedifferent types types of HAdV has excreted been difficult in the due feces for of the black detection children in oncogenic HPV risk in patients aged 20-50 years attend that living in Quilombola communities in Espírito Santo for routine gynecologic exams in Belém / PA. For results comparison, the Pap test and Hybrid Capture and sequenced. Between August 2007 and May 2010, second generation (CH2) molecular method are used. 212state, fecal the hexonsamples gene were was amplifiedobtained byfrom PCR 133 (nt (62.7%)21-322) For diagnosis, 184 samples were tested. In the CH2 children with (symptomatic) and 79 (37.3%) without tests, a total of 19.5% (36/184) were positive for HPV (asymptomatic) diarrhea. The overall frequency of being 12% (22/184) positive for high risk HPV; 3.8% positivity was 27.8% (59/212), with 30.1% (40/133) (7/184) tested positive for low risk HPV and 3.2% and 24.1% (19/79) among the symptomatic and (6/184) positive samples are mixed infections caused asymptomatic children, respectively. Fifty samples were by types of high and low risk or more than one viral sequenced. Thirty three samples of the symptomatic type. In the analysis of cytological examinations of 184 cases belonged to the following species: 3 (9.1%) A, 6 samples, 176 tests are considered without cellular (18.2%) B, 18 (54.5%) C, 2 (6.1%) D, 4 (12.1%) F; E and atypia (usual default), being 17% (30/176) of these G were not found. Among the C species, types 1, 2, 5 and specimens revealed positive for HPV in CH2 (56.6% 6 were found. The HAdV-C (n=27) was found in the feces HR / LR 20% / 23.4% mixed infections); two samples with a frequency of four times that of the enteric HAdV-F

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(n=7). The HAdV-A and HAdV-F species accounted for identity was observed with strains detected from approximately 20% of the total of the HAdV types and Denmark (96.3%), and France (96.1%). The occurrence infected only children up to 5 years of age and 70% of of aseptic meningitis caused by EVs in newborns is rarely these samples were present in the stools of symptomatic children. The HAdV-F41 was excreted equally between clinical samples for conducting viral research. In this symptomatic and asymptomatic. This study showed a study,detected, we probablydocument duethe rare to the occurrence difficulty of in a collectingCV-B5 in very large diversity of HAdV in human feces. neonate. This result draws attention to the importance of an etiologic diagnosis in early disease. Furthermore, HV169 - ASEPTIC MENINGITIS IN NEONATE BY it demonstrates the importance of routine laboratory COXSACKIEVIRUS B5 GENOGROUP B IN BRAZIL diagnosis of EVs contributing to the management of Carmona, R. de C.C.1; Vieira, H.R.1; Machado, B.C.1; neurological disease, the broader genomic knowledge Sousa, C.A.1; Lo, D.S.2; Ibidi, S.M.2; Gilio, A.E.2; Alves, of EV, and epidemiological study of the circulation of M.R. de M.1; Carmona, R. de C.C.1 CV-B5 in the pediatric population. Financial support: 1. IAL - Instituto Adolfo Lutz, Av. Dr. Arnaldo, Fapesp 2012/50234-5. FINANCIAL SUPPORT: FAPESP 355 - Cerqueira César, São Paulo - SP, 01246-000 2012/50234-5. 2. HU/USP - Hospital Universitário da HV173 - DETECTION OF RARE REASSORTANT G5P[6] Universidade de São Paulo, Av. Prof. Lineu Prestes, 2565 - Cidade Universitária, São Paulo - SP, 05508-000 ROTAVIRUS STRAIN IN CHILD WITH DIARRHEA, BRAZIL Enteroviruses (EVs) comprise a large genus in the Carmona, R. de C.C.; Cilli, A.; Luchs, A.; Morillo, S.G.; Picornaviridae family, with more 100 serotypes Gregório, D. de S.; Carmona, R. de C.C.; Timenetsky, delineated into four species (A–D) based mostly on M. do C.S.T. their phylogenetic relationships. Coxsackievirus B5 (CV- B5) belongs to the species Enterovirus B, considered IAL - Instituto Adolfo Lutz, Av. Dr. Arnaldo, 355 - one of the most prevalent serotypes in humans, often Cerqueira César, São Paulo - SP, 01246-000 associated with sporadic cases of neurological diseases Group A rotavirus (RVA) genotype G5, which are and epidemics of meningitis aseptic. EVs neonatal common in pigs but also detected in horses and cattle, infections are associated with a broad spectrum of signs and symptoms, ranging from febrile illness not reportedwere first in identified children inwith humans severe in diarrheaBrazil in theworldwide. 1990´s. SinceFollowing 90´s, its G5 first RVA detection, strains has this been genotype rarely hasisolated also beenfrom specific to a multisystem disease potentially fatal. The aseptic meningitis in neonate by CV-B5 in Brazil, and humans and commonly found in combination with P[8] aim of this study was to report the first detection of to understand of the genetic relationships with strains genotype. The aim of this study was to analyse the rare detected in others countries by phylogenetic analysis of collected from a 12-years-old boy in May 2013, state of neonate with suspect of meningitis aseptic was collected Goiás,G-P combination Western Brazil. G5P[6], RVA genotype identified G5P[6] in a stoolwas detected sample the protein VP1 gene. The cerebrospinal fluid (CSF) from and subjected to cell culture isolation. Subsequently, using a commercial immunoenzymatic assay, PAGE, genotyped by RT-PCR and sequencing of the VP4 and VP7 genes. The comparison of the VP7 sequence of IAL3029 enterovirus-positive was identified as serotype CV- assay (IFA), and carried out RT-PCR and sequencing strain showed 94-95% of nucleotide (nt) identity B5 (IAL-E1186) by indirect immunofluorescence the VP1 region. Genetic analysis of IAL-E1186 strain compared to human Brazilian G5 strains of 90´s decade, revealed that the VP1 displayed a great relatedness to including prototype IAL28 (Lineage I) (bootstrap value 99%). Comparing with others human and animal VP7 genogroup B. The VP1 sequence of IAL-E1186 strain sequences from Europe, America, Asia, and Africa, the other selected human CV-B5 strains already defined to displayed the highest nucleotide sequence identity to similarity varies from 78-88%nt. The VP4 sequence of STU27/DEU/09 strain (96.5%), which was detected in IAL3029 clustered within Lineage I showing the highest Germany in 2009. Also, the highest nucleotide sequence nucleotide identity (95%nt) compared to Brazilian

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

143 Human Virology: HV porcine strains (PGRV33 and 898/07) and ranged from 82 to 92%nt comparing to animal RVA P[6] strains from transmission detected, around 61,864 suspected cases werevirus Chickungunyareported by Pan in America. American Since Health the first Organization autoctone human G5P[6] genotype supports the hypothesis of a until the epidemiological week 20 of 2014. Meanwhile, dynamicother countries. interaction The identificationbetween human of a and rare animal reassortant RVA, in the same region Dengue epidemics still happen and are most related to the introduction of new serotypes among co-circulating human and animal RVA. Recently, or the ones not in circulation recently. In this context, uncommonindicating that RVA there strains is a constantwith similarity flux of genetic to animal materials strain the city of Rio de Janeiro, which is hosting major events, have been detected in Brazil and selective vaccine and increasing foreigners visiting, is vulnerable to pressure could increase the circulation of unusual import arboviruses. The aim of this study is to monitor strains. The continued RVA surveillance in Brazil is vital mosquitoes infected by arboviruses in the region where to monitoring the possible reemergence of strains, such it is being built Olympic Village, residence of athletes as genotype G5, after the introduction of the vaccine during the Olympic and Paralympic Games in 2016. against RVA. In addition, simultaneous surveillance of Adult mosquitoes are collected in traps BG Sentinel ® animal RVA infections is necessary for understanding and Aspirator. Insects are separated by gender, species, the evolution of RVA. FINANCIAL SUPPORT: INSTITUTO sex and analyzed by RT-PCR for detection of Dengue ADOLFO LUTZ CTC/53-2005. virus, Chikungunya and other arboviruses. From October 2013 to January 2014, 2,056 mosquitoes were collected HV188 - DETECTION OF ARBOVIRUS IN MOSQUITO 32 Aedes sp. (7 males and 25 females) and 2.023 Culex POPULATION OF RIO DE JANEIRO CITY: MONITORING sp. (538 males and 1,486 females). The area investigated IN PREPARATION FOR 2016 OLYMPICS presented a prevalence of mosquitoes of the genus Ferreira, D.F.; Santos, L.L.R.; da Silva, J.L.1; dos Reis, Culex, however, it was possible to detect within the A.S.1; Dias, C.M.G.2; Ribeiro, M.S.3; Meire, G.L.S.4; sampling Aedes sp a positive female for Dengue virus Chanasit, J.S.5; Campos, R.M.4 serotype 4. The production of information concerning 1. Gerencia de Pesquisa em Antropozoonoses - rates of infection vectors is a pioneering activity in GPA/LACEN/SVS/SESRJ. health services in the country and may contribute to the 2. Gerencia De Doenças Transmitidas Por assertiveness of the models used to predict outbreaks Vetores E Zoonoses - GDTVZ/DTI/CVE/SVS/SESRJ and epidemics scenarios and decision making in relation 3. Subsecretaria de Vigilância em Saúde - SVS/ to vector control actions. FINANCIAL SUPPORT: FAPERJ; SESRJ BNI; INBEB 4. Laboratório de Interação Vírus Célula, Departamento de Virologia, Instituto Microbiologia Prof. HV196 - FREQUENCY OF SEROLOGICAL MARKER FOR Paulo de Góes. Centro de Ciência e Saúde, Universidade HUMAN T-CELL LYMPHOTROPIC VIRUS (HTLV-I/II) Federal do Rio de Janeiro. IN INMATE POPULATION OF A CITY OF PERNAMBUCO 5. Instituto Bernhard Nocht para Medicina Morais, V.M.S.1; Lopes, T.R.R.1; Cahu, G.G. de O.M.1; Tropical – Colaborador da OMS para Arboviroses e Febres Silva, D.M.2; Rabelo, D.C.C.2; Lucena, W.A.T.2; Lima, P.C. Hemorrágicas de S.2; Albuquerque, A.C.C.2; Coelho, M.R.C.D.2 The last two decades have been marked by the large 1. UFPE - Universidade Federal de Pernambuco, number of human diseases caused by arboviruses. Av. Prof. Morais Rego, 1235 - Cidade Universitária, Recife - An arboviral depends on the replication in two hosts: PE, 50670-901 vertebrate and arthropod vector. In this scenario, 2. ASCES - Associação Caruaruense de Ensino climatic changes, the increasing area of deforestation, the Superior e Técnico, Av. Portugal, 584, Bairro Universitário- increasing speed in transportation of hosts and vectors Caruaru - PE, 55016-400 and the disorganized urbanization are contributing Prisons are known to be favorable environments for the factors for the emergence and re-emergence of arboviral transmission of sexual and blood diseases. The inmate diseases in various regions of the world. One example population is considered high risk for infections such as was the recently introduction (December of 2013) of the September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

144 Human Virology: HV

HV206 - HUMAN GROUP C ROTAVIRUS IN C and sexually transmitted diseases. Factors such as HOSPITALIZED CHILDREN FOR GASTROENTERITIS marginalization,human immunodeficiency illicit drug virus,use and hepatitis low socioeconomic B, hepatitis IN NORTHERN REGION, BRAZIL: EPIDEMIOLOGICAL, level contribute towards dissemination of infectious CLINICAL AND PHYLOGENETIC ANALYSIS and contagious diseases in prisons. However, the inmate Lobo, P. dos S.2; Guerra, S. de F. dos S.1; Siqueira, population has needed to investigate other virus, such J.A.M.1; Soares, L. da S.1; Gabbay, Y.B.1; Linhares, A. da as human T-cell lymphotropic virus (HTLV-I/II). The C.1; Mascarenhas, J.D.P.1 HTLV-1 can be transmitted by horizontal, vertical and parenteral route, staying asymptomatic in approximately 1. IEC - Instituto Evandro Chagas, Rodovia BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 97% of patients. In Brazil, data based on the prevalence 2. Centro Nacional de Primatas of blood donors, it was estimated that 2.5 million people are infected with HTLV-I in 2002. On the other hand, Acute gastroenteritis (AG) is a major cause of morbidity a study realized in Minas Gerais found a frequency of and mortality and rotavirus is one of the causative 1, 6% for HTLV, however, there are few studies of the agents of AG and it is responsible for about 38.3% of prevalence of HTLV in the inmate population of a city the hospitalizations, resulting in 197,000 deaths of of Pernambuco. The aim the study was to estimate the frequency of positive anti-HTLV I/II markers in male countries. Rotavirus belongs to the family, children under five years annually, mainly in developing population of a prison in the state of Pernambuco, in the genus Rotavirus, have genome composed by 11 period May to July 2011. The datas from each individual segments of double-stranded RNA (dsRNA) and are were collected through an interview, after 5 mL peripheral blood was collected in without anticoagulant H). Most of the cases belongs to the group/species A classified into eight groups/different species (A to tube. The samples were centrifuged at 1500 rpm for (RVA), however rotavirus group/species C (RVC) has 10 minutes to separate serum, after, the samples were assumed importance in gastroenteritis and is usually forwarded to the Department of Virology of Laboratory relate to self-limiting childhood diarrhea, with possible Immunopathology Keizo Asami (LIKA) of Universidade transmission from pigs. This study aims to detect the Federal de Pernambuco (UFPE) to perform the anti- RVC in children less than three years of age hospitalized with AG in Belém city, Pará state, Brazil. From May 2008 The serology was realized in 1085 samples and the to April 2011, an intensive surveillance for AG was frequencyHCV by the of commercial the anti-HTLV kit I/II(Murex was 0.72%HTLV I+II, (8/1085). DiaSorin). The performed in a pediatric hospital of Belém. A total of 279 population study has less 30 years old, 75% (6/8) were fecal samples with negative results to RVA and norovirus married, 37.5% (3/8) had tattoos and have used cocaine. had been tested for RVC. The nucleic acid was extracted None inmates used injection drugs or had sex with from a 10% suspension using silica methodology. The another man, 50% (4/8) did not use condom, 25% (2/8) cDNA was obtained by reverse transcription and after had a sexually transmitted disease and 12.5% (1/8) had received blood transfusions. The frequency of anti-HTLV amplified by polymerase chain reaction (PCR) using (I/II) was lower in this population than in Minas Gerais primer pairs specific for the VP7 gene (G8S/G8A); for State, this may be due to the low frequency of risk factors, VP6 (C1/C4); for VP4 (T434/T435); and NSP4 (+)/ such as injection drugs and use cocaine. However, data Cowden) and negative control (water RNAse free) (-) specific for NSP4 gene. Positive control (prototype about HTLV among Brazilian prisoners is still scarce, and were used in all the tests. Automatic sequencing and it draws attention to the need for epidemiological studies subsequent phylogenetic analysis were performed. Data and policies to prevent transmission of infectious and were analyzed using statistical chi-square partition, contagious diseases during incarceration. FINANCIAL SUPPORT: CNPq. 0.05. The positive rate for RVC was 2.1% (6/279) by PAGE Fisher’s exact test and linear regression with p values ≤ samples as I2 (VP6), G4 (VP7), P[2] (VP4) and E2 (NSP4) genes.and RT-PCR. The frequency The sequencing of RVC was classified higher in these the age positive group between 24 to 36 months (5.7%) with p= 0.0493. Male September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

145 Human Virology: HV were more affected by RVC with a frequency of 83.3%. Caraya A. and C. penicillata were positive by HI. From the Signs and/or symptoms most frequent in hospitalized children with RVC were fever (80%), vomiting (83.3%) of the following viruses: Bussuquara (n = 3), Mayaro (n and dehydration (100%). In summary, in this study it =total 1), samples,Cacipacore five (n (20.8%) = 2), Oropouche were positive (n = for3) oneand orDengue more was possible the characterization and phylogenetic (DEN-2) (n = 2). Noteworthy is the fact that one of the analysis of RVC VP6, VP7, VP4 and NSP4 genes, providing important data regarding the presence of these agents in Virus (VORO), featuring an animal not checked out the hospitalized children with AG in the metropolitan region urbananimals region was positiveand origin for of specific Goiânia, antibody which may Oropouche suggest of Belém, Pará. the existence of this movement as arboviruses epizootic promoted by a competent vector for the transmission of HV211 - DETECTION OF ANTIBODIES FOR YELLOW FEVER VIRUS AND OTHER ARBOVIRUS IN NON- in NHP in the region, and the record of positivity for HUMAN PRIMATE AT THREE URBAN PARKS IN antibodiesthe virus. Thisagainst is theVORO first in Goiás investigation state becomes of arboviruses relevant GOIÂNIA-GO in the context of public health. FINANCIAL SUPPORT: Badr, K.R.A.1; Gibrail, M.M.1; Vasconcelos, P.F.C.2; CNPQ Fiaccadori, F.S.1; Guissoni, A.C.P.1; Cardoso, D.D.P.C.1 HV214 - A HIGH-THROUGHPUT DNA MICROARRAY 1. IPTSP/UFG - Instituto de Patologia Tropical PLATFORM FOR THE DIAGNOSIS OF VIRUSES e Saúde Pública/ Universidade Federal de Goias, Rua 235 - TRANSMITTED BY ARTHROPODS AND RODENTS s/n, Setor Universitário, Goiânia - Goiás, 74605050 1 1 1 2 2. IEC - Instituto Evandro Chagas, Rodovia Trabuco, A.C. ; Khan, M.J. ; Alfonso, H.L. ; Badra, S.J. ; BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 Figueiredo, L.T.M.2; Quintana, V.H.A.1 Arboviruses are transmitted by hematophagous 1. FCFRP/USP - Faculdade de Ciências arthropod vectors. They are maintained in nature through Farmacêuticas de Ribeirão Preto/ Universidade de São Paulo, cycles involving vectors, hosts and their reservoirs, Avenida do Café, s/nº Ribeirão Preto - SP, 14040-903 such as vertebrates, and are thus present in the wild 2. FMRP/USP - Faculdade de Medicina de environment. In Goiânia, an outbreak of yellow fever in Ribeirão Preto/ Universidade de São Paulo, Av. Bandeirantes, 2007/2008, with records of epidemics, infections and 3900 - Monte Alegre, Ribeirão Preto - SP, 14049-900 death in the population of non-human primates (NHP’s) Brazil is a tropical country where several viruses urban parks in the township occurred. In this context, the transmitted by arthropods (arbovirus) and rodents present study aimed to carry one sero-epidemiological (robovirus) are circulating. Considering the public health research on NHP’s the city of Goiania. The study Were involved all species present in NHP Screening Center for for the development of rapid diagnostic methods. Wild Animals of the Brazilian Institute of Environment Thesignificance DNA microarray of these viruses,platform there has isemerged a great recently interest and Renewable Natural Resources - Regional Goiânia as a high-throughput method for pathogen detection. (CETAS-IBAMA), Thus, blood samples were collected by The objective of this study was the development of a femoral venipuncture, or brachial vein from 24 animals. DNA microarray platform (RoboArbovirusChip) for After processing and storage, the samples were sent the detection of roboviruses and arboviruses. Based to LACEN-GO for forwarding to the Instituto Evandro on the sequences of 466 viruses (roboviruses and Chagas - IEC. The samples were subjected to reactions of arboviruses) deposited in the GenBank, 4715 probes the Hemagglutination Inhibition (HI) using antigens from with 60 nucleotides were designed. Approximately 10 19 different arboviruses: Eastern Equine Encephalitis; probes per virus were designed. The microarray slide Western equine encephalitis; Mayaro; Mucambo; Guaroa; was prepared by Agilent (USA) in an eight array per Maguari; Tacaiuma; Yellow Fever; islanders; Icoaraci; slide format. Each array contained the 4715 probes Utinga; Sao Luis; Cacipacore; Bussuquara; Rocio; Belem; with at least three replicates, plus positive and negative Caraparu; Oropuche; Catu and Dengue virus (DEN-1-4). controls. The DetectiV software (http://detectiv. NHP’s species investigated, only the species C. libidinosus

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posterssourceforge.net/) - Human Virology: HV was used to analyze the fluorescence XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

146 Human Virology: HV data. To evaluate the RoboArbovirusChip, 15 viruses The information was collected in the interview in order to was tested: 15 laboratory viruses from 4 families were complete a questionnaire prepared by researchers, based detected: Flaviviridae – Dengue virus type-1 (DENV-1), on the symptoms of individuals with suspected dengue. Dengue virus type-2 (DENV-2), Dengue virus type-3 Results: From January 2012 to January 2013, 96 patients (DENV-3), Dengue virus type-4 (DENV-1), Saint Louis were recruited, of which, 47 were treated in a tertiary encephalitis virus (SLEV), Rocio virus (ROCV), Yellow unit and 49 in two primary health centers in Fortaleza, fever virus (YFV), Bussuquara virus (BSQV), Iguape Brazil.The mean duration of disease of the patients virus (IGUV) and Ilheus virus (ILHV) – Alphaviridae registered in tertiary healthcare was approximately – Mayaro virus (MAYV) – Rabdoviridae – Piry virus seven days, while primary units, was three days.The (PIRV) – and Bunyaviridae – Oropouche virus (OROV), basic units, the main symptoms of inclusion had the Rio Mamoré virus (RIOMV) and Guaroa virus (GROV). following frequencies in descending order: headache: The RoboArbovirusChip was also able to detect DENV- 81.63%(40), retroorbital pain: 76.65%(37), myalgia: 1, DENV-2, DENV-3 and DENV-4 from human serum 72.80%(36), vomiting: 42.85%(21) nausea: 38.77%(19), samples. No cross-hybridization was observed with RNA arthralgia: 32,04%(16) and rash: 15.38%(8). And the obtained fro C6/36 cells, mouse brain or dengue negative tertiary unit, the main symptoms include headache patients. These results strongly suggest the potential of were 80.85%(38), myalgia 76.59%(36), arthralgia this platform for the used as a rapid diagnostic method 63.82%(30), retroorbital pain 59.57%(28), nausea: for high-throughput monitoring of roboviruses and 57.44%(27), vomiting: 55.31%(26), rash 44.68(21) and arboviruses.FINANCIAL SUPPORT: FAPESP. petechiae 37.5%(18). With respect, the other clinical manifestations that may be associated with dengue HV217 - COMPARISON OF SYMPTOMS OF INCLUSION patients recruited in the basic units had the following AND OTHER CLINICAL DENGUE DRIVE BETWEEN percentage: 30.61% diarrhea(15), cough 26.53%(13), PRIMARY AND TERTIARY HEALTH UNIT odynophagia 20.40%(10), rhinorrhea 16.32%(8), Pontes, C.M.L.1; Monta, M.J.1; Neto, L.L.S.1; Igreja, meningism 8.16%(4), 10.20% sputum (5) and pruritus R.R.1; Filho, F.A.X.M.1; Souza, N.V.2; Parente, A.M.B.3; 4.08%(2).While, in tertiary units, 51% patients had Colares, J.K.B.4; Lima, D.M.1 cough (24), pruritus 35.41%(17), diarrhea 35.41%(17), 1. UNIFOR - Universidade de Fortaleza, odynophagia 29.16%(14), meningism 25%(12), 20.83% Avenida Washington Soares, 1321 - Edson Queiroz, Fortaleza sputum (8) and rhinorrhea 12.50%(6).Conclusion:The - CE, 60811-905 principal criterion for cases of suspected dengue 2. Departamento de Patologia e Medicina recommended by the Ministry of Health (2013) is Legal/ UFC - Universidade Federal do Ceará, Rua Monsenhor fever, and with respect to this signal, it was observed Furtado, s/n, Rodolfo Teófilo, Fortaleza - CE, 60441-750 that patients seek hospital when they are with a longer 3. MSC/UNIFOR - Mestrado em Saúde Coletiva disease than patients recruited the basic health unit. da Universidade de Fortaleza, Av. Washington Soares, 1321, Regarding the inclusion criteria, no differences between Edson Queiroz, Fortaleza - CE, 60811-905 units, demonstrating the importance of follow the new 4. HSJ - Hospital São José de Doenças Infecciosas, Rua Nestor Barbosa, 315, Parquelândia, Fortaleza have been added, such as vomiting and nausea that had - Ce, 60455-610 representativeclassification forprevalence. dengue FINANCIAL cases in which SUPPORT: symptoms MCTI; Dengue is an acute infectious disease currently CNPQ; MEC; SISU considered the most important arboviral disease in the world affecting in particular the tropical countries. Thus, the aim of this study was to identify the clinical adopted in Brazil since January 2014, through the comparisonmanifestations, of basedsigns onand the symptoms new WHO presented classification, by patients.Methodology:Cross-sectional study, conducted through interviews of patients with suspected dengue. September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

147 Human Virology: HV

HV220 - IDENTIFICATION OF HUMAN BOCAVIRUS agents on study, moreover, it was found as a co-infection IN STOOL SAMPLES FROM CHILDREN WITH ACUTE etiologic agent. This work contributes to the surveillance GASTROENTERITIS of gastroenteritis affecting children, showing the Sampaio, M.L. da S.1; Brandão, C.J. de F.2; Sardi, S.I.1; presence of HBoV as a possible viral etiologic agent. Campos, G.S.1; Dorea, A.1; Tigre, D.M.1; Paula, F.L.1; FINANCIAL SUPPORT: FAPESB ( Fundação de Amparo à Andrade, A. de S.1; Costa, L.F. de M.1 Pesquisa do Estado da Bahia) 1. UFBA - Universidade Federal da Bahia, Rua HV223 - RESPIRATORY INFECTIONS CAUSED BY Augusto Viana , s/n, Palácio da Reitoria, Canela , Salvador - PARAINFLUENZA VIRUS IN HOSPITALIZED CHILDREN BA, 40110-909 Ocadaque, C.J.; Pereira, S.A.R.; Oliveira, F.M.S.; 2. Hospital Aliança, Avenida Juracy Magalhães Florencio, C.M.G.D.; da Silva, F.E.R.; Alves, A. de A.; Junior, 2096 - Rio Vermelho, Salvador - BA, 41920-900 Moura, F.E.A. Gastroenteritis is an important cause of mortality UFC - Universidade Federal do Ceará, Avenida da and morbidity among children worldwide, especially Universidade, 2853 - Benfica, Fortaleza - CE, 60020-181 in children. This disease can be caused by bacteria, fungi, protozoa and virus infections. Among the viral The hospital respiratory infections (HRI) pediatric infections include: Norovirus (NoV), Human Rotavirus are responsible for high rates of infant morbidity and mortality. Due to non-implementation of routine the Human Bocavirus (HBoV). The HBoV has a laboratory methods for detecting virus little is known single(HRV), strandedAdenovirus DNA, (AdV) non-enveloped, and, more recently belongs identified, to the about the role of these agents in the etiology of HRI. Parvoviridae family, genus Bocavirus and encodes four International studies have highlighted several virus proteins: two structure proteins (VP1 and VP2), one nonstructural protein (NS1) and one unknown function virus (HPIV) type 3. Objective of the study is to verify associated with these infections, including parainfluenza nucleoprotein (NP1). The objective of this study was the detection rate of types 1, 2, 3 and 4 of HPIV in to detect HBoV in stool samples of children under 5 hospital respiratory infections in hospitalized children years old with clinical manifestation of gastroenteritis. Hospital Infantil Albert Sabin, located in Fortaleza-Ce. Initially, stool samples were tested for the presence Nasopharyngeal aspirate samples collected were used in of NoV, AdV and HRV by ELISA using the kits of the study from January to December 2013, hospitalized RIDASCREEN® 3rd. Generation Norovirus (R-Biopharm, and started a children of respiratory infection for at least Germany), RIDASCREEN® Adenovirus (R-Biopharm, 72 hours after your hospital stay. The samples were Germany) and RIDASCREEN® Rotavirus (R-Biopharm, analyzed by the technique of polymerase chain reaction preceded by reverse transcription (RT) for detecting the of HBoV was made by extraction of viral DNA in the stool HPIV 1, 2, 3 and 4 plus chain respiratory syncytial virus samplesGermany), with respectively. the QIAamp Subsequently DNA kit (QIAGEN, the identification Germany) and submitted to Nested-PCR using the Pan bocavirus FluB) and adenovirus (without RT). A total of 120 cases of HRI(RSV), were metapneumovirus, included in the study Influenza and, of A these, and B 31 (FluA (32.2%) and bp region of the gene encoding the polyprotein, VP1- were positive for HPIV, and 5 HPIV-1 (5/31), no HPIV-2 VP2.primer The that results amplifies of these a fragment tests demonstrated of approximately that from 576 (0/31), 14 HPIV-3 (14/31) and 12 HPIV-4 (12/31). The 105 samples analyzed, 41.9% (44-105) were positive for co-detections of HPIV with other virus were found in 10 HBoV, 19.04% (20-105) for NoV, 3.8% (4-105) for AdV and 3,8% (4-105) for HRV . Among the positive samples and 1 to CoVH-OC43. Patients that were detected HPIV cases, 4 with adenovirus; 3 with RSV, 2 with influenza A for HBoV, it was detected co-infection in 27.3% (12-44) had been hospitalized for neurological diseases (40%), stool samples, of which 91.6% (11-12) were co-infection lung (13.33%), heart (8.33%) and immunosuppression with NoV and 8.4% (1-12) with AdV; in contrast, the (8.33%). Children up to 24 months were the most HRI HBoV infection was detected exclusively in 72.7% (32- presented by HPIV. Infections were predominantly 44)patients. The results of this study show that HBoV has diagnosed as upper respiratory tract (82.67%), and high incidence of infection compared to the other viral pneumonia (10.33%) and bronchiolitis (7%) were September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

148 Human Virology: HV predominant in infections of the lower airways. Three presented the most promising antiherpes activity, with children with positive samples for HPIV (two with HPIV- an IC50 value of 39.20 µg/mL and no toxicity (>50 µg/ 3 and 1 to VPI-4) had complications from pneumonia mL) against the cells used in the antiviral assay. This and died. The occurrence of IPV-3 was predominant in preliminary parallel screening directed the selection the driest months of the year as already described. The of the most promising species. Phytochemical studies VPI-4 showed greater movement when compared to the are now being carried out to support the bioguided HPIV-1 and 2. This study shows that the HPIV can join fractionation of these samples, in order to isolate the HRI commonly the case in our city. FINANCIAL SUPPORT: natural products responsible for the biological activities FUNCAP-PPSUS 2009 experiments. FINANCIAL SUPPORT: CNPq and CAPES. HV231 - SCREENING OF MULTIPLE BIOLOGICAL identified, and further mechanistic in vitro/in vivo ACTIVITIES OF MARINE INVERTEBRATES AND ALGAE HV233 - MOLECULAR EPIDEMIOLOGY OF HUMAN FROM SOUTH AMERICAN COAST ADENOVIRUS IN ACUTE CHILDHOOD RESPIRATORY Boff, L.; Kratz, J.M.; Almeida, M.T.R.; Costa, D.T.M.; INFECTIONS IN FORTALEZA-CEARÁ, AN EQUATORIAL Gomes, M.; Sarda, F.N.; Steindel, M.; Reginatto, F.H.; AREA OF BRAZIL, BETWEEN 2001-2011 Schenkel, E.P.; Simões, C.M.O. Ocadaque, C.J.1; Pereira, S.A.R.1; Florêncio, C.M.G.D.1; Harsi, C.M.2; Marinheiro, J.C.2; Moura, F.E.A.1 UFSC – Universidade Federal de Santa Catarina, Campus Universitário Reitor João David Ferreira Lima - 1. UFC - Universidade Federal do Ceará, Trindade, Florianópolis - SC, 88040-900 Avenida da Universidade, 2853 - Benfica, Fortaleza - CE, Natural products remain an important source of active 60020-181 substances, even though the development of high- 2. USP - Universidade de São Paulo, Av. Prof. Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 throughput screens based on molecular targets proved valuable for drug discovery. The shift away from large Human adenovirus (HAdV) is commonly viral agent combinatorial libraries towards more diversity-oriented associated with infections of the respiratory tract, both small compounds collections, containing natural upper and lower, mainly in children under the age of products features, highlight their potential as drug leads. Marine natural products in special represent divided into 54 types. HAdV are important respiratory a spring of unique structures with diverse biological pathogens,five. HAdV arefound classified in between into seven 2 and species 27% (A-G)of acute and properties. In this study, we evaluated multiple in vitro respiratory infection (ARI) cases. Few studies have biological activities of 39 marine samples collected off analyzed the diversity of species and types of HAdV South American coast, containing crude extracts and associated with ARI in Brazil. The purpose of this study fractions from marine invertebrates (sponges, corals was to determine the circulation patterns of the different and ascidians) and algae. Cytotoxic potential was HAdV species and respective types associated with ARI evaluated on A549 cells (lung tumor) by sulforhodamine in children in the city of Fortaleza, northeastern Brazil. B assay. Antiviral activity (herpes simplex virus type 1, Nasopharyngeal aspirates were collected from children KOS strain) was investigated by plaque reduction assay. with ARI symptoms and were submitted to the indirect Antifungal and antibacterial activities were investigated by disk diffusion method against several pathogens. Antiprotozoal activity (Leishmania amazonensis levelimmunofluorescence of species and type assay through (IFA) for PCR, initial nested-PCR detection and of amastigotes) was assessed by a colorimetric assay sequencingHAdVs. Then of thethe HAdVhypervariable strains were regions identified of the tohexon the employing parasites expressing beta-galactosidase. gene (HVR1 to HVR6). HAdV were detected 290 (3.41%) Results were expressed as mean percentage of inhibition of the 8,517 samples, 60 (20.69%) of those found in or 50% inhibitory concentrations (IC50) of three coinfection with one, two or three viruses. A total of independent experiments. Results showed a very diverse 190 (65,52%) HAdV were molecularly characterized: 162 HAdV belonging to species B (85.72%), 21 to hydroalcoholic fraction from the sponge Raspailia sp. HAdV- C (11.11%) and 6 to HAdV-3 (3, 17%), getting profile of activities. Regarding the antiviral activity, the September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

149 Human Virology: HV

wild and peridomestic rodents can be involved in viral throughout the period the predominance of species B transmission. The aim of this study was to investigate ina strainalmost without all years identification. analyzed. Seven The different species types circulated were the knowledge of a rural population which lives in an endemic rural area to BV occurrence, evaluating period. The predominant types were 3 (67.73%) and perceptions about virus spread in environment, as well 7identified (17.80%). circulating HAdV-3 was in Fortalezafound in all during the years the analysisand the as measures to prevent infections. It was conducted HAdV-7 was not observed in years 2006, 2010 and 2011. an epidemiological survey and 240 individuals were interviewed about BV outbreaks in region and exposure and the only representative of the species and the HAdV- factors. The population comprises 127 men (52.9%) Only in 2011, all kinds of species identified C circulated, and 113 women (47.1%), aged from 5 to 90 years. A 2007. The detection rate of HAdV in this study is similar total of 124 individuals (51.7%) know or heard about 4, began to be identified in our population only from BV. Of these individuals, 100 (80.7%) have contact with The predominance of HAdV-B, type 3, observed in this dairy cattle and horses, 46 (19.2%) have contact with study,to the findingsis similar of otherto that studies reported using in IFA recent to detect studies HAdV. in wild environment, 67 (54%) practice milking. All of Colombia and South Korea. In turn, in southeastern Brazil there are reports of the predominant circulation People who never had heard outbreaks were asked of HAdV-C and HAdV-B, with type 7 being the major aboutthese variablesmain form were they statistically know BV, oversignificant, the radio p < (2.5%), 0.001. type. Prevalence of HAdV-C over HAdV-B has been or TV (2.9%), if a veterinarian or health professionals reported in many other Latin American countries. In explain to them (2.9%), over the internet (0.4%) or in their neighborhood (7.9%). After smallpox eradication, in the pediatric population of Fortaleza, associated with the vaccination that confers immunity against OPV differentconclusion, respiratory we identified complaints, seven different in a period types of of11 HAdVs years. was discontinued and several outbreaks have been FINANCIAL SUPPORT: CNPQ described worldwide. In Brazil, BV occurs in several regions of the country, but little is known about its HV234 - DO YOU KNOW BOVINE VACCINIA? epidemiology. At the present moment, outbreaks are PERCEPTIONS AND KNOWLEDGE OF associated to rural environments affecting dairy cattle ORTHOPOXVIRUS INFECTIONS IN RURAL BRAZIL and humans. Therefore, individuals located in these Calixto, R.S.1; Costa, G.B.1; Calixto, R.S.1; Augusto, areas are vulnerable. Knowledge regarding BV infection L.T.S.1; Bonjardim, C.A.1; Ferreira, P.C.P.1; Abrahão, and/or transmission is helpful to avoid virus spread in J.S.1; Moreno, E.C.2; Kroon, E.G.1; Trindade, G. de S.1 cattle herds and neighborhood, preventing outbreaks or 1. IMA - Instituto Mineiro de Agropecuária, R. even its contention. FINANCIAL SUPPORT: CAPES, CNPQ, Piauí, 639, Poços de Caldas - MG, 37701-024 FAPEMIG, MAPA, PPG-MICROBIOLOGIA UFMG. 2. HEMOMINAS, Rua Grão Pará, 882 - Santa HV236 - IN VITRO ANTIHERPES EFFECTS OF Efigênia - Belo Horizonte - MG, 30622-020 3. Departamento de Microbiologia/ICB/ A STANDARDIZED EXTRACT OBTAINED FROM UFMG - Laboratório de Vírus localiza-se no Departamento CECROPIA GLAZIOVII SNETHL de Microbiologia, no Bloco F4, sala 258, do Instituto de Silva, I.T.; Santos, T.C.; Silva, I.T.; Battisti, M.A.; Ciências Biológicas da Universidade Federal de Minas Gerais, Ortamnn, C.F.; Reginatto, F.H.; de Campos, A.M.; Av. Antônio Carlos, 6627, Pampulha, Belo Horizonte, MG, Simões, C.M.O. 31270-901 UFSC – Universidade Federal de Santa Catarina, In Brazil, Vaccinia virus (VACV), which is included Campus Universitário Reitor João David Ferreira Lima - in Orthopoxvirus (OPV) genera, is responsible for a Trindade, Florianópolis - SC, 88040-900 disease called Bovine Vaccinia (BV), characterized by Herpes Simplex Viruses cause mild to severe labial and vesiculopustular lesions in dairy cattle and horses, genital infections and represent a good enveloped virus as well as in humans. Infections can occur after direct model for antiviral in vitro research. Natural products contact with animals naturally infected. Furthermore,

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postershave - Human been Virology: evaluated HV due to the efficacy as an alternative XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

150 Human Virology: HV for chemical antiviral agents. Pharmacological studies HV237 - MOLECULAR AND SEROLOGICAL DETECTION using Cecropia glaziovii (embaúba) have described OF CYTOMEGALOVIRUS INFECTION IN CHILDREN ATTENDING DAY CARE CENTER Santos, W.K. da S.; Costa, M.A.; Joventino, K.M. de S.; antiviral activity, and quantification of the chemical major phenolic compounds. Thus, the increased Miranda, F.J.A.; Costa, M.C.M.; Araujo, L.; Costa, A.P.F.; constituents showed C-glycosylflavonoids as the need for predicting the optimal extractive conditions Souza, L.M.S.; Souza, L.B.F.C.; Rocha, N. de S.P.D.; has become a growing challenge in the search for Farias, K.J.S.; Machado, P.R.L. phytopharmaceuticals with high level of uniformity, reproducibility, stability, and biological activity. The UFRN - Universidade Federal do Rio Grande do Norte, Campus Universitário Lagoa Nova, Natal - RN, 59078-970 preparation of a standardized extract obtained from the Cytomegalovirus (CMV) infection is the most prevalent leavesaim of ofthis embaúba study was as well to optimizeas to evaluate the efficiency the cytotoxicity of the in the world with variable incidence according to the and the antiherpes activity. Standardized extract (SE) population studied. The aim of this study was to evaluate was prepared by the turboextraction technique. The the prevalence of CMV in children attending day care Response Surface Methodology (RSM) design was used center of Natal city, Rio Grande do Norte, Brazil. The to evaluate the effects of the variables: (A) solvent type prospective study included 106 children with 1-4 years (water, ethanol 20% and 40%), (B) stirring speed (9500 of age, 50% males and 50% females, besides twelve and 13500 rpm) and (C) extraction time (5, 10 and 15 daycare educators, with median of thirty-one years min). The SE was evaluated in terms of its phytochemical old. Anti-CMV IgG and IgM antibodies were determined markers, isoorientin (ISOO) and isovitexin (ISOV), by using ELISA and CMV DNA was detected by nested PCR HPLC. The cytotoxicity of the SE was investigated on using viral DNA extracted from peripheral blood cells. VERO cells using a trypan blue exclusion assay, and Viral DNA was extracted from serum with AxyPrep Body antiviral activity was tested against HSV-1 (KOS strain) by viral plaque number reduction assay. Results showed to molecular detection inside peripheral blood cells. Questionnairesfluid viral DNA/RNA recollected miniprep epidemiologic-clinical kit from positive samples data. on the content markers, where there was an increasing ofthat the the markers Factor Aextraction had significantly using ethanol influence 20% (p<0.0001) (similar positive anti-CMV IgG, and 17%, negative. As what results were obtained for 40%) (ISOO=8.14 mg/g and concernsOf the studied the detection sample, 83%of anti-CMV children wereIgM antibodies, confirmed 34.9% were positive, and 64% negative. The twelve on the ISOV content (p<0.0184) and Factor C had no daycare educators presented anti-CMV IgG antibodies. ISOV=4.12 mg/g). Factor B exhibited a significant effect No difference emerged from the comparison of positive the ideal extraction conditions using ethanol 20%, and negative child groups in the research of anti-CMV 9500influence. rpm and The 5 experimental min. (desirability design = 1). allowed Cytotoxicity to predict and IgG antibodies as what respects sex, age, income, mother antiviral activity results were expressed as 50% cytotoxic school degree, and water and nutrition status. At the concentrations (CC50) and 50% viral replication molecular detection, 6.6% samples viral DNA obtained inhibitory concentrations (IC50), respectively, in order from peripheral blood cells were positives and no to calculate the selectivity index (SI=CC50/IC50). SE sample of serum was positive. This study found a high inhibited HSV-1 replication showing a SI value of 50.13. prevalence of infection by CMV in children attending day These results indicate that SE might be a promising care center, and viral detection in peripheral blood cells candidate for the development of new pharmaceuticals in children with asymptomatic infection. FINANCIAL for the treatment of herpetic infections. FINANCIAL SUPPORT: Ministério da Educação (MEC). SUPPORT: CAPES/CNPQ

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HV238 - PREVALENCE OF EPSTEIN-BARR VIRUS of EBV infection in gastric adenocarcinoma was 1%. INFECTION IN GASTRIC ADENOCARCINOMA IN PARA There wasn’t concordance in results of qPCR and ISH STATE techniques. There isn’t association of EBV detection Costa, I.B.1; Paixão, L.C.F.1; Barros, I.C.1; Santos, tests with sex, histological type and primary site. In our L.F.P.1;Polaro, A.A.1; Monteiro, T.A.F.1; Burbano, study, we found a high frequency of lymphoid label, but it R.M.R.2 was not possible to infer if these infected cells have some

1. IEC - Instituto Evandro Chagas, Rodovia is suggested to make further studies to understand the BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 roleinfluence of these on thecells development and if it can ofproduce gastric effects cancer. in Then, tumor it 2. UFPA - Universidade Federal do Pará, Rua tissues. FINANCIAL SUPPORT: MS/SVS/IEC. Augusto Corea, nº 1, Belem Do Pará - Para, 68440-000 Epstein-Barr virus (EBV), formally named Human HV239 - DENGUE TRANSMISSION IN A MEDIUM- hespesvirus 4 (HHV-4), primarily infects B lymphocytes SIZED CITY FROM BRAZIL, 2008 – 2012: DESCRIPTIVE and epithelial cells and is present in over 90% of the EPIDEMIOLOGY adult population. The virus is associated with about Mondini, A.1; Ferreira, A.C.1; Neto, F.C.2 10% of gastric adenocarcinomas, ranging according to 1. FCFAR/UNESP - Faculdade de Ciências geographic region. Estimates by the National Cancer Farmacêuticas da Universidade Estadual de São Paulo, Institute indicate 20,390 new cases of gastric cancer in Rodovia Araraquara - Jaú Km 1, Araraquara - SP, 14801-902 Brazil in 2014, ranking the sixth most common cancer 2. FSP/USP - Faculdade de Saúde Pública da in the Brazilian population. In the North Region, is the Universidade de São Paulo, Av. Dr. Arnaldo, 715 - Consolação, second most frequent cancer in men and the third in São Paulo - SP, 01255-000 women. Gastric adenocarcinomas can arise in different Dengue has been a public health problem in tropical regions for decades but autochthonous transmission histological types: intestinal and diffuse. This study aimed has now been reported in countries like USA, France and toregions show ofthe the EBV stomach prevalence and arein gastric classified adenocarcinomas, into two main Croatia. In 2013, 2.35 million dengue cases were reported compares two detection techniques and associate the in the Americas and Brazil alone was responsible for more results with epidemiological characteristics. Was used than 50%. The country has been presenting outbreaks 307 samples of gastric adenocarcinomas. Slides from for more than three decades and important lessons can be learned. Thus, a detailed analysis of dengue hybridization technique (ISH), which was performed transmission in Araraquara, a medium-sized city at the withparaffin one commercial embedded tissuekit according were preparedto the manufacturer’s for in situ central portion of São Paulo, which is a critical area for instructions. The DNA extracted from the tumors was dengue transmission, may clarify transmission patterns used in quantitative PCR (qPCR) for detection of EBV, and entail effective control measures. Dengue has been using the kit “EBV Q-PCR Alert”. Of the 307 samples, 252 circulating in the city since 1990s at low incidences. had results in ISH, being two (1%) positive samples, However, the number of cases has increased in recent without labeling. For qPCR, 228 results were obtained: 133230 (91%)(58%) showwere lymphoidpositive and infiltrate 95 (42%) label undetectable.and 20 (8%) fromyears 2008 and we to 2012will be were describing recovered the fromscenario the Informationof five years The results by the techniques of ISH and qPCR did not Systemof transmission on Diseases herein. of OfficialCompulsory data onDeclaration. dengue reports Data achieve an agreement (p <0.0001) use McNemar test. from 5,253 reported cases were analyzed. The majority The association between the results of ISH or qPCR and variables such as sex, histological type and primary dengueoccurred or at dengue the first with five complications months of the was year a rare - January event toin is considered gold standard for studies of association theMay city. -which The isincidence final portion in Caucasians of the rainy (78%) season. was Severehigher ofsite EBV were and not cancer, statistically and in significant this study (p>it was 0.05). considered The ISH than in other ethnic groups, a pattern that has been described worldwide. Another important observation the presence of the virus in tumor cells. The prevalence for determining prevalence. It is much more specific for September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

152 Human Virology: HV is that dengue transmission has become endemic in the clinical diagnosis of dengue. These are preliminary data from a study that is being conducted in Araraquara to the year, with the exception of 2009, which was atypical detect DENV in mosquitoes and in humans. incity, the with whole cases state being of São officially Paulo. reportedThis is part in ofall an months ongoing of project that is also focused on classical and spatial HV245 - INTERLEUKIN-6 PROMOTER epidemiology involving a survey of risk factors such as POLYMORPHISM IN RENAL TRANSPLANT PATIENTS socioeconomic and climatic variables. Corresponding INFECTED BY CYTOMEGALOVIRUS author: [email protected] FINANCIAL SUPPORT: Santos, W.K. da S.1; Minnicelli, C.1; Souza, L.M.S.1; FAPESP 2013/02338-9 Joventino, K.M. de S.1; Costa, A.P.F.1; Freitas, J.C. de O.C.1; Pereira, M.G.1; Quintana, V.H.A.2; Farias, K.J.S.1; HV241 - MOLECULAR SURVEILLANCE OF DENV IN Lemos, T.M.A.M.1; Hassan, R.3; Machado, P.R.L.1 MOSQUITOES FROM A MEDIUM-SIZED CITY OF THE 1. UFRN - Universidade Federal do Rio Grande CENTRAL PORTION OF SÃO PAULO STATE, BRAZIL do Norte, Campus Universitário Lagoa Nova, Natal - RN, 1 1 1 2 Gusmao, A.F. ; Guioti, F. ; Mota, T.Q. ; Bergo, E.S. ; 59078-970 1 1 Ferreira, A.C. ; Mondini, A. 2. USP - Universidade de São Paulo, Av. Prof. 1. FCFAR/UNESP - Faculdade de Ciências Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 Farmacêuticas da Universidade Estadual de São Paulo, 3. INCA - Instituto Nacional do Câncer, Praça Rodovia Araraquara - Jaú Km 1, Araraquara - SP, 14801-902 Cruz Vermelha, 23, Centro, Rio de Janeiro - RJ, 20230-130 2. Superintendência de Controle de Endemias – The cytomegalovirus (CMV) infection is an important SUCEN/SR-06 cause of morbid-mortality in solid organ transplant Infections by the four dengue serotypes (DENV 1-4) patients. CMV reactivation from latency occurs after have been reported in more than 100 countries and it is immunosuppression, thus immune surveillance is considered one of the most important arthropod borne required to maintain constant persistent infection viruses in terms of public health. Aedes aegypti is the under control. The single nucleotide polymorphism main vector of DENV in Brazil due to its adaptation to the (SNP) located at position -174 in the promoter region urban environment. Araraquara, at the central portion of interleukin (IL)-6 gene is associated with differential of São Paulo State/Brazil, has a high quality of life (HDI expression of this cytokine. The aim of this study was to 0, 830), which is comparable to developing countries. investigate the role of IL-6 -174C/G with CMV infection However, no studies on DENV circulation were available in patients undergoing renal transplantation at the for the city. The aim of this study is to evaluate the University Hospital Onofre Lopes (HUOL), Natal, RN, presence of DENV 1-4 in samples of mosquitoes collected Brazil. From August 2012 to January 2014 samples were in Araraquara from February 2014 to January 2015. We collected from the peripheral blood of organ recipients have installed 150 ovitraps in a preestablished grid of after transplantation. DNA was extracted from peripheral census tracts that covered the entire municipality. The blood mononuclear cells (PBMC) and serum. Genotyping of IL-6 -174C/G was performed using a validated allele the eggs in cardboard paddles were hatched. Mosquitoes Green. Clinical ovitraps were collected five days after installation and information were obtained from medical records of ⎕ species and gender. A Multiplex-Nested-RT-PCR was patients.specific PCRA total in realof 52 time patients, using 27 SYBR (51.9%) male, were usedwere toidentified detect DENV and pooled in our accordingmosquitoes. to Socollection far, we havesite, included in the study with median age= 45y (22-69). Of collected 4041 eggs that produced 2712 adults and them, 1 (1.9%) and 48 (92.3%) were CMV IgM and IgG 336 pools. We have assessed 20 pools (10 pools of each gender). We could not detect the presence of DENV in (10.7%) presented rejection to the graft. Viral DNA was any of the pools. It is important to notice that DENV is detected+, with similar in PBMC frequencies of 20/52 (38.5%) for donors. and in serum Three of patients 10/52 actively circulating in the city and not even 10% of the (19.2%) patients. A possible association was observed pools were analyzed. We are still collecting eggs and between detection of CMV in recipients PBMC and the we will be collecting samples from febrile patients with

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersserological - Human Virology: status HV of donors with all the 20 CMV + being XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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Cytopathological examinations were performed of all 1.49). The frequencies of genotypes CC, CG and GG were samples and the treatment was made as recommended. 30related (57.7%), IgG + 32 donors (61.5%) (p= and 0.068; 0 (0%), OR= respectively,1.2; 95% CI= Hardy- 1.04 – DNA was extracted using QIAamp ® DNA Mini Kit Weinberg equilibrium (HWE) ( ²= 7.5, p= 0.006, df= 1). commercial kit, following manufacturer’s instructions. The CC genotype was present 7 (70%) of the 10 patients AAV DNA was investigated by PCR and nPCR and HPV by ⎕ with CMV infection in serum and in 23 (54.8%) from PCR and hybrid capture. AAV and HPV genotyping was the 42 patients negative for the virus in serum, although performed by RFLP and RLB, respectively. About 50% of cases referred to the colposcopy clinic had normal Fisher test). In this cohort, the SNP-174C/G genotypes cytology, followed by low-grade lesions and fewer, high- werethis difference not associated was not to statisticallyserological significantor molecular (p= status 0.48, grade lesions. Out of the normal group, 78% remained of CMV or clinical symptoms of reactivation. A possible with normal cytology, while 22% progressed to cervical reason for the HWE deviation found in our study is in lesion; 74% of the low-grade group regressed to normal the small number of patients and the low frequency of cytology, and 78.6% of the high-grade lesion group the G allele already described worldwide. A possible regressed to low-grade or normal cytology. Prevalence association was observed between serological status of AAV2 was 30% at both times of collection and HPV of donors and viral reactivation in recipients of renal in the second moment, 36.5% were positive. The PCR population that requires further investigation. testwas showedpositive goodfor 56% correlation of the samples, with hybrid at first capture visit, andfor transplant which describes a specific panorama of our HPV detection. Using hybrid capture and RLB, HR-HPV HV249 - AAV-HPV COINFECTION AND THE were the most frequent, with HPV16, 58, 51, 52 and 53 EVOLUTION OF THE INTRAEPITHELIAL CERVICAL LESIONS the study. Logistic regression analysis demonstrated the Freitas, L.B.; Volpini, L.P.B.; Boldrini, N.A.T.; Miranda, presencethe genotypes of AAV mostly was inversely identified related in the to two the momentsprogression of A.E.; Spano, L.C. of HPV-induced cervical lesions. FINANCIAL SUPPORT: UFES - Universidade Federal do Espírito Santo, Av. CAPES, FAPES Fernando Ferrari, 514, Goiabeiras, Vitória - ES, 29075-910 HV250 - VIRUS SURVEILLANCE IN PATIENTS WITH The cervical cancer is one of the most incident CLINICAL DIAGNOSIS OF DENGUE FROM A MEDIUM- types of cancer in women worldwide, being human SIZED CITY OF RONDONIA STATE, BRAZIL papillomavirus (HPV) recognized as the etiologic Pereira, W.V.E.G.; Fagotti, A.; Guioti, F.; Ferreira, A.C.; agent. Some cofactors related to viruses and hosts Mondini, A.

The adeno-associated virus (AAV) is a parvovirus that FCFAR/UNESP - Faculdade de Ciências Farmacêuticas dependsmay influence on the the helper progression virus for of productive malignant infection lesions. da Universidade Estadual de São Paulo, Rodovia Araraquara to occur, and HPV was the helper virus reported - Jaú Km 1, Araraquara - SP, 14801-902 more frequently in the genital tract. A bidirectional The infection by any of the four serotypes of the dengue interaction between these viruses was demonstrated, virus (DENV 1-4) can be asymptomatic or produce which AAV could repress the gene expression of HPV, a disease that ranges from a mild febrile illness to life threatening hemorrhagic manifestations. In Brazil, the parvovirus in the development of HPV-induced cervical majority of dengue disease diagnostics are obtained by carcinoma.subsequently Therefore, may reflect this on study a protective aimed to role investigate for this serology and clinical and epidemiological criteria. Virus the role of the coinfection AAV-HPV in the progression of intraepithelial lesions of the cervix in women attended reaction or the detection of the nonstructural protein at the University Hospital of Espírito Santo. To identify 1isolation, are usually identification performed ofin DENVreference by polymeraselaboratories chainfor a the persistence / clearance of the viral infection and the limited number of cases. No further tests to detect other progression / regression of the lesion, two samples of arthropod borne viruses (arboviruses) are performed each woman were collected with one year of interval. in febrile patients. In our study, we are testing samples

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

154 Human Virology: HV with clinical diagnostic of dengue from a medium sized HPV infection in men. The HPV Infection in Men (HIM) city from the west part of Brazil for DENV 1-4 and Study is a prospective study of the natural history of HPV infection in men from Brazil, , and the USA. At alphaviruses. We collected blood samples from febrile baseline, 22% of the genital specimens HPV-positive by patientsother flaviviruses, that had clinical along with diagnostic orthobunyaviruses of dengue from and August 2013 to January 2014 in Ji-Paraná (Rondonia), which is allegedly an area with the circulation of Roche Linear Array were considered HPV unclassified several arboviruses. Viral RNA was extracted and a HPVbecause types the was viral further type observed could not using be identified.a PCR-sequencing Among protocol,these unclassified and multiple samples, Beta-HPV a high types prevalence were found of Beta- in to detect members of the Flavivirus, Alphavirus and most specimens by a bead-based multiplex genotyping Multiplex-Nested-RT-PCR with genus-specific primers methodology. Our aim was to identify the prevalence to identify DENV 1-4, yellow fever virus, mayaro virus, and distribution of Beta-HPV types detected in samples oropoucheOrthobunyavirus virus and genera seven and other species-specific viruses was performed. primers concurrently collected from the genitals, anal canal, We were able to collect 103 blood samples for the study. and oral cavity of the HIM Study participants. Samples So far, we have tested 12 samples and we could not detect were analyzed using a bead-based multiplex genotyping DENV or any other arboviruses. However, 89% of the methodology capable of simultaneously identifying samples still remain untested. It is undeniably important 43 distinct Beta-HPV types. We analyzed 198 genital, to provide differential diagnostic for febrile illnesses, not anal, and oral samples concurrently collected among only for a better management of the patients but also to 66 participants (22 men per country). We observed understand the epidemiology of the viruses that may be that the overall prevalence and detection of multiples circulating in a certain location and the control measures infections by different Beta-HPV types was higher in the that should be taken to control their spread. genital region from the 3 study sites. Multiple infections were less common in the USA. Mexican men presented HV253 - PREVALENCE AND DIVERSITY OF BETA- the highest prevalence of the same HPV type across all PAPILLOMAVIRUS IN ANOGENITAL AND ORAL three anatomic sites analyzed. Concomitant detection SAMPLES OF MEN IN THE HIM STUDY of the same HPV types in the anal canal and oral cavity Nunes, E.M.1; Ferreira, S.1; Campbell, C.M.P.2; Gheit, was rare. Beta-HPVs were highly prevalent in the male T. 3; Tommasino, M.3; Baggio, M.L.1; Galan, L.4; Silva, genital region. The detection of the same Beta-HPV R.J.C.; Ponce, E.L.5; Giuliano, A.R.; Villa, L.L.6; Sichero, simultaneously in the 3 anatomical regions analyzed is L.1 suggestive of digital transmission or auto-inoculation. 1. Center of Translational Oncology, Cancer Further research is needed to determine how these Beta- Institute of São Paulo HPVs are transmitted. FINANCIAL SUPPORT: Ludwig 2. Department of Cancer Epidemiology, H. Lee Institute for Cancer Research; FAPESP [Grants numbers Moffitt Cancer Center and Research Institute 13/01440-4 to LS, 13/ 20470-1 to EMN and 08/57889- 3. International Agency For Research On 1 to LLV]; CNPq [Grant number 573799/2008-3 to Cancer (IARC) LLV]. The infrastructure of the HIM Study cohort was 4. Ludwig Institute for Cancer Research, São supported through a grant from the NCI-NIH to ARG (CA Paulo Branch R01CA098803). 5. National Institute of Public Health 6. Center of Translational Oncology, Cancer Institute of São Paulo; Department of Radiology and Basic Oncology, School of Medicine of The University of São Paulo and HPV Institute, School of Medicine, Santa Casa de São Paulo Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide. However little is known about the natural history of September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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HV263 - LACK OF TRANSMITTED MAJOR HIV-1 HV264 - SPATIOTEMPORAL DYNAMICS OF INTEGRASE RESISTANCE MUTATIONS AMONG HIV-1 DISSEMINATION OF NON-PANDEMIC HIV-1 SUBTYPE INFECTED CHILDREN AND ADOLESCENTS FROM RIO B CLADES IN THE CARIBBEAN REGION DE JANEIRO Gonzalo, B.; Cabello, M.; Mendoza, Y.Y. Azevedo, S.S.D.; Fernandez, J.C.; Morgado, M.G. FIOCRUZ - Fundação Oswaldo Cruz, Av. Brasil, 4365 FIOCRUZ - Fundação Oswaldo Cruz, Av. Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 - Manguinhos, Rio de Janeiro - RJ, 21040-900 Raltegravir, an integrase inhibitor (INI), is one of the epidemic in the Caribbean region is mostly driven salvage therapy options offered by the Brazilian Ministry byThe subtype Human B; immunodeficiency but information about virus type-1the pattern (HIV-1) of of Health for HIV-1 infected children from six years old viral spread in this geographic region is scarce and different studies point to quite divergent models of antiretroviral therapy (HAART). As this drug has been viral dissemination. In this study, we reconstructed inand use adolescents in Brazil sincefailing 2009, first and in thissecond study, line we highly evaluated active the spatiotemporal and population dynamics of the the potential occurrence of INI transmitted resistance HIV-1 subtype B epidemic in the Caribbean. A total of mutations and the HIV-1 subtype diversity among 72 1,806 HIV-1 subtype B pol sequences collected from HIV-1 seropositive children and adolescents from Rio de 17 different Caribbean islands between 1996 and Janeiro, Brazil. The viral RNA was obtained from plasma 2011 were analyzed together with sequences from the samples of 24 drug-naïve and 48 failing HAART children United States (n = 525) and France (n = 340) included and adolescents from Rio de Janeiro. Integrase region as control. Maximum Likelihood phylogenetic analyses revealed that HIV-1 subtype B infections in the Caribbean and automatically sequenced. Subtype was determined are driven by dissemination of the pandemic clade was amplified by nested polymerase chain reaction (BPANDEMIC) responsible for most subtype B infections mutations was performed using the HIV Drug Resistance across the world, and older non-pandemic lineages by phylogenetic analyses and the profile of resistance (BCAR) characteristics of the Caribbean region. The region as subtype B (73.5%), followed by F1 (10%), C non-pandemic BCAR strains account for > 40% of HIV- database. Most samples were classified in integrase 1 infections in most Caribbean islands; with exception URF_BF (7%) and one as URF_BU (1.3%). No differences of Cuba and Puerto Rico. Bayesian phylogeographic in(5.5%) HIV-1 and subtype A1 (2.7%). frequencies Five samples were observed were classified between as analyses indicate that BCAR strains probably arose in drug-naïve and failing HAART groups, except for subtype the island of Hispaniola (Haiti/Dominican Republic) around the middle 1960s and were later disseminated INI resistance mutations were not observed in the two to Trinidad and Tobago and to Jamaica between the late groups,F1 that wasbut not accessory identified mutations in the drug-naïve were detected, group. Major with 1960s and the early 1970s. In the following years, the higher frequency in the drug-naïve patients (33%). The BCAR strains were also disseminated from Hispaniola most prevalent mutation was V151I, present in 20.8% and Trinidad and Tobago to other Lesser Antilles in drug-naïve group and 8.3% in failing HAART group. islands at multiple times. The BCAR clades circulating in Hispaniola, Jamaica and Trinidad and Tobago appear to as HIV-1 subtype B. Mutation R263K was present in 8% have experienced an initial phase of exponential growth, andThis 4% mutation among was drug-naïve only detected and failing in samples HAART classified groups, with mean estimated growth rates of 0.35-0.45 year-1, respectively. The mutation L74V (4%) was only detected followed by a more recent stabilization since the middle drug-naïve group, while mutations E157Q (12.5%) and 1990s. These results demonstrate that non-pandemic G163K (2%) were only detected in failing HAART group. subtype B lineages have been widely disseminated The absence of major resistance mutations in both through the Caribbean since the late 1960s and account groups emphasizes the importance of INI in the salvage for an important fraction of current HIV-1 infections in therapy and these results highlight the relevance of the the region. continuous surveillance of integrase genetic diversity and transmitted mutations in the country. September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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HV265 - EVALUATION OF A POSSIBLE SENTINEL use of mosquito traps associated with RT-PCR does not SURVEILLANCE SYSTEM FOR DENGUE TRANSMISSION work as a sentinel event for early detection of DENV IN A NEIGHBORHOOD FROM SÃO JOSÉ DO RIO human infections but it can be used as an important PRETO (SP): EPIDEMIOLOGICAL AND MOLECULAR instrument for the entomological and viral surveillance. APPROACHES Parra, M.C.P.1; Favaro, E.A.1; Dibo, M.R.1; Neto, F.C.2; CAPES, CNPQ. EVOLVING INSTITUITIONS: FAMERP, FINANCIAL SUPPORT: FAPESP, PRONEX, INCT-DENGUE, Eiras, A.E.4; Kroon, E.G.4; Mondini, A.3; Nogueira, M.L.1 UNESP, UFMG E USP 1. FAMERP - Faculdade de Medicina de São HV266 - EXPERIMENTAL MODEL OF INFECTION BY José do Rio Preto,Av. Brigadeiro Faria Lima, 5416 - Vila São OROPOUCHE VIRUS IN MICE Pedro, São José do Rio Preto - SP, 15090-000 Mamani, P.R.; Souza, W.M.; Badra, S.J.; Figueiredo, 2. USP - Universidade de São Paulo, Av. Prof. L.T.M. Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 3. UNESP - Universidade Estadual Paulista, Virology Research Center, School of Medicine of The Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010 University of São Paulo in Ribeirão Preto, Brazil. 4. UFMG - Universidade Federal de Minas Oropouche virus (OROV) of the family Bunyaviridae, Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, genus Orthobunyavirus, serogroup Simbu, and is Belo Horizonte - MG, 31270-901 transmitted to mammals and birds by Aedes serratus, Dengue (DENV 1-4) is the most important vector- Culex quinquefasciatus and the Culicoides paraenses borne virus worldwide and Aedes aegypti mosquitoes is the primary vector in urban cycle. The OROV is the are responsible for the urban transmission of dengue. etiological agent of Oropouche fever in humans, which São José do Rio Preto (SJRP), a medium-sized city is clinically characterized as an acute febrile disease, located at the northwestern region of São Paulo State, that is endemic in north of Brazil and all Amazon region has been presenting high incidences of the disease The OROV is the second arbovirus most important, for more than 20 years and 2013 was not different. where estimated more than 500,000 cases has been Geographical Information System (GIS) and spatial reported in Brazil only supplanted by dengue virus. analysis tools may aid understanding how dispersion of We show here preliminary results of an experimental mosquitoes can impact dengue incidences. Spatial data model of infection by OROV in young adult Swiss albino concerted with traps for capturing adult mosquitoes mice (Mus musculus) by intraperitoneal inoculation. may enable the analysis of fundamental variables in The OROV strain BeAn 1999 was grown in C6/36 dengue transmission. In our study, we propose the use of adult traps associated with molecular diagnosis as a sentinel surveillance system for the early detection (Aedes albopictus) cells by 4 days, it was confirmed by of dengue cases in a region of SJRP. MosquititoTM titratedimmunofluorescent by detection assay. of cytopathic The stock effect was (CPE)centrifugation in serial and BG-SentinelTM traps were installed weekly from 10-foldat 5,000xg, dilutions filtered inoculated through ain 0.22 quadruplicates μm membrane of Vero and epidemiological week 15 of 2012 until epidemiological week 22 of 2013. The mosquitoes were tested with 107 plaque forming units (PFU) in Vero cells per ml. A Hemi-Nested-Multiplex-RT-PCR for the presence of numbercell cultures, of 24 and female obtained Swiss a albinofinal titer mice, of OROVwith 3 of weeks 1.5 x DENV. TerraView and ArcGIS softwares were used to old, were inoculated intraperitoneally with 3 x 106 perform spatial analysis and to produce thematic maps. PFUs of OROV in 200 ul of the virus stock and observed We were able to analyze 893 mosquito pools and 2.8% for 14 days. The develop of clinical symptoms were were positive for DENV-4. Thematic maps demonstrate observable after 6 days post inoculation, the animals clusters of vector infestation. However, they indicate started to show signals of encephalitis characterized that traps associated with molecular surveillance in by loss of equilibrium, circular march and seizures, mosquitoes were not suitable for the early detection of and a mortality rate of 50%. This experimental model, DENV circulation once human cases in the study area is a necessity to understand pathogenesis of OROV, and were detected previously in health facilities. Thus, the

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

157 Human Virology: HV is very important for preclinical testing of vaccine or therapeutic intervention for OROV. orientation, 40% (4/10) declared to be bisexual. No use ofcould injected be classified drugs was as reported.classes B andHaving C. Regarding had a tattoo sexual and HV269 - SEROPREVALENCE AND RISK FACTORS OF piercing were reported by 80% and 60%, respectively. HCV IN PEOPLE LIVING WITH HIV The frequency HCV/HIV coinfection in this study was Cahú, G.G.O.M.; Silva, D.M.; Lopes,T.R.R.; Morais, lower than in others Brazilian regions, this may be due V.M.S.; Silva, J.L.A.; Coêlho, M.R.C.D. to the low frequency of risk factors, such as injectable UFPE - Universidade Federal de Pernambuco, Av. Prof. drugs. FINANCIAL SUPPORT: SCHOLARSHIP FROM Morais Rego, 1235 - Cidade Universitária, Recife - PE, 50670- CNPQ. 901 HV271 - IMPACT OF A MULTIPLEX MOLECULAR Coinfection by hepatitis C virus (HCV) in people living PANEL FOR DIAGNOSIS OF RESPIRATORY VIRAL INFECTIONS often observed due to the similar transmission route, Sena, A.; Oliveira, C.P.; Silva, A.L. de S.; Trindade, particularlywith the human in relation immunodeficiency to the parenteral virus route, (HIV) illicit is A.C.G.; Nogueira, G.B.; Laurindo, M.F.L.; Bueno, V.D.C.; injecting drug use and blood transfusions. It is estimated Neto, M.M.; Moura, M.E.G.; Ricci, E.; Lazari, C. dos S.; that approximately 30% of all HIV-positive individuals Granato, C. are coinfected with HCV in the United States and Europe. In Brazil, prevalence depends on the geographic area, FLEURY - Fleury Medicina e Saúde ranging from 4.1% to 54%. The prevalence of cirrhosis is Viral infections are amongst the main causes of three times higher in patients with HCV/HIV coinfection respiratory diseases. They are usually diagnosed using than in patients exclusively infected with HCV. The aim of the present study was to estimate the prevalence of detects only a few number of pathogenic viruses and HCV among HIV-positive individuals and evaluate the doesdirect not fluorescence include emergent assays (DFA). ones. However,Conversely, this there method are risk factors associated with the coinfection. This cross- molecular panels currently available, which identify sectional study evaluated individuals attended at the a large amount of viral agents, with higher sensitivity University Hospital of Pernambuco, Northeastern Brazil, and faster performance. In order to evaluate the between November 2013 and July 2014. The patients impact of simultaneous molecular detection of multiple answered a standard questionnaire about socio- respiratory viruses for etiologic diagnosis of respiratory economic data, like the socio-economic status based infections, we analyzed 372 samples from respiratory on CritérioBrasil, which considers very poor people, tract processed in a private laboratory from 2011 classes D and E, whose income is under U$ 150.00 per October to 2013 July. A commercial kit has been used month. The samples were tested for HCV antibodies (CLART PneumoVir®, Genomica, Spain), based on a byenzyme-linked immunosorbent assay (ELISA) using multiplex polymerase chain reaction (PCR) step, and the Murex anti-HCV kit (version 4.0; AbbottDiagnostics Division), according to the manufacturer’s instructions. A total of 500 individuals were screened, of whom 181 simultaneously,subsequent analysis adenovirus, of the amplified bocavirus, material coronavirus on a type low (36.2%) were female and 319 (63.8%) were male, with density microarray. This platform detects and identifies, ages ranging from 18 to 98 years (mean = 43.1 years); H1N1, H3N2) B and C, metapneumovirus A and B, 29.2% were white, 21.4% were black, and 49.4% 229, enterovirus, influenza A (pandemic and seasonal representing other races. Regarding sexual orientation, syncytial virus (RSV) A and B. Of all samples, 56% 28.4% declared to be homosexual or bisexual. The wereparainfluenza positive for1, 2, at 3, least 4a and one 4b, virus, rhinovirus, a larger respiratoryproportion overall prevalence of HCV antibodies was 2% (95% CI compared to the 30% positivity of DFA found in previous 1.02%–3.77%) in people living with HIV. This serological studies performed in the same institution. Rhinovirus marker was observed in 8/10 (80%) males, the age was the most frequent (34% of positive samples), group more frequent was from 40 to 49 years (4/10, followed by RSV (19%), metapneumovirus (9%) and 40%) and the socio-economic status in 8/10 (80%)

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersinfluenza - Human Virology: A pandemic HV H1N1. Thirteen co-infections had XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

158 Human Virology: HV been detected (6% of positive samples). Additionally, genotype 1, 4.32% for genotype 2, 18.65% for genotype 3 and 0.73% for genotype 4. The distribution of HCV by DFA up to 14 days before, which detects adenovirus, genotypes was different among Brazilian regions. In the we identified 43 samples which had also been analyzed Northeast states, subtype 1b was found in 35.79% of with negative results. The molecular panel was able to samples, followed by subtype 1a, with 21.05%. Subtype detectinfluenza at least A and one B, virus parainfluenza in 28 of them 1, 2(65%), and 3, identifying and RSV, 3a was found in 16.84% of the samples and 2b in 1.05% not only viruses which were not detectable by DFA (15 in this region. Other subtypes as such 2a, 3b and 4 were samples) but also those which DFA should have been not detected. In the South states, in 26.64% of samples able to detect (16 samples). There were 4 co-infections subtype 3a was detected, followed by 1a in 21.62%, 1b amongst these samples (14% of positive samples). We in 16.22%, 1a/1b in 2.32%, 2b in 10.04%, 2a in 1.93%, concluded that molecular assays which detect multiple and 4 in 0.39%. In the Southeast states, subtype 1b was viruses simultaneously may have positive impact for present in 27.80% of samples, followed by 1a in 23.11%, diagnosing respiratory infections, once they detect a 1a/1b in 5.38%, 3a in 16.36%, 2b in 1.49%, 2a in 0.92%, larger variety of viral agents and, in addition, they are 4 in 0.92%, and 3b in 0.11%. Although genotype 1 has more sensitive when compared to DFA, including cases predominated in all regions, a larger proportion of of co-infection. Future studies with larger number of genotype 3 was seen in South region. These results are samples may show even more robust epidemiologic data. similar to those found in a previous survey in Brazil (Campiotto et al, 2005), which analyzed 1688 samples HV273 - GEOGRAPHIC DISTRIBUTION OF HEPATITIS from 1995 to 2000 and demonstrated a 64.9% frequency C VIRUS GENOTYPES IN BRAZIL, 2013-2014 for genotype 1, 4.6% for genotype 2, 30.2% for genotype Reys, C.; Sacramento, P.R.; Moreira, C.M.; Lazari, C. 3, 0.2% for genotype 4. In all regions, genotype 1 was the dos S.; Granato, C.; Bueno, V.D.C.; Moura, M.E.G. most frequent (51.7% to 74.1%), while genotype 3 was FLEURY - Fleury Medicina e Saúde more common in the South (43.2%). Hepatitis C virus (HCV) is endemic worldwide and HV276 - VIRAL CONJUCTIVITIS CAUSED BY HUMAN infects more than 185 million people around the world. ADENOVIRUS: COMPARISON AND STANDARDIZATION In Brazil, a prevalence of 1.82% has been reported. HCV OF MOLECULAR DETECTION METHODS IN EYE SWAB is a positive-stranded RNA-enveloped virus which has SAMPLES Bonon, S.H.A.; Baldi, C.; Costa, S.C.B. major genotypes and over 100 subtypes that vary with regarda high to genetic their geographic variability distribution and is classified and response into seven to University of Campinas/Faculty of Medical Sciences/ treatment. The aim of this study was characterize HCV Laboratory of Virus genotypes circulating in some states of Brazil. For this Adenovirus (AdvH) is a major pathogen associated project, 1218 sequential samples from HCV infected with eye infections worldwide. Therefore, a pattern for patients were analyzed, from January 2013 to June detecting AdvH in ocular swab specimens is desirable and may aid in therapeutic management to minimize the polymerase chain reaction (RT-PCR), and the 5’ UTR impact of these infections. Thus, we analyzed 175 samples were2014. sequenced HCV-RNA wasusing amplified Big Dye® by Direct reverse Cycle transcriptase- Sequencing collected from 79 patients who presented signs and Kit (Applied Biosystems, USA). The samples were symptoms of viral conjunctivitis to the Ophthalmology obtained in different cities from several Brazilian States, Department at the Clinics Hospital/UNICAMP. Three eye with the following distribution: 874 samples from swab samples were collected from 48 patients: one on Southeast region (São Paulo, Rio de Janeiro and Minas Gerais states); 259 samples from South region (Paraná and Rio Grande do Sul states); and 85 samples from the day of the first visit (day 0), one on the second day Northeast region , (Bahia, Pernambuco and Maranhão of the visit (day +5), and one on the third day of the visit states). Genotypes 1, 2, 3 and 4 were found among these of(day dexamethasone +10). This study 0.1% used a/ randomized povidone-iodine design which0.4%, samples, and the general frequencies were 52.93% for was created by ophthalmologists to study the efficacy

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersadministered - Human Virology: on HV the days +5 and +10, in the treatment of XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

159 Human Virology: HV patients with acute viral conjunctivitis. The main goal of this work was the standardization the detection of AdvH (low [LSIL] or high grade [HSIL] squamous intraepithelial infections in eye swab samples using a Polymerase Chain lesions)Precancerous and histological cervical disease stages is (cervicalclassified intraepithelial by cytological Reaction (PCR) with the primers H1 and H2 and a Nested neoplasia [CIN] grades 1 to 3). There are about a dozen Polymerase Chain Reaction (Nested PCR) with the HPV types associated with the development of cervical primers externals AdTU7 e AdTU4 and internals AdNUS cancer, but a major role is attributed to the high-risk HPV e AdNUA. This was done to determine the prevalence types 16. Integration of the viral genome into the host of infections caused by AdvH in patients with signs and DNA is related to lesion persistence and progression symptoms of conjunctivitis viral and to compare the and is considered a late event in cervical carcinogenesis. Integration usually occurs within fragile areas of the PCR detection for HSV-1, HSV-2, VZV, CMV and EBV to viral E1 and E2 ORF, with the latter being the most verifyefficiency the incidence of both techniques. of other herpesvirus Was performed in all samples Nested frequently described. Elimination of E2 expression on the day 0. AdvH detection by PCR was positive in results in transcriptional over-regulation of the viral oncogenes E6 and E7, leading to increased expression 12/48 (25%) and 5/48 (10.4%), respectively. Nested of both viral oncoproteins that target tumor suppressor 29/48 (60.4%) on the day 0 and on the days +5 and +10, proteins, p53 and pRb respectively, among others, resulting in loss of cell cycle control, DNA alterations and ThePCR wasremaining positive 31 in patients 37/48 (77%) had only day 0,a andsingle day visit, +5 and on telomerase activation. In order to evaluate the physical the+10, day 18/48 0, because (37.5%) they and skipped 10/48 the (20.8%), others days respectively. of visits state (episomal or integrated) of HPV 16 genome, a PCR combining 8 primers pairs were used, covering the with the 48 patients on the day 0 to verify the prevalence E1-E2 region, as described by Vernon et al (1997). Our of(days conjunctivitis +5 and +10). viral, This being patients 33/79 were(41.7%) taken positive together for preliminary results involved 45 samples from patients AdvH, 4/79 (5%) positive for HSV-1 and 79/79 (100%) infected by HPV16, harboring cervical lesions in different negative for the others herpesvirus tested (HSV-2,VZV, stages of progression. Among them, 8 (18%) presented CMV and EBV). Of accord with the seasons, the major episomal HPV16, 17 (38%) mixed forms and 20 (44%) occurrence of conjunctivis viral was in the spring 34/79 were integrated. The upper region E1a were the most (43%), being 23/34 (67,7%) positive for AdvH. Nested frequently absent (disrupted) (19/42%), as well as for PCR was more effective that PCR for ocular detection of mixed states (18/40%); followed by the downstream AdvH on the three days of the collected, with standard E2c region, counting for 11 (24%) cases of complete

forms. It is interesting to observe that literature points deviation of 1on the days +5 and +10, and 0 on the day outdisruption a predominance and 8 (18%) of disruption combined inepisomal+integrated E2 region but our 0.HV279 The results - MAPPING were statistically THE INTEGRATION significant (p SITES ≤ 0.0001). E1-E2 results suggested a highly prevalent E1 inactivation. The OF HPV-16 AS A TOOL TO EVALUATE DIFFERENT STAGES OF CERVICAL DISEASE PROGRESSION that can successfully map the HPV 16 genome fragile Mello, F.C. do A.1; Carestiato, F.N.1; Mello, F.C. do A.1; areas.approach Data here are described being analyzed is a very in specific order tomethodology search for Cordeiro, T.I.1; Silveira, F.A.2; Cavalcanti, S.M.B.1 statistical correlation between integration and severity 1. UFF - Universidade Federal Fluminense, of the lesion but it has been observed that E1-E2 absences Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 were suggestively frequent in HSIL and cancer. In a few 2. UFRJ - Universidade Federal do Rio de cases, episomal forms were observed in cancer samples, Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de suggesting additional biomarkers as responsible for Janeiro - RJ, 21941-901 carcinogenesis. Persistent infection with oncogenic genotypes of genital Human Papillomavirus (HPV) is associated cause of morbidity and mortality of women worldwide. with the development of cervical cancer, a significant September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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HV281 - EVALUATION OF REAL TIME QPCR ASSAY FOR laboratory in São Paulo State. FINANCIAL SUPPORT: ENTEROVIRAL MENINGITIS DIAGNOSIS IN PUBLIC Fapesp 2012/50234-5 HEALTH LABORATORY FROM SÃO PAULO STATE HV282 - MOLECULAR DETECTION OF FLAVIVIRUSES Machado, B.C.1; Vieira, H.R.1; Alves, M.R.M.1; da Silva, IN TICKS (ACARI: IXODIDAE) FROM CAPYBARAS AND M.V.2; Carmona, R. de C.C.1 ENVIRONMENT IN AN URBAN PARK OF UBERLÂNDIA, 1. IAL - Instituto Adolfo Lutz, Av. Dr. Arnaldo, MINAS GERAIS, BRAZIL 355 - Cerqueira César, São Paulo - SP, 01246-000 Vieira, R. de S.; Pascoal, J. de O.; Pascoli, G.V.T.; 2. Instituto de Infectologia Emílio Ribas, Av. Dr. Oliveira, T.F. de M.S.; Yokosawa, J.; Szabo, M.P.J. Arnaldo, 165 - Cerqueira Cesar, São Paulo - SP, 01246-900 UFU - Universidade Federal de Uberlândia, Av. João Enterovirus (EV) genus is the most frequent etiological Naves de Ávila, 2121 - Santa Mônica, Uberlândia - MG, agent involved in viral meningitis cases worldwide. 38408-100 Implementation of a fast method to diagnose enteroviral meningitis became a very important requirement in public The genus Flavivirus of Flaviviridae family comprises health services, because it quickly provides information more than 70 virus species including many arthropod- to clinicians allowing better medical management. The borne viruses. Tick-borne encephalitis virus (TBEV), a aim of this work was to evaluate preliminary results of pathogen that infects human central nervous system, has the real-time reverse transcription-PCR (RT-qPCR) assay spread extensively in some regions of the world. Human implantation for detection of EV based on the conserved cases of TBEV infections have increased in the past 30 years dramatically posing a danger to public health in (CSF) samples were selected from meningitis cases, sent several European countries. The main hosts for the virus to5’untranslated the Enteric Diseases region. A Laboratory, total of 616 Adolfo cerebrospinal Lutz Institute fluid are small rodents and humans are accidental hosts. In for EV diagnostic between 1998 and 2013. These samples endemic areas, recreational or occupational exposure to have been stored at – 70ºC. The RNAs were extracted rural or outdoor settings is a potential risk for infection directly from CSF by QIAamp® Viral RNA Mini Kit, and from infected ticks. The Flaviviridae includes several other tick-borne viruses affecting humans that are closely assay was applied. Evaluation was made by comparing related to TBEV. In Brazil the survey for virus in ticks is thea TaqMan® RT-qPCR single-tube results with fluorogenic it previously 5’ nuclease obtained RT-qPCR by cell culture viral isolation method. EV RNA was detected in ticks, potentially responsible for zoonoses, in an urban overall scarce. We herein report a survey for flavivirus in 94 of 616 (15.2%) samples using RT-qPCR assay and park in Uberlândia city, state of Minas Gerais, Brazil. EV was isolated from 58 of 616 (9.4%) samples by viral Ticks were collected from natu¬rally infested capybaras and from environment and stored at -70ºC. RNA was predictive value and negative predictive value of 89.7%, extracted from 60 ticks by using Trizol (Invitrogen) and 92.4%,culture. 55.3% RT-qPCR and had 98.9%, a sensitivity, respectively. specificity, Accuracy positive was polymerase chain reaction (PCR), in two rounds, using cDNA (RNA reverse transcribed into complementary viral isolation and RT-qPCR showed a Kappa value of DNA) was performed. The assay utilized degenerate 0.78,92.2% demonstrating and incorrect a good classification and substantial 7.8%. Cellagreement. culture In our study, RT-qPCR assay was slight superior to viral RNA-dependent RNA polymerase) and detects a range primers targeting the flavivirus NS5 gene (encoding culture for detecting EV from CSF samples. These results demonstrate that TaqMan RT-qPCR assay is a fast and tick produced amplicons of approximately 242 bp and of flaviviruses. Two samples of Amblyomma dubitatum sensitive assay for detection of EV infection. The test is were submitted to sequencing. Nucleotide sequences easily performed, which makes it an excellent alternative were edited using Lasergene program (DNAstar) and to the laborious and time-consuming viral culture. EV compared with those available in the GenBank database RT-qPCR with CSF samples was highly sensitive and using the BLAST algorithm. The sequences obtained were not similar to any known sequence. Other tick samples to the epidemiological surveillance of viral meningitis diseasespecific andwith has incorporation a potential intoto contribute routine publicsubstantially health will be investigated for the presence of flavivirus. In September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersaddition, - Human Virology: the isolation HV and purifica¬tion of the virus in XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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Vero cell culture will be attempted. Further research is that this age group is the most affected, as well as the important to determine potential emerging tick-borne highest percentage of positive cases are in the group zoonotic pathogens in Brazil. FINANCIAL SUPPORT: with clinical manifestation. The genotype found (GII.I) FAPEMIG AND CNPQ. in this study was also detected in Belém, in samples of diarrheic children collected in different years (1998- HV283 - DETECTION AND GENOTYPING OF 2000 and 2003) and in Parauapebas in 2006, both in SAPOVIRUS IN FECAL SAMPLES FROM CHILDREN Pará state. Therefore, the results of this research are HOSPITALIZED IN SÃO LUÍS, MARANHÃO, BRAZIL, relevant as they demonstrate the circulation of SaVs in FROM JUNE 1997 TO JUNE 1999 São Luís and reinforce that further studies should be Resque, H.R.1; Costa, L.C.P. das N.1; Portal, T.M.1; developed, as there is still lack of information regarding Siqueira, J.A.M.1; Linhares, A. da C.1; Luz, C.R.N.E. da2; the epidemiology of this virus. Financial Support: PIBIC/ Gabbay, Y.B.1; Resque, H.R.1 CNPq Keywords: sapovirus, gastroenteritis, children, 1. IEC - Instituto Evandro Chagas, Rodovia São Luís BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 HV284 - CIRCULATION OF NOROVIRUS PANDEMIC 2. UFMA - Universidade Federal do Maranhão, VARIANT GII.4 SYDNEY 2012 AND NEW ORLEANS Av. dos Portugueses, 1966 - Anjo da Guarda, São Luís - MA, 65080-805 2009 IN CHILDREN FROM MANAUS, AMAZONAS, BRAZIL Acute gastroenteritis (AGE) is a major cause of childhood Gabbay, Y.B.; Hernandez, J. das M.; da Silva, L.D.; morbidity and mortality worldwide. Among the viruses Rodrigues, E.A.M.; Bandeira, R. da S.; Lucena, M.S.S. that cause AGE the (SaVs), members of the de; Soares, L. da S.; Mascarenhas, J.D’A.P.; Resque, Caliciviridae family, seems to be very important since H.R.; Gabbay, Y.B. they are associated with sporadic cases and outbreaks of AGE occurring in schools, day care centers and cruise IEC - Instituto Evandro Chagas, Rodovia BR-316 km 7 ships. The most frequently observed symptoms on a SaV s/n, Levilândia, Ananindeua - Pará, 67030-000 infection are diarrhea, vomiting and abdominal pain. Norovirus (NoV) is a enteric pathogen known to cause The virus transmission occurs by the fecal-oral route, pandemics of acute gastroenteritis worldwide. In primarily through contact person-to-person, intake developing countries, its estimated cause 200,000 deaths of contaminated water and/or food, and aerosolized in children under 5 years old. In the EUA, some authors particles generated during episodes of vomiting. The related that after the increased use of rotavirus vaccine, study aimed to detect and genotype SaVs in fecal NoV become the primary cause acute gastroenteritis. samples from hospitalized children with and without Due NoV suffer a dynamic process of mutation and gastroenteritis in São Luís, MA, during the period of June recombination, providing a great genetic diversity and 1997 to June 1999. Nucleic acids were extracted from SaVs this is the basis for its adaptability. These events have by the silica method and were subsequently tested by RT- led to the emergence of variant strains, mainly of linhage PCR using the primers pair P289 and P290. Specimens GII.4, which has caused epidemics globally. In 2012, were considered positive when showing amplicons of 331 bp. The positivity rate was 8.1% (11/136), with years old from the city of Manaus, Amazonas, Northern 15.2% (7/46) in the diarrheal specimens and 4.4% Brazil.fecal specimens The samples were werecollected tested from for children NoV initially until five by (4/90) in non-diarrheal ones (p <0.04). One sample EIE and the positive samples by reverse transcription- polymerase chain reaction (RT-PCR) using a pair of cases, 27.3% were associated with fever, vomiting and primers with target the polymerase gene. The positive ,was sequenced and 18.2% and with classified fever, asanorexia GII.1. Ofand the abdominal positive pain. The presence of asymptomatic cases reinforces the sequenced using the primers EVP2F and EVP2R. These suggestion that even in the absence of clinical symptoms primerssamples wereidentified designed as GII.4 to amplify NoV were the antigenictested by regionPCR and of the virus continues to spread. SaVs detection in samples the viral capsid (VP1 protein), subdomain P2, allowing of children under two years corroborates published data

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersidentification - Human Virology: of HV variants. Samples were sequenced in XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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Brazil, have shown approximately 10% of seropositivity Foster City, USA), aligned and analyzed by the Bio to Piry virus. However, the disease produced by Piry EditABI Prismprogram 3130XL (v.7.1.3.0) DNA Sequencer and compared (Applied with Biosystems, others virus has not been reported, except for one accidental registered in GenBank. A dendrogram was generated by case in the laboratory. There is a poor knowledge on the neighbor-joining method, with 2,000 replicates in the human infection and the disease by vesiculovirus the bootstrap analysis, using MEGA 6.0 software. From as well as a limitation on the diagnosis which is based the 51 samples positive by PCR (polymerase region), 16 in serologic tests and performed in few laboratories. were characterized by the capsid region, demonstrating Ten years ago, our group reported a conventional RT- the predominance of the strain GII.4 Sydney 2012 PCR to vesiculovirus. As we show in this on going study, (62.5%) in the amazonian population, but the GII.4 New the conventional RT-PCR evolved into a SYBR green Orleans 2009 was also detected in lower percentage (37.5%). Nucleotide analysis showed difference of 1% gene of Brazilian vesiculovirus. Our preliminary results among the Sydney ones and 7% among the all positive showI-based that real-time it was able RT-PCR to amplify that amplifies genomes 221 of bp4 Brazilian of the G samples. When compared with the other GII.4 variants, Vesiculovirus (Piry, Carajas, Alagoas and Indiana) with as Farmington Hills 2002 and the DenHaag 2006b a higher sensitivity than the conventional RT-PCR. The prototypes, the nucleotide difference was 15%. The melting curve of the real time RT-PCR, 81.41 ± 0.50 °C, results demonstrate the circulating of the pandemic strain Sydney 2012 in Manaus and are relevant if considered the limited data about the variants of NoV products.has shown The that sera it is ofspecific 165 patients for Brazilian with vesiculovirusacute febrile in the North region and also in Brazil. The surveillance illness,and do from not generateSinop city, primer-dimers in Mato Grosso or non-specificState, were of NoV variants is also important and necessary collected during the dengue outbreak of 2011 - 2012 and because each two to three years, it is being observed tested by the real time RT-PCR and no positive samples a new variant that replace the antecessor. FINANCIAL were found. This real time RT-PCR is reproducible, SUPPORT: Evandro Chagas Institute, PIBIC/IEC/CNPq, PPGV/IEC/FAPESPA. of infections. It could also be used in a surveillance programspecific, and to better could becomeevaluate a theuseful importance tool for the of diagnosisBrazilian HV286 - DEVELOPMENT OF A REAL TIME-PCR vesiculovirus in human disease. FINANCIAL SUPPORT: BASED IN SYBR GREEN FOR RAPID DETECTION OF FAPESP - Fundação de Amparo à Pesquisa do Estado de BRAZILIAN VESICULOVIRUS São Paulo Nº. 13/14929-1, Fellowship Nº. 13/02256-2, Tolardo, A.L.1; Romeiro, M.F.1; Souza, W.M.1; Siqueira, 12/02836-6, 12/24150-9. C.E.H.2; Bronzoni, R.V. de M.3; Figueiredo, L.T.M.1 HV288 - INFLUENZA A H1N1 PDM09: LABORATORY 1. FMRP/USP - Faculdade de Medicina de SURVEILLANCE OF SUSPICIOUS DEATHS OF SEVERE Ribeirão Preto/ Universidade de São Paulo, Av. Bandeirantes, ACUTE RESPIRATORY SYNDROME IN THE STATE OF 3900 - Monte Alegre, Ribeirão Preto - SP, 14049-900 SAO PAULO, BRAZIL - 2013 2. Laboratório Municipal de Análises Clínicas , SINOP, MT Cirqueira, C. dos S.; Martines, R.B.; Iglezias, S.D.A. 3. Universidade Federal do Mato Grosso, Kanamura, C.T. Ramos, A. da C.; Aguiar, A. SINOP, Mato Grosso, Brazil IAL - Instituto Adolfo Lutz, Av. Dr. Arnaldo, 355 - Vesiculovirus is a genus in the family Rhabdoviridae. Cerqueira César, São Paulo - SP, 01246-000 In the Americas, Indiana and New Jersey serotypes of vesiculovirus, are both endemic and responsible for vesicular stomatitis, a disease of cattle, horses and pigs, In April 2009 a new influenza A virus called responsible for important economic losses. In Brazil, it 5,700H1N1pdm09 deaths. virus After was this identified time the in pandemic humans resulting activity have been reported other vesiculovirus, such as Piry, isin thedeclining, first influenza however, pandemic the transmission of the 21st centuryof the viruswith Carajas, Cocal, Alagoas and Maraba. Curiously, serologic remains. The disease presents clinical and transmission surveys in human populations of different regions of

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posterscharacteristics - Human Virology: similarHV to those of seasonal influenza XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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Dengue is a major challenge to public health in Brazil So its vigilance with seasonal period and the risk of co- and worldwide. Dengue virus (DENV) is an arbovirus of and keeps as an etiological agent of flu cases in Brazil. risk of the emergence of new viruses become important four antigenically distinct serotypes DENV-1-4. Infection laboratorymovement withsurveillance other influenza along with viruses, cases as of well suspected as the withthe family one of Flaviviridae, these serotypes genus may Flavivirus, causes a diseaseclassified whose into deaths from severe acute respiratory syndrome (SARS). spectrum ranges from clinically asymptomatic or a The objective of the study is demonstrate the prevalence mild and self limited febrile illness called dengue fever to severe clinical forms. In Brazil, the co-circulation of four serotypes has led to successive epidemics and Goias of etiological diagnosis of influenza A H1N1 pdm09 in state, usually present high number of dengue cases. In deathsSão Paulo of inSARS. 2013 Routine in tissue sampleshematoxylin-eosin fixed in formalin stains 2013 were related alarming statistics, with an increase wereand embeddedperformed infor paraffin histopathological (FFEP) of suspectedevaluation. fromThe of 401% in the number of reported cases and 25% in the number of deaths. The Epidemiological Surveillance pdm09 was performed on 3-mm sections of FFPE lung System plays an important role in the continuous immunohistochemical assay for Influenza A H1N1 monitoring infections, nevertheless, it has been shown H1N1pdm09 antibody, appropriate positive and negative controlsand airways were tissues run in using parallel. a monoclonal The results anti-influenza were evaluated A of new cases. To increase the detection of DENV in early and the data interpreted toghether with clinical, infections,the relevance the of NS1 laboratory research diagnosis was introduced in the confirmation in Public epidemiological and laboratory information available. Health Laboratories. However, the time of infection has Immunohistochemical staining for H1N1pdm09 was seen in 21 of 198 suspected deaths to SARS, with 28.5% of choice. Therefore, the present study evaluated the of those elucidated only by IHC. 75.4% were male and occurrencea decisive influence of DENV on infection the results in regardingGoiania, Goias the method in the 24.6% female, no pregnant woman; Ages ranged from 15 epidemic period of 2012-2013 among the population to 70 years. Concomitant bacterial infectious agent was with clinical suspected of dengue infection (up to seven observed in 28.57% of cases. Other viral etiologic agents days) using a combination of serological and molecular methods. Serum samples were collected from 278 symptomatic patients who were attended in basic health in 19 cases of Influenza A not subtyped, 1 case positive centers between October/2012 and May/2013. The for both influenza H1N1 pdm09 and not subtyped samples were submitted to the research of serological influenza A, 2 deaths by influenza B and 2 deaths from markers NS1, IgM and IgG using a commercial kit pdm09,hantavirus even were in 2013also identified.detected the Although transmission we observed of the (DuoTest-Bioeasy), as well as viral RNA detection by virusa decrease in the in state the ofincidence Sao Paulo of casesand its of associationH1N1 influenza with RT-PCR for the C-prM region. The positivity for infection mortality. The vaccination and laboratory surveillance was 43.9% (122/278), with rates of 30.5%, 13.6% and must be maintained and the sustained transmission 20.5% for NS1, IgM and RNA markers, respectively. In of the disease may enable genetic recombination and 24.5% of DENV infection cases, NS1 was the only marker virulence alteration. detected, demonstrating its advantage mainly favoring early diagnosis with higher rate of positivity between 4 HV296 - INVESTIGATION OF DENGUE VIRUS and 5 days. The IgM was detected singly in 16.4% with INFECTION IN GOIANIA, GOIAS, BY MOLECULAR AND SEROLOGICAL METHODS For the RNA, the highest rate was observed between 1 de Oliveira, T.S.; Guimarães, V.N.; Cunha, M. dos P.; andstatistical 3 days. significance The combination for the of sixth these and three seventh markers, days. Souza, M.B. de L.D.; Cardoso, D. das D. de P.; Carneiro, notably constitute a powerful tool in the diagnosis R. dos S. Fiaccadori, F.S. DENV. Monitoring of dengue epidemics by laboratory IPTSP/UFG - Instituto de Patologia Tropical e Saúde Pública/ Universidade Federal de Goias, Rua 235 - s/n, Setor system, it is relevant to establish a complete data base to Universitário, Goiânia - Goiás, 74605050 confirmation, with an active and effective notification

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

164 Human Virology: HV be used for surveillance studies. FINANCIAL SUPPORT: and HPV-31 (7.2%). Among samples for which it was CNPQ; FAPEG four different molecular variants belonging to four HV298 - PREVALENCE OF HPV-16 VARIANTS IN branchesfeasible to of analyzegeographical intratype and phylogenetic variability, we relatedness. identified REPRODUCTIVE AGED WOMEN ATTENDED AT European variants (87%) were the most prevalent and PRIMARY HEALTH CARE UNITS IN BOTUCATU, SP, diverse group, followed by the Asian-American (6.4%), BRAZIL African (4.8%) and North-American (1.6%). Within the Candeias, J.M.G.1; Kurissio, J.K.1; Silveira, A.C.1; European branch, 75.0% of the samples were prototype Ferreira, S.2; Bolpetti, A.3; Sichero, L.2; Silva, M.G.3; and the remaining 25.0% were B-12. Although we Villa, L.L.4,5; Candeias, J.M.G.1 conducted cytology examination in all samples, we did 1. INSTITUTO DE BIOCIêNCIAS/UNESP - not observe correlation to any lesion grade and molecular Instituto de Biociências da Universidade Estadual Paulista, variants detected. However, since this is an ongoing Instituto de Biociências, Distrito de Rubião Junior S/N, study where multiple samples are being collected from Botucatu - SP, 18618-970 the same women, we hope to obtain more information 2. ICESP - Instituto do Câncer do Estado de São Paulo, Av. Dr. Arnaldo, 251 - Sumaré, São Paulo - SP, 01255-000 regardingHV301 - DETECTION this specific OF question. JC POLYOMAVIRUS (JCV) AND 3. FMB/UNESP - Faculdade de Medicina de HUMAN CITOMEGALOVIRUS (HCMV) IN GLIOMAS Botucatu da Universidade Estadual de São Paulo, Av. Prof. Silva, B.F.G.; Stangherlin, L.M.; Castro,L.F.F.; Silva, Montenegro Bairro: Distrito de Rubião Junior, s/n, Botucatu - M.C.C. SP, 18618970 4. FM/USP - Faculdade de Medicina da UFABC - Universidade Federal do ABC, Rua Catequese, Universidade de São Paulo, Av. Dr. Arnaldo, 455 - Cerqueira 242 - Jardim, Santo André - SP, 09090-400 César, São Paulo - SP, 01246903 The John Cunningham virus (JCV) infects a large 5. FCMSCSP - Faculdade de Ciências Médicas proportion of the population worldwide. The virus da Santa Casa São Paulo,Rua Doutor Cesário Motta Júnior, usually remains latent on the kidney cells but can 61 - Vila Buarque, São Paulo - SP, 01221-020 reactivate under immunosuppressive conditions and It is known that cervical cancer is the second migrate to the brain, resulting in progressive multifocal leading cause of death in women worldwide usually leukoencephalopathy, a fatal demyelinating disease due to persistent infection with high-risk Human of the central nervous system. JCV can transform cells Papillomavirus (HPV). Among those, HPV-16 molecular in culture and is oncogenic in laboratory animals. In variants have been shown to be differentially involved addition, viral DNA sequences have been detected in in viral persistence and development of cervical lesions. several kinds of human malignancies, including brain The aim of this study was to determine the prevalence tumors of glial origin, medulloblastomas and colon of HPV type and HPV-16 molecular variants in a cohort carcinoma, suggesting a possible relationship between consisting of 1601 women aged 18-50 years attended at JCV and cancer, Intringly, a study has demonstrated Primary Health Care Units in Botucatu, Brazil. Ecto and that the Human Cytomegalovirus (HCMV), a Beta- endocervical samples were taken during physical exam herpesvirus possibly implicated with brain cancer using a non-lubricated speculum. HPV was detected by malignancy, is capable of induce JCV replication in vitro, polymerase chain reaction (PCR) and HPV genotyping in cells not permissive for this virus. In the present study was conducted by Linear Array HPV Genotyping Test we aimed to verify the presence of JCV and HCMV viral (Roche Molecular Systems Inc.). HPV-16 intratypic variation in single or mixed infections was evaluated by and fresh tumor samples were tested for the presence sequencing a fragment of the viral long control region ofDNA HCMV sequences and JCV in glial by brainreal time tumors. PCR Paraffin-embedded and nested PCR. (LCR). The overall frequency of HPV DNA detection was From 19 samples analyzed HCMV DNA was detected 33.0% and HPV-16 (17.0%) was the most frequent HPV in all samples (100%) and JCV in 13 samples (68%). genotype followed by HPV-58 (8.3%), HPV-52 (8.2%) Despite a low number of specimens tested, the results

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165 Human Virology: HV suggest a positive association between HCMV and JCV HV304 - SEROPREVALENCE OF HEPATITIS E VIRUS in glioblastomas. We are currently testing more samples AMONG SCHISTOSOMIASIS PATIENTS IN RECIFE, PE, for the presence of viral DNA and RNA in tumor tissues. BRAZIL Funding: UFABC Sena, A.1; Passos, A.M.1; Reinaldo, M.R.1; Ferraz, M.L.1; Lopes, E.2; Granato, C.1 HV302 - VALIDATION OF TWO SINCICIAL RESPIRATORY VIRUS RAPID TESTS 1. UNIFESP - Universidade Federal de São da Silva, E.R.M.; Lazari, C.S.; Granato, C.F.H. Paulo, R. Sena Madureira, 1500 - Vila Mariana, São Paulo - SP, 04021-001 Grupo FLEURY 2. UFPE - Universidade Federal de Pernambuco, Acute respiratory infections caused by respiratory Av. Prof. Morais Rego, 1235 - Cidade Universitária, Recife - syncytial virus (RSV) are important health burdens that PE, 50670-901 affect infants worldwide. RSV infections are a common Hepatitis E virus (HEV) infection is a worldwide disease cause of pneumonia and bronchiolitis in children up usually presented as an acute self-limiting hepatitis, but to 5 years. Currently, RSV infection is diagnosed using in immunocompromised patients it may cause chronic laboratorial facilities and trained staff. Conversely, moderate endemic to HEV, with seroprevalence from immunochromatographicdirect fluorescence assays test (DFA), (ICT) which for antigen require detection specific 1%infection to 5% with in the cirrhosis. general Brazilpopulation has beenand 10% classified in renal as may be useful as a point-of-care method of simple transplant recipients. Schistosomiasis may play a execution, which may provide rapid and reliable results. role in virus infection by altering the immune system. In order to validate this method in our population, However, the seroprevalence of HEV in this population we have evaluated two different ICT commercial kits group is unknown. The aim of this study was to evaluate – BinaxNow® RSV Card (Alere, USA) and RSV Test the prevalence of past or present HEV infection in Bioeasy® (Bioeasy, Brasil) – and compared to results of schistosomiasis patients in Recife, PE. One hundred nineteen schistosomiasis patients were enrolled in this study. Clinical and laboratory data were available Adenovirusa DFA panel (AD). which Sixty-six detects respiratory RSV, Influenzavirus tract samples A (IVA)were for eighty individuals. The mean age of patients was tested.and B Compared (IVB), Parainfluenzavirus to DFA, BinaxNow® 1, 2, has 3 shown (PIV1-3) 90.4% and 50 years old (median 51, range from 14 to 78 years sensitivity (19/21 RSV-positive samples in DFA). Twenty old) and 52 (65%) were female. Serum samples were RSV-negative samples were tested using BinaxNow®, 10 tested for the presence of anti-HEV IgG antibodies of which were negative and the other 10 were positive by enzyme immunoassay (Mikrogen, Germany) and to other viruses in the DFA panel. The internal control for the presence of HEV RNA using real time reverse of the ICT did not work in three of them. The result was transcriptase-polymerase chain reaction with primers targeting the HEV ORF2 and ORF3. A third set of primers of 76.4%. Four false-positive results were detected, and probe targeting the human RNAse P gene was used suggestingnegative in cross-reactivity, 13, corresponding in positive to an overall samples specificity to IVB, PIV3 and AD, according to DFA. Regarding Bioeasy®, specimen quality and extraction. Anti-HEV IgG was 84.2% sensitivity was demonstrated (16/19 RSV- positiveas endogenous in 7 (6%) internal of the amplification 119 serum control samples to tested.certify positive samples in DFA). All of 30 RSV-negative samples None of the samples showed the presence of HEV RNA. tested with Bioeasy® remained negative, showing 100% Seropositive patients aged from 35 to 70 years old (mean 56). The mean AST and ALT in patients without which were positive to other viruses in DFA. We did not IgG antibodies were 31 U/L (range from 11 to 132 U/L) havespecificity, to discard without any test any of cross-reactivity this brand due to in inconclusive 10 of them and 33 U/L (range from 6 to 132 U/L). Patients with IgG results. We concluded that RSV Test Bioeasy® has better performance, with acceptable sensitivity and superior lowerantibodies in patients did not with show IgG significant antibodies increase (83,667) in ofwhen ALT use in children healthcare settings. comparedor AST levels. to Thepatients mean withoutplatelet levelIgG was(138,535) significantly (p = specificity; therefore, it is appropriate for point-of-care September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

166 Human Virology: HV

0.03). The same was observed for the mean leucocyte RT-qPCR.Minimum infectious rate(MIR) was calculated levels, 3,533 in patients with IgG and 4,993 in patients for positive species.Between 11.090 mosquitoes, without IgG (p = 0.02). Alkaline phosphatase, gamma- glutamyl transpeptidase, albumin and protein levels pools, 1/137(MIR=1.04) of Aedes aegypti positive for were also decreased in patients with IgG antibodies, DENV-14.556 females and 1/67(MIR=2,6) classified in 13 Culex species sp. constituted for SLEV.For 593 MAYV,10/67(MIR=26,2) Culex sp.,7/162(MIR=3.7) the seroprevalence of HEV may be higher in patients Cx. quinquefasciatus,4/137(MIR=4.1) Ae. aegypti and withalthough schistosomiasis not significantly. than This in studysome demonstratesimunocompetent that populations in Brazil. Also, schistosomiasis patients with more impaired immune system and more severe disease 4,5/54(MIR=24) 55/137(MIR=57,1) Cx. comp. Ae. pipiens aegytpi,34/54(MIR=162.7) were confirmed by RT- seem to be more at risk of HEV infection. Hepatitis E Cx.comp.qPCR and pipiens,68/162(MIR=36) 1 pool was confirmed by Cx. isolation.For quinquefasciatus, DENV- should be further investigated among schistosomiasis 46/67(MIR=120,4) Culex spp.,1/6(MIR=142.8) Limatus patients. FINANCIAL SUPPORT: FAPESP 2012/22925-3 sp.,2/4(MIR=500) Psorophora spp.,3/7(MIR=375) E 2013/03701-0 Ps. varipes albigenu,1/1(MIR=1000) were positive.In previous studies,SLEV,MAYV and the 4 serotypes of DENV HV307 - MOLECULAR INVESTIGATION OF NATURAL INFECTION BY FLAVIVIRUSES AND ALPHAVIRUSES IN during a large outbreak of dengue.The SLEV genotype ADULT MOSQUITOES CAPTURED IN CUIABÁ, MATO V-A,were identifiedprevioulsy in patientsreported from in thehumans urban areafrom of Cuiabá, Cuiabá GROSSO, BRASIL Serra, O.P.1; Cardoso, B.F.1; dos Santos, F.A.L.1; Heinen, species presented log of 3.52-4.08 copies/µL of MAYV. L.B. da S.1; Pacheco, T. dos1; Zuchi, N.1; Ribeiro, A.L.M.2; Experimentaly,was identified these in a Culexspecies sp. have Female.Culex been demonstrated and Aedes as Rodrigues, J.S.V.1; Miyazaki, R.D.1; Slhessarenko, R.D.1 competent vectors for the virus, but their participation 1. UFMT - Universidade Federal de Mato in the epidemiological cycle of MAYV transmission is Grosso, R. Guadalajara, 1 - Jardim das Américas Cuiabá - MT unclear.DENV-4, responsible for the majority of the 78060-624 2. HUJM - Hospital Universitário Júlio Müller, several species, possibly due to hematophagy in humans. Av. Fernando Corrêa da Costa, nº 2367, Bairro Boa Esperança, Cuiabáhuman presentscases in MTa variety during of 2012-2013, culicidae species, was identified associated in Cuiabá - MT, 78060-900 to environmental conditions favorable to the occurrence Arboviruses belonging to Flavivirus and Alphavirus of arboviral outbreaks,emphasizing the importance of genus are considered an important public health entomological and virological surveillance. FINANCIAL issue in Brazil, mainly in tropical areas.The aim of the SUPPORT: CAPES, FAPEMAT E UFMT study was to identify the frequency of hematophagous HV309 - OCCURRENCE OF PICOBINAVIRUS GENOGROUP I IN WORKERS OF PIG FARMS FECES IN alphaviruses in Cuiabá, MT. Culicidae specimens were THE WESTERN REGION OF PARANA capturedmosquitoes in naturally3 locations infected from 200 by censitary flaviviruses sectors and Gallego, J.1; Bittencourt, L.H.F.B.2; Kunz, A.F.1; Macedo, R.1; Lustosa, J.M.1; Evers, F.1; Navarro, I.T.2; Takiuchi, allocated in pools(1-10 mosquitoes) according to E.1 sex,from species, Cuiabá indata 2013, and identifiedcollection withpoint.Minced dichotomy pools key, were subjected to total RNA extraction, duplex-RT- 1. UFPR - Universidade Federal do Paraná, PCR followed by multiplex-semi-nested-RT-PCR to 5 Rua XV de Novembro, 1299 - Centro, Curitiba - PR, 80060- 000 Saint Louis encephalitis(SLEV), dengue 1(DENV-1) and 2. UEL - Universidade Estadual de Londrina, alphaviruses and 11 flaviviruses.Positive samples for Rodovia Celso Garcia Cid, Km 380 - Campus Universitário, PCR and subjected to nucleotide sequencing. A region of Londrina - PR, 86057-970 mayaro(MAYV) viruses were amplified by single RT- (PBVs) are small, non-enveloped, bisegmented double-stranded RNA genomic viruses of the SLEV envelope gene was amplified for phylogenetic analysis.PositiveSeptember/October 2014 pools Volume for 19 MAYV – Supplement were confirmed2 - Abstracts/Posters by - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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carcinoma. Approximately 20% of HCV infections are occurred in children’s feces and pigs with diarrhea, their spontaneously resolved, and the mechanism of HCV rolevertebrate as etiologic hosts. agent Despite in this first pathogenesis detections are of still PBV unclear. have clearance is associated with polymorphisms in immune response genes. HCV can prevent the innate immune agents primarily affecting immunocompromised. Human response of the host through the action of the NS3/4A In humans, PBVs are classified as emerging opportunistic protease. A catalytic interaction between amino acid genogroups, denominated genogroup I and genogroup II. Cys-508 of the MAVS protein and S139 of the NS3/4A ComparativePBV strains studies are currently of PBV classifiedgenome sequences into two detected distinct protease can impair the interferon cascade, and in humans and pigs has been suggested the possibility mutations at Cys-508 can confer the ability to resolve of inter-species transmission, highlighting the zoonotic HCV infection in some individuals. The aim of this study potential associated with this virus. The intent of this was to search for substitutions in the coding sequence study was to detect PBV in workers feces and contacts from MAVS and investigate demographic and risk factors associated with HCV infection in an attempt to feces samples were collected from workers between correlate the spontaneous resolution of hepatitis C with 2012of pig andfarms 2013, in western in the WesternParaná. OneRegion hundred of Paraná. sixty Thefive the presence of mutations at position 508. The region PBV diagnosis was carried out in fecal samples by silver- of the human MAVS gene containing residue 508 was stained polyacrylamide gel electrophoresis (SS-PAGE). were collected from chronic and spontaneously resolved the primer pair PicoB25/PicoB43 to genogroup I gene patients,amplified andand sequenced.the data were Demographic subjected and to riskhypothesis factors thatThe positiveencodes samples the RNA-dependent-RNA-polymerase- were amplified by RT-PCR using protein. Of the 165 samples processed 7.3% (12/165) were positive for PBV from the PAGE technique. Of gendertesting and with venereal a 95% disease confidence with patients interval, who revealing achieved a spontaneousstatistically significanthepatitis C resolution. association Because between all patients female carried a cysteine at position 508, it was not possible to PCRthese, may 9 samples belong towere the amplified PBV genogroup in RT-PCR, II. Considering generating establish a relationship between hepatitis C clearance the closeproduct interaction of 201pb. between The samples humans not and amplified pigs in swinein RT- and the presence of mutations at position 508 of the production chain of the western region of Paraná and MAVS protein in the patient groups selected for this the possibility of cross-species infection, the need for analysis. FINANCIAL SUPPORT: FAPESP, CNPQ E CAPES. further investigation must be reinforced to elucidate the epidemiology and pathogenesis of PBV in humans and HV324 - SEROSURVEY OF HANTAVIRUS INFECTION animals. IN SINOP, MT da Silva, D.J.F.; Vieira, C.J. da S.P.; Siqueira, C.E.H.; HV314 - INVESTIGATION OF FACTORS THAT Barreto, E.S.; Moreli, M.L.; Bronzoni, R.V. de M. INFLUENCE THE SPONTANEOUS RESOLUTION OF 1. UFMT - Universidade Federal de Mato HEPATITIS C VIRUS INFECTION Grosso, R. Guadalajara, 1 - Jardim das Américas Cuiabá - MT 1 1 1 Provazzi, P.J.S. ; Miura, V.C. ; Carvalho, L.R. ; Rosa, 78060-624 1 2 3 3 P.C.R. ; Nogueira, M.L. ; Grotto, R.M.T. ; Silva, G.F. ; 2. UFG - Universidade Federal de Goiás, Av. Rahal, P.1 Esperança, s/n - Setor Itatiaia, Goiânia - GO, 74001-970 1. Sao Paulo State University - UNESP, Hantaviruses are rodent borne pathogens included in the Department of Biology genus Hantavirus (Bunyaviridae family), and transmitted 2. São José do Rio Preto Medical School, to humans through aerosols of infected wild rodents Laboratory of Virology excreta. Brazil has reported in the last two decades 3. Sao Paulo State University - UNESP, nearly 1,800 cases of hantavirus cardiopulmonary Department of Internal Medicine syndrome (HCPS). Mato Grosso state has reported The hepatitis C virus (HCV) is associated with chronic 15.2% of these cases within the same period, with a and active hepatitis, cirrhosis, and hepatocellular case-fatality rate of ~40%. Sinop municipality is located

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

168 Human Virology: HV in the middle north of Mato Grosso state, in a region may also favors the emergence of HIV-1 drug-resistant covered by Amazon rainforest. Since its foundation, in variants, due to low genetic barrier of some drugs as 1974, the landscape has undergone intense deforestation well as poor adhesion to treatment regimens, among due to population growth, lumber commerce, and others. These variants may limit the therapeutic options agroindustry development. It has been suggested that available for people who were newly infected with them. such changes favors the emergence of hantaviruses The aim of this study was to evaluate the viral diversity in the human population. Sinop is located in a region and prevalence of transmitted drug resistance mutations considered as endemic for the disease, though there (TDRM) among ARV-naive HIV-1-infected pregnant are no epidemiological studies performed in the city. women followed at Hospital Geral de Nova Iguaçu (HGNI) From January 2011 to February 2012, we developed in Rio de Janeiro – Brazil, between 2012 and 2013, and an arboviruses surveillance study, and sera from 198 to compare the results with a similar previous study patients with acute febrile illness were collected. These including pregnant women recruited from 2005-2008. sera were also assessed for anti-hantavirus antibodies The viral RNA was extracted and the HIV-1 protease by ELISA using Araraquara virus recombinant N protein and reverse transcriptase (PR/RT) regions of the pol (ARAV rN) as antigen. Thirty-nine (19.7%) clinical gene were sequenced from 41 plasma samples using samples were IgG positive, with titers from 100 to 3,200, an in house genotyping method. HIV-1 subtypes were but none IgM positive. Mean age of seropositive patients determined by phylogenetic analyses and TDRM were (29 female, 10 male) was 32.9 years (95% CI: 29.9- detected using the Calibrated Population Resistance 35.9). Serological surveys of Hantavirus, using the same methodology, were performed in all Brazilian regions, 58.5% of the sequences, representing a decrease in the showing 0.6% to 13% seropositive individuals in general prevalenceTool-CPR v.6.0. found The in HIV-1the previous subtype Bperiod was identified(81%). The in frequencies of the subtypes F1 (12.2%) and C (2.4%) remained similar, while there was a 3-times increase in populations. Since the first record of HCPS in Brazil, the recombinant forms frequency (from 8.0% to 26.8%) serological14 cases were evidence notified of inHantavirus Sinop, however infection they in werehumans all since the previous study. The overall prevalence of any fromconsidered Sinop importedmunicipality, cases. where Our findingsthese viruses demonstrate can be HIV-1 TDRM was 12.2%, in accordance to the previous causing minor or subclinical infections. Deforestation estimates (10.7%). TDRM for nucleoside reverse due to population growth and extensive agricultural transcriptase inhibitors remained similar (from 5.6% activity in Sinop could be associated with this high to 4.9%), while protease inhibitors TDRM increased seroprevalence. Furthermore, public health policies from 3.0% to 7.3%. No TDRM for nonnucleoside targeting to raise population awareness about the disease are necessary. analyzed samples collected between 2012 to 2013, contrastingreverse transcriptase with the 2%-prevalence inhibitors were previously identified found. in the HV326 - HIGH HIV-1 DIVERSITY AND MODERATE PREVALENCE OF TRANSMITTED DRUG RESISTANCE periods. These results indicate an increase in the HIV-1 MUTATIONS AMONG ANTIRETROVIRAL-NAIVE HIV- Multidrug-resistant strains were not identified in both INFECTED PREGNANT WOMEN IN RIO DE JANEIRO, Rio de Janeiro between the two periods. The moderate BRAZIL prevalencediversity and of a HIV-1probable TDRM change in inthis the population TDRM profile could in Delatorre, E.1; Pilotto, J.H.2; Morgado, M.G.1 1. Laboratório de Aids e Imunologia Molecular, ARV regimens to prevent HIV vertical transmission, affect the virological outcome of the standard first-line Instituto Oswaldo Cruz reinforcing the importance of continuous monitoring of 2. Hospital Geral de Nova Iguaçu, Ministério the HIV-1 genetic diversity and TDRM in Brazil. da Saúde The increase in the access of antiretroviral (ARV) drugs in Brazil has strong impact in the reduction of morbidity and mortality of HIV-1 positive individuals. However, it

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

169 Human Virology: HV

HV327 - ESTIMATING THE ORIGIN OF THE HIV-1 HV329 - GENOTYPING OF HPV IN SAMPLES OF HIGH CRF02_AG LINEAGES CIRCULATING IN BRAZIL GRADE CERVICAL INTRAEPITHELIAL NEOPLASIA Delatorre, E.1; de Castro, C.A.V.2; Pilotto, J.H.3; Bello, Dias, M.C.1; Calmon, M. de F.1; Provazzi, P.J.S.1; Camilo, G.1; Morgado, M.G.1 H.P.1; Badial, R.M.1; Kobayashi, M.T.2; Quintana, S.M.2; Yamamoto, A.Y.3; Rahal, P.1 1. Laboratório de Aids E Imunologia Molecular, Instituto Oswaldo Cruz 1. Laboratório de Estudos Genômicos, Instituto 2. Laboratório de Virologia, Departamento de de Biociências, Letras e Ciências Exatas – UNESP Patologia Clínica, Instituto Fernandes Figueira 2. Departamento de Ginecologia e Obstetrícia, 3. Hospital Geral de Nova Iguaçu, Ministério FMRP-USP da Saúde 3. Laboratório de Virologia Clínica, HCFMRP- The HIV-1 CRF02_AG is one of the most prevalent USP circulating recombinant forms (CRF) in the world and The human papillomaviruses (HPV) infect the mucosal is responsible for at least 8% of the HIV-1 infections and squamous epithelium, remaining asymptomatic worldwide. This CRF is distributed mainly in West Africa or causing disease. Persistent infection with HPV is and, to a lesser extent, in the Middle East and North responsible for almost all cervical cancers, as well as Africa. Due to the migrations from these endemic regions, other anogenital carcinomas, therefore, it has a large CRF02_AG has been reported recently in countries expression in public health. During the development where this recombinant is not native, including Brazil. of cervical carcinoma, multiple premalignant stages In a previous study, including six CRF02_AG samples can be distinguished, including cervical intraepithelial neoplasia of low (CIN I) and high grade (CIN II and III). least four introductions of this clade in Brazil, probably In this way, the present study aimed the genotyping of fromidentified Western in Rio African de Janeiro countries. from 2004-2011, As more we CRF02_AG found at the human papillomavirus present in 95 samples of high grade cervical intraepithelial neoplasia (CIN II aimed to review the geographic origin and to date the and III) and correlates the HPV types detected in the introductionsamples have of been some identified lineages since circulating then, in in this Brazil study, using we samples with the clinical and pathological data of the a larger dataset. A total of 20 Brazilian (18 from Rio de lesions. Genotyping was made by PCR with PGMy09/11 Janeiro and two from São Paulo) and 1,505 African HIV-1 CRF02_AG pol sequences were analyzed using Maximum according to Sanger method. In the sequence analysis, Likelihood (ML) and Bayesian phylogeographic methods. theand electropherograms GP5+/6+ primers, were followed observed by in the sequencing BioEdit The ML analysis showed that the 20 CRF02_AG Brazilian software, and the sequence quality was assessed by

The Bayesian phylogeographic analysis of the Brazilian using the BLAST tool in the NCBI website. To calculate sequences wereand distributedtheir most inclosely five different related lineages.African thePhred/Phap/Consed. correlation between The genotypes HPV types versus were clinical identified and sequences (n = 212) placed the origin of all Brazilian pathological data from samples, the Minitab software lineages in West Africa, probably Ghana (lineages BR-I, BR-II and BR-III), Senegal (BR-IV) and Nigeria (BR-V). as CIN III were predominant and that 86 samples were Lineages BR-I and BR-II comprise >1 sequence, all from positivewas used. for The HPV findings and nine showed samples that were lesions negative. classified The the Rio de Janeiro state, and their dates of origin were HPV16 was the most common type and proved to be estimated at 1985 (95% highest posterior density: 1979 correlated with CIN III, whereas HPV33 proved to be – 1992). These results support the existence of at least correlated with CIN II. Among the evaluated samples, 17 showed co-infection with HIV, but a positive correlation from West Africa into Brazil and further indicate that at was not found between HIV and CIN II or III. On the other leastfive independent two of these introductions lineages have of beenthe CRF02_AG disseminated lineage in hand, CIN II lesions were positively correlated with the Rio de Janeiro state for about 30 years. patients aged between 18 to 28 years. Some patients had follow-up samples and the same genotype of HPV as well as different genotypes in different samples from

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

170 Human Virology: HV the same patient was observed. In this way, the analysis The dehydration step was performed using 70% acetone allowed to observe the prevalence of HPV genotypes and in 1% uranyl acetate followed by 90% acetone and 100% their relationship with high-grade lesions, together with acetone. Cells were included in epoxy resin and kept at clinical and pathological data. These data are important 60C to complete polymerization. Ultra-thin sections, for the formulation of preventive measures, such as 50nm thick, were performed to electron microscope vaccines against the HPV types most commonly found observation. Semi-thin sections, 0.5 µm thick, were in cervical cancer, thus helping prevent the development performed to optical photonic microscopy. The ultra- of the disease. FINANCIAL SUPPORT: FAPESP, CNPQ, thin sections were stained with uranyl acetate and lead CAPES. citrate, the semi-thin sections with methylene blue. By optical photonic microscopy we observed in both cell HV337 - MOLECULAR DETECTION IN HPV-16 AND lines similar features such as presence of very large HPV-18 NATURALLY INFECTED CELL LINES, SIHA nuclei with highlighted nucleoli, reduced cytoplasm, AND HELA RESPECTIVE ULTRASTRUCTURAL CELL MORPHOLOGY interconnected cells by desmosomes. Very electrodense Simoes, R.S.Q.S.1; Fernandes, A.T.G.2; Carvalho, cellscell membranes were detected presenting by electron filopodia, microscopy few vacuoles presenting and M.O.O.1; Silva, M.A.N.1; Silva, L.M.1; Braga, R.M.1; Roma, well developed mitochondria and rough endoplasmic E.H.2; Almeida, M.G.B.2; Barth, O.M.1 reticulum, many vesicles and ribosomes in HeLa and 1. Laboratory of Viral Morphology and Morphogenesis, Instituto Oswaldo Cruz, FIOCRUZ, Pavilhão PCR method using different amount of template (1-5µL). Helio e Peggy Pereira TheseSiHa cell preliminary lines. Furthermore, results suggested HPV-DNA awas strong amplified cellular by 2. Aboratory of Immunology and activation in these cervical keratinocytes. More studies Immunogenetic, Evandro Chagas National Institute of are needed to evaluate the morphological alterations Infectious Diseases, FIOCRUZ induced by HPV in those cell lines. FINANCIAL SUPPORT: Human papillomavirus (HPV) is the primary factor of CAPES/PROGRAMA DE PÓS-GRADUAçãO EM MEDICINA HPV-types 16 and 18 cervical cancer causing an average TROPICAL/BRASIL SEM MISÉRIA, IOC AND INI-FIOCRUZ of 70% of cases worldwide. Prophylactic quadrivalente HV339 - STANDARDIZATION OF A PCR PROTOCOL IN HPV vaccine against HPV-types 6 and 11 (non-oncogenic) CERVICAL SQUAMOUS INTRAEPITHELIAL LESIONS and HPV 16 and 18 (oncogenic types) was developed. PREVIOUSLY GENOTYPED USING HPV TYPE 3.5 LCD- Few studies have assessed the transmission electron ARRAY microscopy in different cells lines. The present study Simoes, R.S.Q.S.1; Carvalho, M.O.O.1; Fernandes, investigated molecular detection by PCR using consensus A.T.G.2; Rocha, N.2; Russomano, F.B.3; Barth, O.M.1; primers in standard samples of SiHa and HeLa cell lines Roma, E.H.2; Almeida, M.G.B.2 and respective ultrastructural cell morphology. In order to optimize the current cell lines, a screening process of 1. Laboratory of Viral Morphology and 3x106 cells was counted and DNA extracted using DNA Morphogenesis, Instituto Oswaldo Cruz, Fiocruz, Pavilhão blood mini kit (Qiagen). For molecular detection, DNA Helio E Peggy Pereira of SiHa and HeLa cells naturally infected with HPV 16 2. Laboratory of Immunology and and 18, respectively, were used as positives controls and Immunogenetic, Evandro Chagas National Institute of Infectious Diseases, FIOCRUZ control. MY09/11 consensus primers that amplify a 450 3. National Institute of Women, Children and the amplification misture without DNA as a negative Adolescent Healthy Fernandes Figueira, FIOCRUZ positive standard samples. For ultrastructural analysis, Human papillomavirus (HPV) is critical in the pathogenis thebp DNA SiHa sequence and HeLa specific cells were for HPVembedded L1 ORF in were epoxy used resin, to of cervical cancer, the second most common cancer tetroxide. Later steps followed by washes in cacodylate as high-risk and low-risk depending upon the associated bufferfixed in 0.2 1% M glutaraldehyde in sodium sucrose and post-fixed0.7% and distilledin 1% osmium water. diseaseamong women intensity. in Brazil.This study The mucosalwas designed types areto evaluate classified a

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

171 Human Virology: HV standard PCR protocol in positive samples previously HV341 - GENETIC DIVERSITY OF INFLUENZA VIRUS genotyped. DNA was extracted from eight samples of STRAINS CIRCULATING IN THE AMAZON REGION, cervical biopsies using DNA tissue mini kit (Qiagen). BRAZIL Samples with squamous intraepithelial lesions of cervix Santos, M.C.1; Barbagelata, L.S.1,2; Ferreira, J. de low grade (LSIL) and high grade (HSIL) were previously A.1; Júnior, E.C.S.1; de Pinho, R.A.P.1; Gomes, É.R.1; genotyped using HPV type 3.5 LCD-Array kit (Chipron). Filizzola, E.M.A.1; Gonçalves, M. dos S.1; Brabo, M.V.V.1; Rodrigues, S.V.1,2; Mello, W.A.1; Medeiros, R.1,2 capture probes according to the manufacturer’s protocol. AMultiple PCR protocol HPV types using per sampleconsensus were primers detected MY09/MY11 from specific 1. IEC - Instituto Evandro Chagas, Rodovia BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 that amplify a 450-bp fragment of the L1 open reading 2. NÚCLEO DE MEDICINA TROPICAL/ UFPA - Núcleo de Medicina Tropical da Universidade Federal frame of genital HPV was proposed. Amplification was do Pará, AV. Generalíssimo Deodoro, 92, Belém - Pará, 66055- 2,5mM dNTPs, 50mM MgCl2, 10µM each primer, 5 unit 240 ofdeveloped Taq polymerase, in a 25µL and reaction 5µL of mixturesample). (10X After PCR activation buffer, run. Each cycle included a desnaturizing step at 94ºc for affects the respiratory tract and the etiologic agent that Flu or influenza is a highly contagious viral disease that 1step min, at an94ºC annealing for 5 min, step 35 at cycles 55ºC offor amplification 30 s, and a chainwere elongation step at 72ºC for 1 min using Gene Amp PCR Orthomyxoviridae family. The high genetic diversity of thesecauses viruses it is the provides Influenza antigenic viruses, changes, which allowing belong to them the min was done. DNA isolated from HPV-16 positive SiHa to escape from host defenses. Such mutations occur and(Applied HPV-18 Biosystems). positive HeLa A final cells elongation were used at 720Cas positives for 10 mainly on the glycoproteins surface, hemagglutinin (H) controls. The integrity of the DNA isolated from cervical and neuraminidase (N). In this context, the constant monitoring of mutations, associated to the variability primers (GH20/PC04) that amplify a 268-bp, and all samplesspecimens showed was confirmed positive detection. using a β-globinHPV was genedetected set inof obtaining data to subsidize interventions for prevention of the Influenza viruses plays an important role in 83.3% (5/6) of positive samples and negative in 100% and control of the infections by this pathogen. In order of negative samples. The discordant sample was positive Amazon region in the year 2013, as well as analyze the occurrenceto characterize of genetic Influenza rearrangement strains circulating among circulating in the werefor β-globin performed gene andin the specific standardized HPV-DNA protocolmay be atusing low strains and verify the incidence of HA and NA genes thelevel common and could set not of beprimers detected. (MY09/MY11) Several modifications for HPV mutations associated to escape the host immune response, pathogenicity and antiviral resistance, a total and annealing time. Adjustment in concentrations of of 2198 samples of patients with signs or symptoms reagentsdetection, in such the asPCR the reaction modification mix was of thealso extension applied. Therefore, the results showed that the protocol used was of Acre, Amapá, Amazonas, Pará and Roraima was suggestive of flu, attended by health units in the States able to detect HPV in accordance of previous commercial analyzed, from January to December 2013. The samples were analyzed by extraction and detection of viral requires a gold standard protocol able to detected HPV nucleic acid by Polymerase Chain Reaction preceded evenPCR in protocol. the absence Clinical of cytological identification alterations. of HPV Molecular types by real-time reverse transcription (qRT-PCR). Positive epidemiological studies are need to identify HPV types in samples were subjected to conventional RT-PCR for general population before and after the HPV-vaccination. FINANCIAL SUPPORT: CAPES, INI AND PROGRAMA DE gene amplification of HA and NA and then sequenced. PÓS-GRADUAÇÃO EM MEDICINA TROPICAL/BRASIL Influenza virus was detected in 401 samples, being SEM MISÉRIA B. The HA gene analysis showed the genetic similarity 229 A (H1N1) pdm09, 51 A (H3N2), and 121 Influenza

of the Influenza A virus strains circulating in our region with the flu vaccine strains. Most strains of September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - InfluenzaHuman Virology: B virus HV belongs to Victoria lineage, however XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

172 Human Virology: HV the contemplated one in the vaccine was Yamagata. plasma DNA obtained from healthy individuals was It was also demonstrated the presence of amino acid substitutions in the HA gene, related to increased eight replicates of a standard curve (1,000 to 31.25 UI/ml)applied. were In order diluted to definefrom standard the limit plasma of HBV (3rd detection, WHO Relating to NA analysis, in the A (H1N1) pdm09 strains International Standard for HBV, NIBSC code 10/264). itvirulence was found in Influenza the I106V A replacement, (H1N1) pdm09, which A (H3N2) is associated and B. Results. The ability of the primers to detect all known with decreased sensitivity to antiviral drugs. However, no important substitution in A (H3N2) samples was HBV alignment. Two commercial extraction kits (A and HBV genotypes was confirmed by their search in a global virus strains circulating in our region, as seen in strains analyzedverified. Our worldwide. study highlights Amino the acid variability replacements of Influenza that andB) were the compared.earlier cycle We thresholdverified that (Ct) kit observedB provided during more were presented, which was related to pathogenicity efficient extraction due to the higher quantity of DNA and antiviral resistance, reinforce the need for also compared the most suitable samples for HBV-DNA monitoring these viruses. FINANCIAL SUPPORT: SVS/ extraction:real-time PCR plasma, amplification ultracentrifuged of the sameplasma samples. and buffy We MS,CNPQ,PPSUS,FAPESPA coat. Ultracentrifuged plasma was chosen because it allowed HBV detection, with lower Ct, in all samples of HV348 - OPTIMIZATION OF A TAQMAN MOLECULAR PLATFORM FOR HEPATITIS B VIRUS (HBV) a concentration of 500nM for primer and 250nM for DETECTION IN BLOOD DONORS probe.HBV patients. A commercial For the detectionreaction ofbuffer HBV DNA,tested we was defined also Santos, D.F.1; Junior, M.C.R.1; Rodrigues, E.S.1; Slavov, able to reduce the Ct. Moreover, genotypes A, B, C, D, E, S.N.1; Salustiano, S.G.1; Azevedo, R.1; Martinelli, A. de and F were detected by real-time PCR. The sensitivity of L.C.2; Covas, D.T.1; Kashima, S.1 the reaction was 225.84 UI/ml of DNA, using the Probit 1. Regional Blood Center of Ribeirão Preto, analysis. Conclusion. The optimization of the real-time Faculty of Medicine of Ribeirão Preto, University of São Paulo PCR demonstrated that it detects all known genotypes. 2. Division of Gastroenterology, Department However, a suitable validation must be still performed in of Clinical Medicine, Faculty of Medicine of Ribeirão Preto, order to improve its sensitivity. University of São Paulo HV354 - VIRAL METAGENOME OF THE HUMAN Although Nucleic Acid Technologies (NAT) have been INTESTINAL TRACT – PRELIMINARY RESULTS implemented in the routine practice for the detection Silva, P.A.; Nogueira, R.; de Lima, L.M.P.; Nagata, T. of different viruses threatening blood transfusion, such as HCV and HIV in Brazil, there is no NAT technique for UCB - Universidade Católica de Brasília, W5 Norte, HBV in this platform. Our aim was to optimize a real- Brasília - DF, 70790-160 time TaqMan® PCR test for detection of the major HBV Diarrheal diseases are major public problem worldwide, genotypes (A-F). Materials and Methods. Primer and especially in developing countries. The knowledge of probe sequences for the conserved region among all HBV these etiological agents is important for the treatment genotypes (gene S) were obtained from previous studies. and epidemiological studies. In order to identify viral population and new viral pathogens including the DNA from plasma was extracted using two commercial non-cultivable, the intestinal metagenome aims the Their specificity was confirmed by sequencing analysis. kits (A and B). Several concentrations of primers (250 sequencing of the entire genetic material present in and 500nM) and probe (100–400nM) were evaluated the fecal sample. The study proposal is a metagenomic investigation of the human intestinal tract viruses from The reaction was controlled using HBV DNA standards 12 Brazilian individuals to analyze the viral population in order to obtain a better efficiency of amplification. from Worldwide HBV DNA Performance Panel PHD301 and identify new potential viruses that cause diarrheal (BBI Diagnostics) containing the genotypes A, B, C, diseases. Methodology: A previous experiment was done D and E. Genotype F was obtained from plasma of a with one sample from a healthy individual. The fecal patient with chronic hepatitis B. As a negative control, sample (~ 500 mg) was suspended in PBS and centrifuged

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

173 Human Virology: HV at 9700g for 10 minutes, the supernatant was collected infectious conditions in children, such as the lower respiratory tract disease and asthma exacerbation. at 30.000 rpm for 2.5 hours and the genome form the Therefore, this study aimed to identify the prevalence of pelletand filtered was extracted in 0.45μm using and PureLink0,2μm, and ® ultracentrifugedViral RNA/DNA HRV in samples collected from children with symptoms Mini Kit (Invitrogen). The reverse transcriptase and of respiratory infection. Methods: From February to December 2013, 195 nasal secretion samples were collected from 0-6 years old children who have presented theamplification library constructed using the with illustra the GenomiPhiTruSeq DNA V2 Sample DNA symptoms of respiratory infection at the Hospital Infantil Preparationamplification kit kit V2 (GE (Ilummina). Healthcare) For was the sequencing performed, was and Cosme e Damiao (HICD) considered as a reference in the used HiSeq 2000 paired end (Ilummina), To alignment state of Rondonia, Brazil. The biological samples were the sequences was used Bowtie aligner. Results: The analysis of sequencing in comparison with the viral data viruses by real-time PCR using the SYBR Green system. base resulted in 25.032.073 reads. 8,548 reads were Results:submitted Molecular to molecular detection techniques rate of forHuman identification Rhinovirus of aligned with viral sequences. The alignment there was was 86% (168/195). The more frequent symptoms a greater number of alignments with baculoviruses and related to patients HRV positive were cough with 90.4%, bacteriophages, without the presence of enteric viral followed by rhinorrhea (76.7%), pulmonary secretions pathogens. Conclusion: There was no detection of any (71.4%) and nasal obstruction (70.2%). Children who are virus pathogens and this fact can be explained by the from 0 to 1 years old were more susceptible to infection rarity of sequences of eukaryotic viruses in samples of by HRV. The seasonal distribution during the months viromes gut of healthy individuals and the detection from February to December 2013 has showed peaks in of baculovirus is the result of contamination during the warmer months, specially from June to September. ultracentrifugation. The study with the other samples is Conclusion: Data has demonstrated that the detection in progress. Perform viral metagenome of samples from the intestinal tract is an innovative initiative, considering found. These data are really relevant once there is no that it is a country that has high biodiversity, feeding publishedof HRV was works significant, in the observed region. It by is theimportant high prevalence to know habits and natural life and may bring results completely the causative agent of infection that is of great value to aid in prognosis and therapeutic management of these children. Therefore, the development of prospective newHV355 to the - MOLECULAR scientific community. IDENTIFICATION OF HUMAN RHINOVIRUS AND CLINICAL MANIFESTATIONS IN A severity of the disease in children is needed. Financed CHILD POPULATION IN THE STATE OF RONDONIA by:studies FIOCRUZ-RO, to define FAPERO,the epidemiology CNPQ. of this virus and the Alves, F.A.G. dos S.1,2,3; dos Santos, A. de O.1,2,3; Baffini, V.R.1,2,3; Souza, L.F.B.1,2,3; Tarboda, R.L.M.1,2; Salcedo, HV356 - INVESTIGATION OF HEPATITIS E VIRUS IN J.M.V.1,2; Vieira, D.S.1,2,3 CASES OF ACUTE FLACCID PARALYSIS OF UNKNOWN ETIOLOGY 1. UNIR - Universidade Federal de Rondônia, 1 1 2 2 Av. Presidente Dutra, 2965, Centro, Porto Velho - RO, 76801- Morgado, L.N. ; Vitral, C.L. ; da Silva, É.E. ; Pinto, M.A. 974 1. UFF - Universidade Federal Fluminense, 2. CEPEM - Centro de Estudo e Pesquisas da Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 Mulher, R. Barão de Lucena, 71 - Botafogo, Rio de Janeiro - RJ, 2. FIOCRUZ - Fundação Oswaldo Cruz, Av. 22260-020 Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 3. FIOCRUZ - Fundação Oswaldo Cruz, Av. Hepatitis E is transmitted by the fecal-oral viral infection Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 and is considered as the main cause of acute liver Human Rhinovirus (HRV) is among the leading causes of infection in several areas of Asia, Africa, and the Middle infections in the upper respiratory tract. The HRV is an East. Brazil is considered as an area of low endemicity for hepatitis E virus (HEV). The possibility of circulation of C. Generally, the infection is involved with more serious HEV in our country has been demonstrated in numerous RNA virus, which is classified in three species: A, B, and September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

174 Human Virology: HV seroepidemiological studies, which observed prevalence of infection rates ranging generally 2-8%. It was also virus infection has been described as an infection reported an acute case of the disease by researchers at outbreakaddition tousually the risk starting of pandemics. in April In in Brazil the Influenzanorthern the Instituto Oswaldo Cruz (Fiocruz). Complementing equatorial region and gradually spreading to the other regions of the country. In this context, this study aims antibodiesthese findings, and the HEV possibility genome of sequenceszoonotic infection in different with speciesHEV was of animals, also confirmed especially by pigs, the in detection which the of infection specific populationto determine segments the pattern located of in circulation the North ofand influenza Northeast A showed almost universal. Hepatitis E usually presents ofviruses Brazil. (H3N2 From June and 2013 H1N1pdm09) to June 2014, and the influenza Respiratory B in itself indiscriminately of other viral hepatitis and Virus Laboratory of Instituto Evandro Chagas, belonging progresses to healing spontaneously after a few weeks of disease onset. However, some cases may progress to more accredited as Reference Laboratory by World Health severe forms, including the appearance of neurological Organizationto National (WHO) Influenza received Surveillance 5,432 clinical Network, specimens and syndromes, with a predominance of peripheral nerve (nasopharyngeal aspirate or swab combined) of disorders such as Guillain-Barré syndrome (GBS) and brachial neuritis. Each year, the IOC Enterovirus Laboratory, which represents a National Reference samplespatients were presenting from states signs in or the symptoms North region of influenza, (AC, AM, Laboratory of the Ministry of Health of Brazil, receives AP,for investigationBP, RR) and Northeast / confirmation region of (CE, viral MA, etiology. PB, PE, These RN). around 500 fecal samples from individuals with acute Diagnosis was received by detection of viral genome involving the extraction of viral RNA (RNAv), using the with a large proportion (> 80%) of these cases remains PureLink TM Viral RNA / DNA Mini Kit (Invitrogen) and undeterminedflaccid paralysis etiology. (AFP) or The Guillain-Barré objective of syndrome this project (SGB), is to investigate the HEV research by the viral genome in preceded by Reverse Transcription (qRT-PCR) in real Time,the amplification using SuperScript of RNAv byIIITM Polymerase One-step Chain RT-PCR Reaction with were negative for the presence of enteroviruses as well Platinum ® Taq - (Invitrogen Life Technologies). Of the asfecal those samples in which from enterovirusescases of acute wereflaccid detected. paralysis Fecal that total patients analyzed, 877 (16.1%) were positive for samples from healthy individuals without clinical signs and symptoms of neurological syndromes were used as a control group. influenza viruses, 394 (45%) were positive for Influenza withA (H1N1pdm09), coinfection (one 241 H1N1pdm09 (27.5%) for / influenza B, two H3N2 B, 218 / HV359 - CIRCULATION OF INFLUENZA A AND B (26%) for Influenza A (H3N2), three (0.34%) patients VIRUS IN THE NORTH AND NORTHEAST REGIONS OF BRAZIL, FROM JANUARY 2013 TO JUNE 2014 B) and 22 patients presented inconclusive influenza A Gomes, E.R.1; Barbagelata, L.S.1; Ferreira, J. de A.1; typing. In this study, all types of human influenza viruses Santos, M.C.1; de Souza, E.M.A.1; Gonçalves, M. dos S.1; ofcirculated all infections), more intensively contrary to in what the wasfirst expected,half of 2013 since (65% we Medeiros, R.2; de Melo, W.A.1 justof all experience infections) the compared World Cup to the in our first country half of 2014and a (35% large 1. IEC - Instituto Evandro Chagas, Rodovia number of people (locals and foreigners) circulated in BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 the territory under study. The peak prevalence occurred 2. NÚCLEO DE MEDICINA TROPICAL/ in April and May both in 2013 and in 2014 with relevance UFPA - Núcleo de Medicina Tropical da Universidade Federal do Pará, AV. Generalíssimo Deodoro, 92, Belém - Pará, 66055- 240 extendedin May 2013, its peakwhen by Influenza the end Aof infection June 2013. (H1N1pdm09) FINANCIAL SUPPORT:represented IEC/SVS/MS 16% (n = 142) of cases, and the Influenza B in health authorities, due to its high transmissibility andInfluenza impact is aon viral morbidity disease that and generates mortality major rates, concerns which increase substantially during seasonal epidemics in

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175 Human Virology: HV

HV361 - GENETIC DIVERSITY AND TRANSMITTED have been integrated in the local epidemiology. This DRUG RESISTANCE OF HIV-1 IN MOZAMBIQUE might probably due to the high number of emigrants Vubil, A.2; Jani, I.2; Bhatt, N.2; Gudo, E. S.2; Mabunda, mainly from Central Africa and neighboring countries N.2; Ismael, N.2; Bila, D.2; Morgado, M.1; Fernandez, (Tanzania and Malawi) where HIV diversity is known to J.C.C.1 be high. Considering the HIV-1 diversity and the rapid expansion of TARV in Mozambique, continuous molecular 1. IOC/FIOCRUZ - Instituto Oswaldo Cruz da epidemiology surveillance is important to monitor HIV-1 Fundação Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, Rio infections, that might impact in the dynamics of the AIDS de Janeiro - RJ, 21040-360 epidemic in Mozambique. 2. INS-MISAU Sub-Saharan Africa accounts with 69% of global HV372 - EX VIVO RHINOVIRUS REPLICATION IN HIV/ADS infections. The HIV-1 epidemic in African HUMAN TONSILLAR EXPLANTS countries is characterized by high degree of genetic Junior, R.B.M.; Criado, M.; Gagliardi, T.; Escremim, F.; diversity. Subtype C followed by subtype A1 are the Silva, M.; Carenzi, L.; Tamashiro, E.; Valera, F.; Lima, most prevalent and occurring respectively, in South and W.A.; Arruda, E. Central Africa. In addition, transmitted drug resistance FMRP/USP - Faculdade de Medicina de Ribeirão mutations are increasing in many countries as a result Preto/ Universidade de São Paulo, Av. Bandeirantes, 3900 - of the universal access to antiretroviral therapy (TARV) Monte Alegre, Ribeirão Preto - SP, 14049-900 and lack of adherence to treatment. The aim of this study is to improve knowledge about HIV-1 genetic Acute respiratory infections (ARI) caused by human diversity and transmitted drug resistance (TDR) of the rhinovirus (HRV) have great impact in public health, viruses circulating in different regions of Mozambique. but little is known about HRV persistence/latency. In a METHODS: Blood samples were collected from 200 previous study we detected HRV by TaqMan Real Time HIV-1 positive blood donors in three cities in northern PCR (qPCR) in 38% of children with chronic tonsillar Mozambique, between November 2009 and June hypertrophy without ARI symptoms, who underwent 2010. Genotyping of HIV-1 resistance was performed tonsillectomy at the Otorhinolaryngology Clinic, using Trugene HIV-1 Genotyping assay. Phylogenetic University of São Paulo Hospital, in Ribeirão Preto. High inferences using maximum likelihood were applied on frequency of HRV detection in tonsils in the absence of protease and reverse transcriptase segments derived ARI symptoms suggests that these agents may persist from the genotyping of HIV-1 resistance. RESULTS: A in human tonsils. The present study is based on two total of 95 HIV-1 genotyping sequences were analyzed, experimental approaches: immunohistochemistry (IHC) of tonsillar tissues naturally infected with HRV and ex A1 (10.5%), recombinant forms CA1, DA1, DU and vivo inoculation of tonsillar explants with stocks of HRV- CU79% (5.3%), was classified 3.2% subtypeas subtype D C.and Followed 2.1% subtypeby subtype G. 16 (major receptor group) and HRV-1A (minor receptor Transmitted drug resistance mutations associated to group). Roughly 50% of all IHC tested with antibody for the non-nucleoside reverse transcriptase inhibitors VP1 capsid protein, indicating the presence (NNRTI) were detected in 4.2% of the samples and of virus capsid protein in lymphoid tissue, within and one individual show protease inhibitor (PI) resistance outside of the lymphoid follicles of tonsils and adenoids, mutation associated to typranavir. CONCLUSIONS: This and also in epithelial cells in adenoids. This prompted us study shows that subtype C remains the most prevalent to try a model of ex vivo infection of adenoid explants. in northern of the country and supply evidences of low Tissue fragments of ~3 mm3 were placed in 6-well levels of TDR in Mozambique. Phylogenetic analysis has tissue culture plates, and cultured over 7 days in RPMI- shown striking genetic similarity between South African 1640 with 10% BSA. Explants were considered viable when epithelial outgrowth developed around the tissue in Mozambique, suggesting a continuous introduction fragment. A 5µL droplet (HRV-16 at 107and HRV-1A andisolates circulation and autochthonous of closely related subtype strains C strains in the identified country. at 106,25 TCID50/mL) of virus stock was carefully However, new subtypes such as D, G and recombinants inoculated on top of the tissue. After 24 hours, tissue was September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

176 Human Virology: HV washed and re-incubated at 37ºC in a 5% CO2 atmosphere

software.2010 were The amplified amino acidby RT-PCR. sequences The and 731-bp the alignmentsegments VP1.for 3 In more addition, days. Aftera different that, fixation, experimental dehydration approach and ofwere them sequenced were obtained and classified by BioEdit as E2 software. genotype The by results RotaC wasparaffin taken, embedding with ex vivo were infection done for of dissociatedlater IHQ with lympho- anti- showed the prevalence of variations at antigenic sites epithelial cells from adenoids and palatine tonsils with II and III, VP4-binding domain, enterotoxin domain rhinovirus HRV-16 and HRV-1A. HRV capsid protein VP1 and at ECM-protein binding domain. Some nucleotide polymorphisms led to amino acid changes in all strains 5 post infection. Results indicate that lymphoid cells collected in a given year. Only samples collected in 2006 fromwas detectedtonsillar bytissues indirect are susceptible immunofluorescence and permissive on day to infection and may function as possible reservoirs of HRV. same kind of change was observed at positions 136 Assays to quantitate continuous progeny production encoded different amino acid at position 87 (E→D). The in lymphocytes are underway. FINANCIAL SUPPORT: FAPESP and CNPq. 2007(V→M) and and 2009, 139 respectively, (T→I) for strains and these from were 1994. maintained Positions for103 the (V→I) later and years. 131 Finally, (Y→H) in had position amino 135 acid the changes samples in HV375 - COMPARISON OF THE NSP4 DEDUCED from 1994 has the same amino acid as RV reference strain AMINO ACID SEQUENCES FROM GROUP A HUMAN DS-1 (M), and the samples from other years have the ROTAVIRUS G2P[4]E2 STRAINS CIRCULATING IN SÃO same amino acid as RV patterns TB-Chen and B1711 (I). PAULO, BRAZIL Since DS-1 and TB-Chen strains were collected in 1976 Bertol, J.W.1; Fregolente, M.C.D.2; Caruzo, T.A.R.1; and 1996, respectively, this mutation probably occurred Gatti, M.S.V.1 between 1994 and 1996. Most of the variation in NSP4 1. UNICAMP - Universidade Estadual de amino acids occurs in C-terminal region, including Campinas, Cidade Universitária Zeferino Vaz - Barão the VP4 binding domain. This fact reinforce the idea Geraldo, Campinas - SP, 13083-970 that somehow NSP4 is involved in morphogenesis and 2. UNICID - Universidade Cidade de São pathogenesis activities and more studies are necessary Paulo, Rua Cesário Galeno, 448/475, Tatuapé - São Paulo - to understand how these variation may interfere in SP, 03071-000 structural conformation on interaction sites between Rotaviruses (RV) are the main agents of gastroenteritis NSP4, VP4 and VP6 of RV.Financial support: FAPESP in humans and animals. Their non-structural protein HV376 - EVALUATION OF DETECTION OF VIRAL NSP4, encoded by segment 10, is a viral enterotoxin and RESPIRATORY INFECTION CAUSED BY RESPIRATORY is involved in the viral morphogenesis and pathogenesis. SYNCYTIAL VIRUS (RSV) The gene encoding NSP4 has been characterized into Pedrosa, C.F.; Campos, A.D.; Carraro, E. 15 genotypes (E1-E15) and this diversity of observed amino acid sequences could alter human RV-A (HuRV-A) UNICENTRO - Universidade Estadual Centro Oeste, R. strains virulence. It is unclear whether NSP4 should be Padre Honorino João Muraro, 875 - Santa Cruz, Guarapuava included in RV vaccination strategies; however, NSP4 - PR, 85015-430 does appear to play a role in immunity and protection. Human respiratory syncytial virus (RSV) is an The aim of this study was to determine the deduced important viral agent that causes serious diseases of the amino acids sequences of NSP4 from G2P[4] HuRV-A. respiratory tract in the paediatric population worldwide From 113 positive stool samples for HuRV-A by enzyme especially in children under 5 years of age. Cause immunoassay or polyacrylamide gel electrophoresis, 40 samples were characterized as G2P[4] using semi- cases of severe respiratory disease, such as bronchiolitis nested multiplex RT-PCR (samples from 1994, 2005 withsymptoms recurrent ranging wheezing from common and pneumonia. influenza framesMethods: to and 2006) or using semi-nested RT-PCR (samples from Collection of respiratory samples occurred in the city 2009 and 2010). The NSP4 gene of 13 HuRV-A strains of Guarapuava PR, in winter the years 2013 and 2014. from 1994, 17 from 2006 to 2007 and 10 from 2009 to Inclusion of individuals was about the acute respiratory

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

177 Human Virology: HV disease and nasopharyngeal secretion collection, with Infectious Mononucleosis (IM) is characterized by people of all ages. To detect RSV applied RT-PCR test a triad of high fever, pharyngitis and tonsillitis and in front of RSV controls strain, known to be positive, cervical lymphadenopathy. The clinical symptoms are obtained from the laboratory of Virology of the UNIFESP, mild/moderate in most cases. The transmission is oral- who had the genetic material extracted “QIAamp RNA oral route through contact with the saliva of an infected host, being nasopharyngeal infection a primary event. It RSVAB-GTCTTACAGCCGTGATTAGG (5’- 3’) and reverse: affects both sexes, all ages, especially children under 3 RSBAB-GGGCTTTCTTTGGTTACTTCmini kit (Qiagen). Used amplification (5’- primers 3’), amplifying forward: 838 base pair fragment. Reverse transcription reaction 15 to 25 years and is uncommon after age 30. Epstein- (RT-PCR) with RNA extracted from the sample, dNTPs, Barryears, virus with (EBV) significant is the numbers etiologic also agent in theof IM. range The between present Buffer, molecular grade H2O, forward and reverse study aimed to describe the seroepidemiology of EBV primers, reverse transcriptase enzyme “Moloney infection in patients suspected of IM. The study included Murine Leukemia Virus (MMLV-RT) and RNase inhibitor, 1,666 serum samples from patients with suspected IM incubated 42°C for 1 hour and 72°C for 15 minutes (SIM) from January to December 2013, sent to the EBV in Multigene clusters TC 9600 thermal cycler G. The Laboratory, at Evandro Chagas Institute, State of Pará, Brazil. The serological diagnosis was carried out by (PCR) cDNA had addition Taq DNA polymerase, dNTPs, enzyme immunoassays (EIA) for the semi-quantitative reaction of amplification polymerase chain reaction determination of IgM anti-VCA, following the by manufacturer’s instructions. EIA shoed that 23.40% of reaction-MgCl2, Buffer,in thermal primers, cycler, final with volume 95°C for of 5 25µl minutes, reaction 40 the samples were positive for IgM antibodies, 70.70% cyclescompleted to 94°C with per molecular 30 seconds, grade 54°C water. for 45 Amplificationseconds, and were negative and 5.9% were indeterminate. Among the 72°C for 1 minute. Last step 72°C for 7 minutes. A result positives (n = 390), 48.46% were from male patients. Overall, 47.17% of positives were in the age range of 0-5 material to 2% and stained with ethidium bromide for years, 27.43% between 11-30 years, 21.02% in the group visualizationconfirmed by in electrophoresis ultraviolet light. in Results agarose and gel Discussion: amplified above 30 years of age, and 4.38% lacked information The reaction was optimized for each primer in relation to the gain in the detection capability of the dilutions of had higher prevalence of fever (55.12%) followed by strains controls. The hybridization temperature of the headacheon age. Regarding (21.02%), clinicallymphadenopathy findings, positive(13.84%), patients cough primers with best performance was 54ºC. RT-PCR for (11.79 %) and myalgia (11.28%). The present study RSV was held in 50 nasopharyngeal samples collected, showed the low positivity for EBV IgM class antibodies and there was no detection of RSV in none of the samples anti-VCA, important marker used as a diagnosis of IM. The analyzed. Conclusion: The standardization of PCR for high prevalence of infection in children and young adults the detection of RSV control strain showed maximum suggests that the infection occurs early in life, which is capacity strains detection controls, however there was in accordance with other populations of developing no detection among the 50 samples collected for the countries, and lower in patients older than 30 years. completion of the study. Financial support: Capes.

HV384 - SEROEPIDEMIOLOGY OF EPSTEIN-BARR lymphadenopathy,There was no sex difference.Ascough and myalgia. to clinical The findings, occurrence the VIRUS INFECTION IN PATIENTS WITH SUSPECTED ofprofile childhood found isinfection suggestive tends of IM, to such be asasymptomatic fever, headache, or INFECTIOUS MONONUCLEOSIS REFERRED TO EVANDRO CHAGAS INSTITUTE, ANANINDEUA, PARÁ, young adults that leads to the development of classical BRAZIL IMclinically in most not cases. specific, FINANCIAL with an SUPPORT: infection MS/SVS/IEC occurrence in Barros, I.C.; Polaro, A.A.; Raissa, L.; Monteiro, T.A.F.; Costa, I.B. IEC - Instituto Evandro Chagas, Rodovia BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

178 Human Virology: HV

HV386 - INVESTIGATION OF THE EPIDEMIOLOGY OF HEPATITIS A IN THE MUNICIPALITY OF CAMPOS DOS in relation to water spout, number of people living in the GOYTACAZES-RJ BEFORE THE INTRODUCTION OF housemulatto larger ethnic, than contact 5, educational with flood water,level of use the bottled mother water and THE VACCINE IN THIS CITY father and bath in the river, pond and marsh.There was Kury, C.M.H.1; Vitral, C.L.1; Cruz, O.G.2; Pinto, M.A.2; de no statistical difference in gender, type of sewage device, Oliveira, J.M.2; Merlone, M.P.1; Kury, C.M.H.4; Freixo, wages of family grouping and type of district property H. de O.3; Santos, T.V.3; Lima, G.A.3; Araujo, B.R.3; of the individual, among others. Multivariate analysis Menezes, I. de Q.3 age, non-white ethnic, maternal educational level and 1. UFF - Universidade Federal Fluminense, numberdemonstrated of household a statistically members.The significant results correlation of this study for Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 support the decision of the municipality of Campos to 2. FIOCRUZ - Fundação Oswaldo Cruz, Av. vaccinate children against hepatitis A before the start of Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 school activity and corroborate data from other Brazilian 3. FMC - Faculdade de Medicina de Campos, Av. Alberto Torres, 111 - Centro, Campos dos Goytacazes - RJ, seroprevalence studies showing that a large proportion 28035-580 4. Secretaria Municipal De Saude infection of children under five years of age are susceptible to HAV Brazil is currently considered a country of intermediate HV387 - HIGH FREQUENCY OF ASSOCIATION OF endemicity for hepatitis A virus (HAV).The prevalence HUMAN RHINOVIRUS AND BOCAVIRUS ASSOCIATED of HAV infection has decreased in several Brazilian WITH BACTERIA IN PATIENTS WITH CHRONIC regions, a fact that has led to an increase in the number RHINOSINUSITIS of susceptible individuals at risk for HAV infection. Prates, M.1,2; Souza, J.1; Paula, F.E.1; de Jesus, B.L.S.; The introduction of vaccination against HAV in this Júnior, R.B.; Saturno, T.H.; Gagliardi, T.B.1; Leite, M.2; Valera, F.C.P.2; Tamashiro, E.2; Arruda, E.2; Anselmo- Lima, W.T.2 Brasilepidemiological that implemented profile wouldthe vaccine be of against great importance.HAV, which 1 startedCampos in dos march Goytacazes 2011 to all is children the first under and unique the age cityof two in Departments of Cell Biology and Otorhinolaryngology 2 years old. This study aims to evaluate the epidemiology and Head and Neck Surgery , University of Sao Paulo School of Medicine, Ribeirao Preto, SP, Brazil, 14049-900 of hepatitis A in this municipality so that the impact of vaccination can be further evaluated.From august, 2011 to july 2012, individuals under 19 years old were of paranasal sinuses mucosa with great public health randomly selected in schools from all of 14 districts of impact.Chronic rhino-sinusitisPrevious studies (CRS) have is anshown inflammatory the presence disease of the municipality. Calculation of sample size was based viruses and bacteria in paranasal sinuses of CRS patients, on an estimated prevalence of HAV in the order of 40%, but the simultaneous detection of viruses and bacteria has not been investigated. In the present study secretions than 99%.After informed consent, capillary blood and tissue samples from CRS patients undergoing an accuracy rate of 5% and a level of confidence greater surgery at the University of Sao Paulo Hospital in detection.Each participant or legal guardian underwent Ribeirao Preto, Brazil, were tested simultaneously for ansamples interview on filter using paper a standardized(DBS) were obtained questionnaire. for anti-HAV The viruses and common bacteria. A total of 481 samples EpiData ® version 3.1 software and the program “R were tested from 103 CRS patients (including 31 Archive Network ®” were used for univariate and patients with nasal polyps) and 34 patients without multivariate data analysis Analysis of 585 children and CRS who underwent rhinoplasty, as a control group. 377 adolescents children showed an overall prevalence Real-time PCR was used to test for a panel of common of anti-HAV of 20.6% and 94.1% of children under 5 respiratory viruses and bacteria. Of 137 patients 37 years were seronegative.Univariate analysis showed that (27%) were negative for all tested bacteria, including 15 (10.9%) who were also negative for any virus. Twenty- factors associated with seropositivity were:age, black or two patients were positive only for viruses, with higher the statistically significant correlation between the risk September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

179 Human Virology: HV frequencies of human metapneumovirus (HMPV) CD4 values for patients with positive and negative samples. Regarding the group of SC, the median age was (16.2%). One hundred patients had at least one sample 3.8 years, and the result showed a positivity of 25.7% positive(24.3%) for and bacteria, human and parainfluenza most of them virus had 3 only (HPIV3) CRS for HRSV. In the AC group the presence of the virus was (n=25) or CRS with nasal polyp (n=61). The viruses most detected in 7.7% of samples. Comparing the AC and SC frequently detected in association with bacteria were groups, we observed that 22.7% of the caregivers of human rhinovirus (HRV) (36%) and human bocavirus children infected with HRSV were also infected with the (HBoV) (23%). Staphylococcus aureus was detected in virus. The odds ratio showed that having a child infected 27 patients, and 11 of them (40.7%) were negative for with HRSV at home increases the chance of infection for viruses; Streptococcus pneumonia was detected in 30 the caregiver in 12 times [OR 12.1 (95% IC 3.1 – 46.6)]. patients, and 9 of them (30%) were negative for viruses; The HCW that have children infected with HRSV at home

16 of them (31.4%) were negative for viruses; Moraxella chain. The presence of children infected with HRSV at catarrhalisHaemophilus was influenza detected was in detected 16 patients in 51 (37.5%)patients, and homeseems canto play represent a significant an important role on the factor HRSV fortransmission the virus none of them had viruses detected. Finally, 58 patients acquisition, even for subjects without symptoms. These were positive for Pseudomonas aeruginosa, including 18 (31%) only positive for this bacteria. The present study and hospital visitors informed about the prophylactic has shown a high frequency of bacteria in patients with measuresfindings show against the the need virus, to maintainmainly in thethe medicalHRSV season staff CRS associated with respiratory viruses, mostly HRV and in order to prevent the onset of outbreaks coming from HBoV. the community.

HV388 - DETECTION OF RESPIRATORY SYNCYTIAL HV389 - EVALUATION OF THE ROLE AND IMPACT VIRUS FREQUENCY IN DISTINCT GROUPS OF OF ACUTE RESPIRATORY VIRAL INFECTIONS IN THE ASYMPTOMATIC PATIENTS CITY OF GUARAPUAVA PARANÁ Moreira, L.P.; Watanabe, A.S.A.; Camargo, C.N.; Campos, D.A.; Pedrosa, F.C.; Carraro, E. Granato, C.; Bellei, N. UNICENTRO - Universidade Estadual Centro Oeste, R. UNIFESP - Universidade Federal de São Paulo, R. Sena Padre Honorino João Muraro, 875 - Santa Cruz, Guarapuava Madureira, 1500 - Vila Mariana, São Paulo - SP, 04021-001 - PR, 85015-430 The human respiratory syncytial virus (HRSV) has a Acute respiratory infections are the most common disease great impact as a causative agent of acute respiratory infection in young children, immunocompromised as the etiology of more than 50 of the acute respiratory patients and the elderly. Individuals with asymptomatic infectionsin all individuals. around Influenza the world. A and The B have characteristic been reported of infection could play an important role in the virus transmission chain. In this study a Real-time PCR suffer, puts him in a prominent position among emerging technique was used to evaluate the frequency of HRSV influenza virus in frequent and unpredictable variants in 534 samples from distinct groups of patients who important for infection control measures and to optimize attended in Sao Paulo hospital during the period of 2009 treatment.diseases. The Objectives: laboratory To evaluate confirmation the role of influenzaand impact is to 2012 with high risk of infection acquisition. Of all the of acute respiratory infection caused by respiratory samples analyzed, 94 were from health care workers viruses, identifying and characterizing the respiratory (HCW), 100 from patients with immunosuppression due viruses that infect the people of the city of Guarapuava to HIV infection (HIV), 171 from symptomatic children Paraná frequenters of the Village Health Center Hospital (SC) and 169 from their asymptomatic caregivers (AC). Laboratory Carli and Santa Teresa with symptoms of In the HCW group, the median age was 33.5 years, and a respiratory infection, and so assess the clinical and HRSV frequency of 1% was found. In the HIV group, the epidemiological data Association of the disease with median age was 46 years and the frequency found was between 2013 and 2014 in 50 patients with symptoms of the virus identified. Methods: We conducted a study 4%,September/October with no statistically 2014 Volume significant 19 – Supplement difference 2 - Abstracts/Posters between - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

180 Human Virology: HV respiratory infection, associated with seasonal climatic phylogenetic tree was build using the MEGA software. variables such as temperature, precipitation and relative Among the samples, 65,67% were from Boa Vista , humidity. We use samples of nasal secretion and these 5,97% from Mucajaí, 4,48% from São João da Baliza and were tested by reverse transcriptase polymerase chain 23,88% from the other counties of Roraima. Thirteen reaction (RT-PCR), which was chosen as the method for samples also shown positives results in the sequencing. were analyzed and of these, two were positive for the Thesamples evolutionary were positive model toused fragment to build amplification, the tree was thisthe viral identification. Results: All 50 samples collected were characterized by the presence of mild symptoms with 1000 of bootstrep. Two phylogenetic trees were ofrespiratory upper respiratory virus Influenza tract, the A. most Respiratory common infectionsof which madeMaximum based Likehood in the found based genotypes on the Tamura-Nei(genotypes A (G+I) and were runny nose and cough. In these two years of study, D), both rooted in the genotype C, the oldest genotype. most cases of infection occurred in autumn and winter Our data presented various similarities in relation to the with more infections in the months of May to July, mas samples; we found two samples similar to genotypes os vírus respiratórios foram detectados ao longo de todo A1 and A2 from Caribe and one next to American strain. o período do estudo. Conclusions: Our results show that This can be explained by the geographic proximity of respiratory infections have certain impact on population, these countries with Roraima State. Inside to Brazil, we Since this region is rather cold, with mild temperatures found samples similar to Brazilian south, southeast and extreme being a factor susceptible to respiratory Amazonas state. This diversity of the same genotype infections, This suggests that more attention should be in Roraima can be related to 49% of its population paid to viral pathogens, increasing healthcare during the being made by immigrants, we can point this last data FINANCIAL SUPPORT: Capes as the main factor to this high diversity in the same genotype. Based on the analysis of the genotype D tree, fluHV390 season. - PHYLOGENY OF HEPATITIS B VIRUS (HBV) we obtained only one sample that shown similarity of CIRCULATION IN RORAIMA STATE 100% with a Colombian strain, and forming a clade with Granja, F.1; Júnior, W.P.L.1; Barros, J. de A.1; de Sousa, sequences from Colombia, Costa Rica and Amazonas. We D.D.1; de Souza, V.C.2; Acosta, P.O.A.1; Naveca, F.G.2 can suggest that, considering the geographic proximity 1. UFRR - Universidade Federal de Roraima, between Roraima and Colombia, the studded strain has Avenida Capitão Ene Garcez, 2413 - Aeroporto, Boa Vista - as origin in the neighboring country. The circulating RR, 69310-000 HBV’s strains of Roraima demonstrated many origins 2. ILMD/ FIOCRUZ pointing the state as an important area to study the viral The HBV belongs to family, dispersion. genus; it has a circular DNA partially HV391 - FREQUENCY OF SEROLOGICAL MARKER FOR double strand with approximately 3.2 kilobases that HEPATITIS C IN INMATE POPULATION OF A CITY OF encode four viral genes. The virus has a large genetic PERNAMBUCO diversity that divides it into 10 genotypes named from Cahu, G.G. de O.M.1; da Silva, D.M.1; de Morais, V.M.S.1; A to J and further divided into numerous subgenotypes. da Silva, D.M.2; Rabelo, D.C.C.2; de Lucena, W.A.T.2; This project objective was trace the phylogenetic origin de Lima, P.C. de S.2; de Albuquerque, A.C.C.2; Coêlho, of the HBV’s strains that circulate in Roraima State. M.R.C.D.1 The extracted samples were submitted to nested-PCR to amplify a segment from the S gene. The sequenced 1. UFPE - Universidade Federal de Pernambuco, fragments were edited and submitted to BLAST to identity Av. Prof. Morais Rego, 1235 - Cidade Universitária, Recife - PE, 50670-901 database containing sequences from many localities with 2. ASCES emphasisconfirmation in adjacent and genotype places to obtaining. Roraima. WeThe createddatabase a The inmate population is characterized by social and this study’s sequences were submitted to Jmodeltest exclusion, drug use and terrible conditions of software to choose the best evolutionary model and the

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersconfinement - Human Virology: resulting HV in a high prevalence of infectious XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

181 Human Virology: HV and contagious diseases. The hepatitis C virus (HCV) is HV409 - DETECTION OF RESPIRATORY VIRUSES AND a causative agents of these diseases and the parenteral BACTERIA IN SECRETIONS FROM THE MIDDLE EAR route is their primary route of transmission, moreover the AND THE RESPIRATORY TRACT FROM PATIENTS virus is to cause more common of liver transplantation. WITH CHRONIC SECRETORY OTITIS MEDIA AND The inmate population is considered at high risk for HCV TONSILLAR HYPERTROPHY infection, because prison conditions, marginalization, Saturno, T.H.1; Buzzato, G.P.1; Gagliardi, T.2; Modena, drug abuse and low socioeconomic status contribute J.L.P.2; Carenzi, L.R.1; Prates, M.1; Tamashiro, E.1; to the spread of HCV in prisoners. In Brazil, studies Valera, F.1; Lima, W.T.A.1; Arruda, E.2 conducted in different countries showed frequencies ranging from 6.3% to 41%, however, there are not 1. Departamento de Oftalmologia, Otorrinolaringologia e Cirurgia de Cabeça e Pescoço, reports of the frequency of HCV infection in the inmate Faculdade de Medicina de Ribeirão Preto populations in the State of Pernambuco. Objective: 2. Departamento de Biologia Celular e The aim the study was to estimate the frequency of Molecular, Faculdade de Medicina de Ribeirão Preto positive anti-HCV markers in male population of a prison of a city of Pernambuco, in the period May Chronic tonsillar hypertrophy (CTH) is a chronic to July 2011. Material and Methods: The datas from each individual were collected through an interview, is frequent in children worldwide, characterized by inflammation of palatine tonsils and adenoids that after 5 mL peripheral blood was collected in without the presence of recurrent infections and tonsillar anticoagulant tube. The samples were centrifuged at hypertrophy. CTH may be associated with nasal 1500 rpm for 10 minutes to separate serum, after, the obstruction, recurrent sinusitis, snoring, auditory samples were forwarded to the Department of Virology tube dysfunction, otitis media, obstructive sleep apnea of Laboratory Immunopathology Keizo Asami (LIKA) and altered facial growth. Some CTH patients develop of Universidade Federal de Pernambuco (UFPE) to secretory otitis media (SOM), with accumulation of effusion in the middle ear that frequently requires anti-HCV 4.0). Results: The serology was realized in surgery. SOM pathogenesis is not entirely clear, but 1085perform samples the anti-HCV and the byfrequency the commercial of the anti-HCV kit (MUREX was chronic tubal obstruction by hypertrophic adenoid and 1.66% (18/1085). The mean age of these was 36.11 the presence of viral infections may be contributing years (±12), 83.3% (15/18) were married, 55.56% factors. The high frequency of respiratory viruses and (10/18) had tattoos, 16.67% (3/18) used injection drugs, 44.44% (8/18) have used cocaine, 11.1% (2/18) adenoids prompted the present study. Samples from bacterial biofilm recently recognized in hypertrophic had sex with another man, 55.56% (10/18) did not palatine tonsils, adenoids, nasopharyngeal aspirates use condoms during intercourse, 27.78% (5/18) had and middle ear secretion from 37 patients with CHT a sexually transmitted disease and 16.67% (3/18) had and SOM were tested by real-time PCR (qPCR) for: received blood transfusions. Conclusion: The frequency human rhinovirus (HRV), human adenovirus (HAdV), of HCV infection was lower in this population than in human bocavirus (HBoV), human enterovirus (HEV), previous studies, this may be due to the low frequency human metapneumovirus (HMPV), human respiratory of risk factors, such as injectables drugs. However, data syncytial virus (HRSV), human coronaviruses (HCoV), on HCV among Brazilian prisoners is still scarce, and it draws attention to the need for epidemiological studies (HPIV), and for the bacteria Streptococcus pneumoniae, human influenza virus (Flu), human parainfluenza virus and policies to prevent transmission of infectious and Staphylococcus aureus, Pseudomonas aeruginosa, contagious diseases during incarceration.FINANCIAL SUPPORT: SCHOLARSHIP FROM CNPQ most frequently detected virus in middle ear secretion wasHaemophilus enterovirus influenzae (22%), but and the Moraxella other respiratory catarrhalis. viruses The were also detected. Of note, HRV species C was detected in 3 patients (8%). S. pneumoniae was the most frequently detected bacterium, in 35% of patients. Curiously, in only 11 (29%) patients there was agreement between September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

182 Human Virology: HV viruses and bacteria detected in adenoid and middle dsRNA of samples were extracted from fecal suspensions ear secretion, and in only 5 (13%) there was agreement and submitted to RT-PCR and seminested PCR, using between middle ear and nasopharyngeal secretions. The primers to G and P genotypes not usually detected. PCR data strongly indicate that SOM is not directly caused by any of the viruses or bacteria tested, but their presence is Results: For G, 56 samples were possible typed by subjectedproducts wereto either subsequently seminested-PCR purified andor sequencing sequenced. to the mechanical obstruction of the Eustachian tube bylikely hypertrophic due to stasis adenoids. of secretions Financial and biofilm support: consequent FAPESP, n=2), G3 (1.8%, n=1) and G12 (89.3%, n=50). P genotype CAPES and CNPq. wasreaction characterized we identified: in 27 G1 samples types by(5.4%, sequencing n=3), G2 reaction, (3.6%,

HV416 - GENOTYPIC CHARACTERIZATION OF (11.1%, n=3), P[8] (44.5%, n=12) and P[9] (14.8%, ROTAVIRUS STRAINS IN CHILDREN HOSPITALISED n=4).with the The following dual specificities:characterization P[4] (29.6%,was determined n=8), P[6] FOR ACUTE DIARRHEA IN BELÉM, PARÁ, BRAZIL IN for 67 samples (88.2%), among which the unusual POST-VACCINE INTRODUCTION PERIOD combinations G12P[6] (68.7%, n=46), G12P[9] (3%, Fecury, P.C.M.S.1,2; de Oliveira, A. do S.L.1; Soares, L. da S.1; Bezerra, D.A.M.1; Justino, M.C.A.1; Linhares, A. da C.1; Mascarenhas, J.D’A.P.1; Guerra, S. de F. dos S.1 notn=2) andpreviously G3P[9] (1.5%,typed n=1)underscores were identified. the Conclusion:importance The identification of unusual genotypes among samples 1. IEC - Instituto Evandro Chagas, Rodovia of continuous monitoring and characterization of BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 circulating RV strains, particularly in the current 2. FAMAZ - Faculdade Metropolitana da scenario of post-vaccine introduction in Brazil. Possible Amazônia, Avenida Visconde de Souza Franco, nº 72, bairro circulation of new or unusual genotypes may represent Reduto (Doca), Belém - PA, 78600-000 a challenge to current vaccination strategies. FINANCIAL Gastroenteritis is the second leading cause of death, being SUPPORT: FAPESPA, IEC rotavirus (RV) is the most common enteropathogen, HV420 - LOSS OF DISEASE CONTROL AFTER accounting for an estimated 453,000 deaths in children SUPERINFECTION OF A LONG TERM NON <5 years in 2008. The RV is devoid of envelope with PROGRESSOR HIV-1 POSITIVE INDIVIDUAL FROM genome composed of 11 segments of double-stranded RIO DE JANEIRO, BRAZIL RNA (dsRNA) that codes for 12 proteins. The VP4 and Caetano, D.G.1; Côrtes, F.H.1; Vorsatz, C.2; Grinsztejn, VP7 proteins make-up the outer layer shell of the RV B.2; Veloso, V.G.2; Bello, G.1; Morgado, M.G.1 The public health impact of RV disease, its genotypic 1. Laboratório de Aids e Imunologia Molecular diversityparticle and and define the need 37 Pfor and assessing 27 G genotypes, vaccine effectiveness respectively. IOC/FIOCRUZ - Instituto Oswaldo Cruz da Fundação are among current priorities. In addition, the monitoring Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, Rio de Janeiro - of circulating strains rotavirus vaccine introduction is RJ, 21040-360 strongly recommended, since new or emerging strains 2. Instituto de Pesquisa Clínica Evandro Chagas may pose a threat to vaccination strategies. Objective - Fiocruz, Av. Brasil, 4365, Rio de Janeiro, RJ, 21040-360 (s): Genotypic characterization of previously untyped Long Term Non Progressor (LTNP) patients are a rotavirus strains obtained from children hospitalized for diarrhea after countrywide introduction of rotavirus vaccine. Material and Methods: Stool specimens were infectionrare segment in the of absence the HIV-1+ of antiretroviral population therapy. that maintain Some obtained during a case-control study to assess vaccine high levels of T-CD4+ cells for more than 8 years after effectiveness in Belém, Pará, Brazil, from May 2008 to viral lineage with divergent effects in pathogenesis, May 2011. A total of 122 previously G and/or P untyped, characterizedstudies identified by maintenance infected LTNPs or withloss moreof progression than one were selected. Of these we could examine 76 in this control. Here we report the case of a LTNP diagnosed study, 44 of which only were not G-typed, 16 only were in year 2000 and that has been shown control of the not P-typed and 16 were not typed for G and P. The viral

September/October 2014 Volume 19 – Supplement 2 - Abstracts/PostersT-CD4+ - Human Virology: cell levels HV (median of 1093 cells/mm3; IQR XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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960-1215) and viral load (median of 216 copies/ml; Dengue is a febrile condition caused by an arbovirus IQR 150-345) along 12 years of follow-up. In mid-2012, of the genus Flavivirus, which is transmitted by Aedes the patient started to show an increase in viral load, aegypti mosquitoes and occurs predominately in tropical reaching about 10.000 copies/ml in early 2014. The genetic diversity of the viral quasispecies was studied similar antigenic characteristics. The virus genome is longitudinally in 3 moments (2009, 2011 and 2014) by acountries. positive-sense All four RNA identified with serotypes10,644 nucleotides (DENV 1-4) that show is translated into a polyprotein, which is subsequently using DNA extracted from cryopreserved PBMC samples cleaved into three structural – Capsid (C), pre- andthe amplificationlimiting dilution, of the to C2-C4assess envelopesingle sequences region (env),from Membrane (prM), and Envelope (E) – and seven non- the viral population. Phylogenetic analysis showed structural proteins. Two of the structural proteins, prM that, in 2009, 100% of the viral population (n=28 and E, are the primary antigens and therefore represent sequences) formed a single monophyletic group that clinically important targets for vaccine production. we denominated cluster A. However, the quasispecies There is no commercial vaccine available against obtained in 2011 were distributed in 2 monophyletic dengue virus, especially because antibodies against one groups with genetic distance of 16,8%. The previously serotypes into the cells and can lead to increased viremia. obtained (n=24 sequences) and a new cluster B with Thespecific Schneider serotype 2 (S2)can enhancecell line, thederived entry from of heterologous Drosophila 17%identified of remaining cluster sequences A grouped (n=5 83% identical of the sequences) sequences melanogaster embryos, is often used as a machinery to produce recombinant proteins, due to the ability to incorporate heterologous DNA into their genome. withwas identified. a change inThe their analysis relative of samplesprevalence: from the early cluster 2014 A In addition, a metal-induced promoter assures high containedconfirmed 9%the persistenceof obtained ofsequences the 2 viral (n= populations, 3 sequences) but protein expression without compromising the regular while cluster B had 91% of the remaining ones (n=30 cell cycle. In this study, we examined the expression of sequences). Together, the data obtained allow us to the recombinant complex prM-sE protein of dengue conclude that loss of progression control in this LTNP serotype 4, that can be used to improve diagnose and coincides with the detection of a new viral population, further vaccine studies. After induction with copper which could be explained by a reinfection event between sulfate, the protein was found mostly in the supernatant 2009 and 2011. The switch of the proportions of cluster A to B indicates a progressive substitution of the original column (that allows separation of HIS tagged proteins) virus by the new variant, which could be associated and it was purified with a pre-packed nickel affinity separation). To verify the expression and purity of this A. These results alert for the negative consequences of protein,and a gel Western filtration Blot Superdex (using anti-his200 column antibody) (size exclusionand SDS- reinfectionwith a better to fitnessdisease of pathogenesis, variant B compared even in to patients variant Page gels were performed. The results showed a high with a long history of control of immunity, viremia and yield protein expression and purity. In addition to this disease progression, reinforcing the need of continued use of adequate measures to prevent new infections. dengue virus, using a commercial ELISA assay (IgM FINANCIAL SUPPORT: CNPQ/FAPERJ andstudy, IgG) we and identified conventional samples PCR. of patientsIn further infected studies, with the positive samples will be analyzed against the prM-sE HV421 - EXPRESSION IN INSECT CELLS OF PROTEIN expressed in S2 cells and the results will be compared PRM AND E FROM DENGUE-4 VIRUS FOR USE IN with the commercial available assay. Financial Support: VACCINE AND DIAGNOSTICS FAPESP and CNPq. Abrão, E.P.; Moraes, F.M.; Esposito, D.L.A.; Fonseca, B.A.L. USP - Universidade de São Paulo, Av. Prof. Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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HV425 - GENETIC CHARACTERIZATION OF SEROTYPE found four amino acid substitutions, two of them (E428 1 DENGUE VIRUS ISOLATED IN RORAIMA, BRAZIL, e E436) which did not change the conformation of the FROM 2008 TO 2010 envelope (Leucine x Valine; Isoleucine x Valine), and Sousa, D.D.1; Granja, F.1; Silva, G.A.V.2; Naveca, F.G.2; the other two substitutions (E227 e E338) with high Acosta, P.O.A.1 probability of changing the envelope (Proline x Serine; Leucine x Serine). These results may indicate an adaptive 1. UFRR - Universidade Federal de Roraima, evolution of DENV-1 or the entry of a new lineage in Avenida Capitão Ene Garcez, 2413 - Aeroporto, Boa Vista - the state, emphasizing the importance of molecular RR, 69310-000 monitoring strains of DENV circulating in the region. 2. Centro de Pesquisas Leonidas e Maria Deane Financial support: CNPq - UFRR - FIOCRUZ - AM Roraima is highlighted as a dengue virus hyperendemic HV433 - ANALYSIS HCV AND HIV VIRAL LOADS, AND state, with the four serotypes circulating and with a high THE CD4+ AND CD8+ T LYMPHOCYTES COUNTING incidence of this disease. Futhermore, its geographic HIV/HCV CO-INFECTED AND HIV MONO-INFECTED localization has an important part on the entry of new PATIENTS BEFORE AND AFTER THE USE OF HIGHLY genotypes/serotypes of dengue in Brazil.DENV-1, after ACTIVE ANTIRETROVIRAL THERAPY (HAART) a brief incursion in the state in 1981, was reintroduced Klein, T.M.; Abrão, E.; Miquelitto, F.; Fonseca, B.A.L. in 2000 and it has been one of most isolated serotypes USP - Universidade de São Paulo, Av. Prof. Almeida until 2011. Nevertheless, there is little data that shows Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 the occurrence of genetic variability or any evolution in genetic composition of this serotype on infections Because HIV and HCV share the same route of occurred in different years. The objective of this project transmission, approximately 30% of the world’s was to perform a molecular characterization of the population infected with HIV is co-infected with HCV. With E gene from DENV-1 isolated on infections occurred between 2008 and 2010 in the state. The samples were in the surveillance of AIDS patients, and nowadays the the onset of the HAART, there was a significant increase inoculated in C6/36 lineage cells from Aedes albopictus, C Hepatitis has becoming one of the most important causes of death among these patients. The pathogenesis Hemi-Nested-PCR techniques. For the obtainment of of hepatitis C occurs through of the host immunological anand amplicom identified from by indirect the envelope immunofluorescence region, it was made and RT- an response to viral infection that is mediated mainly for with 1724 bp. The amplicons were sequenced and the CD4+ and CD8+ T lymphocytes. However, the HIV infection consensusRT-PCR test sequences with specific were primers, obtained generating using the a program product and consequently a deregulation of the immunological causes a decrease in the CD4+ T lymphocytes counting Geneious v.5.5.4. For the molecular analysis, the response. Some studies have demonstrating that the sequences were compared to reference sequences from HIV/HCV co-infection accelerate the AIDS progression, accelerate too the liver damage, the HCV viral load of DENV-1 from different parts of the world available on is higher when compared with HCV mono-infected GenBank.the four serotypes The phylogenetic of dengue virus,reconstruction and to five wasgenotypes made patients, and co-infected patients using HAART have a by the Maximum-likelihood method. All of the isolates were grouped in genotype V of DENV-1. The sequences why, this research aimed to analyze the viral loads of HIV compromised recovery of CD4+ T lymphocytes. That’s used in this study were grouped on a phylogenetic tree and HCV in 13 patients co-infected with HIV/HCV before forming a subclade with strains of DENV-1 from neighbor and after the use of HAART. The HIV viral load and the countries and states and Caribbean region, indicating Roraima as possible entry route of new strains of dengue also in the same way. We compared all the results with CD4+ and CD8+ T lymphocyte counting were analyzed in Brazil. The analyzed Brazilian sequences formed three the results obtained of 11 HIV mono-infected patients. distinct lineages. The isolates of this study were grouped Two serum samples of each patient (one before the use in lineage III, different from the strain that circulated in of antiretrovirals and another after the beginning of the state in 2001, which belonged to lineage II. It was September/October 2014 Volume 19 – Supplement 2 - Abstracts/PostersHAART) - Human Virology:were used. HV The HIV viral load and the CD4+ and XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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sub-subtype F1 by shotgun sequencing strategy in a medical records. There was no statistical difference NGS plataform. For this purpose, we analyzed 10 clinical betweenCD8+ T lymphocytes the HCV viral counting load before were tookand ofafter the HAARTpatient isolates of HIV-1 sub-subtype F1 collected in the State of in both HIV mono-infected and HIV/HCV co-infected Rio de Janeiro, Brazil in 2012. Each of these DNA samples groups. The HIV viral load was the same for both groups were subjected of an env gene nested PCR and sequencing before HAART, and after HAART, as expected these viral generating a fragment of 2,500 bp by ABI PRISM® 3100 loads decreased in both groups becoming undetectable Genetic Analyzer (Applied Biosystems), which was used for the most of the patients. It was observed a higher as reference. The same PCR env protocol was applied, before and after the treatment. Both groups increased NGSPREP) and further fragmented (NEBNext® dsDNA CD4+ T lymphocyte counting in the mono-infected group the products were purified by magnetic beads (MagSi- the recovery of these cells in the co-infected group was their CD4+ T lymphocytes counting after HAART, but andFragmentase). assessed for These quality products using Agilent were quantifiedTechnologies by for the both groups before and after HAART. Our results Bioanalyzerfluorescence-based before measurementslibrary preparation (Qubit, with Invitrogen) adapters suggestlower. The that CD8+ the HIVT lymphocytes therapy doesn’t counting have was any theeffect same on for NGS in GS Junior Roche 454 Life Sciences. We initially the HCV viral load. HAART is being effective in to inhibit software, evaluated quality at NGS QC Toolkit Software, in mono-infected patients and the absence of alteration subsequentlyeliminated PCR used amplification the QuRe bias Software by using forCD-HIT-454 capture HIV replication. A major CD4+ T lymphocytes recovery minority variants, based on coverage and diversity to the literature data. attempt to reconstruct all the individual sequences of the in the CD8+ T lymphocytes counting corroborate with viral quasispecies. In this study, over 60 Mb of sequence HV434 - DETERMINATION OF HAPLOTYPES OF THE data and > 150 haplotypes were generated. The number ENVELOPE OF HIV-1 IN INDIVIDUALS INFECTED of haplotypes varied with a median of 19 (IQR=8- WITH SUB-SUBTYPE F1 BY NEXT-GENERATION 50) per sample with a median of 60% (20%-80%) of SEQUENCING frequency for majority population of quasispecies. The de Almeida, D.V.; Junqueira, R.M.; Guimarães, M.L. IOC/FIOCRUZ - Instituto Oswaldo Cruz da Fundação definition of workflow and of parameters used in tools Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, Rio de Janeiro data will facilitate the analysis for better understanding - RJ, 21040-360 quasispeciesfor checking, diversity filtering and and evolution, mapping helping haplotypes in a vaccine of NGS conception. The high degree of HIV-1 genetic diversity and the structural complexity of its envelope glycoproteins HV438 - IGG ANTIBODIES OF HIGH AVIDITY TO were obstacles to the designing of an effective vaccine HANTAVIRUS IN HUMAN POPULATION FROM THE candidate, requiring the improvement of reagents STATE OF ALAGOAS able to induce a broad immune response. The HIV-1 Borges, A.A.; Santos, F.M.; Medeiros, N.P.T.; Santos Jr, diversity estimation is currently restricted to single- J.A. dos; Melo, D.M. de; Borges, A.A. nucleotide variants; to hence knowledge on quasispecies populations comprehensively required novel methods LAPEVI/ICBS/ UFAL - Laboratorio de Pesquisas em that allow complete reconstruction of the individual viral Virologia e Imunologia/Instituto de Ciências Biológicas e da haplotypes, which now is available by Next-Generation Saúde/Universidade Federal de Alagoas, Av. Lourival Melo Sequencing (NGS). HIV-1 subtype B is currently the most Mota, Ciudad Universitaria - AL, 57072-900 spread in the aids pandemic, as well as the subtype C is the Hantaviruses are zoonotic viruses associated with wild most prevalent, thus most of the generated information rodents, belonging to the family Bunyaviridae, that can concern this subtypes. In Brazil, sub-subtype F1 and cause human illnesses with a range of severities. Cases of human hantavirus infection are rarely reported in the Northeast region and the state of Alagoas is considered BF1 recombinants have a significant relevance. Thus, such silent area for hantavirus. However, our previous the aim of this study was to develop a workflow capable toSeptember/October characterize and2014 defineVolume 19 quasispecies – Supplement population2 - Abstracts/Posters of - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

186 Human Virology: HV results obtained with volunteers from the state of In immunocompromised patients, reactivation of BKV in kidney leads to infection in the urogenital tract, especially to hantavirus in 5,36% of them and suggested that in renal transplant patients. The BKV disease is quite someAlagoas hantavirus (n=1324) could showed be circulatingthe presence in thisof specific state. TheIgG variable, and this variability is determined by the quality IgG-avidity test is a very useful tool for differentiating and quantity of immune host factors. Despite having been between recent and past infections, besides to reinforce described for many years, BKV nephropathy had not been clinically observed until the middle of last decade. antigen in the ELISA test. Thus, our antibody avidity However, in recent years there has been an increased assaythe specificity was used of to the test binding the positive of the serum antibodies samples on thefor incidence of cases of nephropathy. This increase is anti-hantavirus IgG from participants of the serological attributed to improved diagnostic techniques and mainly survey performed in Coruripe, Palmeiras dos Índios and by the increasing capability of immunosuppression. OlhoD’água do Casado counties from Alagoas. Then, 79 Objectives: This work aims to use PCR and nested PCR positive sera samples collected during the survey were (N-PCR) to detect polyomavirus DNA in samples of used to detect by enzyme immunoassay (ELISA) IgG plasma and urine from renal pediatric transplantation antibodies against N protein of Araraquara hantavirus patients and with glomerulopathy to assist the treatment (rN ARAV) after a treatment with a caotropic agent such as of this infection. Patients and Methods: Were studied 81 urea. For this, in the ELISA test performed, after the stage biological samples, 66 patients with glomerulopaty and of serum incubation with the antigen rN ARAV (tested 15 renal transplantation, samples of plasma and urine in quadruplicate), two wells/sample were washed three of each patient was collected in different periods. DNA times with 6M urea, while others two wells/sample extraction was done using commercial kits (Axygen were washed just with buffer solution. At the end of test, PCR to detect viral DNA using primers that identify density (OD). The relative avidity index was calculated theScientific, two types Inc.),and of polyomavirus subsequently (BK was and made JC). aThe simple PCR bycolor dividing development the OD wasof the quantified urea-washed by reading wells theby opticalthat of is considered the standard method to perform the the wells washed with buffer solution and expressed as results, only 1/81 (1.2%) samples was positive for BK high avidity and that with index between 30 and 60% virus.detection Conclusion: and identification viremia and of disease viruses. can Results: be caused As by a werea percent. considered Samples intermediatewith an index ≥60%avidity. were From considered the 79 BKV. In the genital tract can produce failure renal and samples tested, 77 (97,47%) was considered of high rejection to trasplanted organ. The rapid detection is avidity with mean index of 93,18%. Only 2 samples very important to early treatment and the monitoring of (2,53%) were considered of intermediate avidity. In these patients in relation to poliomavirus infections.

HV440 - MOLECULAR DETECTION OF DENGUE VIRUS areconclusion, present with in serathe use from of IgG-avidity individuals test that we hadconfirmed never IN SERA SAMPLES FROM PATIENTS WITH FEBRILE livedthat IgG or travelledantibodies out specific of state to of hantavirus Alagoas, demonstratingof high avidity ACUTE ILLNESS IN MACEIÓ, STATE OF ALAGOAS, evidence for the presence of hantavirus in this region. BRAZIL Financial support: CNPq, FAPEAL, CAPES. Borges, A.A.1; Silva, J. de M.1; Lira, E.L.1; Muller, V.D.M.1; Melo, D.M. de1; Botelho, J.A.1; da Silva, A.A.S.1; Aquino, HV439 - SURVEILLANCE OF ACTIVE POLIOMAVIRUS V.H.2; Borges, A.A. INFECTION IN GLOMERULOPATY AND PEDIATRIC 1. LAPEVI/ICBS/ UFAL - Laboratorio de RENAL TRANSPLANTATION PATIENTS Pesquisas em Virologia e Imunologia/Instituto de Ciências Menoni, S.; Bonon, S.; Costa, S.; Torres, S. Biológicas e da Saúde/Universidade Federal de Alagoas, Av. UNICAMP - Universidade Estadual de Campinas, Lourival Melo Mota, Ciudad Universitaria - AL, 57072-900 Cidade Universitária Zeferino Vaz - Barão Geraldo, Campinas 2. FCFRP/USP - Faculdade de Ciências - SP, 13083-970 Farmacêuticas de Ribeirão Preto/Universidade de São Paulo, Avenida do Café, s/nº Ribeirão Preto - SP, 14040-903

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Dengue virus (DENV) infection is the most important 1. Oswaldo Cruz Institute arthropod-borne viral disease in the world. DENV are 2. Laboratory of Clinical Immunology, Federal positive single-stranded RNA viruses that belong to University of Mato Grosso do Sul the family Flaviviridae, genus Flavivirus. There are 3. Laboratory of Clinical Immunology, Federal four serologically distinct dengue virus serotypes University of Mato Grosso do Sul,Oswaldo Cruz Foundation – (DENV-1, DENV-2, DENV-3, DENV-4). Dengue virus Mato Grosso Do Sul infection causes a spectrum of illness ranging from Since the beginning of the AIDS epidemic in Brazil, asymptomatic or mild febrile illness to classic dengue fever (DF) and to severe and fatal hemorrhagic disease. prevalence of HIV-1 subtype B followed by sub-subtype F1molecular and BF epidemiologyrecombinants studiesin almost have the identified entire country. a high dengue can be accomplished through the detection of The exception is the South region where a co-circulation theSpecific presence laboratory of antibodies, tests performed virus isolation,in the diagnosis detection of of the virus genome by reverse transcription followed prevalence of these subtypes varied according to the by polymerase chain reaction (RT-PCR), NS1 antigen geographicof subtype C,region, B and genomic BC recombinants region and is studied verified. group The detection, and immunohistochemistry. In the State of involved in the study. In order to add a new piece in Alagoas, the diagnosis is performed only by serology and this puzzle we aimed to investigate the genetic diversity virus isolation. Thus, the aim of this study was to perform and the transmitted resistance mutations in individuals the molecular detection of DENV in sera samples from from Mato Grosso do Sul, Central Brazil. For this purpose individuals with febrile acute illness and clinical suspect 131 HIV-1 ART-naïve positive individuals were enrolled of dengue admitted to hospitals from Maceió/AL, whose since November 2009 to July 2011. DNA were extracted, illness has comprised between 0-7 days following the onset of symptoms. Sera from 55 patients were collected were edited in DNASTAR software and then aligned with and were tested both by one-step real time RT-PCR and referenceamplified sequences in PR/RT regionusing Clustal and sequenced. W. Neighbor Sequences Joining by RT-PCR conventional, for detection of the 5’UTR phylogenetic inferences with Tamura-Nei substitution region and for detection of parts of the C, prM and NS5 model and recombination analyses applying Bootscan genes at the viral genome, respectively. Of the total were performed in Mega 6 and Simplot programs, samples, in 15 (27.27%) was detected DENV genome in respectively. In order to investigate transmitted drug at least one of the used tests. The viral serotype could mutations sequences were submitted to the Stanford be determined in 9 samples (60%), of which 8 (88.9%) HIV Database for Transmitted DRM (TDRM/CPR Tool) presented DENV-4 and 1 presented (11.1%) DENV-3. The Code Version 6.0. Based on these analyses, 68.7% was overall positivity was considered low for a region were dengue epidemics occur annually. This data suggests that as C and 1.5% as D. All BF1 recombinant samples other members from genera Flavivirus may be causing classified as subtype B, 11.5% as F1, 6.9% BF1, 11.5% infections in Maceio, in spite of the patients are receiving high percentage of non-B subtypes (31.3%) has been a clinical diagnosis of dengue. However, more studies are previouslywere classified described as uniquein this geographic recombinant region. forms. We Thisalso highlight a temporal trend of increasing of subtypes C innecessary progress to in confirm these thissame supposition. samples. Financial For this purpose,support: Moderate transmitted drug resistance mutations were Ministériodetection tests da Saúde/CNPq/SESAU-AL/ for the genomes of other FAPEAL, flaviviruses CAPES are and sub-subtype F1 even not statistically significant. mutations, 3.9% of NNRTI mutations and 2.3% of PI. HV442 - HIV-1 MOLECULAR EPIDEMIOLOGY AND Thisverified found in is 11.5% in agreement of our samples; with previous being studies 7.8% of in NRTIART- PREVALENCE OF TRANSMITTED DRUG RESISTANCE naïve Brazilian samples. Taken together the data herein MUTATIONS AMONG INDIVIDUALS FROM MATO generated add new pieces in the Brazilian molecular GROSSO DO SUL STATE,BRAZIL epidemiology of HIV-1 and emphasizes the importance 1 2 2 3 Leite, T. ; Tanaka, T. ; Zacalusni, S. ; Castro, A.R. ; of monitoring drug resistance patterns among ARV- 1 Guimarães, M. naïve individuals in this region of Brazil.

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HV449 - EPIDEMIOLOGICAL AND SPATIAL ANALYSIS is greater than the considered acceptable <0.99%. We OF DENGUE CASES OCCURRING IN THE MUNICIPALITY observed that the rainfall and temperature rates were OF TRES LAGOAS- MS IN THE TRIENNIUM 2012-2014 similar in the 3 years studied and is directly related to Machado, A.M.1; Gotardi, A.H.B.1; Silva, M.H.R.1; the increased of positives cases. Also observed that the Machado, A.R.S.R.2; Oda, J.M.M.1; Mirandola, P.H.1; IAI (Dengue positive) remained high throughout the Guerra, O.G.1; Machado, A.M.1 triennium. Thereat we hypothesized that the decrease in the number of positive cases, in the year 2014, is related 1. UFMS - Universidade Federal de Mato to circulation of the same serotype during all this period. Grosso do Sul, Cidade Universitaria - Universitaria, MS, Therefore, we conclude that the entry of a new serotype 79090-900 of dengue might lead to a major outbreak, because the 2. FMRP/USP - Faculdade de Medicina de city has favorable characteristics of the circulate and Ribeirão Preto/ Universidade de São Paulo, Av. Bandeirantes, 3900 - Monte Alegre, Ribeirão Preto - SP, 14049-900 maintenance of the vector and the dengue. Dengue is the most important arboviral disease HV458 - OCCURRENCE OF HUMAN RHINOVIRUS found in Brazil and responsible for thousands of cases SPECIES AMONG DIFFERENT POPULATIONS DURING annually. Três Lagoas - MS in recent years, the dengue EIGHT YEARS has become a serious public health problem. We aimed Watanabe, A.S.A.; Moreira, L.; Silva, E.; Camargo, C.; to analyze retrospectively and spasso temporally all Kamikawa, J.; Granato, C.; Bellei, N. the cases of Dengue in Três Lagoas, in the triennium UNIFESP - Universidade Federal de São Paulo, R. Sena 2012-2014 relating them to different socioeconomic Madureira, 1500 - Vila Mariana, São Paulo - SP, 04021-001 and infrastructural areas. For this, the data of all dengue cases, occurred in the period were obtained from Human rhinoviruses (HRV) are the major etiologic Epidemiological Surveillance and the indices of Aedes agent of the common cold. HRV infections are common aegypti infestation (IAI) obtained by the Vector Control among persons of all ages and account for 25–50% of Department. The data were geocoded using SPRING 5.1.5 respiratory illnesses among individuals presenting ® system. Simultaneously the temperature and rainfall data, for the period, were obtained and correlated to techniques improved the molecular epidemiology of influenza-like illness (ILI). Advances in molecular dengue cases. In 2012 we observed 1050 positive cases, HRV, resulting in description of new HRV C specie in with 404 men and 646 women and mean age 20-34 2006. The aim of the present study was to describe years (32.66%), with highest number of cases occurred the molecular epidemiology of the HRV species among between the months of January to April (84.1%). In 2013 different populations during 8 years. Nine distinct was observed an increase in numbers of cases, with 3855 populations were analyzed, including children, adults, positive cases (1714 men and 2141 women) and mean elderly, hospitalized patients, and immunossupressed age 20-34 years (30.6%) with the highest number of cases patients. Severity parameters (presence of ILI, wheezing, occurred between the months of March to May (85.5%). ICU hospitalization, and mortality) were evaluated. Two In the year 2014, until the present time, we observed a distinct diagnostic tests were used for HRV detection: marked decrease in the number of cases with only 48 conventional PCR and real time PCR. Amplicons were positive cases (20 men and 28 women) which, differently from the previous years, occurred mainly in the months method. Multiple sequences were aligned using Clustal purified and sequenced with Sanger´s sequencing of May and June (87.5%). Most cases of Dengue fever (75%) occurred in the western and southwestern region subjected to phylogenetic analyses using TOPALi (v2.5) X (v2.0.11). The multiple-sequence alignment was of the city, especially in neighborhoods considered of software. HRV was detected in 25.3% (304/1202) middle class, where are found countless vacant lots and of studied samples. Species distribution was: 51.7% numerous deposits of vector. The IAI (Dengue positive) (134/259) HRV A, 14.3% (37/259) HRV B and 34.0% in the city ranged from 1.58% to 2.73%, however in (88/259) HRV C. Among all studied populations the neighborhoods with higher incidence of dengue positive species distributions follow this pattern (Table), except cases this rates ranged between 2.35% to 14.6%, which for children, with higher frequency of HRV C, followed

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

189 Human Virology: HV by HRV A and HRV B, and renal transplant outpatients was detected using degenerate primers MY09/MY11 (HRVA>HRV B>HRV C). Severity parameters were statistically similar among HRV species. Frequency of codetection with other respiratory viruses using direct toand genotypes general primers 6, 16 GP5+/GP6and 18. RESULTS: +. Viral genotyping Smoking wasand alcoholperformed consumption using multiplex was reported PCR with by 85.13% specific (74/106) primers of patients and 56.6% (60/106) used other drugs. virus,fluorescence human adenovirus assay and/or and conventional respiratory syncytial PCR resulted virus Positive results were found in 83.02% (88/106). An werein 42/1353 frequently (3.10%) found of HRVin codetection. positive samples. The molecular Influenza HPV prevalence rate of 90.24% was detected in young distribution of HRV species were similar with the patients aged between 11 and 30 years. Among patients described by others authors, with higher frequency taking multiple drugs, 86.67% (52/60) were HPV of HRV A, although our study excluded the previous positive and 23.08% (12/52) samples with multiple reported association between HRV C and severe infection, including wheezing children. prevalence was 78.26% (36/46) and two HPV types type infections were identified. Among “non users” HPV HV467 - PREVALENCE OF HUMAN PAPILLOMAVIRUS The prevalence of HPV found in this study differs (HPV) IN HEALTHY ORAL MUCOSA OF USERS AND markedlywere identified from studies in 13.89% conducted of samples. around CONCLUSIONS: the world, in NON-USERS OF DRUGS IN A STATE IN NORTHEASTERN which a lower HPV prevalence has been reported. Few BRAZIL data are available on the oral HPV infection frequency Ribeiro, M.G.M.1; Marcolino, L.D.2; Miranda, E.A.3; for the Brazilian population and multiple drug users, Jain, S.A.1; Santos, I.G. de A.1; Trento, C.L.2; da Silva, notably the crackers, exhibit risky sexual behavior that M.G.2; Dolabella, S.S.1 increases the exposure to sexually transmitted diseases. 1. LEPAT/UFS - Laboratório de Entomologia e Additional studies about HPV prevalence between drug Parasitologia Tropical da Universidade Federal de Sergipe, Av. users are needed for better knowledge of their exposure Marechal Rondon, s/n Jardim Rosa Elze, São Cristóvão - SE, to the virus and development of prevention strategies. 49100-000 FINANCIAL SUPPORT: Fundação de Apoio a Pesquisa e à 2. FMB/UNESP - Faculdade de Medicina de Inovação Tecnológica do Estado de Sergipe (FAPITEC) e Botucatu da Universidade Estadual de São Paulo, Av. Prof. PROMOB CAPES/FAPITEC. Montenegro Bairro: Distrito de Rubião Junior, s/n, Botucatu - SP, 18618970 HV468 - LACK OF INTRA-HOST VIRAL EVOLUTION 3. DOL/UFS IN HIV CONTROLLERS: A 10-13 YEARS FOLLOW-UP STUDY Infection with Human Papillomavirus (HPV) is the 1 1 2,3 sexually transmitted viral disease most prevalent in Caetano, D.G. ; Côrtes, F.H. ; Vorsatz, C. ; Grinsztejn, 2,3 2,3 1 1 world. The infections can range from asymptomatic B. ; Veloso, V.G. ; Bello, G. ; Morgado, M.G. establishment to induction of squamous cell carcinomas. 1. Laboratório de Aids e Imunologia Molecular The aim of this study was to evaluate and compare the IOC/FIOCRUZ - Instituto Oswaldo Cruz da Fundação prevalence of HPV and its genotypes in healthy oral Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, Rio de Janeiro - mucosa of users and non-users of drugs in a state in RJ, 21040-360 Northeastern Brazil by PCR technique. METHODS: One 2. IEC - Instituto Evandro Chagas, Rodovia hundred and six patients with healthy oral mucosa, BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 between 11 and 79 years visiting the Dentistry Clinic 3. FIOCRUZ - Fundação Oswaldo Cruz, Av. of the Universidade Federal de Sergipe and alcohol and Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 drug users frequenting the rehabilitation institutions HIV-1 elite controllers (EC) are a very rare fraction located in the state of Sergipe were evaluated. The samples were collected by exfoliation of oral mucosa undetectable levels of viral RNA in plasma (<50 copies/ using sterile cytology brushes. For quality control of ml)(<1%) for ofseveral HIV-1+ years infected in the absence patients of that antiretroviral maintain DNA extraction beta-globin PCR was used. DNA-HPV therapy. Analysis of the HIV-1 genomes integrated in

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

190 Human Virology: HV host cells revealed that most EC patients shown HIV- HV469 - HIGH PREVALENCE OF HUMAN 1 quasispecies with low diversity and extremely low PAPILLOMAVIRUS (HPV) IN ORAL LESIONS IN THE divergence rate, compatible with a true evolutionary STATE OF SERGIPE, BRAZIL latency. Those longitudinal studies, however, analyzed Ribeiro, M.G.M.1; Marcolino, L.D.3; Miranda, E.A.2; the viral evolution over relatively short time periods Jain, S.A.1; Santos, I.G. de A.1; Piva, M.R.2; Trento, C.L.2; da Silva, M.G.3; Dolabella, S.S.1 of the HIV-1 quasispecies in three EC patients, two of them(≤5 years). with occasionalHere we described viral load the blips long-term (ECB42, evolution ECB46) 1. LEPAT/UFS - Laboratório de Entomologia e Parasitologia Tropical da Universidade Federal de Sergipe, Av. and one with undetectable viral load along the follow- Marechal Rondon, s/n Jardim Rosa Elze, São Cristóvão - SE, up (EC52). The genetic diversity of the viral quasispecies 49100-000 was studied longitudinally over an interval between 10 2. DOL/UFS - Departamento de Odontologia da Universidade Federal de Sergipe, Av. Marechal Rondon, s/n C2-C4 envelope region (env), using DNA extracted from Jardim Rosa Elze, São Cristóvão - SE, 49100-000 and 15 years (2000-2013) by the amplification of the cryopreserved PBMC samples and limiting dilution. 3. FMB/UNESP - Faculdade de Medicina de Phylogenetic analysis of viral quasispecies revealed the Botucatu da Universidade Estadual de São Paulo, Av. Prof. existence of some long branches that were associated to Montenegro Bairro: Distrito de Rubião Junior, s/n, Botucatu - hypermutation probably due to the action of the cellular SP, 18618970 enzyme APOBEC3G. Hypermutated sequences represent Among the infectious agents, HPV infection in oral mucosa 16% of sequences of patient ECB46, 9% of sequences of is often associated with the onset and/or development patient ECB42 and 0% of sequences of patient EC52. All of malignancy. Detection rates of HPV in oral cavity hypermutated sequences were then removed from the reported in the literature varies markedly around the subsequent genetic distance estimations. Analysis of world and make the relationship between HPV and oral viral quasispecies of patient ECB42 at years 2000/2001 carcinogenesis still controversial. The aim of this study (n = 15), 2004 (n = 11) and 2011 (n = 9) revealed a was to evaluate the prevalence of HPV infection and its stable diversity between 1 and 2% and an increase in genotypes in patients with oral lesions in the state of the viral divergence from 0.5% to 1.7%. Analysis of viral Sergipe, Brazil by PCR technique.METHODS: Analyses quasispecies of patient ECB46 at years 2000 (n = 18), included 39 patients frequenting the Dental Clinic of the 2004 (n = 20) and 2011 (n = 13) revealed a decreasing Universidade Federal de Sergipe. aged between 2 to 83 trend of both diversity (from 1.8% to 0.8%) and years, with clinically detectable lesions in oral mucosa divergence (from 1.5% to 0.5%) over time. Analysis of The samples were collected by exfoliation of oral mucosa viral quasispecies of patient EC52 at years 2000/2001 (n using sterile cytology brushes. For quality control of = 20), 2004/2005 (n = 19) and 2013 (n = 16), revealed, DNA extraction beta-globin PCR was used. DNA-HPV for this patient, an extremely low level of viral diversity was detected using degenerate primers MY09/MY11 (0.1%) and no increase in viral divergence over time. This study revealed that HIV-1 variants integrated into cellular genomes of EC patients displayed undetectable genotypesand general 6, primers 16 and GP518. RESULTS:+/GP6 +. ViralOur studygenotyping evaluated was or extremely low turnover over long time intervals and pre-malignantperformed using (dysplasias), multiplex PCR malignant with specific (Oral primers Squamous for that most variation seems to be due to hypermutation Cell Carcinomas-OSCC), and benign lesions (hyperplasia, papillomas and other lesions), having found the virus presenting occasional blips. These results also support related to cellular restriction factors, as verified for those in all groups. HPV prevalence was 76.92% (30/39). a very low rate of decay of the HIV-1 reservoirs in EC. The most common virus type HPV-6 was present in FINANCIAL SUPPORT: CNPQ/SAPERJ 56.67%(17/30) positive samples, followed by HPV- 18 in 26.67%(8/30) and HPV-16 in 6.67 %(2/30). Multiple infection was detected in 26.67% (8/30) of samples. In 40% (12/30) samples the virus type was

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersnot - Human identified. Virology: The HV relative prevalence of HPV according XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

191 Human Virology: HV to samples histopathology were 66.67% in papilloma many animal species, including the wild rodents, in the cases; 83.34% (5/6) in OSCC; 100% in hyperplasia cases search for food and the conditions associated with the (1) and 72.23% (13/18) in samples with other types production system, possible the largest animal / human of lesions. All samples with dysplasia were positive. contact. The objective of this study was to analyze the Patients who were also associated with papilloma spatial distribution of positive cases of hantavirus in or hyperplasia showed a HPV prevalence of 50%. In the state of Mato Grosso between the years 2008-2012. patients with other associated lesions the rate was Data were taken from DATASUL tabulated in Excel 2007, 100%.CONCLUSIONS: A high prevalence of HPV in oral analyzed and identifying risk areas using the program lesions was found in our study. This differs from other MapInfo Professional 7.0 software. 20 municipalities in studies conducted in Brazil where HPV rates observed ranging from 0% to approximately 30% in oral lesions. the largest number of infected in the municipalities of Because HPV was found in all lesions groups evaluated, Sinopthe state 14.81% with 108 (16), confirmed Tangara cases da Serra for hantavirus, 13.89% (15),with additional studies concerning HPV distribution in oral lesions in this country are needed, to assess the role of Feliz Natal 8.33% (09) and Peixoto de Azevedo 8.33% human papillomavirus in the development of lesions (09).Campo The Novo infected Parecis were 11.11%mainly males were 70.37% identified (76) (12), and in oral mucosa. FINANCIAL SUPPORT: Fundação de residents of rural areas with 57.40% (62). The cases Apoio a Pesquisa e à Inovação Tecnológica do Estado de occurred in Mato Grosso focused on the Middle North, Sergipe(FAPITEC) e PROMOB CAPES/FAPITEC. predominantly agricultural region and grain producer, who suffered change your cerrado biome to large tracts HV472 - SPATIAL DISTRIBUTION OF POSITIVE CASES of monoculture plantations was observed that the high OF HANTAVIRUS IN THE STATE OF MATO GROSSO frequency region of the affected people were males BETWEEN THE YEARS 2008-2012 and residents of rural areas, which can be explained Godoy, H.P.1; Cruz, C.A.1; Santos, R.F.1; Siqueira, A.B.1; by the fact that they are the predominant sex as rural Pimenta, S.T.S.2; Buzinaro, M.G.1 workers, increase the risk of contact between humans 1. UNESP - Universidade Estadual Paulista, and infected rodents. Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010 HV475 - ADENOVIRUSES PREVALENCY IN CHILDREN 2. UFMT - Universidade Federal de Mato WITH ACUTE GASTRENTERITIS BEFORE AND AFTER Grosso, R. Guadalajara, 1 - Jardim das Américas Cuiabá - MT 78060-624 INTRODUCTION ROTAVIRUSES VACCINATION IN BRAZIL Hantavirus disease is an emerging disease transmitted Muller, E.C.A.; Oliveira, A. do S.; da Silva, L.S.; Guerra, by the inhalation of excreta of wild rodents and S. de F.; Oliveira, D.B.; Mascarenhas, J.D’A.P.; Gabbay, mostly occurs in individuals who have contact with Y.B.; Linhares, A. da C. the countryside with these rodents. This is a serious disease, with a mortality rate above 50%, which can lead IEC - Instituto Evandro Chagas, Rodovia BR-316 km 7 to respiratory failure and shock. Hantavirus belongs to s/n, Levilândia, Ananindeua - Pará, 67030-000 the family Bunyaviridae and is enveloped viruses that Gastroenteritis is one of the major causes of childhood measure approximately 120 nm, have single-stranded illness worldwide. Adenoviruses may play a role in RNA and negative polarity. In Brazil, cases were reported the etiology of gastroenteritis. Adenovirus contains a in 14 states noting that most of these cases were double-stranded DNA and is non-enveloped. It belongs detected in the south and the states of Minas Gerais, to the Adenovidae family, genus . They São Paulo, Mato Grosso and the Federal District. In are divided into six species (A-F) and 51 serotypes. Epidemiological studies have detected adenovirus in 2 to the State Department of Health The state of Mato to 22% of cases of acute diarrhea in pediatric hospitals Mato Grosso, the first cases occurred in 1999, according Grosso is composed of 141 municipalities, is the largest and emergency departments. The main purpose of this grain producer in Brazil, and the pursuit of productive land, resulted in the destruction the natural habitat of of adenoviruses in stool samples from 1,160 children study was to detect the presence and define the types September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

192 Human Virology: HV under three years of age including hospitalized patients and outpatients during the rotavirus pre-vaccination genetic reverse system and grown in embryonated period (March to September 2003) and post-vaccination chickenRecombinant eggs. influenzaThe viruses vaccines are generated are produced by cell using co- period (May 2008 to April 2009). This was a surveillance transfection with cloned cDNA of hemagglutinin (HA) carried out by Institute Evandro Chagas these children and neuraminidase segments from wild-type strain were hospitalized with gastroenteritis in Belém, Pará, Brazil. Our study placed emphasis on 40 and 41 strain. However, theses clones are obtained by use of adenovirus (AdE) serotypes (species F). We used EIA plus six segments from influenza A/PR/8/34 (PR8) and immunochromatography for screening, and PCR homologousspecific restriction recombination sites and in in yeast vitro ligation,(HRY) cloning which and typing. The adenoviruses (Ad) were found in 7.24% sometimes are difficult to perform. In advantage, the (84/1160)and nucleotide of samples. sequencing AdE was for presentmolecular in 5% identification (58/1160) fragments containing homologous ends with the vector of samples, corresponding to 69% (58/84) of positive cantechnique be directly is an clonedefficient using and simplein vivo process,recombination. where DNA The cases. Overall, 3.3% (25/760) were found during the pre- vaccination period and 8.2% (33/400) during the post- the egg supply in pandemic situation, e.g. as occurred in vaccination period study. Our data suggest an increase 2009currently (pnd2009). influenza Additionally, vaccine also isthe limited WHO byrecommends capacity of in the prevalence of AdE after introduction of the Vero cells as an alternative substrate for recombinant vaccine Rotarix®. As based on nucleotide sequencing, species F was characterized as the more prevalent in grow sub optimally in this cell line. Recently, were our region, accounting for 70% (21/30) of isolates, with reportedinfluenza the vaccine enhanced production, growth of however H1N1 in some Vero virusescell by type 41 accounting in 85.7% (18/21) of positive cases. changing an amino acid residue in HA (117N>D). Thus, epidemiology when comparing samples from the pre andThere post-vaccination were no significant period. variations Ad predominated in the molecular in the to overcome the difficulties of producing recombinant age group of 18-24 months during the pre-vaccination pandemic influenza vaccine, the objective this work ofwas the the HA/117N>D development mutation of the (by first HRY) reverse in a chimeric genetics could be found during the post-vaccination period. virussystem expressing of influenza HA madefrom byisolated HRY andpandemic the insertion (2009) period; no clustering of Ad into a specific age group capable of improved its replication in Vero cells. For relation to gender and the temporal distribuition. Our this, the pJG-HW2000-2013 vector was constructed resultsFurthermore, provide no strong significant evidence differences of adenovirus could bebelonging seen in to species F being a cause of severe gastroenteritis among children under 3 years of age. byand HRY the mutationfrom pJG-HW2000 effect was firstto pJG-HW2000-2013. evaluated in prototype The pJG-HW2000-2013/PR8influenza PR8 strain. The was PR8 transfected genome wasin HEK subcloned 293T/ 476 - INFLUENZA: DEVELOPMENT OF A GENETIC MDCK co-culture and recombinant PR8 (IC-PR8) was REVERSE SYSTEM BY YEAST-BASED HOMOLOGOUS generated. After, the HA/117N>D mutation was inserted RECOMBINATION AND ITS APPLICATION IN THE by HRY into pJG-HW2000-2013/PR8 and mutant virus IMPROVED VACCINE PRODUCTION IN CULTURE CELL (IC-PR8/HA-117N>D) was generated. Plaque assay Silva Jr, J.V.J.1; Cruz, F. da S.P.1; Bertani, G.R.1; Machado, performed in Vero cell showed replication larger in IC- A. de M.V.2; Gil, L.H.V.G.1 PR8/HA-117N>D than in IC-PR8. After, the HA segment 1. CPQAM/FIOCRUZ - Centro de Pesquisas Aggeu Magalhães da Fundação Oswaldo Cruz, Av. Professor HW2000-2013 and the clone was co-transfected with Moraes Rego, s/n - Campus da UFPE - Cidade Universitária, pJG-HW2000-2013/PR8of 2009 influenza isolate andwas clonedchimeric by virusHRY intoIC-PR8/ pJG- Recife - PE, 50.740-465 HApnd2009 was generated. The mutation HA/117N>D 2. CPqRR/FIOCRUZ MINAS - Centro de was inserted by HRY in IC-PR8/HApnd2009 and mutant Pesquisas René Rachou/ Fundação Oswaldo Cruz, Av. Augusto virus (IC-PR8/HApnd2009-117N>D) was recovered. de Lima, 1715 - Barro Preto, Belo Horizonte - MG, 30190-002 Comparison of the plaque assay and viral growth

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

193 Human Virology: HV kinetics between IC-PR8/HApnd2009-117N>D and IC- (1.0±1.1), more frequently inside the stroma (2.3±2.3) PR8/HApnd2009 in Vero cell is in progress. FINANCIAL and in perivascular (1.5±1.2) areas. The migration of LC SUPPORT: CAPES, CNPq, Fiocruz into the epithelium presenting HPV lesions is important

HV478 - RARE PRESENCE OF LANGERHANS CELLS recruiting immune cells to eliminate the HPV-infected WITHIN INTRAEPITHELIAL CERVICAL LESIONS keratinocytes,to induce a pro-inflammatoryinitiating an effective microenvironment acquired immune by INDUCED BY HPV INFECTION response. Moreover, studies on LC function in cervical Carvalho, M.O. de O.1; Simões, R.S. de Q.2; Rodrigues, lesions and cancer are necessary for understanding the F.C. das C.1; Portari, E.A.3; Barth, O.M.S.2; Russomano, mechanisms involved in HPV clearance, the immune F.B.3; Fernandes, A.T.G.1; de Almeida, M. da G.B.1 evasion favoring the cancer progression and to further 1. INI - Instituto Nacional de Infectologia discovery of new drugs and chemotherapies. Evandro Chagas, Av. Brasil, 4365 - Manguinhos, Rio de HV484 - HERPESVIRUS AND PAPILLOMAVIRUS DNA Janeiro - RJ, 21040-360 DETECTION IN ADULTS AND ELDERLY PATIENTS 2. IOC - Instituto Oswaldo Cruz, Av. Brasil, 4365 - Manguinhos, Rio de Janeiro -RJ, 21040-900 WITH HEAD AND NECK CANCER 3. IFF - Instituto Nacional de Saúde da Mulher, Bonon, S.H.A.1; Torres, S.V.S.1; Sbegue, A.2; Menoni, da Criança e do Adolescente Fernandes Figueira, Av. Rui S.M.F.1; Costa, S.C.B.1 Barbosa, 716 - Flamengo - Rio de Janeiro - RJ, 22250-020 1. UNICAMP - Universidade Estadual de Cervical cancer (CC) was the 4th cause of cancer death Campinas, Cidade Universitária Zeferino Vaz - Barão and is the second most frequent cancer in women Geraldo, Campinas - SP, 13083-970 worldwide. HPV is associated with cervical cancer and 2. PUCCAMP - Pontifícia Universidade plays a crucial role in tumor formation. Over 170 types Católica de Campinas, Prédio H13 - Portão 2. Rod. Dom Pedro, km 136. Parque das Universidades, Campinas - SP, tropism to a particular location. Around 45 types of HPV 13086-900 infectof HPV the have genital been tract identified, and may eachbe divided with preferentialin low and high oncogenic risk types. Of these, HPV-16 and -18 are for a large proportion of these diseases have also been the most frequently associated with anogenital cancer. associatedViruses that with have various been malignant identified states. as causative Concomitantly, agents Despite these infections being considered a risk factor, the number of cases of oral cancer (considered to factors associated with HPV infection may favor the reportedly has been increasing among young adults. The developmentthey are not of sufficient cervical squamous to induce intraepithelial CC. However, lesion other oncogenicoccur usually potential around of orherpesvirus after the fifthand papillomavirus decade of life) (C_SIL), a cancer precursor lesion. The cervical cellular and their possible role in the development of malignant immune response is responsible for control of HPV conditions, in particular cancer of head and neck has lesion progression. Langerhans cells (LC), a professional been described. Material and Methods: The aim of this antigen-presenting cell, play an important role in the study is to evaluate the presence of DNA of human immune response surveillance against viral infections. herpesvirus directly in biopsies removed surgically from However, several studies are controverse about the role twenty-six patients, aged 45-75 years. Fresh biopsies of LC in cervical HPV infection. The present study aims from 16 patients with head and neck cancer and 11 fresh to detect LC presence in C_SIL. Patients, 19 to 50 years biopsies from a healthy control group were studied. DNA aged, were recruited from IFF-Fiocruz. Nine cervical extraction was executes and nested PCR technique was with high C_SIL diagnosed biopsies were taken and used for DNA detection in lesions to analyzed for the processed for immunohistochemistry analysis using an presence of DNA of Epstein-Barr virus (EBV), Human anti-langerin antibody for LC detection. Positive stained Cytomegalovirus (HCMV), Human Herpesvirus 6 (HHV- cells were evaluated in the epithelium, basal layer of 6), Human Herpesvirus 7 (HHV-7), Human Herpesvirus 8 (HHV-8) and Papillomavirus (HPV). Results: Biopsies detected in all samples, rarely inside the epithelium from cancer patients showed 55,5 % positive samples epithelium, stroma and perivascular fields. LC were September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

194 Human Virology: HV for virus in relation to 22,2 % control group. HPV astrocytomas (ACT) (Grades I-III), 10 glioblastomas occurred in 8/16 (50%) cancer biopsies and only patient (GBM) (Grade IV), and 15 oligodendromas (OLD) (Grades of control group was positive. HHV-6 and HHV-7 was I-III) were tested for the presence of HCMV DNA, RNA detected in 22% from cancer group and HHV-7 was the and protein. Viral DNA was detected by quantitative real most prevalent with four individuals study group and time PCR (q-PCR), using primers for the UL83 ORF, in one of the control group, followed by EBV with two of 18/27 cases of ACT (66,6%), 06/10 cases of GBM (60%) the study group and the control group and one patient and 10/15 cases of OLD (66,6%). The immediate early in the study group showed co-infection of EBV and transcript (IE1) was detected by in situ hybridization HHV-6 and eight patients in the study group had HPV against four in the control group. No CMV virus DNA was cases of ACT (48,1%), 04/10 cases of GBM (40%) and detected in both groups. Conclusion: HPV were the most 07/15(ISH), usingcases of a fluorescein-conjugatedOLD (46,6%). The IE1 viral probe, protein in 13/27 was prevalent virus found in biopsies of cancer followed by detected by immunohistochemistry (IHC) in 10/27 cases of ACT (37%), 4/10 cases of GBM (40%) and 8/15 cases of ODG (53,3%). These results suggest that theEBV virus and HHV-7,in these and lesions there can were lead statistically patients tosignificant a worse qPCR is a more sensitive method of viral detection. In prognosis,differences as between well as virusgroups. infections This finding may be suggests an etiologic that addition, the data indicate that the presence of HCMV agent of cancer. Financial Support: CAPES

HV492 - DETECTION OF HUMAN CYTOMEGALOVIRUS samplesdoes not demonstrateddiffer significantly that inthe different viral load types increases of gliomas. with IN DIFFERENT TYPES OF GLIOMAS theInterestingly, grading of however, tumors, quantificationup to grade III, of and viral decreases DNA in the in Castro, L.F.F.1; Stangherlin, L.M.1; Kimura, L.M.2; GBM (grade IV). Funding: FAPESP Guerra, J.M.2; Shirata, N.K.2; Nonogaki, S.2; Medeiros, R.S.S.3; Silva, M.C.C.1 HV497 - IDENTIFICATION OF RESPIRATORY VIRUS AND COINFECTIONS IN SAMPLES FROM CHILDREN 1. UFABC - Universidade Federal do ABC, Rua ASSISTED BY THE CHILDREN HOSPITAL COSME Catequese, 242 - Jardim, Santo André - SP, 09090-400 DAMIAO – PORTO VELHO, RONDONIA 2. IAL - Instituto Adolfo Lutz, Av. Dr. Arnaldo, 1,2,3 2,3 355 - Cerqueira César, São Paulo - SP, 01246-000 Alves, F.A.G. dos S. ; Santos, J. das V. ; dos Santos, 3. FM/USP - Faculdade de Medicina da A.O.1,2,3; Baffini, V.R.; Pereira, A.V.C.2,3; Souza, L.F.B.1,2,3; Universidade de São Paulo, Av. Dr. Arnaldo, 455 - Cerqueira Lima, N.C. da S.2,3; Matos, N.B.1,2,3; Salcedo, J.M.V.1,2,3; César, São Paulo - SP, 01246903 Vieira, D.S.1,2,3 Human Cytomegalovirus (HCMV), a member of 1. UNIR - Universidade Federal de Rondônia, the Herpesviridae family is a ubiquitous pathogen Av. Presidente Dutra, 2965, Centro, Porto Velho - RO, 76801- associated with several human malignancies in 974 immunocompromised, transplanted recipients and 2. FIOCRUZ - Fundação Oswaldo Cruz, Av. newborns. In addition, evidence suggests that HCMV Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 is associated with tumor malignancy based on sero- 3. CEPEM - Centro de Estudo e Pesquisas da epidemiologial studies as well as on the presence of Mulher, R. Barão de Lucena, 71 - Botafogo, Rio de Janeiro - RJ, HCMV DNA, RNA and antigens in tumor tissues. In 22260-020 particular, we and others have detected the viral genome Acute respiratory infections (ARIs) are an important and viral proteins in glioblastoma multiforme (GBM), cause of childhood morbidity and mortality worldwide, the highest-grade glioma (Grade IV) and most malignant which present greatest impacts in developing countries. form of astrocytoma. Studies searching for HCMV in ARIs are caused by different etiologic agents like different types of gliomas are still scarce. Therefore viruses and bacteria that are the major causatives of the aim of our work was to verify the HCMV presence technique the major respiratory viruses and check grades I-IV. Fifty-two tissue samples consisting of 27 forARIs. potential Objective: bacterial To identify coinfections by immunofluorescence in samples from and viral load in paraffin-embeded sections of glioma September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

195 Human Virology: HV children treated at the Hospital Cosme Damiao - Porto 1. IEC - Instituto Evandro Chagas, Rodovia Velho, Rondônia. Methods: Sixty samples were collected BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 by nasopharyngeal swab from children (0-6 years old) 2. NÚCLEO DE MEDICINA TROPICAL/ who had symptoms of acute respiratory infection from UFPA - Núcleo de Medicina Tropical da Universidade Federal February to December 2013. These samples were tested do Pará, AV. Generalíssimo Deodoro, 92, Belém - Pará, 66055- 240 of the following viruses: adenovirus, Human Respiratory Acute Respiratory Infection (ARI) are characterized by by immunofluorescence technique for the identification respiratory tract infections, sudden onset, and variable 1, 2 and 3 (HPIV) using a commercial panel. Besides that, severity, one of the major public health problems samplesSyncytial were Virus tested (RSV), in the influenza Microbiology A and laboratoryB, parainfluenza for the worldwide. Viruses are the leading causes of ARI since it following bacteria: Escherichia coli, Delftia acidovorans, is estimated that 90% of those infections are related to Rothia mucilaginosa, Streptococcus sp., Neisseria other viruses also play an important role in the induction Corynebacterium sp., Moraxella catarrhalis, Kocuria sp. ofthose gravity pathogens. in respiratory In addition infections to Influenza such A and as B viruses,Human andmeningitidis, Streptococcus Staphylococcus parasanguinis aureus, through Neisseria the following flava, Virus (PIV), Human Coronavirus (HCoV) and Human to optochin, bile solubility, PCR and sequencing. Results: BocavirusRhinovirus (HBoV). (HRV), The Adenovirus similarity (AdV), of the Parainfluenza symptoms Untiltests: thisantibiogram, moment thirty-fourCAMP, catalase, samples biofilm, were susceptibility analyzed by laboratory diagnosis. In this context, this study aims to to be positive. Among the positive samples there was make difficult the clinical diagnosis, becoming essential immunofluorescence, of these 88.2% (30/34) were found SARI in Belém, State of Pará, Brazil, determining which virusdetect is non-influenzathe most frequently viruses involved in hospital one; and patients to describe with found the following viruses: 30% (9/30) Parainfluenza the seasonality of infection by those agents in Belém. A 1, 23.33% (7/30) Parainfluenza 2, 13.33% (4/30) total of 271 samples were collected from patients with Adenovirus.Parainfluenza Multiple 3, 16.66% infections (5 / 30) were Respiratory observed Syncytial in nine ARI, both sexes, and different ages, treated at hospitals Virus, 6.66% (2/30) Influenza A and 20.58% (7/30) in Belém, from January 2013 to January 2014, screened samples, two samples were positive for HPIV-1 + HPIV-3, one sample HPIV-1 + Adenovirus, one sample HPIV-2 + extractedby Influenza from Surveillance the clinical specimen Network, by but a commercial diagnosis kit. for Influenza A, one sample HPIV-1 + RSV, one sample RSV + Theinfluenza detection virus of was viral negative. genome The was viral performed nucleic acid by real- was Adenovirus, one sample Adenovirus + Influenza A, one time polymerase chain reaction (qRT-PCR), using probes positivesample HPIV-1for respiratory + HPIV-2 viruses + Adenovirus present andcoinfected one sample with bacteria.Conclusion:HPIV-2 + HPIV-3 + RSV The infections.presence of Five viral of and the bacterial samples the 271 samples tested, 95 (35%) were positive for one or coinfection highlights the necessity for further research moreand specific viruses oligonucleotides investigated. And primers 56 (58.9%) for those were virus. positive Of for rhinovirus; eight (8.4%) for adenovirus; seven (7.3%) It is important to know the causative agent of the infectionthat can define since theit serves epidemiological as an aid inprofile the prognosis in this region. and HKU1; four (4.2%) for coronavirus OC43; three (3.1%) management of these children. for parinfluenzabocavirus; two virus (2.1%) type 3; for five coronavirus (5.3%) for coronavirus 229E; two (2.1%) for coronavirus NL63. Eight co-detections for HV511 - DETECTION OF NON-INFLUENZA rhinovirus were also found in seven of them. Regarding RESPIRATORY VIRUS IN HOSPITAL PATIENTS WITH seasonality, circulation of those virus was predominant SEVERE ACUTE RESPIRATORY INFECTION (SARI) IN BELEM, STATE OF PARA, BRAZIL is usually associated with the period of highest rainfall 1 1 1 Barbagelata, L. ; Vergueiro, M. ; Santos, M. ; Ferreira, in theour firstregion. half Theof the indicated year, mainly data Mayshow and the June, important which J.1; Gomes, E.1; Souza, E.1; Medeiros, R.2; Mello, W.1 of respiratory infection, highlighting the rhinovirus, role of non-influenza viruses for inducing severe cases September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

196 Human Virology: HV once was only indicated as an inducer of the common our region, accounting for 70% (21/30) of isolates, with type 41 accounting in 85.7% (18/21) of positive cases. region.cold. Another FINANCIAL important SUPPORT: finding IEC/SVS/MS is the demonstration of epidemiology when comparing samples from the pre the first evidence of HCoV HKU1, NL63 in the Amazon andThere post-vaccination were no significant period. variations Ad predominated in the molecular in the HV513 - PREVALENCE ADENOVIRUS AMONG age group of 18-24 months during the pre-vaccination CHILDREN WITH ACUTE GASTROENTERITIS PRE AND POST ROTAVIRUS VACCINE INTRODUCTION IN could be found during the post-vaccination period. BRAZIL period; no clustering of Ad into a specific age group Muller, E.C.A.1; Oliveira, A. do S.1; da Silva, L.S.1; relation to gender and the temporal distribuition. Our Guerra, S. de F.1; Mascarenhas, D.O.J.D’A.P.1; de Sousa, resultsFurthermore, provide no strong significant evidence differences of adenovirus could bebelonging seen in M.S.2; Linhares, A. da C.1 to species F being a cause of severe gastroenteritis 1. IEC - Instituto Evandro Chagas, Rodovia among children under 3 years of age. BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 HV515 - CO-CIRCULATION OF TWO DENV-1 LINEAGES 2. UFPA - Universidade Federal do Pará, Rua IN SÃO JOSÉ DO RIO PRETO, SÃO PAULO, BRAZIL Augusto Corea, nº 1, Belem Do Pará - Para, 68440-000 Silva, G.C.D.1; Périco, J.M.B.1; Vedovello, D.1; Colombo, Gastroenteritis is one of the major causes of childhood T.E.1; Pinheiro, T.M.1; Pires, L.M.1; Silva, G.C.D.1; illness worldwide. Adenoviruses may play a role in Ullmann, L.S.1; Junior., J.P.A.2; Drumond, B.P.3; the etiology of gastroenteritis. Adenovirus contains a Nogueira, M.L.1 double-stranded DNA and is non-enveloped. It belongs to the Adenovidae family, genus Mastadenovirus. They 1. FAMERP - Faculdade de Medicina de São are divided into six species (A-F) and 51 serotypes. José do Rio Preto,Av. Brigadeiro Faria Lima, 5416 - Vila São Epidemiological studies have detected adenovirus in 2 Pedro, São José do Rio Preto - SP, 15090-000 to 22% of cases of acute diarrhea in pediatric hospitals 2. UNESP - Universidade Estadual Paulista, and emergency departments. The main purpose of this Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010 3. UFJF - Universidade Federal de Juiz de Fora, R. José Lourenço Kelmer - Martelos, Juiz de Fora - MG, 36036- of adenoviruses in stool samples from 1,160 children 330 understudy wasthree to years detect of theage presenceincluding andhospitalized define the patients types and outpatients during the rotavirus pre-vaccination Dengue fever represents an important public health period (March to September 2003) and post-vaccination problem in Brazil. São José do Rio Preto (SJRP) has period (May 2008 to April 2009). This was a surveillance been presenting a hyperendemic circulation of dengue carried out by Institute Evandro Chagas these children virus (DENV) since 2008, when three serotypes (I, II were hospitalized with gastroenteritis in Belém, and III) started circulating in the city. The serotype 4 Pará, Brazil. Our study placed emphasis on 40 and 41 was introduced in SJRP in 2011, and nowadays the four adenovirus (AdV) serotypes (species F). We used EIA serotypes already had been detected in the city. Dengue and immunochromatography for screening, and PCR virus 1 (DENV-1) serotype re-emerged in São José do Rio Preto in 2008 (1,83% of cases), after over 10 years and typing. The adenoviruses (Ad) were found in 7.24% without its detection. In the subsequent years (2009- (84/1160)and nucleotide of samples. sequencing AdV was for presentmolecular in 5% identification (58/1160) 2014), DENV-1 was the predominant serotype in Dengue of samples, corresponding to 69% (58/84) of positive cases in the city, except for the year 2013, when DENV- cases. Overall, 3.3% (25/760) were found during the pre- 4 was the main circulating serotype. The literature vaccination period and 8.2% (33/400) during the post- reports the circulation of multiple DENV-1 strains in vaccination period study. Our data suggest an increase some countries, including Brazil, and DENV-1 clade in the prevalence of AdV after introduction of the diversity was previously associated with an increase in vaccine Rotarix®. As based on nucleotide sequencing, the abundance of this serotype and enhanced mosquito species F was characterized as the more prevalent in transmission. Thus, the aim of this study was to identify

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197 Human Virology: HV the predominant genotype strains (or clades) of DENV-1 that may have contributed to the large number of cases After started the vaccination several studies showed of DENV1 infection in SJRP. This study included serum human rotavirus vaccine with antigen specificity G1P8. samples sent to the Laboratório de Pesquisa em Virologia hospitalizations and mortality caused by rotavirus. In of FAMERP for Dengue diagnosis between 2008 and contrast,the benefits the Norovirus in reducing has thebeen number emerging of as infections, the main May 2014. In this period, of 1162 samples positive for etiological agent. Few studies in Brazil have investigated DENV, 490 (42%) were positive for DENV1. Of these, the circulation of Norovirus, and most of them are in we have sequenced the complete E gene directly from closed places (hospitals, nursing homes, and day care serum of 29 infected patients previously diagnosed centers), and few of them report the circulation in the with DENV-1 infection by capillary DNA sequencing community. The objective of this study was to investigate using the BigDye v3.1 in ABI3130 automatic sequencer the infection caused by Rotavirus and Norovirus in (Applied Biosystems) or by Illumina technology (Illumina). Consensus DENV-1 E gene sequences were system of with gastroenteritis in Guarapuava - PR. On obtained using Accelrys Gene 2.5 software (Accelrys) hundredchildren untiltwenty to stoolfive years samples of old were enrolled included in theour health study and alignment (MUSCLE) and phylogenetic analysis between the months of March 2011 through February using Mega 6.0 software, using previously published were applied for Rotavirus and Norovirus detection, E(Maximum gene sequences Likelihood, from GenBank. TN93+G+I) The were results performed showed respectively.2012. RT-PCR The assays children for VP7 include and VP1 had genes a mean amplification of 2.8 and that all DENV-1 belong to genotype V and are grouped a median of 3 years, of old and the average of rotavirus in two separate clades, indicating the existence of two vaccination rate was 83.85%. No rotavirus infection DENV-1 lineages in SJRP. We detected the co-circulation cases were detected and Norovirus was detected in 19 of these lineages at least for three years (2010-2012). samples (15.8%) of stool investigated. The months with the largest Norovirus circulation were September and of the co-existence of these two lineages described October 2011, suggesting the possibility of an outbreak hereAlthough is still the not clinical clear, these and epidemiologicalresults highlight significancethe genetic during this period. This result suggests a reduction diversity of circulating DENV-1 in SJRP and the potential of rotavirus infection caused by rotavirus in children for DENV-1 lineages to persist in a hyperendemic city. with high vaccination coverage rate and pointed that FINANCIAL SUPPORT: INCT/CNPQ-DENGUE; FAPESP; Norovirus is circulating in the community children as an CAPES; FAMERP/FUNFARME;SECRETARIA MUNICIPAL agent of gastroenteritis, should now be considered in the DE SAÚDE differential diagnosis and in the institution of measures to control transmission. HV522 - NOROVIRUS CIRCULATION IN COMMUNITY CHILDREN WITH HIGH ROTAVIRUS VACCINATION HV523 - CHARACTERIZATION OF THE GENOTYPIC COVERAGE PROFILE OF HEPATITIS DELTA VIRUS: HDV Carraro, E.; Ambrosini, V.A.; Semaan, L.M.; Campos, GENOTYPE-1 ISOLATION IN THE REGION OF D.A.; Pedrosa, F.C. WESTERN AMAZON, BRAZIL Souza, L.F.B.1,2; Vieira, D.S.2,3; dos Santos, A. de O.1,2; UNICENTRO - Universidade Estadual Centro Oeste, R. 2,3 2,3 Padre Honorino João Muraro, 875 - Santa Cruz, Guarapuava Pereira, A.V.C. ; Salcedo, J.M.V. - PR, 85015-430 1. UNIR - Universidade Federal de Rondônia, Diarrheal disease is a common cause of morbidity and Av. Presidente Dutra, 2965, Centro, Porto Velho - RO, 76801- mortality worldwide, estimates in the top 5 causes 974 of deaths, with most occurring in young children in 2. CEPEM - Centro de Estudo e Pesquisas da Mulher, R. Barão de Lucena, 71 - Botafogo, Rio de Janeiro - RJ, nonindustrialized countries. The group A rotavirus 22260-020 was considered the most important as a cause of 3. FIOCRUZ - Fundação Oswaldo Cruz, Av. gastroenteritis in children. In Brazil, in March 2006, Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 the National Immunization Program included oral

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198 Human Virology: HV

Hepatitis Delta Virus (HDV) is a hepatotropic subvirus and determines advanced liver disease in young patients. which is dependent on the hepatitis B virus. Viral genetic Financial support: FIOCRUZ/CEPEM diversity is related to the geographical origin of the HV524 - MOLECULAR DIAGNOSTIC OF HUMAN T-CELL LYMPHOTROPIC VIRUS IN WHOLE BLOOD SAMPLES levelsisolates, of andendemicity yet eight are genotypes the center were and identified north Africa, and OF PATIENTS WITH ANTIRETROVIRAL TREATMENT theclassified Amazon from Basin, HDV-1 Eastern to HDV-8. European, The regions Mediterranean, with high FOR HIV IN PERU Middle East and parts of Asia. Methods: This study Huatuco, E.M.M. ; Bustamante, F.C.2; dos Santos, has included sera from 54 patients with HBV/HDV T.A.4; Barbosa,M.L.3; Oliveira, D.B.3; Durigon, E.L.3; do infection whose HDV-RNA and HBsAg were detectable. Rosario, J.S.C. 4 Clinical and epidemiological data were obtained from 1. UNMSM - Universidad Nacional Mayor de San Marcos, Calle Germán Amézaga N° 375 - Edificio Jorge physician. HDV-RNA was extracted and converted to Basadre, Ciudad Universitaria, Lima 1, Lima - Peru the Medical Records Outpatient filled by the specialist 2. INS - Instituto Nacional de Salud, Avenida PCR-RFLP. Twelve HDV-3 and four HDV-1 samples were calle 26 No. 51-20 - Zona 6 CAN. Bogotá, D.C. sequencedcDNA. The cDNAby nested was amplifiedPCR-RFLP. and Processing, genotyped editing by nested and 3. ICB/USP - Instituto de Ciências Biomédicas production of the consensus sequences were performed da Universidade de São Paulo, Edifício III USP - Administração using Phred, Phrap-Consed. The consensus sequences - Av. Prof. Lineu Prestes, 2415 - Butantã, São Paulo - SP, 05508-900 was generated by Maximum Likelihood method using 4. FMH/USP - USP - Universidade de São MEGA5were aligned based using on theHKY-substitution Clustal X2. A phylogeneticmodel. Results: tree Paulo, Av. Prof. Almeida Prado, nº1280 - Butantã, São Paulo Fifty-two samples were positive in Nested PCR-RFLP. - SP, 05508-070 A prevalence of 92.3% for HDV-3 and 7.6% for the Human retrovirus HTLV and HIV share similar routes of HDV-1 has also been observed. In the analysis of the transmission but cause different diseases. In common evolutionary lineage it was observed from 16 sequenced samples, twelve of them belonged to HDV-3 and four to system change .Increased co-infections in South HDV-1, supporting the nested PCR-RFLP results. The Americashare tropism is evident. for CD4It is +an lymphocytes,emerging health the problem immune patients infected with HDV-1 had become symptomatic, in patients with antiretroviral treatment in Peru. The two of whom had splenomegaly. They have showed antiretroviral therapy has decreased mortality and values of 52 and 53 respectively for AST and ALT and morbidity in patients with HIV-1 prolonging its life, with three of them showed Chronic Liver Disease signs on consequences that have resulted in an increased number Abdominal Ultrasonography and esophageal varices at endoscope as well. Only one patient underwent liver the accurate diagnosis of HTLV-1. This work aims to reportof inflammatory the progress syndromes, in diagnosing these HTLV-1 processes of 23 difficult cases Metavir system. Conclusion: Earlier studies in patients of patients living with HIV-1 and antiretroviral therapy withbiopsy hepatitis and has Delta showed in the State fibrosis=2 of Rondonia and activity-1 have shown in HAART during 2012.-2013 at the National Institutes higher prevalence of HDV-3 in Amerindian population, of Health. Serological studies were performed for the and in those studies no patient was indigenous. The detection of anti-HTLV-1 antibodies in serum with ELISA cases series shows a relatively young age with relatively advanced Chronic Liver Disease, since 3 of 4 patients used WESTERN BLOT additional test (Innogenetics had already signs of portal hypertension with evidence INNO-LIA-HTLV-I/II).(HTLV-I + Bioelisa II5.0-Biokit). In the investigation For confirmation of the viral was of esophageal varices. We reinforce the thinking that the hepatitis Delta represents a public health problem in blood was used the kit protocol (invitrogem Purelink many countries worldwide. However, we cannot neglect Genomicgenome and DNA), the and proviral the genomic DNA purification material was from stored whole at the presence of HDV-1 that is generally diagnosed late -20 ° C. Test for amplifying proviral DNA segment was used (Nested-PCR). The consensus primers were used

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV for amplification of the virus HTLV-1in the first run SK43 XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

199 Human Virology: HV and SK44, and for the second run were used tax1 and fragmenttax 2.Verificando length polymorphism agarose gel 3%. (RFLP) B-actin were was evaluated used to (Taq1confirm New the England-Biolabs). presence of DNA. NucleasesThe analysis hydrolyze of restriction using easily detectable by differences in their migration electrophoretic.specific sequences We report in DNA are resulting serologically from positive digestion, for HTLV 43.5% (10/23) and 56.5% (13/23) indeterminate cases. In PCR 26.09% (6/23) of cases were positive for HTLV. The 73.91% (17/23) were negative for HTLV. In 100% (23/23) of samples evidenced positive B-actin. The protocol RFLP that were used allowed us to determine the 33.33% (2/6) of HTLV-1 positive not found HTLV-2. Subtypes could not be determined befits a man of 63 years and a woman of 40years, we induce signals that trigger the activation of cytokines, chemokinesentirely possibly that can due interfere to HIV-1 diagnosis infected and CD4 even + T more cells may be related to the emergence of different phenotypes of HIV-1 that modulates the cellular microenvironment, livingdifficult with to HIVdifferentiate and antiretroviral subtypes. treatment,In the capital we assumeof Peru that(Lima) we was should confirmed continue circulation investigating, of HTLV-1 to contribute in patients to the improvement of new diagnostic systems in public health.

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV IMMUNOBIOLOGICALS IN VIROLOGY - IV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

201 Immunobiologicals in Virology: IV

IV34 - EXPRESSION OF HEPATITIS A VIRUS PROTEINS IV98 - EVALUATION OF IMMUNOGLOBULIN IGA AND IN BACULOVIRUS EXPRESSION VECTOR SYSTEM IGG DETECTION AGAINST HPV da Silva Jr, H.C.; de Azevedo, M.L.B.; Galler, R.; Costa, A.P.F.1; Machado, P.R.L.1; Souza, L.B.F.C.1; Medeiros, M.A. Freitas, J.C. de O.C.1; Eleutério Jr, J.3; Giraldo, P.C.2; Gonçalves, A.K. da S.1 FIOCRUZ - Fundação Oswaldo Cruz, Av. Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 1. UFRN - Universidade Federal do Rio Grande The hepatitis A virus (HAV) is the primary etiologic do Norte, Campus Universitário Lagoa Nova, Natal - RN, agent of acute viral hepatitis and is estimated to cause 59078-970 2. UNICAMP - Universidade Estadual de tens of millions of new infections each year worldwide. Campinas, Cidade Universitária Zeferino Vaz - Barão Currently, there are commercially available vaccines Geraldo, Campinas - SP, 13083-970 against HAV based on inactivated viruses. However, 3. UFC - Universidade Federal do Ceará, the high cost of production hinders the introduction of Avenida da Universidade, 2853 - Benfica, Fortaleza - CE, these vaccines into the routine of developing countries. 60020-181 In this context, the use of recombinant proteins of The interest in HPV seropositivity has increased HAV may represent an alternative model to existing considerably since HPV vaccines have become available vaccines. Given this, the present study aimed to evaluate worldwide. To assess the performance of ELISA in the expression of VP1 and P1-2A proteins of hepatitis analyzing serum samples provided from women with A virus in the baculovirus expression vector system (BEVS). The VP1 and P1-2A genes were cloned into the polylinker of pFastBac™ Dual vector, under the control IgGand and without IgA genitalrecognizing DNA-HPV HPV-16, infection and 18 confirmed as well byas of polyhedrin promoter. The recombinant baculoviruses virus-likePCR, for detection particles. of Fifty specific women antibodies sexually of active the isotypes female were subsequently generated and used to express the patients, between 18-35 years from the outpatient clinic recombinant proteins in Spodoptera frugiperda 9 (Sf9) at university hospital from August to December 2013, cells. In order to verify the expression and evaluate were enrolled In order to test them, positive controls solubility of VP1 and P1-2A, the infected cells were were obtained from patients with HPV induced lesions harvest, disrupted and analyzed by Western blotting.

assay was used to identify antibodies to HPV virus-like particlesand DNA-HPV by ELISA. positive The samples confirmed were by divided PCR. A into specific HPV P1-2AThe nickel (rP1-2A) affinity were chromatography expressed with (IMAC) molecular was massused toof positive and negative, and an ELISA detecting IgA and aboutpurification. 35kDa The and recombinant 110kDa, respectively. proteins VP1 The (rVP1) rVP1 wasand IgG anti-HPV-VLP was carried out. The effectiveness of detected in both the soluble fraction and in the insoluble ELISA and the Kappa (k) index was obtained from the fraction of the lysate, whereas rP1-2A was detected only values entered in the ROC curves for IgG and IgA. IgG- in the insoluble fraction. The soluble fraction of rVP1 VLP-HPV16 showed a good correlation between ELISA and PCR (k=0.75) and IgG-VLP-HPV18 showed a very was purified successfully, but the recovery was relatively good correlation between ELISA and PCR (k=0.84). While the IgA antibody correlation was also positive, although Theselow, around results 0.5 indicatemg/L of infectedthat solubility culture. Themay difficulty represent of weaker: IgA-VLP-HPV16 was moderate (k=0.45) and aobtaining drawback rP1-2A for obtaining in soluble largeform precludedamounts of purification. structural proteins of HAV. Further investigations are needed to assay concerning IgG was the following: sensitivity, limit the formation of aggregates and optimize extraction IgA-VLP-HPV18 good (k=0.66). The efficacy of the conditions. FINANCIAL SUPPORT: CAPES, FIOCRUZ and 88% to IgG-VLP-HPV16, and 100%, 92% and 94% tospecificity IgG-VLP-HPV18. and accuracy The were,assay respectively,concerning IgA82.3%, was 92% the

respectively, 64.7%, 80% and 73.8% to IgA-VLP-HPV16, andfollowing: 100%, sensitivity,80% and 84.8% specificity to IgA-VLP-HPV18. and accuracy IgG were, and

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Immunobiologicals in Virology: IV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

202 Immunobiologicals in Virology: IV

IgA antibodies against HPV-16 and 18 can be detected in unvaccinated individuals by using the VLP that serve as the basis for bivalent HPV vaccine. The values for ELISA four DENV serotypes and to other flaviviruses. Different assays and the values found for IgG correlate good/ reactivity patterns were identified: flavivirus cross- very good with HPV16 /18 detected by PCR. FINANCIAL reactive, dengue-group specific, dengue subcomplex SUPPORT: FAPESP somespecific mAbs (DENV-1, cross-reacted -3 and with -4 and yellow DENV-2 fever and virus, -3) West and Niledengue virus serotype-specific and Saint Louis (DENV-2 encephalitis or -3). virus. Additionally, None of IV198 - DEVELOPMENT, CHARACTERIZATION AND the mAbs recognized the alphavirus Venezuelan equine APPLICATION OF MONOCLONAL ANTIBODIES encephalitis virus. Furthermore, mAb D3 424/8G was AGAINST BRAZILIAN DENGUE VIRUS ISOLATES successfully conjugated to horseradish peroxidase and Zanluca, C.1,2,3; Mazzarotto, G.A.C.A.1,2; Bordignon, used to develop a capture enzyme-linked immunosorbent J.1,2; dos Santos, C.N.D.1,2 assay for anti-DENV IgM detection in sera from patients 1. FCC - Fundação Carlos Chagas, Av. Prof. with acute dengue. The results were consistent with Francisco Morato nº 1565 Jd. Guedala, São Paulo - SP, 05513- results from the commercially available PanBio IgM 900 2. FIOCRUZ - Fundação Oswaldo Cruz, Av. mAbs against DENV isolates circulating in Brazil to be Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 developedcapture assay and kit. characterized. To our knowledge, These thesemAbs are may the be first of 3. UFPR - Universidade Federal do Paraná, special interest in the development of diagnostic assays, Rua XV de Novembro, 1299 - Centro, Curitiba - PR, 80060- as well as for basic research, and have the potential to 000 Dengue is the most prevalent human arboviral disease, Financial support: Fiocruz, CNPq, CAPES, Fundação increase the specificity of dengue diagnosis in Brazil. and dengue virus (DENV) infection symptoms are not Araucária IV201 - PRODUCTION AND CHARACTERIZATION OF from other acute febrile illnesses. So an early diagnosis MONOCLONAL ANTIBODIES FOR DENGUE VIRUS-4 issufficiently crucial to specific reducing to morbidity allow clinical and mortality differentiation from 1 1 1 dengue hemorrhagic fever and dengue shock syndrome. Andrade, A. de S. ; Sardi, S.I. ; Costa, L.F.M. ; Sampaio, 1 2 1 Although Brazil is a hotspot for dengue, no serological M.L. da S. ; Brandão, C. ; Campos, G.S. diagnostic test has been produced using Brazilian DENV 1. UFBA - Universidade Federal da Bahia, Rua isolates. This study aims to improve the development of Augusto Viana , s/n, Palácio da Reitoria, Canela , Salvador - immunodiagnostic methods for DENV detection through BA, 40110-909 the production and characterization of monoclonal 2. Hospital Aliança, Avenida Juracy Magalhães antibodies (mAbs) against Brazilian DENV isolates. B Junior, 2096 - Rio Vermelho, Salvador - BA, 41920-900 lymphocytes producing antibodies against DENV were Dengue virus (DV) is an arbovirus belonging to family obtained from spleens of Balb/c mice immunized with Flaviviridae, genus Flavivirus, with four serotypes DENV-1 BR-01/MR, DENV-2 BR/01-01 or DENV-3 BR named DENV-1, DENV-2, DENV-3 and DENV-4. In Brazil, the infection by DENV-4 reemerged in 2010 after 30 myeloma cells by PEG (MW 3000-3700), and hybridomes years, exposing the population at high risk for developing 290-02. Spleen cells were fused with P3X63Ag8.653 secreting anti-DENV antibodies were screened by severe diseases because of the co-circulation of the four serotypes. The monoclonal antibodies (MAbs) are cells. Two freeze-thaw cycles and limiting dilutions an important tools due to its potential application to indirect immunofluorescence on DENV-infected C6/36 were performed and 22 stables clones were obtained. viral diagnostics. The aim of this work was to produce and to characterize of MAbs against DENV-4. The and most were raised against the envelope or the pre- methodology to produce MAbs was developed by Köhler membranemAbs include proteins IgG2bκ, of DENV. IgG2aκ To investigate and IgG1κ whether isotypes, the mAbs could be used for diagnostic and epidemiological were hyperimmunized with DENV-4 viral antigen obtainedand Milstein to virus (1975). multiplication Briefly, experimentalin C6/36 culture animals cells

September/Octoberpurposes, mAbs 2014 were Volume assessed 19 – Supplement for specificity 2 - Abstracts/Posters to the - Immunobiologicals in Virology: IV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

203 Immunobiologicals in Virology: IV and subsequent concentration and precipitation with polyethylene glycol 8000. ELISA test was performed to screen hybridoma supernatants for reactivity to DENV- HaemagglutininMethodology and gene specific of A/Victoria/361/2011 primers for each (H3N2)- strain. 4, and the characterization of MAbs was obtained by likeThese virus analyses that was showed recommended a significant to be used change in the in 2013 the season. The deduced amino acid sequence of the HA techniques. A total of ten hybridomas were obtained protein has shown four mutations as compared with the producingIndirect Immunofluorescence MAbs named E4, C11, (IFA) F12, and H12, Western A12, A2, Blot B4, wild virus sequence: R172Q, E206D, S235Y e K262N, C12, D2 and F10. The characterization of MAbs by IFA starting from methionine. Two of these mutations (sites showed a high positive reaction to DENV-4 in infected 172 and 235) were described in egg-adapted prototype cells, especially F10, and weak reaction for C11, C12 but not in the cell-propagated prototype. In addition, it and F12. The characterization of MAb F10 was then has been shown that the mutation in the site 172 was performed by Western Blot showing recognition to viral responsible for alteration in the antigenicity of the strain, protein of approximately 20 kD, compatible with the leading a less effective protection against the circulating molecular weight of protein M (prM). In conclusion, this viruses. Although the circulating virus didn´t change in study shows the use of MAbs produced against DENV- the forthcoming season it was necessary change the egg 4 as a potential diagnostic tools to detect viral antigen adapted H3N2 strain to another that was antigenically or viral proteins. FINANCIAL SUPPORT: FAPESB-Bahia- like the circulating virus and the cell-propagated Brazil prototype virus. This change may cause a delay in the production of the vaccine that could be critical for the IV235 - GENETIC CHARACTERIZATION OF THE manufactures because of the schedule production to SEED LOTS OF INFLUENZAVIRUS FOR VACCINE PRODUCTION cannot be changed. Sequencing shows an important tool Botosso, V.F.; Comone, P.; Oliveira, R. das N.; Tenório, inprovide monitoring the vaccines changes for in the circulating first day strains of the campaignand also E.C.N.; Miyaki, C. Instituto Butantan, Av. Vital Brasil, 1500, Butantã, São FINANCIAL SUPPORT – FUNDAÇÃO BUTANTAN assisting in the production of an effective flu vaccine. Paulo - SP, 05503-900 IV437 - COMPARISON OF Q AND DEAE MEMBRANE Since 2012, the Butantan Institute is responsible for the AND MONOLITH SUPPORTS IN DNA REMOVAL FROM production of part of the vaccine used in the Brazilian HEPATITIS B VACCINE Gouvea, M.N.; Sciani, J.M.; Souza, C.; Rocha,B.A.; Santos, J.R.; Oliveira, D.C.A.; Botosso, V.F.; Tenório, annual influenza vaccination campaign. It is a trivalent split E.C.N. B,and which inactivated have been vaccine grown from in a embryonatedmixture of Influenzavirus hen´s eggs. A, subtypes H1N1pdm and H3N2, and Influenzavirus Instituto Butantan, Av. Vital Brasil, 1500, Butantã, São may be due to the continuous antigenic variation in Paulo - SP, 05503-900 theThe epidemicdifferences strains. in the protectiveBecause of efficacy this, the of compositionthe vaccines Butantan Institute’s hepatitis B vaccine (VRHB) is composed by the virus recombinant surface S-antigen that publishes the recommendations on the strains to (HBsAg), expressed by the transformed yeast Hansenula of the influenza vaccine is reviewed annually by WHO polymorpha. The harvesting of cells is followed by washing and disruption of the cells to release the HBsAg, selectionbe used in and each development influenza season. of optimal A prerequisite candidate vaccine for the viruses,production adapted and supply to grow of anin idealeggs orinfluenza cells, that vaccine gives is high the to eliminate host cell-derived proteins. One impurity yields of the surface antigens (HA and NA). In order to targetedwhich is for purified clearance by severalduring downstream physico-chemical process steps is guarantee the characteristics of the strains included in residual host DNA. The regulatory guidance for our the vaccine production, each Working Seed Lot used was analyzed by nucleotide sequence of Haemagglutinin should be less than 100 pg/dose. DNA is mainly released (HA) and Neuraminidase (NA) genes using the Sanger inproduct cell disruption specifies stepthat DNAand removed content inalong the thefinal following product

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Immunobiologicals in Virology: IV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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Human adenovirus (HAdV) presents 52 serotypes which molecules in DEAE based resin, which is separated from can cause respiratory, gastrointestinal, urogenital, and purification steps, including adsorption of negative systemic infections both in children and adults. The batch procedure shall be replaced by a convective media aim of this study is to produce monoclonal antibodies the semi-purified product by rough filtration. This in (McAbs) against HAdV-2 hexon protein which will be with regulatory issues. Sartorius and BIA Separations chromatographicchromatography supports (flowthrough (Sartobind mode), Q inand compliance Sartobind rabbit antiserum used in IC tests was prepared against applied in a imunochromatografic test (IC). Polyclonal columns) were compared to the DEAE resin (DE52, by the fusion of mouse myeloma cells (Sp2/0-Ag14) Whatman)D membrane used filters; in the DEAEvaccine CIMmultusTM production process. monolithic The withpurified lymphocytes Ad2 hexons. from Hybridoma female BALB/c cell lines mice were immunized prepared starting material was the supernatant from a precipitation with Ad 2 by a standard technique . Hybridomas were screened for antibody production by the indirect ELISA goal of this study was to select the media with the . Selected hybridomas were cloned by limiting dilution. bestintermediate ratio between purification DNA removal/HBsAg step (PS, >80% adsorption purity). The (a of salt concentration in the binding step, PS (2CV) were theFour Ig monoclones subtypes in reactedwere determined specifically ELISA with isotype purified assay. Ad2 drawback due to its low pI). To investigate the influence Allhexon the andMcAbs expanded produced in large from scale. the four After strains purification, were IgG1 subtypes. In this study we showed shows that applied to Sartobind filters or monolithic columns, in andfive NaClelution concentrations peaks were fromanalysed: 0 to 600HBsAg mM, content and bound was 41 by indirect ELISA . This suggested that those Mabs evaluatedmolecules withwere passive eluted with hemagglutination 2 M NaCl. Whole tests flowthrough and SDS- recognizedMcAbs was specifica conformational for adenovirus epitope serotypes on the 2,3,5 protein and PAGE; DNA removal was tested with a threshold assay. that might be conserved in all sorotypes. Mixture of the McAbs not resulted in high reaction. The rapid assay concentrations and incubated with the DE52 resin, in was performed and results were interpreted by the conditionsPS samples similar were alsoto the prepared production with process. same fiveAny NaCl salt presence or absence of visually detectable colored lines concentration had no led to dramatic lower adsorption of . An immobilized polyclonal rabbit capture antibody on HBsAg to the DE52 resin, while concentrations up to 300 a membrane formed colored line at the specimen if the mM NaCl resulted in gradative less retention of HBsAg specimen contained adenovirus antigen. An internal and DNA in DEAE chromatographic supports (Sartorius control line C, containing antimouse antibody that or BIA Separations). Similar behavior was observed in captured the colored conjugate antibody,was also built into the device to serve as a procedural quality. Here, we protein was adsorbed with any salt concentration. With can demonstrate that monoclonal antibodies reacted someSartobind adjusts, Q filtrations both platforms only for may the be DNA used – theto replace HBsAg against several serotypes adenovirus stocks. If so, this the current process. FINANCIAL SUPPORT – BUTANTAN rapid test may provide an advantage over cell culture FOUNDATION in samples containing noncultivatible adenoviruses but further studies evaluating this test still are necessary. IV503 - MONOCIONAL ANTIBODIES AGAINST ADENOVIRUS TYPE 2: PREPARATION AND PRELIMINARY CHARACTERIZATION Paulini, I.J.1; Silva, J.S.2; Thomaz, L.2; Rocha, L.B.2; Harsi, C.M.2; Belley, N.2; Granato, C.F.H.2 1. UNIFESP - Universidade Federal de São Paulo, R. Sena Madureira, 1500 - Vila Mariana, São Paulo - SP, 04021-001 2. USP - Universidade de São Paulo, Av. Prof. Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Immunobiologicals in Virology: IV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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IV525 - STABILITY EVALUATION OF DENGUE VIRUS evaluated for 12 weeks by ELISA capture IgM (INS) using ANTIGEN (DENV-2) PRODUCED IN SUCKLING MOUSE a validated panel of sera. A higher titer of DENV-2 antigen BRAIN FOR APPLICATION IN ELISA IGM TEST produced in the brain of suckling mouse concentration Peña, B.P.1; Huatuco, E.M.M.1; Zapana, E.M.1; Garcia, 1:20 with an average OD of 1.625 for the positive control P. 2 assessment where the antigen with STABILZYME ® 1. UNMSM - Universidad Nacional Mayor de SELECTwas observed. preserved A significant at a temperature difference of 2-8 at ° C 12 maintained weeks of San Marcos, Calle Germán Amézaga N° 375 - Edificio Jorge their initial O.D. readings unlike antigen without applying Basadre, Ciudad Universitaria, Lima 1, Lima - Peru stabilizing preserved at 2-8 ° C. O.D. readings between 2. INS - Instituto Nacional de Salud, Avenida antigens with STABILZYME ® SELECT at temperatures calle 26 No. 51-20 - Zona 6 CAN. Bogotá, D.C. of 2-8 ° C and the antigen without stabilizer kept at -20 Enzyme immunoassays, such as ELISA, are important and analytical methods that are widely used for various mice for antigen production of DENV is a widely used purposes. The proteins have the correct activity when method.° C showed The noantigen significant production differences. technique Using more suckling used its conformation is appropriate, those found in its native is with sucrose-acetone (Clarke and Casals, 1958) (IPK, state, are more stable .There are several factors that cause 2005). The antigen production method with suckling protein denaturation within which was the temperature. mouse brains is a tedious technique. We conclude that Therefore, it is very important that the proteins to be the application of STABILZYME ® SELECT product stored and used in an environment which stabilizes the maintains the stability of DENV-2 antigen produced in native structure. Currently, there is a range of stabilizers suckling mouse brains 2-8 ° C during the evaluation time sold by different marks which ensure the maintenance of of 12 weeks. protein stability. As these products are designed to ensure the conservation of proteins in their native structure. In the present study, the stabilizing ability STABILZYME ® SELECT product applied in Dengue Virus antigen (DENV- 2) produced in suckling mouse brain BALB / C CNPB for a period of 12 weeks was evaluated. These antigens were evaluated in an ELISA system according to the time capture. To assess the stability of dengue viruses (DEN-2) antigen produced in suckling mice brain during a period of 12 weeks were used ELISA Ig M. The Virus inoculation, Suckling mice of 1-2 days old BALB/C CNPB were intracerebrally inoculated with 20 ml of DENV-2 reference strain S168203 from the NIBSC. The purity polymerase chain reaction (RT-PCR) Production of DENV-2of the strain antigen, was For confirmed the production by reverse of antigen transcription from suckling mouse brain, the methodology described by Clarke and Casals in 1958 was used. ELISA antigen titer IgM capture (INS), Serial dilutions of the antigen were performed and compared the optical densities (O.D.) obtained at each concentration was performed. The stability evaluation of the antigens produced was performed using the STABILZYME® SELECT stabilizer preserved at temperatures 2-8 ° C and compared to antigens without applying stabilizer and maintained at temperatures of 2-8 ° C and -20 ° C. All antigens were September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Immunobiologicals in Virology: IV PLANT AND INVERTEBRATE VIROLOGY - PIV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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PIV38 - EXPRESSION OF PARASPORIN PROTEINS 3. LVA/UNICAMP - Laboratório de Virologia FROM BACILLUS THURINGIENSIS IN INSECT CELLS Animal/ Universidade Estadual de Campinas, Rua Monteiro Lamar, D.C.; Correa, R.F.T.; Ribeiro, B.M. Lobato - n° 255, Campinas -SP, 13083-970 4. LTP/UNICAMP - Universidade Estadual UnB - Universidade de Brasília, Campus Universitário de Campinas, Cidade Universitária Zeferino Vaz - Barão Darcy Ribeiro, Brasília - DF, 70910-900 Geraldo, Campinas - SP, 13083-970 Bacillus thuringiensis (Bt) is a pore-forming bacteria This study was aimed to investigate the effect of high able to produce a crystalline protein inclusions during hydrostatic pressure on Tobacco Mosaic Virus (TMV), a sporulation. Some strains exhibit entomopathogenic virus model for immunology and one of the most studied activity which makes Bt a largely-used bioinsecticide virus to date. When subjected to treatment with pressures to control both agricultural insect pests and human disease vectors. Interestingly, somo Bts produce non- epitopes in comparison to sera from immunized mice insecticidal crystalline inclusion-forming proteins called withit was the observed native aform significant of the changevirus. Thesein the recognizedalterations parasporins which are preferentially toxic to tumor cells. were further studied by combining the high pressure treatment with urea or low temperatures and inoculation the baculovirus/insect cell-based expression system. of these altered virions in Balb-C mice, the collected sera Bt proteins are efficiently expressed in insect cells using Therefore, in this report, we have successfully expressed titers were determined by ELISA and cross referenced two Bt-derived parasporin genes using recombinant between the groups tested. The titter obtained showed baculoviruses and insect cells. For this, both parasporin-2 that the antigenicity of viral particles was maintained gene and a newly discovered parasporin-3-related gene after treatments using monoclonal antibodies against (41%) derived from a Brazilian Bt strain were inserted the native form. The epitope prediction algorithms could into a commercial vector pFastBac1™ (Invitrogen) and not infer the some observed changes in the epitope recombinant baculoviruses were constructed using the commercially available Bac-to-Bac system (Invitrogen). structure. The antigenicity of canonical epitopes was Recombinant viruses were used to infect cells and the maintained,profile suggesting although conformational binding intensities changes were in the different protein recombinant proteins were detected by SDS-PAGE and among the treatments used. Patterns of recognition immunobloting. Heterologous proteins will be analyzed from the epitope mapping were then cross checked with by transmission and scanning electron microscopies to the prediction algorithms for the TMVcp amino acid verify possible crystalline formations in the cytoplasm sequence to infer which alterations might have occurred. of insect cells. Moreover, the recombinant proteins are hexahistidine-tag for further toxicity tests against tumor epitopeOur findings mapping suggest using that sera different from mice cleavages immunized sites werewith being purified by affinity chromatography based on a cells. theexposed virus after after the the high treatments; pressure condition this was was verified imposed. via

PIV82 - HIGH HYDROSTATIC PRESSURE EFFECT ON PIV151 - A BEGOMOVIRUS EXISTING AS A COMPLEX THE EPITOPE MAPPING OF THE TOBACCO MOSAIC OF WELL DEFINED SUBPOPULATIONS IN A NON- VIRUS COAT PROTEIN CULTIVATED HOST Lima Neto, D.F.1,2; Barnabé, A.C.3; Caserta, L.C.3; Godinho, M.T.; Xavier, C.A.D.; Lima, A.T.M.; Zerbini, Martini, M.C.3; Rabelo, A.4; Bonafé, C.F.S.4; Arns, C.W.3 F.M. 1. LNBio – Laboratório Nacional de DFP/UFV - Departamento de Fitopatologia da Biociências, Rua Giuseppe Máximo Scolfaro, 10000 Pólo II de Universidade Federal de Viçosa, Campus Universitário, Viçosa Alta Tecnologia de Campinas, Bairro Guará Campinas – SP, - MG, 36570-000 13083-100 DNA plant viruses, particularly the begomoviruses, cause 2. CNPEM - Centro Nacional de Pesquisa em Energia e Materiais, Rua Giuseppe Máximo Scolfaro, 10.000 - serious epidemics in economically important crops Polo II de Alta Tecnologia, Campinas - SP, 13083-970 worldwide. In Brazil several indigenous begomoviruses have been described infecting tomatoes following the

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

208 Plant and Invertebrate Virology: PIV

new isolates reinforce the current importance of the in the mid-1990s. Non-cultivated plants harbor many disease. In addition, there is little information about begomoviruses,introduction of aand novel it speciesis believed of the that these vectorhosts genetic parameters and heritability associated to mosaic may act as reservoirs and as mixing vessels where resistance in sugarcane. In this regard, the present recombination may occur. Begomoviruses also display study aimed to evaluate the resistance of 79 sugarcane nucleotide substitution rates equivalent to those of genotypes (varieties and elite clones) to a severe SCMV RNA viruses. In this work we sampled Sida acuta plants strain and also to estimate genetic parameters associated showing typical viral symptoms in a small area (aprox. 1 with mosaic resistance. Buds were collected from ha) in the municipality of Viçosa, MG, Brazil. Total DNA sugarcane stools without mosaic symptoms, maintained at the IAC Sugarcane Breeding Station, Ribeirão Preto, from which leaf samples of each stool were tested by PTA- ofwas 65 extracted full-length from genomes fifty samples (33 DNA-A and the and viral 32 genome DNA-B ELISA (Plate Trapped Antibody-ELISA) in order to verify components,was amplified from by RCA,26 samples) cloned andwere sequenced. obtained and A total the the virus sanity. Three weeks old seedlings, planted in stiff 89% DNA-A identity threshold established by the ICTV plastic tubes (volume of 50 cm3) in a two complete block was used for taxonomic placements. Sequence analysis indicated that the clones correspond to a novel species proof greenhouse conditions. Symptoms were evaluated for which the name Sida acuta mosaic virus (SAMV) design, were artificially inoculated with SCMV in aphid is proposed. Additionally, the analyses indicated the serological test PTA-ELISA were performed, being six by a grade scale and the confirmation of infection by the mixed infections and pseudorecombination among the same experimental design. Symptom data were them.coexistence We reconstructed of three well-defined the phylogenetic SAMV relationships strains, with submittedmonths later to validatedvariance analysis,under field adopting conditions, the Splitadopting plot for full-length genomic components, CP and Rep genes temporal arrangement. The mean incidence of mosaic using Bayesian inference (BI). Well-supported clades was low under greenhouse conditions, with higher (posterior probabilities higher than 0.99) were observed in all phylogenetic trees representing each distinct SAMV strain. Our results indicate a complex evolutionary xvalues environmental in the field effects.assay. Based The interactionon field results, of sugarcane variance interplay amongst begomovirus isolates even in small genotypesanalysis revealed with days significant of evaluation genotype revealed and a differential genotype populations. FINANCIAL SUPPORT: FAPEMIG, CAPES behavior in mosaic symptom expression, including the AND CNPQ recovery in some of them. The broad-sense heritability at individual level and means based were 19.37% and PIV156 - EVALUATION OF BRAZILIAN SUGARCANE 62.18%, respectively, indicating that the resistance to GENOTYPES RESISTANCE TO SUGARCANE MOSAIC mosaic tends to be a quantitative trait. The combination VIRUS UNDER GREENHOUSE AND FIELD CONDITIONS of symptom evaluation by grade scale with serological Gonçalves, M.C.; Silva, M.F.; Perecin, D.; Xavier, M.A.; Landell, M.G.A.; Pinto, L.R. selection of sources of resistance to mosaic, detecting test ELISA for SCMV detection proved to be efficient for 1. Instituto Biológico, Av. Cons. Rodrigues the virus in symptomless genotypes, and pointing out Alves, 1252 - Vila Mariana, São Paulo - SP, 04014-002 twenty two genotypes as resistant to SCMV strain in 2. UNESP - Universidade Estadual Paulista, study. Financial support: FAPESP (BIOEN 2008/56146- Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010 5), IAC (Instituto Agronômico de Campinas) and CAPES. 3. IAC - Instituto Agronômico, Avenida Barão de Itapura, 1.481, Botafogo Campinas - SP, 13020-902 Sugarcane mosaic virus (SCMV), the causal agent of mosaic, is one of the main viruses infecting sugarcane in Brazil, which is mainly controlled by the use of resistant cultivars. Favorable epidemiological conditions to mosaic dissemination and recent descriptions of

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

209 Plant and Invertebrate Virology: PIV

PIV159 - SEQUENCING AND PHYLOGENETIC ANALYSIS OF PROTEINS P1 AND HC-PRO OF ISOLATES OF SUGARCANE MOSAIC VIRUS theBioEdit viral program. genome correspondingAnalyses of the to sequences proteins P1 confirmed and HC- that the designed primers allowed the amplification of Gonçalves, M.C.; Bueno, F.C.; Harakava, R.; Gonçalves, Pro. Sequences were deposited in “GenBank” providing M.C. community. Multiple alignments and phylogenetic Instituto Biológico, Av. Cons. Rodrigues Alves, 1252 - analysisinformation by Neighbor generated Joining, in the based project on to P1 the and scientific HCPro Vila Mariana, São Paulo - SP, 04014-002 sequences of different SCMV isolates, showed that In Brazil, sugarcane, maize and sorghum are quite Brazilian isolates studied in this work showed higher affected by Sugarcane mosaic virus (SCMV), responsible sequence identity and common origin with Australian for the mosaic disease in these crops. The introduction of and Argentinean isolates. Financial support: FAPESP new maize hybrids and its cultivation in off-season near (BIOEN 2008/56146-5). FCB was recipient of a CNPq PIBIC fellowship. strains in the country. The P1 and HCPro SCMV proteins playsugarcane an important fields has role resulted in the virusin further infection, spread replication of virus PIV190 - MOLECULAR CHARACTERIZATION OF and transmission. The objective of this work was to better A NOVEL SIDA-INFECTING BEGOMOVIRUS FROM understand the genomic organization and variability SOUTHERN BRAZIL of sugarcane and corn isolates of SCMV by means of Ferro, C.G.1; Silva, J.P.1; Pinto, V.B.1; Mar, T.B.1; Godinho, sequencing and sequence analysis of these proteins. The M.T.1; Lima, A.T.M.1; Lau, D.2; Zerbini, F.M.1 infected plant material of sugarcane and maize were 1. UFV - Universidade Federal de Viçosa, maintained in dehydrated and frozen at -20oC plant Avenida Peter Henry Rolfs, s/n - Campus Universitário, Viçosa tissue. Isolates SCMV “PIR-2”, “RIB-1” and “M-Campinas” - MG, 36570-000 were transmitted by mechanical inoculation to sorghum 2. EMBRAPA Centro Nacional de Pesquisa de “Rio” seedlings using sodium phosphate buffer 0.01 M, Trigo, Rodovia BR-285, 3081, Passo Fundo - RS, 99001-970 pH 7.2, 0.1 % of sodium sulphite; symptoms of infection became evident about six weeks after inoculation, DNA plant viruses) are among the most damaging and total RNA was extracted using Trizol reagent. pathogensBegomoviruses causing (whitefly-transmitted, epidemics in economically single-stranded important crops worldwide. The incidence of begomovirus by synthesizing a cDNA template with a oligo dT and infections in crops increased in Brazil during the 1990s reverseAmplification transcriptase of the viral (RT), genome, followed was by firstlya regular targeted PCR. following the introduction of Bemisia tabaci Middle Fragments of 1210, 1274 nt corresponding to the target East-Asia Minor 1 (MEAM1, previously Bemisia tabaci biotype B). It is believed that MEAM1 transmitted proteins were amplified with primer pairs 5UTR+HCP1R begomoviruses from non-cultivated plants to crops with analyzedand HCP1R+HCP3R by electrophoresis respectively, on 1.2% designed agarose in this gel. work.After The products were properly identified, aliquot and Non-cultivated plants harbor many begomoviruses, andgreater it is efficiency believed than that thethese indigenous hosts may B. tabaciact as species.mixing vessels where recombination may occur resulting sequenced.identification Nucleotide of the target sequences fragments were under analyzed UV light,and in novel species adapted to new hosts. In this study therespective consensuses bands were were submitted excised from to BLAST the gel, (http://blast. purified and we molecularly characterized a novel begomovirus ncbi.nlm.nih.gov/Blast.cgi) algorithm for comparison infecting Sida sp. plants showing typical symptoms to homologous sequences. The generated sequences of begomovirus infection, collected in the state of Rio and the obtained from GenBank were aligned using the Grande do Sul in March 2010. The viral genome was

and sequenced. The DNA-A is 2601 nucleotides (nt) long andamplified has a genomic by rolling-circle organization amplification similar to (RCA),those of cloned other begomoviruses, except for the presence of an additional

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

210 Plant and Invertebrate Virology: PIV open reading frame (ORF) in the complementary strand (AC5). Pairwise sequence comparisons indicated a maximum nucleotide sequence identity of 81.5% with chosenwas confirmed for protein by expression Western is blotting effective. using The improved antibody Tomato dwarf leaf virus (ToDLV, GenBank access number solubilityagainst HisTag. of these These proteins results is ongoing confirmed interest that atthe moment. system JN564749). Phylogenetic analysis placed this isolate in Financial support: CNPq. a monophyletic cluster with other begomoviruses from the Americas. No reliable recombination events were PIV232 - MOLECULAR CHARACTERIZATION detected by the RDP4 program. Therefore, based on OF BACULOVIRUS IDENTIFIED FROM CULEX the criteria established by the International Committee QUINQUEFASCIATUS LARVAE IN SÃO PAULO STATE, on Taxonomy of Viruses, this isolate represents a novel BRAZIL begomovirus species for which the name Sida chlorotic de Carvalho, I.M.V.G.1; Benites, H.G.2; Bernardino, mottle virus (SiCMoV) is proposed. The continuing J. de S.T.3; Queiroz, A.T.L.4; Oliveira, I.B.R.2; Araújo detection of new begomovirus species highlights the Coutinho, C.J.P. da C.2 remarkable genetic diversity of this group of viruses, as 1. Instituto Butantan, Av. Vital Brasil, 1500, well as the power of the RCA technique in assessing this Butantã, São Paulo - SP, 05503-900 diversity. FINANCIAL SUPPORT: CAPES, CNPQ, FAPEMIG 2. SUCEN - Superintendência de Controle de Endemias, R Paula Sousa, 166 - Centro, São Paulo, SP, 01027- PIV215 - EXPRESSION OF REPLICATION COMPLEX 000 DOMAINS OF TOMATO BLISTERING MOSAIC VIRUS 3. UNIFESP - Universidade Federal de São (TOBMV) IN ESCHERICHIA COLI Paulo, R. Sena Madureira, 1500 - Vila Mariana, São Paulo - Junqueira, B.R.T.1; Vasconcelos, K. de O.1; Oliveira, S.2; SP, 04021-001 Dantas, G.1; Nagata, T.1 4. Centro de Pesquisas Gonçalo Moniz, R. Waldemar Falcão, 121 - Candeal, Salvador - BA, 40296-710 1. UnB - Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília - DF, 70910-900 Some mosquito species are humans and animals 2. UniCEUB - Centro Universitário de Brasília, pathogens vectors, such malaria, encephalitis, dengue, Campus do UniCEUB - Asa Norte - Brasília - DF, 70790-075 yellow fever, and heartworm disease. A broad spectrum Tomato blistering mosaic virus is a tymovirus member larvicides and adulticides have been used to combat and possesses a single-stranded RNA genome in these insects. However, the environmental harmful positive polarity. Tymovirus infection causes small effect and the drug-resistance rate increasing in vesicles along the chloroplast periphery. These vesicles mosquitoes population forces new vector-control in chloroplast were shown to be the replication site, approach development. The baculovirus use could be an forming viral replication complex (VRC). Despite alternative to chemical methods. Culex quinquefasciatus knowing the replication site, little is known about the tymovirus replication mechanism. In order to elucidate The objective was to perform parcial molecular baculoviruses were identified in São Paulo, Brazil. the replication process, the overall aim of this project characterization of helicase, polymerase, odv-e27, vlf-1 is antibody production for the major replication and lef-8 baculovirus genes from São Paulo (Brazil). The complex proteins as metyltransferase, helicase and RNA dependent RNA polymerase (RdRp). For this purpose, Samples DNA was amplified, the sequences were aligned, the expression of domains of these three proteins was polymerase, odv-e27, vlf-1, and lef-8 from reference using ClustalX 2.0, with partial sequences of helicase, performed using Escherichia coli system via Gateway baculoviruses genes of other insect orders (Lepidoptera technology (Invitrogen). The three domains were and Hymenoptera) deposited in GenBank database. cloned in the entry plasmid pENTR2B and recombined Phylogenetic analysis was performed. Previous genes to destination plasmid pDEST17. E. coli BL21AI analysis results showed that the viruses found in the six larvae in this study showed 98% similarity with Culex sequencing. The protein expression of RdRp, Helicase nigripalpus NPV, the only species from andwas Metyltransferase transformed with at selected2 and 4 cloneshours post-induction confirmed by genus previously described. This specie shows dipteran

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

211 Plant and Invertebrate Virology: PIV infection and high mortality, showing remarkable infection of other targets potentially compromises the potential as biological control. FINANCIAL SUPPORT: metabolic function of the hindgut, on absorption of 2012/23947-0 water and minerals and in the formation and elimination of feces. PIV299 - BOMBYX MORI COLON RESISTANCE TO PIV300 - CYTOPATHOLOGY OF THE TRACHEAL SYSTEM Baggio, M.P.D.1; Vessaro Silva, S.A.2; Ribeiro, L.F.C.3; OF BOMBYX MORI(LEPIDOPTERA:BOMBYCIDAE) Brancalhão, R.M.C.3 CAUSED BY ALPHABACULOVIRUS Baggio, M.P.D.1; Senem, J.V.2; Ribeiro, L.F.C.2; Torquato, 1. UnB - Universidade de Brasília, Campus 2 2 Universitário Darcy Ribeiro, Brasília - DF, 70910-900 E.B. ; Brancalhão, R.M.C. 2. UEM - Universidade Estadual de Maringá, 1. UnB - Universidade de Brasília, Campus Avenida Colombo, 5790 - Jardim Universitário, Maringá - PR, Universitário Darcy Ribeiro, Brasília - DF, 70910-900 87020-900 2. UNIOESTE - Universidade Estadual do 3. UNIOESTE - Universidade Estadual do Oeste do Paraná, R. Universitária, 1619 - Universitário, Oeste do Paraná, R. Universitária, 1619 - Universitário, Cascavel - PR, 85819-110 Cascavel - PR, 85819-110 Bombyx mori is an insect of the order Lepidoptera Caterpillars of Bombyx mori (Lepidoptera, Bombycidae) that is only found in germplasm banks; it is used in build a cocoon of silk, from which is extracted yarn, used in the production of various tissues. This productive this case, the silk cocoon, which is produced at the activity, known as sericulture, presents a major endscientific of the research 5th larval and instar, for is commercial used in the purposes. production In economic, social and environmental impact in the of various yarns and fabrics, constituting the sericicola state of Paraná. However, the same can be affected by industry. One factor that affects the national sericulture, nuclear polyhedrosis disease, caused by Bombyx mori compromising the commercial production of cocoon, nucleopolyhedrovirus (BmNPV), an entomopathogenic is a nuclear polyhedrosis disease caused by the virus (, Alphabaculovirus), which infects virus from the Baculoviridae family, Bombyx mori B. mori, compromising the production of cocoons and multiple nucleopolyhedrovirus (BmMNPV), genus causing damage to the productive chain. Studies have Alphabaculovirus (AlphaBV. Studies have proved that demonstrated that BmNPV is poliorganotrophic and BmMNPV is polyorganotropic and there are several target various are the target-organs; however, some have organs, such as the tracheal system; however, details of shown to be resilient to pathogens and the present study its cytopathology aren´t known. The tracheal system is aims to analyze the behavior of the colon region, in the responsible for the aeration of the tissues of the insect. hindgut, of B. mori to BmNPV, providing support to the The study described the cytopathology of the tracheas establishment of the viral infectious cycle. The colon of hybrid larvae of B. mori, infected experimentally with is an important region of the hindgut that acts in the BmMNPV, isolated geographically in the state of Paraná. absorption of water and salts and in the formation and Fifth instar hybrid larvae were divided into two groups; elimination of fecal pellets in the insect. Caterpillars of B. one control, and the other inoculated. After ingestion, mori 5th instar were inoculated with a viral suspension and on different days post-inoculation (dpi), from the 2nd to the 9th dpi, the larvae were anesthetized and segments were processed for light microscopy. The dissected. Segments of organs containing branches of the sectionsof BmNPV, obtained and 2̊ to were 9̊ day stained post inoculation by the cytochemical (dpi) colon for transmission electron microscopy. On the 2 st dpi, revealed that colon cells were not susceptible to BmNPV, freshtrachea, hemolymph were collected analysis and was fixed conducted in Karnovsky to determine modified technique Azan, for viral identification. The analysis at all times examined. However, viral occlusion bodies the susceptibility of the hemocytes. The results revealed were observed in adipose, tracheal tissue and into the that the hemocytes were infected from the 2 nd dpi and extracellular medium, indicating the viability of the the epithelial cells of the trachea were infected from the inoculum used. Thus, despite the resistance to BmNPV, 4th dpi. The cytopathology of the tracheal cells showed

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

212 Plant and Invertebrate Virology: PIV hypertrophic nucleus, containing the viroplasm, the site LMV-Cf P3 nucleotide sequence with the similar genomic of the synthesis of the nucleocapsids. Subsequently, the region of other LMV isolates, available in the GenBank, formation and development of the polyhedra occured, ranged from 93% to 97%, which was very similar accentuating the nuclear hypertrophy and culminating to those observed among the LMV isolates used for in cell lysis. Virions were also observed, immersed in comparison. However, there was a deletion of one amino the basal lamina of the trachea, which appeared to be acid in the LMV-CF sequence, positioning it in a separate disorganized. Thus, the cytopathology of the trachea was branch of the phylogenetic tree. Another difference was consistent with the infection caused by AlphaBV, and the found in the nucleotide sequence of 6K2 protein coding- data that was obtained provides a better understanding gene, which usually is highly conserved among the LMV of the infectious cycle of BmMNPV in the body of the isolates from the database, showing 100% identity. The insect. The time of infection, later for the hemocytes, and 6K2 nucleotide sequence of LMV-Cf showed only 92% the presence of virions in the basal lamina of the trachea, identity when compared with those LMV isolates. Since indicated that this system is a secondary target for there are several evidences of the involvement of P3 infection, and also that the hemolymph is an important and 6K2 proteins in the symptoms expression by host dispersant of viral infection. plants, they are considered candidates to be investigated regarding their probable correlation with the atypical PIV338 - GENOME ANALYSIS OF AN ATYPICAL symptoms of closed head showed by the infected plant. ISOLATE OF LETTUCE MOSAIC VIRUS (LMV) Site-directed mutagenesis and protein expression in host Duarte, P. de S.G.; Lucas, M.A.; Figueira, A. dos R.; plants are required to get this information. FINANCIAL Costa, S.B.F.G. SUPPORT: CAPES, CNPQ, FAPEMIG UFLA - Universidade Federal de Lavras, Câmpus PIV342 - CIRCULAR SSDNA SATELLITES ASSOCIATED Universitário, Lavras - MG, 37200-000 WITH BEGOMOVIRUSES IN THE AMERICAS Virus diseases are considered a key challenge for the Castillo, J.N.1; Olive, E.F.1; Mar, T.B.2; Ferro, C.G.2; Silva, production of lettuce (Lactuca sativa L.) in Brazil, where J.P.2; Lau, D.3; Moriones, E.1; Zubiaur, Y.M.4; Zerbini, the losses in infected plants can reach 100%, depending F.M.2 on weather conditions and cultivars. The Lettuce mosaic virus (LMV) is currently considered the most important 1. IHSM - Instituto de Hortofruticultura virus infecting lettuce in Brazil, and the symptoms Subtropical y Mediterránea, 29750 Algarrobo-Costa, Málaga observed in infected plants are mosaic, leaf distortion - ESPAÑA 2. DFP/BIOAGRO/UFV - Departamento and even death of the more susceptible cultivars. In this de Fitopatologia e Instituto de Biotecnologia Aplicada à study, an isolate of LMV, named LMV-Cf, was partially Agropecuária da Universidade Federal de Viçosa, Campus sequenced and analyzed in order to investigate the Universitário, Viçosa - MG, 36570-000 genomic variations which could potentially be linked to 3. EMBRAPA/Centro Nacional de Pesquisa de the induction of atypical symptoms in infected lettuce Trigo, Rodovia BR-285, 3081, Passo Fundo - RS, 99001-970 plants cv. Regina 579, characterized by closure of the 4. CENSA - Centro Nacional de Sanidad Agropecuaria, San José de Las Lajas, Apdo Postal 10, were designed based on the nucleotide sequences Mayabeque, Cuba ofhead. LMV For isolates, genome amplificationavailable in theby RT-PCR,GenBank, the andprimers the Begomoviruses (genus Begomovirus, family Geminiviridae) are plant ssDNA viruses that sequencing. The 5’UTR, P1, HC-Pro, P3, 6K1, CI, 6K2, NIa VPg,amplified CP and genome 3’UTR fragments regions were were completely purified and sequenced sent for Begomoviruses cause serious diseases in economically and analyzed. Among those sequences, two regions importantare transmitted crops in by tropical the whiteflyand subtropical Bemisia regions. tabaci. Two types of circular ssDNA satellites half the size of the LMV-Cf P3 protein, which showed a deletion of three the helper virus components have been described: nucleotides,showed significant resulting changes: in the exclusion the first oneof a glutamicwas found acid in betasatellites and alphasatellites. Betasatellites are in the C-terminal of the protein. The comparison of the associated with monopartite begomoviruses from the September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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Old World and are dependent on them for replication, movement in plants and transmission. Betasatellites Insecta class, Diptera order, suborder Nematocera and consist of an A-rich region, a non-coding satellite CulicomorphaThe Culicidae familyinfraorder, insects also are known flies belonging as mosquitoes. to the conserved region and a single ORF coding for a These insects transmit disease agents in humans, some multifunctional protein. Alphasatellites, also typically of which can cause high mortality and morbidity of associated with Old World begomoviruses, contain a endemic or epidemic form.Material and Methods: Four single ORF coding for a replication-associated protein excursions were conducted in the Mocambo forest, with similarity to those of and an A-rich EMBRAPA, Belém city, Pará state, Brazil (August and region. Unlike typical satellites, alphasatellites are capable of self-replication in host plants but require days each. The arthropods collection was conducted a begomovirus for movement within the plant and for byOctober protected 2013, and January enlightened and March human 2014), attraction lasting in fivesoil insect transmission. In screening symptomatic non- cultivated plants collected in Brazil and Cuba for the and separated in inoculation groups and stored at -70 °and C freezer. canopy The modalities. groups were Arthropods inoculated were in newborns identified albino Swiss mice, and alternatively in VERO and ofpresence ssDNA of satellites begomoviruses half the by sizerolling of circlebetasatellites amplification and C6/36 cells. Results: In the excursions it was collected (RCA), we identified two alphasatellites and a novel class 2.772 culicidae, divided into 185 groups for virus Euphorbia heterophylla and Sida sp. plants sampled in isolation attempted. In total 12 genera with 36 species Chapadaalphasatellites. (state Theof Rio alphasatellites Grande do wereSul, Brazil) amplified infected from by the bipartite begomovirus Euphorbia yellow mosaic species were considered,as follows. Constants during virus and were found to be phylogenetically related to theof Mosquitoesexcursion time: were Aedes identified. hortator Among and Aedes the identified serratus, alphasatellites recently reported from the state of Mato among others; some of the Accessory: Johnbelkinia Grosso do Sul (Brazil), Cuba and Venezuela. The small longipes, Limatus durhamii; Accidental: Aedes fulvus, ssDNA satellites were found associated with bipartite Aedes taeniorrhynchus, Aedes oligopistus, among others. begomoviruses infecting malvaceous species in Cuba. The species caught, Coquillettidea venezuelensis were They do not possess any ORFs, contain an A-rich region, and share a short conserved region with betasatellites. arribalzagae, as Subdominant had Aedes serratus; Our results extend the diversity and geographical range Eventually:classified as Mansonia Eudominant; titillans as species; Dominant: Finally Coquillettidea had some of ssDNA satellites associated to bipartite begomoviruses Rare species like Sabethes belisarioi, Coquillettidea in the Americas, suggesting that they may be widespread albicosta and Aedes fulvus. Groups of inoculated insects in the continent. Financial support: CAPES, CNPq into newborn mice tested shows negative. However (fellowship Pesquisador Visitante Especial to JNC) and there was viral isolation of a new arbovirus of the FAPEMIG (fellowship Pesquisador Visitante to EFO). Bunyaviridae family, Orthobunyavirus genus, a group of Aedes fulvus (strain AR800584) inoculated into PIV344 - FAUNA STUDY AND VIRUS ISOLATION C6/36 cells. Conclusion: Important arboviruses vectors ATTEMPTS IN CULICIDAE CAUGHT IN THE MOCAMBO were collected as Sabethes belisarioi, Coquillettidea AREA, EMBRAPA, BELEM, PARÁ, BRAZIL venezuelensis, Haemagogus janthinomys, Sabethes Neto, J.P.N.1; Castro, K. da S.1; Monteiro, H.A. de O.1; glaucodaemon, Sabethes cloropterus, among others. A Castro, F.C.1; Saraiva, H.A.C.1; Segura, M. de N.O.1; new arbovírus was isolated belonging to the Bunyaviridae Júnior, J.W.R.1; Nascimento, B.L.S.1; Carvalho, V.L.1; family, Orthobunyavirus genus. The study demonstrates Tesh, R.B.2; Vasconcelos, P.F. da C.1 the need to conduct further research into the Mocambo 1. IEC - Instituto Evandro Chagas, Rodovia forest, emphasizing the arboviruses main vector species, BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 entomological monitoring and performing viral isolation 2. UTMB - Department of Pathology, The attempts. Financial support: PIBIC/CNPQ/IEC University of Texas Medical Branch at Galveston,301 University Boulevard, Galveston, Texas, 77555-0144

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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PIV360 - NEXT-GENERATION SEQUENCING APPLIED we found highly divergent contigs (6,596 and 2,674 bp) FOR IDENTIFICATION OF VIRUSES IN WATERMELON related to Bunyaviridae L and S segment, respectively. (CITRULLUS LANATUS) PLANTS Overall, we were able to identify viruses already Quirino, M.S.1; Ribeiro, B.M.1; Aguiar, R.W. de S.2; described infecting watermelon as well as novel viral Orílio, A.F.1; Melo, F.L.1 species that are currently been characterized in our laboratory. 1. UnB - Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília - DF, 70910-900 PIV366 - DETECTION OF POTATO VIRUS Y (PVY) 2. UFT - Universidade Federal do Tocantins, STRAINS IN PLANTS WITH SINGLE AND MIXED Avenida NS 15, 109 Norte - Plano Diretor Norte - Palmas - INFECTION TO, 77001-090 Costa, S.B.F.G.; Santos, B.A.; Figueira, A. dos R.; Duarte, The culture of watermelon (Citrullus lanatus Thunb) P. de S.G. belongs to the Cucurbitaceae family and it is susceptible UFLA - Universidade Federal de Lavras, Câmpus to several viral pathogens. These viral infections do not Universitário, Lavras - MG, 37200-000 cause obvious symptoms in its host plants and, frequently, the viral particles occurs in low concentrations. However, Potato virus Y (PVY) is currently one of the most the capacity to analyze asymptomatic viral infections important viruses in potato worldwide, mainly due to or coinfections increased with the appearance of high throughput sequencing. Thus, this study was designed appearance of new genetic variants. The detection and its high field dissemination capacity and the constant to identify viruses present in symptomatic watermelon plants. The watermelon leaves with viral symptoms were Among the mechanisms responsible for the genomic field management of PVY has been increasingly complex. collected from September to December 2013 in three variability, the recombination is the main one, probably producing regions of Tocantins state. The plant material this study, the DAS and TAS-ELISA and RT-PCR multiplex due to the occurrence of mixed infections in the field. In centrifugation through a sucrose cushion. Total RNA technique were compared, aiming at determining its was subjectedextracted tofrom semi-purification this viral enriched of the fraction viruses using “RNeasy Plant Mini Kit” (Qiagen). Next, the RNAseq strains present in Brazil (PVYO, PVYN-Wi and PVYNTN), efficiency for diagnosing and discriminating the main library was prepared and sequenced by Illumina HiSeq in single and mixed infections. In order to get plants 2000 platform. Forty million reads were generated, and with single and mixed infections, Nicotiana tabacum after quality trimming and de novo assembly using CLC cv. Turkish plants were inoculated with these strains, individually and in combination. In addition, probes contigs were obtained. All contigs were submitted to blastxGenomics against Workbench the viral RefSeq software, database nearly and fifty 4,912 thousand contigs strains by real time PCR (qPCR). Willing to detect mixed were developed for detection and quantification of these these results, we submitted these selected contigs to were collected and analyzed by DAS and TAS-Elisa and infections in the field, ninety potato tubers PVY infected blastxproduced against hits withthe viralGenBank sequences. non-reduntant To further database confirm RT-PCR multiplex. The N. tabacum plants inoculated (nr database), and only the plant viruses were selected. with the different strains reacted with the expected We were able to identify viral genomes belonging to the symptoms, ranging from light mosaic to necrosis, families , and Bunyaviridae. according to the strain. However, in all combinations The largest contig (10,371 bp) showed 91% identity in which the PVYO was inoculated with the necrotic with Papaya ringspot virus. One contig of 1,704 bp contig strains, the plants did not present the expected vein presented 98% identity with Zucchini yellow mosaic necrosis, indicating a predominance of the symptoms virus. We also found two contigs of 1,575 and 1,606 induced by the common strain. The serological tests bp related to the segments 1 and 2 of Partitiviridae. revealed a tendency of higher concentration of strains Interestingly, these contigs presented an overall identity with O serology (PVYO and PVYN-Wi) when N. tabacum of 60%, suggesting these contigs represent a new viral presented mixed infections. The RT-PCR was capable of species belonging to the Partitiviridae family. Moreover, discriminating the tree strains, presenting the patterns September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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plasmid DNA and automated Sanger sequencing. Ribo- tested 5 were negative, 11 were positive for the PVYNTN, probes were labeled with digoxigenin (Dig RNA labeling andof bands 74 were specific positive to each for of thethem. common Among and/orthe 90 tubersWilga mix, Roche) by in vitro transcription with T7 and SP6 RNA strain, with 8 being positive for mixed infections. The occurrence of mixed infections and highly favorable The detection sensitivity of the probes was measured climate for the multiplication and dissemination of the bypolymerases dot-blot analysisaccording of to serial manufacturer’s dilutions specifications.of total RNA PVY could explain the high genomic variability of this extracted from leaf tissue of Nicotiana benthamiana virus derived from recombination among virus isolates. and D. stramonium infected with TSWV and GRSV The probes and primers designed were effective in (Groundnut ringspot virus) and healthy plants. Probes discriminating each strain by qPCR, either in single or mixed infections. As qPCR presents a higher sensitivity, detection was 20 ng of total RNA. Therefore, molecular the designed probes revealed a good potential to be ribo-probesshowed high were specificity produced to TSWV for each and segment in general and limit strand of orientation of TSWV which will support to distinguish strains in infected potato plants.FINANCIAL SUPPORT: between introduced RNA transcription and that derived Capes,employed CNPq, for Fapemig the detection and quantification of these from viral replication complex of RG system. This work is essential in our RG research strategies and can provide PIV377 - DEVELOPMENT OF MOLECULAR TOOLS TO STUDY TOSPOVIRUS REVERSE GENETICS accumulation and replication of the viral genome. This Leite, F. de S.; Lopes, A.P. de M.; Bertran, A.G.M.; informationimportant data is necessary regarding to strandplace in and time segment the many specific events Resende, R. de O.; Orílio, A.F. that are part of the infectious process, such as cell-to-cell UnB - Universidade de Brasília, Campus Universitário infection, systemic infection, accumulation and activity Darcy Ribeiro, Brasília - DF, 70910-900 of defective-interfering RNAs and their effects over the other viral genomic segments. The use of methods based on recombinant DNA technology such as reverse genetics (RG) to study the PIV385 - MOLECULAR ANALYSIS OF SQUASH MOSAIC role of viral gene products in inducing disease has VIRUS (SQMV) ISOLATES FROM TOCANTINS - BRAZIL increased substantially our knowledge of the complex Edreira, N.1; Figueira, A.1; Nascimento, I.2; Balani, D.; steps and interactions involved in the infectious cycle Duarte, P.S.G.1 of plant viruses. Tospovirus [(-) ssRNA], are worldwide spread pathogens mainly of vegetable, causing severe 1. UFLA - Universidade Federal de Lavras, economical losses, to which a RG system has not been Câmpus Universitário, Lavras - MG, 37200-000 developed yet. Currently, our research line focuses on 2. UFT - Universidade Federal do Tocantins, Avenida NS 15, 109 Norte - Plano Diretor Norte - Palmas - the development of both a mini-genome approach and TO, 77001-090 a classical full-length infectious clone strategy towards tospovirus RG. This work presents the development Several viruses can affect cucurbits causing considerable losses in quality and quantity of production. High to support our RG research. First, primers for RT- incidences of the mosaic caused by Squash mosaic virus of highly specific and sensitive ribo-probes generated regions of the L, M (NSm gene) and S (NSs and N regions, and those incidences depend on numerous (SqMV) have been detected in the field of some Brazilian genes)PCR amplification segments of weretomato synthesized spotted wilt for virus specific (TSWV). RNA Subsequently, total RNA was obtained from Datura In this study the coat protein gene of fourteen Brazilian factors such as temperature, field location and vectors. stramonium plants infected with TSWV (BR-01 isolate) isolates of Squash mosaic virus (SqMV) collected in using TRIzol reagent (Invitrogen) followed by RT Tocantins state, named PTY1, PTY2, PTY4, PTY5, PTY10, reaction with SuperScriptIII (Invitrogen) and standard PTY12, PTY14, PTY15, FA, GR2, LC2, LC3, PN1 and PN2, PCR. The amplicons were cloned in the vector pGEM-T easy (Promega) and positive colonies were selected other isolates available in the GenBank. The identity were amplified by RT-PCR, analyzed and compared with by standard restriction enzyme digestion of extracted between the nucleotides of Brazilian SqMV isolates

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

216 Plant and Invertebrate Virology: PIV ranged from 86% to 100%, while the identity of these isolates with SqMV isolates from the GenBank varied a viral enriched fraction, the leaves were ground in between 86% and 88%. The lowest identity (87%) was experimental field of Embrapa Beef Cattle. To obtain observed among the isolates AB054689 from Japan and cushion. Viral RNA was extracted using RNeasy Mini PTY1 PTY10, PTY12, PTY14, PTY15, FA1, GR2 and LC2, KitPBS following buffer, filtred the manufacturer’s and centrifuged instructions. through aThe sucrose RNA and also among the PN2 and the SqMV isolates from samples were pooled and sequenced at Macrogen INc. the database. The amino acid identities of the Brazilian (Korea) using Illumina HiSeq 2000 technology. We isolates ranged from 95%, among the isolate PN2 and obtained approximately 20,299,626 of reads, which were all the other isolates, to 100% among PTY1 and PTY10, trimmed and assembled de novo using CLC Genomics PTY12, PTY14, PTY15, FA1, GR2 and LC2 isolates; PTY2 Workbench 7.0. The assembled contigs (3,254) were and PTY5; LC3 and PN1. When compared with isolates submitted to blastx against the RefSeq Viral database from the GenBank, identities ranged from 91% (between and the contigs related to plant viruses were selected. isolated PN2 and DQ868881.1 and EU421060.1, both We were able to identify complete genomes of viruses Chinese isolates, and the American isolate AF059533.1) from some families: Potyviridae, to 97% (among several isolates). The phylogenetic tree based on the nucleotide sequence showed two major contigs related to Johnsongrass mosaic virus and two clusters, one containing the Brazilian isolates PTY2, highlyand Tymoviridae. divergent genomes Among related Potyviridae, to Rose yellow we identified mosaic LC3, PTY5, PN1, PN2 and PTY4 and the SqMV isolates and Blackberry virus Y, which probably constitute two from the database, and the second group composed by novel genera within Potiviridae family. Moreover, we the remaining Brazilian isolates. However it changed found genomes related to Maize chlorotic dwarf virus when the phylogenetic tree was constructed based on (Seconviridae) and to a novel species/genus in the family the sequence of amino acids. Several Brazilian isolates showing nucleotide substitutions resulted in amino virus in the surveyed hosts Panicum sp., Brachiaria sp acid changes, characterizing the nom synonymous andTymoviridade. Stylosanthes We sp.design Johnsongrass specific primers mosaic to virus,detect Roseeach yellow mosaic virus, Blackberry virus Y and Maize variability,substitutions genomic type and studies confirming of SqMV the isolates large should variability also RT-PCR Host range and phylogenetic analysis are being includeexisting theiramong RNA2 Brazilian fragment, SqMV inisolates. order Toto confirmprovide thisthe conductedchlorotic dwarf for further virus characterization were already amplified of these backviruses. by necessary support for breeding programs which aim to FINANCIAL SUPPORT: CNPQ/REDE PRO-CENTRO- develop SqMV resistant cucurbits plants. FINANCIAL OESTE, FAP-DF, CAPES SUPPORT: CAPES, CNPQ, FAPEMIG PIV406 - PARTIAL CHARACTERIZATION OF PIV398 - NOVEL VIRAL GENUS AND VIRAL SPECIES BEGOMOVIRUS STRAIN FROM CLITORIA INFECTING FORAGE PLANTS IN BRASIL FAIRCHILDIANA ON RIO DE JANEIRO STATE, BRAZIL Silva, K.N.1; Nicolini, C.1; Melo F.L.1; Silva, M.S.2; Brioso, P.S.T. Fernandes, C.D.3; Nagata, T.1; Resende, R.1 UFRRJ - Universidade Federal Rural do Rio de Janeiro, 1. UnB - Universidade de Brasília, Campus Rodovia BR 465 - Km 7 - Campus Universitário, Seropédica - Universitário Darcy Ribeiro, Brasília - DF, 70910-900 RJ, 23851-970 2. EMBRAPA RECURSOS GENÉTICOS E The Sombreiro (Clitoria fairchildiana) is a plant used BIOTECNOLOGIA, Parque Estação Biológica - PqEB s/ in urban landscaping and for medicinal purposes nº.Brasília - DF, 70770-901 (by extracting rotenoids from seeds that has anti- 3. EMBRAPA GADO DE CORTE, Parque Estação Biológica - PqEB s/nº. Brasília - DF, 70770-901 to identify the viral genus associated with mosaic in Panicum sp., Brachiaria sp and Stylosanthes sp. showing leavesinflammatory of C. fairchildiana, activity). The existingobjective in of Rio this de study Janeiro. was mosaic symptoms on leaves and growth reduction Mechanical inoculation for Chenopodium amaranticolor were collected in the State of Mato Grosso do Sul in the were performed in sodium phosphate buffer pH 7.5

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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weight of the deduced P26 protein was calculated and and 1:20; transmission by grafting cuttings of diseased is presented in the context of their genomic positioning. tocontaining healthy plant; 0.1% transmission sodium sulfite by atseed dilutions originating 1:5, from 1:10 The Bayesian phylogenetic tree obtained from the p26 diseased plant; PCR test with primers for Begomovirus. copies found in NPVs genomes showed four clearly Negative results were obtained in the mechanical inoculation and transmission by seed, however, positive hypothesis for the occurrence of three independent results have occurred in transmission by grafting and in capturedefined events clades of (IA, the IB, p26 IIA gene and by IIB) baculoviruses. supporting The the presence and location of signal peptide cleavage sites in Begomovirus on this plant species. P26 amino acid sequences was analysed and only in the the PCR test. This is the first occurrence in the world of clade IB was found signal peptide. Although the function PIV435 - IDENTIFICATION OF THE P26 GENE AND ITS of P26 is not well understood, the signal peptide may EVOLUTION IN THE BACULOVIRIDAE FAMILY lead to differences in activity of the clade IB proteins Craveiro, S.R.1; Inglis, P.W.2; Grynberg, P.2; Togawa, indicating a possible distinct function from other classes R.C.2; Ribeiro, Z.M.A.2; Castro, M.E.B.2 of P26. However, further investigations are needed for 1. UnB - Universidade de Brasília, Campus a better understanding of this protein in baculoviruses. Universitário Darcy Ribeiro, Brasília - DF, 70910-900 FINANCIAL SUPPORT: Capes/Embrapa 2. EMBRAPA RECURSOS GENÉTICOS E PIV446 - GENOME SEQUENCING OF TWO BIOTECNOLOGIA, Parque Estação Biológica - PqEB s/ nº.Brasília - DF, 70770-901 INFECTING PRICKLY PEAR (OPUNTIA COCHENILLIFERA) The family Baculoviridae comprises a group of arthropods- Lamas, N.S.2; Sanches, M.M.2; Freitas, D.M.T.A.2; Reis, M.B.A.2; Sousa, J.G.A.2; Romano, E.2; Melo, F.L.1; Baculoviruses exhibit rod-shaped nucleocapsids Ribeiro, S.G.2 embeddedspecific viruses in a crystalline with circular protein double-stranded matrix composed DNA. of polyhedrin in nucleopolyhedroviruses (NPVs) and 1. UnB - Universidade de Brasília, Campus granulin in granuloviruses (GVs). The Baculoviridae family Universitário Darcy Ribeiro, Brasília - DF, 70910-900 is composed of genera Alphabaculovirus (lepidopteran- 2. EMBRAPA RECURSOS GENÉTICOS E BIOTECNOLOGIA, Parque Estação Biológica - PqEB s/ nº.Brasília - DF, 70770-901 specific NPVs), (lepidopteran-specific Viruses from the genus Potexvirus are widely spread AlphabaculovirusesGVs), are also divided (hymenopteran-specific into Groups I and in different genera of the family Cactaceae. Prickly Pear IINPVs) based and on Deltabaculovirustheir envelope fusion (dipteran-specific proteins GP64 NPVs).and F, (Opuntia cochenillifera) plants collected in Pernambuco respectively. The p26 gene is present in all Group I and II State were propagated and cultivated in the green house. NPVs. However, the majority of the virus with more than RNA from symptomless plants was extracted from one p26 copy belongs to Group II NPVs. The function of 45 plants and used for whole transcriptome shotgun sequencing, using an Illumina Hi-seq 2000 platform, required for optimal virion occlusion in the polyhedron. aiming to study water stress related genes. De novo the p26 gene is not well-defined, but p26 is thought to be assembly of the reads was made with VELVET program far sequenced and was used bioinformatics tools in and several contigs were found to be potexvirus- orderThe p26 to characterize gene was identified the P26 withinprotein NPVsand elucidate genomes the so derived. To further characterize these virus sequences, evolution this gene in Baculoviridae family. The p26 we used Geneious software to perform Blastn searches. copies found in baculoviruses are conserved in position, Comparisons of the nucleotide (nt) and the predicted adjacent to the p10 gene (P1) in all the NPVs containing amino acids (aa) sequences with other potexvirus a single copy, adjacent to iap-2 gene (P2) in all Group II members were made with SDT and CLUSTALW programs. NPVs containing the second p26 copy and adjacent to the ptp1 and ptp2 genes (P3) in Group I NPVs with the second p26 copy. The isoelectric point and molecular Two distinct genomes were identified. One genome comprises 6.636 bp and was identified as September/October 2014 Volume 19 – Supplement 2 - Abstracts/PostersSchlumbergera - Plant and Invertebrate virus Virology: X (SchVX), PIV with a nt identity of XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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94% for the entire genome sequence. The identities for plants will be inoculated with ToCMoV to check for the nt sequences of the polymerase and CP genes are 93% and 94%, respectively. The predicted aa sequences disease phenotype and virus accumulation. FINANCIAL for the polymerase and CP genes share 97% identity SUPPORT:the influence EMBRAPA, of TMV-RRP CNPQ, INCTIPP, super-expression FAPDF. in the is 6.664 bp. The nt sequence of the entire genome has PIV466 - IN SILICO ANALYSIS OF BACULOVIRUS CORE highestwith SchVX. identities The of other 73% assembled to 74% with genome different (OcPotex) strains GENE PROMOTERS Andrade, M. de S.; Pacheco, T.J.A.; Ribeiro, B.M.; Melo, The nt and aa sequences of OcPotex polymerase and CP F.L. of Cactus virus X (CVX) and Zygocactus virus X (ZyVX). LABORATÓRIO DE VIROLOGIA/UnB - Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília - taxonomicgenes also assignment share similar of OcPotex. identities FINANCIAL with those SUPPORT: of CVX DF, 70910-900 EMBRAPA,and ZyVX, requiringCNPq, FAP-DF additional studies for a conclusive The baculoviruses are large dsDNA insect PIV465 - INVOLVEMENT OF TMV RESPONSE viruses with a high number of described species RELATED-PROTEIN IN THE DEFENSE OF TOMATO TO TOMATO CHLOROTIC MOTTLE VIRUS Alphabaculovirus, Betabaculovirus, Gammabaculovirus andinfecting Deltabaculovirus. several hosts, To classified date, 61 inspecies four genus,have been the Villeth, G.R.C.1; Lacerda, A.L.1; Lacorte, C.1; Brasileiro, completely sequenced and are publicly available in A.C.M.1; Boiteux, L.S.2; Ribeiro, S.G.1 GenBank. The analysis of these genomes revealed 37 1. EMBRAPA RECURSOS GENÉTICOS E core genes shared by all of them. These core genes BIOTECNOLOGIA, Parque Estação Biológica - PqEB s/ participate in basic biological functions, such as RNA nº.Brasília - DF, 70770-901 transcription, DNA replication and the formation of 2. EMBRAPA HORTALIÇAS, Parque Estação the virion structure. Moreover, most baculovirus genes Biológica - PqEB s/nº.Brasília - DF, 70770-901 Begomoviruses currently represent one of the most cellular RNApol II (early genes) and those transcribed serious problems for the tomato cultivation worldwide. bycan viral be classified RNApol in(late two genes). groups: Recently, those transcribed the AcMNPV by In Brazil, breeding for resistance to bipartite tomato transcriptome was published and the transcription start begomoviruses allowed to the development of the sites (TSS) for its ORFs were mapped and putative motifs resistant line ‘LAM-157’ (carrying tcm-1 locus) which is a were described. However, it remains to be determined if near-isogenic line to the susceptible Santa Clara cultivar. the observed pattern is conserved through baculovirus To study the interaction and the differential genotype evolutionary lineages. Therefore, we performed an response to Tomato chlorotic mottle virus (ToCMoV), a in silico analysis of baculovirus core gene putative transcriptomic analysis using RNA sequencing (RNAseq) was carried out. Several genes showed differential of ATG) from all Alphabaculovirus and Betabaculovirus expression in resistant ‘LAM-157’ plants inoculated promoters. Firstly, the core genes (+ 1000 bp upstream with ToCMoV. One of these genes, the TMV response- late (6,4 and 27, respectively) according to AcMNPV related protein (TMV-RRP) gene, putatively involved data.(57) wasThe extractedpredicted and TSS classifiedwere manually in early, annotated early/late for and all in plant response to Tobacco mosaic virus infection, late genes (TAAG motif). Subsequently, we calculated the distance of TSS to ATG and the variation was higher for Alphabaculovirus than for Betabaculovirus. Moreover, timeshowed course significant of virus up-regulationinfection in ‘LAM-157’, (log2 fold highlighting change > the distance variation was higher for those genes with the2.5). possible RNAseq involvement results were of confirmedTMV-RRP in by the qPCR process over of a TSS that does not overlap with another ORF. We screened tomato defense/resistance to ToCMoV. To validate this the early promoter for known insect transcription bactor hypothesis in planta, LAM-157- derived TMV-RRP gene binding sites (TFBS). We found two recurrent TFBS: was cloned and transferred to Nicotiana benthamiana HSF and Dfd, both TFBS described in insects. Finally, we plants by Agrobacterium transformation. Transgenic analyzed the early data set in MEME and CentriMo, and

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

219 Plant and Invertebrate Virology: PIV we found different motif for each early core gene, thus, With this new analysis, the 3’ block of this putative we suggest that the early promoters are more complex polerovirus genome was almost completely recovered. and variable than late promoters and other news motifs Only a small portion of the 3’UTR region is absent. The play an important role in transcription regulation in complete CP, MP and RT proteins sequences are almost addition to TATAA box motif. Overall, the late promoter identical of those of the CLRDV isolates. However, the was more conserved than early promoters, suggesting intergenic region and the almost complete polymerase that the promoters of genes transcribed by host RNA (P2) show identities ranging from 73-82% with CLRDV. polymerase are adapted to the host gene regulatory P2 amino acid sequence share similar ID levels with network. Financial suport: CNPq Brassica yellow virus, Pepper yellow leaf curl virus and Beet western yellows virus. Primers were design based PIV471 - SEQUENCING OF NEW POLEROVIRUS in the genome reconstruct and were used to RT-PCRs COTTON VIRUS FROM BRAZIL USING SMALL RNA using three virus independent isolates. The nucleotide DATASET OBTAINED BY DEEP-SEQUENCING sequence comparison of the amplicons showed the Silva, D.C.P.S.; Silva, A.K.F.; Silva, D.C.P.S.; Vaslin, M.F.S. maximum identity of 73% with CLRDV and 71% with UFRJ - Universidade Federal do Rio de Janeiro, Av. Pepper yellow leaf curl virus. In order to obtain the Pedro Calmon, 550 - Cidade Universitária, Rio de Janeiro - complete genome sequence of this virus, more primers RJ, 21941-901 combinations are been tested. FINANCIAL SUPPORT: FAPERJ AND CAPES recombinants virus isolates infecting cotton plants in PIV474 - THE PHYLODINAMIC AND In a previous work, our group identified three Brazil which symptoms and transmission mechanism are PHYLOGEOGRAPHIC OF TOMATO CHLOROTIC SPOT closely related with the Cotton blue disease (CBD). CBD is VIRUS: VIRAL SPREAD IN THE AMERICAS an important cotton crop pathology present in America, de Almeida, M.M.S.1; Lucas, F.M.1; Martinez, R.T.2; Oliveira, R.1 In Brazil it obligate the cotton farmers to plant only CBD resistantAfrica and cultivars Asia that now causes days. significant The disease economic is transmitted losses. 1. UnB - Universidade de Brasília, Campus by Aphis gossypii and is caused by a Luteoviridae virus, Universitário Darcy Ribeiro, Brasília - DF, 70910-900 genus Polerovirus, the Cotton leaf roll dwarf virus 2. IDIAF - Instituto Dominicano de (CLRDV). Typical symptoms include stunting due to Investigaciones Agropecuarias y Forestales, Evaristo Morales, Santo Domingo, República Dominicana internodal shortening, leaf rolling, intense green foliage. Viruses in the genus Tospovirus (family Bunyaviridae) CLRDV molecular diagnosis, were recovered from plants are plant pathogens transmitted by insects known as showingThe isolates CBD identifiedatypical symptoms. in this previous Analyses works, of a partial using thrips, with a trisegmented single-stranded ambisense polymerase sequence of these virus isolates showed RNA (S, M and L) genome and the ability to replicate that they presented low identity with CLRDV and can in vector to. The diversity of the genus comprises 8 be considered as representatives of a new species of the recognized species with a variable host range and genus Polerovirus. In orther to better characterize this geographic distribution. The genus Tospovirus are new species, named Cotton red leaf virus (CoRLV), we widespread in the Americas, where Tomato chlorotic submitted the isolate CoRLV-P01 to a deep sequencing spot virus (TCSV), Tomato spotted wilt virus (TSWV), by “Illumina”. All small RNAs of an infected plant were Groundnut ringspot virus (GRSV), Chrysanthemum sequenced and “contigs” were constructed. However, the stem necrosis virus (CSNV), Zucchini lethal chlorosis contigs weren’t able to reconstruct the viral genome. So, virus (ZLCV), Impatiens necrotic spot virus (INSV), Iris a new analysis of the small RNA data set libraries was yellow spot virus (IYSV), Alstroemeria necrotic streak performed using the software SearchSmallRNA (Andrade virus (ANSV), Melon severe mosaic virus (MeSMV) & Vaslin, 2014). Using CLRDV Brazilian and Argentine and Bean necrotic mosaic virus (BeNMV) have been in independent analysis as reference genome, we were able to obtain 69% of the complete virus genome. 1990’s, with GRSV. Nowadays, GRSV is present in both reported. TCSV was first noticed in Brazil in the early September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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in the Americas. A phylodinamic study was conducted the surveys for begomovirus infection in soybean- tonew infer world the and TCSVthe old spread. world, whereasThirty-three TCSV issequences find only producingin Argentina. areas. This Samples potential obtained threat hasfrom intensified soybean corresponding to RNA S with coding (N protein) and plants collected at Federal District, showing typical no-coding regions were downloaded from GenBank. symptoms of begomovirus infection were analyzed. The alignment was carried out using MUSCLE software. Viral DNA from infected plants was subjected to rolling- The phylogenetic and phylodinamic relationships were inferred by BEAST package using Bayesian Evolutionary Phylogenetic analyses and sequencing comparisons of Analysis. According to the sampling year, the spatial circle amplification (RCA), cloning and sequencing. phylogenetic reconstruction and the dynamic evolution Sida micrantha mosaic virus (SimMV). Cloned DNA-A were determined by SPREAD program. Probably, TCSV andthe DNA-ADNA-B components and DNA-B clones,were introduced confirmed into infection seedlings by has two different origins in South America, one in Brazil of Phaseolus vulgaris ‘Pérola’, Glycine max ‘Conquista’ (S45325) and the other in Argentina (U49709, U49707), and ‘Williams 82’, Solanum lycopersicum ‘Santa Clara’, in 1996. Brazilian lineage spread to the country to North Nicotiana benthamiana and Sida rhombifolia by a biolistic America and Caribe. Once in Caribe, it was introduced after inoculation, symptoms of begomivrus infection appearedmethod to in confirm almost theirall plants infective species capacity. inoculated, Four onlyweeks S. intra-speciesback in North isolates. America. This The new first isolate introduced has the in S Northand L lycopersicum and Phaseolus vulgaris ‘Pérola’ showed no RNAsAmerica from most GRSV likely and in the 1994, M RNA led fromto the TCSV. first Thereassortant isolates symptoms and no infection. Leaves from soybean and originated from the second introduction in the North Sida rhombifolia exhibited yellow and golden mosaic, America around the year 2006 are so close related to yellow vein, chlorotic mottling, necrosis, blistering, leaf Caribbean isolates, at least 97% identical. Comparing the sampled in 2009, the nucleotide sequence accumulated distortion and dwarfing symptoms; symptoms such as first report of TCSV in Brazil with the last (JQ034525) dwarfing, leaf distortion and blistering were observed in isolate is more related to Argentine lineage sharing 99% andN. benthamiana sequencing plants.of the Theobtained infection amplicon. was confirmed This is the by identity,modifications with resultinga clearly in well93% supportedidentity each phylogenetic other. That PCR amplification using begomovirus universal primers reconstruction. There is not an updated information SimMV and these clones will be a valuable tool to study about the strain that are present nowadays in Brazil. soybean-begomovirusfirst report of infectious interaction.FINANCIAL clones of soybean-infecting SUPPORT: EMBRAPA, CNPQ, INCTIPP, FAPDF. PIV479 - CHARACTERIZATION OF SOYBEAN- INFECTING SIDA MICRANTHA MOSAIC VIRUS PIV485 - HIGH GEMINIVIRUS-INFECTION ON A Freitas, D.M.T.A.2; Fusaro, A.2; Lacorte, C.2; Boiteux, POTATO FIELD GROWN FOR SEED PRODUCTION IN L.S.1; Ribeiro, S.G.2 BRAZIL CENTRAL Lima, M.F.1; Barriolli, C.M.C.2; Santos, D.I.3 1. EMBRAPA HORTALIÇAS, Parque Estação Biológica - PqEB s/nº.Brasília - DF, 70770-901 1. EMBRAPA HORTALIÇAS, Parque Estação 2. EMBRAPA RECURSOS GENÉTICOS E Biológica - PqEB s/nº.Brasília - DF, 70770-901 BIOTECNOLOGIA, Parque Estação Biológica - PqEB s/ 2. UNIP - Universidade Paulista, Av Paulista, nº.Brasília - DF, 70770-901 900 São Paulo - SP, 13720-000 The occurrence of begomoviruses in soybean (Glycine 3. FACULDADE ANHANGUERA, Rua max) in Brazil has been sporadically reported and has Alameda Maria Tereza, 2000 Valinhos, São Paulo - SP, 13278- 181 not been associated with yield losses. However, the list of soybean-infecting begomoviruses it is growing, Virus diseases are one of the main causes of yield losses in and there is a concern that yield losses may increase, potato (Solanum tuberosum L.) production worldwide. since soybean infecting begomoviruses causing The main viruses associated with this crop belong to the moderate to severe losses has been recently reported genera , Polerovirus, and Potexvirus;

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

221 Plant and Invertebrate Virology: PIV however, emerging diseases caused by geminivirus PIV520 - COTTON MICRORNAS GHR-MIR2910 AND GHR-MIRNA162 ARE UPREGULATED DURING tabaci) had been detected affecting this crop over the last COTTON LEAFROLL DWARF VIRUS (CLRDV) (Family Geminiviridae) transmitted by whitefly (Bemisia INFECTION Cupido, Manitou, Faluka, Mustang and Ambition of ca. Vaslin, M.F.S.1; Romanel, E.2; Silva,T.F.2; Silva, D.C.P.S.1; years. In July, 2014, a 50-day-old potato field, cvs. Agata, 50ha showing geminivirus-like symptoms such as strong Corrêa, R.L.1 yellow mosaic, leaf reduction size with the edges turned upwards and stunting of plants, was observed in the 1. UFRJ - Universidade Federal do Rio de Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de State of Goiás, Brazil, one of the most important potato Janeiro - RJ, 21941-901 growers in the country. A variable range (2%-40%) of 2. USP - Universidade de São Paulo, Av. Prof. symptoms incidence was observed depending on the Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 leaf samples were collected from symptomatic plants Small RNAs (sRNAs) are a class of non-coding RNAs andcultivar subjected planted to and total its DNAposition extraction in the field. for polymeraseForty-eight ranging from 20- to 40-nucleotides (nts) that are chain reaction (PCR) using universal primers and rolling present in most eukaryotic organisms. In plants, sRNAs are involved in the regulation of development, besides restriction fragment length polymorphism the maintenance of genome stability and the antiviral (RFLP)circle amplification to access diversity (RCA) of for the geminivirus isolates. In detection,addition, response. Viruses, however, can interfere with and exploit sample were also tested by using serological methods the silencing-based regulatory networks, causing the (DAS-ELISA) for detection of Potato virus Y (PVY), Potato deregulation of sRNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs). To understand the virus (PLRV) using polyclonal antibodies. All samples impact of viral infection on the plant sRNA pathway, we didvirus not X (PVX),react with Potato neither virus of S the(PVS) polyclonal and Potato antibodies leafroll deep sequenced the sRNAs in cotton leaves infected with tested, indicating that none of these viruses was involved Cotton leafroll dwarf virus (CLRDV), which is a member in the cause of the symptoms observed in potato plants. of the economically important virus family Luteoviridae. However, a band of ca. 1,2 kb was visualized on a 1,2% A total of 60 putative conserved cotton miRNAs were agarose gel stained with ethidium bromide from the of these miRNAs were clearly misregulated during identified, including 19 new conserved miRNAs. Some they were geminivirus-infected. These data are of great viral infection, and their possible role in symptom majority of the potato samples collected confirming development and disease progression is discussed. seed production. Also, PCR-positive samples produced Furthermore, we found that the 24-nt heterochromatin- anconcern RCA consideringproduct, indicating that that thepotato high field sensitiveness was grown forof associated siRNAs were quantitatively and qualitatively altered in the infected plant, leading to the reactivation existence of low variability among these geminivirus of at least one cotton transposable element. This is the isolates.the technique. Cloning Restriction and sequencing enzyme of profilesthese isolates suggest are the in progress. These data indicate that despite the existence virus-infected cotton plants. Our results indicate that first study to explore the global alterations of sRNAs in of some reports of occurrence of geminivirus in potato some CLRDV-induced symptoms may be correlated with the deregulation of miRNA and/or epigenetic networks. of these viruses in the crop, considering the presence of Ghr miR2910 was 7 to 7.5 times more expressed during fields, however, is it necessary to keep the monitoring infection. His role is still unknown, and its possible target would be the 18S ribosomal RNA. Besides him, virus sources as well as high whitefly populations in the the GHr miR-171, 157, 172 and 162 expression were field and its ability to colonize many plant species. also increased in the infection. On the other hand, the miRNA miR319, 393, 3476, 2111, 355, 159 have been drastically reduced in the infection. RT-PCR reactions in

expression of these different miRNAs. MiRNAs exclusive real time were carried out and confirmed the differential September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

222 Plant and Invertebrate Virology: PIV cotton, as the 2110 and the 3476 are now being studied State of São Paulo. Symptomatic and asymptomatic to try to understand its function. Dysregulation of samples in 10 counties were collected and submitted metabolic pathways regulated by these miRNAs might to biological, serological and molecular tests. Virus explain the symptoms observed in plants showing the transmission was obtained by mechanical inoculation disease in cotton blue. FINANCIAL SUPPORT: CAPES, and by grafting, being observed typical symptoms on CNPQ AND FAPERJ peanut and other indicator hosts. The enzyme-linked immunosorbent assay (ELISA) and ImmunoStrip tests PIV521 - DETECTION OF TOSPOVIRUSES IN PEANUT showed positive reaction to three tospovirus species ON MAIN PRODUCER AREAS OF THE STATE OF SÃO and the RT-PCR determined the presence of the Tomato PAULO, BRAZIL spotted wilt virus (TSWV), Groundnut ring spot virus Andrade, G.P.1; Carvalho, R.C.P.2; Ribeiro, G.P.1; (GRSV) e Tomato chlorotic spot virus (TCSV). Pantoja, M.B.1; Costa, P.M.G.1; Ferreira, P.G.A.1; Godoy, I.J.3; Santiago, J.C.L.1 1. UFRPE - Universidade Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros, s/n, Dois Irmãos, Recife - PE, 52171-900 2. UnB - Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília - DF, 70910-900 3. IAC - Instituto Agronômico, Avenida Barão de Itapura, 1.481, Botafogo Campinas - SP, 13020-902 The peanut (Arachis hypogaea) is a leguminous plant with a high socioeconomic importance. It constitute a food source rich on protein, vitamin, and minerals, used as cooked or toasted grains and in industrialized products. Furthermore, it has been used for producing cosmetics and biofuels. Worldwide, peanut is produced on tropical and subtropical regions and is considered the generating income and contributing for sustainability of small,fifth leguminous medium and in bigyield. farms. In Brazil, Despite it theis very high important, economic relevance, several factors as pests and diseases affect this crop. The diseases caused by viruses, especially those belonging to the genus Tospovirus, may induce drastic yield losses, nearly 100 per cent, and low quality of the product when highly susceptible cultivars are used in presence of high population of thrips vectors. The tospoviruses in peanut induce a range of symptoms from small reduction on plant height to severe stunting, top necrosis, mild to strong mottling, ring spot, as well as plant death in severe cases. The virus infection may also affect the peanut pods and kernels that become a tospovirus infecting peanut in the world was done in 1915,stunted, in Australia. deformed, In andBrazil, discolored. it was initially The firstfound report in 1941. of The objective of this work was to analyze the occurrence of tospoviruses in the peanut producing areas of the

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV VETERINARY VIROLOGY - VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

224 Veterinary Virology: VV

VV24 - ABSENCE OF CLASS I NEWCASTLE DISEASE between 37 and 38), since birds are apparently healthy. VIRUS IN WILD BIRDS FROM PANTANAL All samples were negative by qPCR NDV L gene class Barboza, J.D.B.1; Ometto,T.1; Araujo, J.1; Seixas, M.1; I. Suspicious samples were also tested by qPCR F gene Meneguin, C.1; Colmanetti, T.1; Pinto, L.B.2; Sabino, U.3; Pinho, J.B.3; de Aguiar, D.M.2; Durigon, E.L.1; pathogenicity and both were negative. Despite the low specific for velogenic strains in order to evaluate the Thomazelli, L.M.1 sampling, we deem that, in the studied locations, there is no class I NDV circulation and there is a low prevalence 1. ICB/USP - Instituto de Ciências Biomédicas of class II low pathogenicity NDV. Financial support: da Universidade de São Paulo, Edifício III USP - Administração FAPESP 2013/12112-8 - Av. Prof. Lineu Prestes, 2415 - Butantã, São Paulo - SP, 05508-900 VV25 - ABSENCE OF CLASS I NEWCASTLE DISEASE 2. HV/UFMT - Hospital Veterinário da VIRUS IN WILD BIRDS FROM ANTARCTIC REGION Universidade Federal de Mato Grosso, Av. Fernando Corrêa Thomazelli, L.M.1; Seixas, M.1; Araujo, J.1; Ometto, T.1; da Costa, nº 2367 - Bairro Boa Esperança. Cuiabá - MT - 1 2 1 78060-900 Meneguin, C. ; Petersen, E. ; Barboza, J.D.B. ; Petry, 2 3 3. IB/UFMT - Instituto de Biociências da M.V. ; Durigon, E.L. Universidade Federal de Mato Grosso, Av. Fernando Corrêa 1. ICB/USP - Instituto de Ciências Biomédicas da Costa, nº 2367 - Bairro Boa Esperança. Cuiabá - MT - da Universidade de São Paulo, Edifício III USP - Administração 78060-900 - Av. Prof. Lineu Prestes, 2415 - Butantã, São Paulo - SP, The Newcastle disease is a highly contagious acute 05508-900 2. LABORATÓRIO DE ORNITOLOGIA birds. Due to potential risk that the disease represents E ANIMAIS MARINHOS/UNISINOS - Laboratório de Ornitologia e Animais Marinhos da Universidade do Vale toviral the illness, economy notifiable, of countries affecting producers wild and and commercial exporters do Rio dos Sinos, Avenida Unisinos, 950 - Cristo Rei, São of chicken, the pathogens responsible are well known Leopoldo - RS, 93022-000 and studied. However, there are two major types of 3. IB/UFMT - Instituto de Biociências da Universidade Federal de Mato Grosso, Av. Fernando Corrêa commonly associated with wild birds and do not usually da Costa, nº 2367 - Bairro Boa Esperança. Cuiabá - MT - lineages termed class I and II. The first (class I) is more have high pathogenicity in poultry, so it is scarcely 78060-900 studied worldwide and no literary records in Brazil. The The Antarctic region is the largest natural wilderness left class II viruses are the most important in the pathology of poultry and therefore becomes essential to have an in all regions of the earth. Therefore, it is also the most active epidemiological surveillance, since Brazil is the remote,on the planet, unknown and andnature, most the preserved freer of human of all continent. influence largest exporter of chicken meat in the world. Wild birds, Between 48 Antarctic and sub-Antarctic seabirds especially migratory, may carry the Newcastle disease species observed in coastal countries of South America, virus (NDV) from endemic areas, acting as transmitting 38 were registered in Brazil. Migratory birds, especially agents in their long routes between wintering and seabirds, may carry the Newcastle disease virus (NDV) breeding areas. Therefore, the objectives were to analyze from endemic areas, acting as transmitting agents in by real-time PCR (qPCR), the presence of both classes their long routes between wintering and breeding areas. NDV in samples of wild birds, migratory or resident, Serological evidence and virus isolation indicate that the in different regions of Brazilian wet lands, Pantanal NDV occasionally circulates in Antarctica, however, our biome, Mato Grosso. Three hundred thirty tracheal - knowledge about the global distribution of this virus, cloacal swabs samples from wild birds were tested, especially class I, in wild bird populations is limited. of about 80 different species, all without any clinical Thus, the aim of the study was to analyze by real-time signs of the disease. By qPCR M gene class II NDV, two PCR (qPCR), the presence of both classes I and II NDV, samples (0.6%) were suspected positive. These samples in samples of seabirds, captured in Elephant Island, on Antarctic region. Five-hundred tracheal - cloacal swabs by DNA sequencing due to a low viral load observed (ct deemed suspicious because they not were confirmed September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV samples from five different species (petrel, skua, dove XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

225 Veterinary Virology: VV cable, Antarctic and Papua penguin), were tested, all zoonotic potential has been proven experimentally. In without any clinical signs of disease. By qPCR M gene class II NDV, two samples (0.4%) were positive. DNA located at State of Sao Paulo. Since then, several reports sequencing of these samples was not possible due to a haveBrazil, demonstrated the first description the presence occurred of BCoV in 16 in otherdairy statesherds low viral load (ct between 35 and 38), since birds are such as Rio Grande do Sul, Paraná, Minas Gerais and apparently healthy. All samples were negative by qPCR Mato Grosso do Sul. The diagnosis is based on detection L gene class I NDV. Positive samples were also tested of antibodies or virological methods, which have already to evaluate the pathogenicity and both were negative. importance to determine the causative agent in diarrhea Therefore,by qPCR F genewe believe specific that for velogenicthere is no strains class in I orderNDV inidentified cattle, the different aim of present BCoV genotypes.study was identify Considering the cause the circulation and there is a low prevalence of class II low of diarrhea in a dairy farm located at Araçatuba region of pathogenicity NDV, in the studied location. However, Sao Paulo state, Brazil with association of different tests. For that, four feces samples from one cow, one heifer and support: FAPESP 2013/05485-2 two calves were sent to Instituto Biologico to perform more studies are needed to confirm these data. Financial the differential diagnosis associating several techniques VV26 - CASE REPORT: BOVINE CORONAVIRUS such as electron microscopy, bacteriological and ASSOCIATED WITH OTHER PATHOGENS IN DAIRY virological tests. The bacteriological result was negative FARM IN STATE OF SAO PAULO, BRAZIL to salmonelosis, but one calf presented opportunists Okuda, L.H.; Tunin, A.; Catroxo, M.H.B.; Nassar, A.F.C.; bacteria of Enterobacter genus while the parasitological Souza, S.F.; Brandão, P.E.; Fernandes, A.M.; Ribeiro, test detected worms of Strongylidae family and the C.P.; Nogueira, A.H.C.; de Stefano, E.; Pituco, E.M. heifer, Eimeria spp. The electron microscopy revealed 1. Instituto Biológico, Av. Cons. Rodrigues particles with morphology consistent with Coronavirus Alves, 1252 - Vila Mariana, São Paulo - SP, 04014-002 genus in four animals. The PCR results to BoHV-1 and 2. Zoetis, Rua Luiz Fernando Rodrigues, 1701, BVDV are both negative. The viral isolation in MDBK Vila Boa Vista, Campinas - SP, 13064798 cells was observed cytopathic effect in Holstein’s cow 3. FMVZ/USP - Faculdade de Medicina which was submitted to RT-PCR with positive result and Veterinária e Zootecnia da Universidade de São Paulo, Av. Duque de Caxias Norte, 225. Pirassununga - SP, 13635-900 mixed infections in the herd, i confirmed by sequencing as BCoV. This study showed Enteritis constitutes a major problem in cattle, affecting VV28 - SEROLOGICAL AND MOLECULAR EVIDENCE mainly young animals. Several pathogens are involved OF ORTHOPOXVIRUS IN URUGUAY such as bacteria, virus and parasites and commonly there is an association of agents causing diarrhea in Luiz, A.P.M.F.; Pereira, A.F.; Alves, P.A.; Trindade, G.T.; animals. The bovine corononavirus (BCoV) belongs Abrahao, J.A.; Kroon, E.G. to order , subfamily Coronavirinae, genus UFMG - Universidade Federal de Minas Gerais, Betacoronavirus of family is considered Avenida Presidente Antônio Carlos, 6627 - Pampulha, Belo one of the most important pathogens in neonatal diarrhea Horizonte - MG, 31270-901 in calves, also associated with respiratory diseases Bovine vaccinia (BV) is a viral disease that affects dairy and winter disentery in adult cattle, causing major economic losses in dairy farming and cutting. The BCoV zoonosis. This disease has as etiologic agent the Vaccinia belongs to Betacoronavirus genus, Nidovirales order, viruscattle (VACV), and milkers, which being belongs classified to the asPoxviridae an occupational family, Coronavirinae sub-family of Coronaviridae family. The Orthopoxvirus (OPV) genus. Infected dairy cattle usually transmission route is fecal-oral by ingestion of water and present ulcerative lesions on their teats and udders. Rural food contaminated with feces or respiratory secretions workers, when infected, presented mainly lesions on and aerosols. The inter-species transmission has been their hands. Therefore, BV has a great economic impact, the subject of several studies, which wild and domestic compromising the milk industry and public health ruminants are characterized as reservoirs of BCoV. The services. The VACV exhibits serologic crossreactivity

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

226 Veterinary Virology: VV with other OPV species and was used during the World two mutations (T200I; M72I), isolated D and E has three Health Organization smallpox eradication campaign. mutations (T200I; M72I; N77D). To compare the CPE, The origin of VACV in Brazil is unknown, although some studies have suggested that VACV strains used during the with lymph node suspension (original PCV2b; group campaign may be related to outbreaks of bovine vaccinia A),ST cellsisolate with B semiconfluent(group B), C (group monolayer C), D were (group inoculated D) and and other studies suggest an autochthonous origin. In the last 14 years, outbreaks of BV have been detected across with only minimum essential medium. The total of 17 Brazil. Despite BV impact in Brazil, the VACV circulation passagesE (group was E). STperformed, cells (control but for flask) some were groups inoculated of cells, in Uruguay was not described. We investigated for the number of passages was lower due to viral action. The presence of OPV-neutralizing antibodies and VACV DNA PCV2 viruses from last passage of groups A, B, C, D and in 125 sera samples from Uruguay. Of the 125 sera, E were genetically characterized by sequencing. Each neutralizing antibodies against OPV were detected in 28 (22,4%); of these, 10 (35,7%) had titers of 100 NU/ml, 11 (39,3%) had titers of 200 NU/ml, 5 (17,9%) had titers flask was stained with Giemsa to assess cell morphology. of 400 NU/ml, and 2 (7,1%) had a titer of >800 NU/ml. usedThe cell for evaluation.counting was One performed mutation (T200I)on ten different was observed fields To detect viral genome, we conducted PCR to amplify inrandomly the sequence selected, of andvirus the from magnification 17th passage of 400X of group was the viral growth factor gene (vgf) and hemaglutinin A. Three mutations (T200I, M72I and N77D) were gene (HA) DNA. Two of the 125 sera were positive in found in the last passages of groups B (13 passage), C PCR assays for vgf gene and one serum was positive for (11 passage), D (4 passage) and E (6 passage). The HA gene. Although no exanthematous VACV outbreaks cells were fusiform to rounded, becoming necrotic have been described in bovines in Uruguay, our results with eosinophilic cytoplasm, condensed chromatin, suggest that herds may be exposed to VACV in areas karyorrhexis and karyolysis. Finally, a decrease was surrounding the Brazilian border and therefore may be observed in the number of cells that were more elongated at risk for VACV infection. and the morphologic characteristics were maintained. The CPE was more severe in the 9th passage of group C VV30 - MUTATION IN THE CAPSID PROTEIN OF and B, 3rd passage of group D and 4th passage of group PORCINE CIRCOVIRUS GENOTYPE 2B (PCV2B) E. The control group and A did not show CPE. This is the DETERMINE CYTOPATHOGENICITY IN SWINE TESTICLE CELLS by PCV2 in ST cells. The two (T200I, M72I) and three Cruz, T.F.1; Ordonez, F.J.P.3; Castro, A.M.M.G.2; (T200I,first report M72I, that N77D) describes amino the acid occurrence mutations of CPEin the caused viral Richtzenhain, L.J.2; Araújo Jr, J.P.1 capsid were observed in groups that shown CPE. The 1. UNESP - Universidade Estadual Paulista, occurrence of one mutation (T200I) did not cause CPE Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010 in ST cell. The CPE observed in group B could be due 2. USP - Universidade de São Paulo, Av. Prof. to the occurrence of three mutations (T200I, M72I and Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 N77D) detected in the 13th passage by sequencing. The 3. UNIVERSIDAD DE CALDAS, Manizales, presence of mutations in the viral capsid likely favoreced Caldas, Colômbia the viral replication, wich caused lower cell survival. Porcine circovirus type 2 (PCV2) is a DNA virus characterized by absence of cytopathic effects (CPE) in cell culture. Thus, the objective of the present study was to describe the occurrence of CPE in swine testicle (ST) cells infected with PCV2 presenting mutations in the viral capsid. Four isolates of PCV2 (B, C, D and E) with amino acid mutations in the ORF2 were used in this study. The isolates were obtained after viral isolation in ST cells. The isolated B has one mutation (T200I), isolated C has

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

227 Veterinary Virology: VV

VV32 - PORCINE CIRCOVIRUS GENOTYPE 2B in the capsid protein. For isolated B (one mutation), C PRESENTING AMINO ACID MUTATIONS IN THE (two mutations) and original PCV2b, the number of CAPSID DETERMINES ENHANCED REPLICATION IN passages performed was higher compared to isolates SWINE TESTICLE CELLS D and E. After the occurrence of CPE, due to cell death, Cruz, T.F.1; de Castro, F.L.2; Ordoñez, F.J.P.3; the viral load was lower in the following passages. These Richtzenhain, L.J.2; Araujo Jr, J.P.1 results from this study suggested that the amino acid mutations alone or collectively were likely responsible 1. IB/UNESP - Instituto de Biociências da for the enhanced PCV2 replication in ST cells. This is the Universidade Estadual Paulista, Avenida 24 A, 1515 Bela Vista, Rio Claro - SP, 13506-900 by PCV2 in ST cells associated with increased viral 2. FMVZ/USP - Faculdade de Medicina load.first report The CPE that observed describes in the group occurrence B could ofbe CPE due caused to the Veterinária e Zootecnia da Universidade de São Paulo, Av. Duque de Caxias Norte, 225. Pirassununga - SP, 13635-900 occurrence of three mutations (T200I, M72I and N77D) 3. UNIVERSIDAD DE CALDAS, Manizales, detected in the thirteenth passage by sequencing (data Caldas, Colômbia not shown). FINANCIAL SUPPORT: FAPESP 06/59002-9 PCV2 is a small, single-stranded DNA virus that belongs VV45 - PROSPECTIVE STUDY OF CORONAVIRUSES IN to the family Circoviridae. PCV2 is characterized by RODENTS IN A RURAL AREA OF MINAS GERAIS absence of cytopathic effects (CPE) and lower viral titer Sacchetto, L.1; Miranda, J.B.2; Amaral, C.D.2; Borges, in cell culture. Thus, the aim of this study was to describe I.A.2; Vieira, F.N.2; Ambrósio, L.L.D.2; Alves, P.A.2; the increased of viral replication in isolates of PCV2 de Siqueira, T.R.1; Paglia, A.P.2; Trindade, G. de S.2; that have amino acid mutations in the capsid protein Drumond, B.P.1 associated with the occurrence of CPE. Four isolates of PCV2 (B, C, D and E) with amino acid mutations in 1. UFJF - Universidade Federal de Juiz de Fora, the ORF2 were used in this study. The isolates were R. José Lourenço Kelmer - Martelos, Juiz de Fora - MG, 36036- 330 obtained after viral isolation in swine testicle (ST) cells. 2. UFMG - Universidade Federal de Minas The isolated B has one mutation (T200I), isolated C Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, has two mutations (T200I; M72I), isolated D and E has Belo Horizonte - MG, 31270-901 three mutations (T200I; M72I; N77D). ST cells with Emerging viruses are an important public health node suspension (original PCV2b; group A), isolated B problem worldwide. Some animals, including small (groupsemiconfluent B), C (group monolayer C), D (group were inoculatedD) and E (group with lymphE). ST mammals, have an important role in the maintenance and transmission of viruses by acting as natural host essential medium. To evaluate viral replication, aliquots reservoirs. Coronaviruses (CoVs) (order Nidovirales, (intracellularcells (control flask)viruses) were were inoculated separated with from only the minimum infected family Coronaviridae, subfamily Coronavirinae) are large and control cell suspensions after trypsinization. enveloped positive-stranded RNA viruses that infect Extracted DNAs were analyzed by quantitative PCR a broad range of mammals. Bats and rodents are rich (qPCR) for ORF2-PCV2. The total of 17 passages was sources of viruses, such as coronaviruses, that can be performed, but for some groups of cells, number of transmitted to humans. Therefore, the aim of this work passage was lower due to occurrence of CPE. The viral was to perform a prospective study of coronaviruses. loads determined for isolates B, C, D and E were higher The capture of small mammals was performed on a rural when compared to original virus (group A). All passages property (forest, pasture and domiciliary areas) located from the control group were negative for PCV2 by in the city of Rio Pomba, Minas Gerais, from September qPCR. The occurrence of CPE more severe coincided 2012 to September 2013. Lung samples were obtained with the peak of viral load (1011 DNA copies/ml ST cell from 59 rodents and preserved in RNAlater. The samples suspensions) in groups B, C, D and E. The CPE resulted in were macerated and total RNA was extracted using cell death. Thus, the number of passages was lower for RNeasy® Minikit (QIAGEN). For cDNA synthesis, RNA isolates (D and E) presenting three amino acid mutations was submitted to RT-PCR with random primers fallowed

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

228 Veterinary Virology: VV by real time PCR (qPCR) assay to detect coronaviruses (a hundred and ninety-one (n=391) samples of serum larger region of the RdRp) using primers that targeted e semen from bulls were collected. All samples were a conserved region of the virus genome using SYBR® obtained from eighteen different farms from Center- Green PCR Master Mix (Applied Biosystems). Plasmids West, Southeast and Southwest regions of RS. The serum containing the genes of interest were used as positive samples were submitted to serum neutralization test, controls. Up to date, RNA extraction was performed while the semen were used for detection of viral DNA by for all samples and qPCR was performed for 18 lung polymerase chain reaction (PCR) targeting the viral DNA samples. So far, none of the tested samples was positive polymerase gene. The results of the serum neutralization for coronavirus. The serum and other organs of the showed the presence of 44 animals positive for BoHV-1 collected rodents are going to be tested in the future. (17.6% of total) corresponding to twelve (66.7%) of the Surveillance studies of animal species, like rodents, are studied farms. Five of the farms had a higher frequency essential to understand the circulation, maintenance and of animals positive for BoHV-1 (above 15%), with several transmission of these viruses as well as their emerging cases of animals with antibody titers above 1:64. BoHV-1 potential. Financial support: FAPEMIG, CNPq, CAPES, PCR detection in semen showed the occurrence of two UFJF, PROPESQ/UFJF. positive samples, precisely in one of the farms with higher BoHV-1 antibody titers. This particular has a history VV62 - DETECTION OF BOVINE HERPESVIRUS 1 IN SEMEN OF BULLS FROM BEEF CATLE FARMS IN RIO possibly being related to the BoHV-1 shedding in semen. GRANDE DO SUL FINANCIALof nutritional SUPPORT: and health FAPERGS, management ULBRA, FEEVALE deficiencies, Henzel, A.1; Mascitti, A.K.2; Salla, P. de F.2; Lunge, V.R.2; Spilki, F.R.1 VV63 - SEROLOGICAL EVIDENCE OF HEPATITIS E VIRUS (HEV) IN SWINES FROM SANTA CATARINA 1. FEEVALE - Universidade Feelave, Av. Dr. STATE, BRAZIL Maurício Cardoso, 510 | Bairro Hamburgo Velho, Novo 1 1 2 Hamburgo - RS, 93510-250 Lopes, K.G.S. ; Silva Júniro, J.V.J. ; Kramer.B. ; Bordin, 2 2 3 2 2. ULBRA - Universidade Luterana do Brasil, L.C. ; Pandolfi, J.R. ; Bertani, G.R. ; Silva, V.S. ; Gil, Rua Martinho Lutero - 301 - Universitário, Cachoeira do Sul L.H.V.1 - RS, 96501-595 1. CPQAM/FIOCRUZ - Centro de Pesquisas Brazilian cattle herd is the second largest in the world, Aggeu Magalhães da Fundação Oswaldo Cruz, Av. Professor with more than 200 million animals. The state of Rio Moraes Rego, s/n - Campus da UFPE - Cidade Universitária, Grande do Sul (RS) is responsible for approximately Recife - PE, 50.740-465 5% of this total with the majority of the beef cattle 2. EMBRAPA SUÍNOS E AVES, Parque herds located in the Center-West, Southeast and Estação Biológica - PqEB s/nº, Brasília, DF, 70770-901 Southwest regions of the state. Some of the most 3. UFPE - Universidade Federal de Pernambuco, Av. Prof. Morais Rego, 1235 - Cidade Universitária, Recife - important respiratory and reproductive cattle diseases PE, 50670-901 are caused by bovine herpesvirus type 1 (BoHV-1), an alphaherpesvirus belong of the genus, The hepatitis E virus (HEV) is the only member of the Herpesviridade family. Viral transmission occurs by family , its viral genome (vRNA) consists of respiratory, ocular and genital secretions. BoHV-1 a single strand RNA (ssRNA), positive-sense, surrounded may also be present in the semen of infected bulls, by a non-enveloped capsid. The HEV has four genotypes (genotypes 1-4), the genotypes 1 and 2 are found insemination. BoHV-1 has been detected in association exclusively in humans and only genotype 3 and 4 have withleading clinical to dissemination cases, but the by effectivenatural matingoccurrence or artificial of this zoonotic potential with the pigs being the primary animal, virus is still unknown under conditions of extensive although the presence of vRNA has also been reported in cattle farming. The aim of this study was to investigate other animal species. The transmission by direct contact the occurrence of BoHV-1 in bulls from beef cattle farm with infected animals or consumption of contaminated located in the main producing regions of the RS. Three food by zoonotic genotypes has increased the concern

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

229 Veterinary Virology: VV of public health agencies with this infection. To evaluate region. This study provides evidence for the occurrence the presence of HEV in swines, serum samples from

Brazil, were submitted to the immunoassay (ELISA) highlightof BoHV-4 the genomes need for in epidemiological samples of follicular surveillance fluid andfor forconfined detection pigs ofand anti-HEV wild boars IgG. from The Santatest was Catarina performed state, BoHV-4semen ofin cattlesuch specimens in Brazil. in In order addition, to prevent these possible findings in duplicate and samples with results very close to the cutoff were retested. About the overall results, 100% reproduction. of wild boars samples (68 samples) were negative for spreading of such agent through techniques of artificial anti-HEV IgG, whereas 50% of domestic pigs samples VV84 - PREVALENCE OF BLUETONGUE VIRUS ANTIBODIES IN BOVINE FROM ARAPONGAS the circulation and absence of HEV in Brazilian herds MUNICIPALITY, STATE OF PARANA, BRAZIL and(16 wild samples) boars werestudied, positive. respectively. These Additionally, results confirm this Bronkhorst, D.E.1,2; Negri Filho, L.C.3; Moraes, E.M.S.1; Katto, S.4; Affonso, M.Z.4; Neto, D.B.4; Headley, S.A.5; boars from Brazil. Thus, these data highlight the risk of Alfieri, A.A.6; Silva, L.C.4; Bogado, A.L.G.1; Barca Junior, zoonoticwork is the transmission first serologic and report contributes about tothe epidemiology HEV in wild F.A.1; Okano, W.4 studies about HEV in Brazil. 1. MV/UNOPAR - Medicina Veterinária da Universidade Norte do Paraná, Av. Paris, 675 - Jd. Piza, VV83 - DETECTION OF BOHV-4 IN FOLLICULAR FLUID Londrina - Paraná, 86041-140 AND BOVINE SEMEN 2. IC/FUNADESP - Iniciação Científica da Albuquerque, N.R.M.1; Finoketti, F.1; Campos, F.S.1; Fundação Nacional de Desenvolvimento do Ensino Superior Brito, M.E.D.1; Paulo, W.M.1; Franco, A.C.1; Firpo, R.M.2 Particular, SCS Quadra 7 - Bloco “A”, Salas 726/728 Torre do 1. UFRGS - Universidade Federal do Rio Pátio Brasil Shopping, Brasília - DF, 70307-901 Grande do Sul, Avenida Paulo Gama, 110 - Farropilhas, Porto 3. BOLSISTA PIBIT CNPQ/UNOPAR Alegre - RS, 90040-060 - Programa Institucional de Bolsas de Iniciação em 2. UFCSPA - Universidade Federal de Ciências Desenvolvimento Tecnológico e Inovação / Universidade da Saúde de Porto Alegre, R. Sarmento Leite, 245 - Centro Norte do Paraná, Av. Paris, 675 - Jd. Piza, Londrina - Paraná, Histórico, Porto Alegre - RS, 90050-170 86041-140 4. Mestrado em Saúde e Produção de Bovine herpesvirus 4 (BoHV-4), a member of the Ruminantes/UNOPAR - Universidade Norte do Paraná, Av. family Herpesviridae, subfamily , Paris, 675 - Jd. Piza, Londrina - Paraná, 86041-140 genus is widely distributed in the cattle 5. PATOLÓGICA VETERINÁRIA/UEL- population, and has been recovered from various tissues Universidade Estadual de Londrina, Rodovia Celso Garcia of reproductive organs as well as from aborted fetuses. Cid | Pr 445 Km 380 | Campus Universitário, Cx. Postal 10011, In this study, a search was made to detect the presence Londrina - PR, 86057-970 6. Mestrado em Saúde e Produção de Ruminante/UEL- Universidade Estadual de Londrina, obtainedof BoHV-4 from genomes ovaries in of follicular females fluids in slaughterhouses and semen of Rodovia Celso Garcia Cid | Pr 445 Km 380 | Campus andcattle. 164 One semen hundred samples and twentywere acquired follicular from fluid differentsamples Universitário, Cx. Postal 10011, Londrina - PR, 86057-970 distributors. Samples were examined for the presence Bluetongue (BT) is an important viral disease of BoHV-4 genome fragments with a nested PCR targeting domestic and wild ruminant that is transmitted by the viral thymidine kinase gene, designed to amplify a hematophagous Culicoides midges, that is caused by fragment of 315 base pairs. Six out of 120 (5%) follicular the bluetongue virus (BTV), member of the genus in the family Reoviridae, included in the OIE diseases list. Cattle and wild ruminants are frequently ampliconsfluid samples obtained and 15 were out of cloned 164 (9%) and semen subjected samples to infected but fatality rates from BT in cattle are low, it has contained amplifiable BoHV-4 genome segments. The important consequences for the dairy and beef cattle in fact corresponded to the expected BoHV-4 genomic sector because it can reduce milk yields as well as cow sequencing, what confirmed that the amplified products September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

230 Veterinary Virology: VV fertility. Malignant catarrhal fever is the main differential during smallpox vaccination campaigns worldwide. diagnosis in cattle. This study aimed to evaluate the After the eradication of smallpox, the vaccination was prevalence of BTV infection in adult dairy cattle. A total suspended due side effects of the infection with VACV. of 218 serum samples from healthy bovines from the 32 Nowadays, outbreaks of Bovine vaccinia (BV), a disease herds from rural settlement “Dorcelina Folador” in the caused by VACV, have been reported in Brazil. This region of Arapongas, Paraná state,were submitted to the disease mainly occurs at rural environments affecting agar-gel immunodiffusion (AGID) test for bluetongue milk cattle and their handlers, who become infected after virus antibodies as recommended by the OIE. The antigen contact with lesions placed in the cow’s teats and udders. used was commercial BTV antibody test Kit VMRD Inc. Infected humans present as a major symptom ulcerative This study was approved by the Ethical Committee for lesions in the hands. Little is known about VACV origin the use of Experimental Animals of the Universidade and which hosts participate at its transmission cycle. Norte do Parana (UNOPAR), Brazil (Protocol 015/13). There are many evidences of the participation of small The prevalence of seropositive animals for BT found was rodents. VACV strains have been isolated from sylvatic 33.5% (73/218). The percents of seropositive herds in rodents and the same virus was isolated from humans, agar gel immunodiffusion test for bluetongue virus were cattle and mice during an outbreak of BV. It has also 65% (21/32). These data indicate a wide BTV circulation been shown that intranasal infected mice can shed viral in bovine and show that infection is widespread among particles in their feces, and these particles remain viable the properties of the Arapongas. In 2001 and 2002, four for some time. Also, viruses belonging to the genus outbreaks of this disease in Paraná State were reported Orthopoxvirus, the same in which VACV is placed have to the OIE, clinical cases occurring in goats and sheep. rodents as reservoirs, like Monkeypox virus and Cowpox The prevalence of seropositive bovine for BT found in virus. To better understand the role of rodents in the the Minas Gerais was 59.1.%, São Paulo 53.7%, Mato circulation of VACV, 14 Mus musculus were captured at Grosso do Sul 42%, Rio de Janeiro 40.9%, Santa Catarina urban areas close to Belo Horizonte, in the state of Minas 37.8% and Rio Grande do Sul 89.7%. The prevalence of Gerais. The animals were trapped in size selective cages infection by the BTV may result from the annual average and baits of hazelnut cream were used. After anesthesia, temperature of Arapongas which is 18°C, reaching 22°C mice were measured and had their sera collected. The in some months, which may have contributed to the organs were also sampled and stored properly. Sera vector propagation and consequent spread of infection. were used for DNA extraction via PCI and this DNA Financial support: This work was supported by PIBIT- used as sample for PCR reactions. The targets for PCR CNPq, FUNADESP, UNOPAR e KROTON. reactions were the genes A56R, A26L and C11R. Two samples were positive for at least one of these genes. VV87 - STUDY OF THE CIRCULATION OF VACCINIA Sequencing of the sample positive in the A26L reaction VIRUS IN SMALL RODENTS OF URBAN AREAS OF THE revealed a similarity between the sequence analyzed and STATE OF MINAS GERAIS the sequences of Brazilian isolates, such as Passatempo Miranda, J.B.1; Apolinário, I.B.1; Müller, U.B.2; Ferreira, P.C.P.1; Bonjardim, C.A.1; Kroon, E.G.1; Howard, J.C.3; participation of small rodents in the VACV cycle, at the Trindade, G. de S.1 samevirus andtime Araçatuba as it raises virus. questions These findingsabout how reinforce there theare 1. UFMG - Universidade Federal de Minas not documented outbreaks of VACV in urban areas if the Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, virus is present at these places. Further experiments are Belo Horizonte - MG, 31270-901 necessary to better understand the relationship of the 2. UNIVERSE OF COLOGNE virus with other brazilian isolates. 3. INSTITUTO GULBENKIAN DE CIÊNCIA, Rua Quinta Grande 6, 2780-156 Oeiras, Portugal Vaccinia virus (VACV) is the prototype virus of the family Poxviridae, which comprises many viruses that can infect vertebrates and insects. VACV was used as vaccine

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231 Veterinary Virology: VV

VV114 - MICROSCOPIC LESIONS AFTER CHALLENGE < 0.0001) was reported between scores of microscopic WITH TWO BRAZILIAN AVIAN INFECTIOUS lesions and ciliary activity (gold standard method) on BRONCHITIS VARIANTS Mores, M.A.Z.1; Okino, C.H.1; Montassier, H.J.2; Mattos, microscopic differences were found between these two tracheal samples at 5dpi, by Pearson test. No significant G.L.M.1; Brentano, L.1; Coldebella, A.1; Esteves, P.A.1; IBV variants tested. The results indicate that, besides Trevisol, I.M.1 the genotypic variation, lesions produced by the IBV variants here studied were similar to those described 1. EMBRAPA SUÍNOS E AVES, Parque for standard IBV strains with respiratory tropism, Estação Biológica - PqEB s/nº, Brasília, DF, 70770-901 producing main lesions in the upper respiratory tract 2. UNESP - Universidade Estadual Paulista, and, renal damage in few birds. FINANCIAL SUPPORT: Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010 PROJETO EMBRAPA 03.12.03.012.0.00 Avian Infectious Bronchitis (IBV) is one of the greatest health challenges on poultry industry. The emergence VV119 - OCCURRENCE OF CANINE HERPESVIRUS of new variants has been frequent recently, therefore INFECTION IN DOGS OF BREEDING KENNELS IN studies reporting pathogenesis are important to TROPICAL CLIMATE ZONE characterize the most representative IBV strains. The de Souza, F.F.; Tavares, D.C.; Kurissio, J.K.; Araujo Jr, aim of this study was to evaluate microscopic lesions J.P.; Toniollo, G.H. in SPF chickens challenged with two Brazilian IBV UNESP - Universidade Estadual Paulista, Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010 Sixty two one-day old SPF chickens were divided into threefield isolatesgroups and previously housed in genotyped positive pressure as IBV isolators. variants. The canine herpesvirus (CHV) is a cause of abortion in Groups A and B were challenged at 28 days of life with dogs, but can be detected in healthy animals. This virus 104.0IED50 of 3735 and 3736 IBV variants, respectively. is associated to different clinical abnormalities as death Group C was kept as mock infected negative control of newborns and lesions in respiratory and reproductive group. Nine birds from each group were euthanized at tracts in adult dogs. There is no vaccine against CHV 1 day-post infection (dpi) and at 5 dpi, remaining six birds at 8 dpi. All birds were necropsied and trachea, importance of virus in breeding kennels. Thus, this in Brazil due to scarcity of studies confirming the sinuses, Harderian gland, lung, kidney and gonads were study aimed to correlate the abortion incidence with percentage of positive dogs to CHV at Ribeirão Preto routine histology. At 1 dpi acute tracheitis was observed region. Adult (1 to 5 years-old) dogs (39 males and incollected, few birds fixed of groups in buffered A and formalin B. Exudation and of processed mucus and by 209 females) from different breeds (Schnauzzer, heterophils; degeneration, necrosis and sloughing of Pug, Poodle, Yorkshire Terrier, Lhasa Apso, Shih-Tzu, Maltese, German Spitz and French Bulldog) from 9 were predominant lesions. At 5 dpi, all birds of groups A commercial kennels were used. Blood samples were andepithelial B had cells chronic and tracheitis,heterophilic with infiltrate complete in theciliary mucosa loss, obtained by jugular vein puncture and placed into vials containing 10% EDTA. The DNA extractions lymphocytes and plasma cells infiltrating the mucosa (Promega, Madison, WI, USA). The extracted DNA were made with Wizard® Genomic Purification Kit theand epithelialsame trend hyperplasia. was observed In sinuses, at 8 dpififty inpercent this tissue. of the was subjected to qPCR using primers directed to the Atbirds 8 fromdpi tracheal groups A mucosa and B had was mild regenerating, chronic inflammation, showing thymidine kinase CHV3 (CGTGGTGAATTAAGCCTAA) and CHV4 (ATGCTATTGGGTGTCTATC), and GoTaq™ mild epithelial hyperplasia. Chronic interstitial nephritis qPCR master mix kit (Promega, Madison, WI, USA) in wasglandular observed degeneration, in few birds of lymphocytic groups A and infiltration B at 8 dpi. Mild and the ABI 7300 (Life Technologies™, SP, Brazil) equipment. As a negative control reaction, nuclease-free water was gland of all birds of challenged groups. No lesions were observedlymphocytic in infiltrationthe tissues wasof groupobserved C and in thein lungsHarderian and gene fragment for thymidine kinase CHV were initial denaturationused. The reaction at 95°C/2 conditions min, followed for amplification by 40 cycles of the of

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV gonads of all groups. Significant positive correlation (P XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

232 Veterinary Virology: VV denaturation at 95°C/30 sec, annealing at 57°C/30 sec, This paper aims to analyze the frequency of rotavirus in and extension at 72°C/30 sec. Although the kennels heifers kept under two different production systems: (i) evaluated in this study presented cases of abortion conventional breeding system in a family basis and (ii) (2.1 to 42.9%/female number) and neonatal death (1.8 outsourced rearing of heifers and calves system. In the to 32.7%/9 kennels), none sample showed positive period from 2012 to 2013, 150 and 95 stool samples were results to CHV. We suggest as cause of these alterations collected (diarrheal and non-diarrheal) from heifers up the presence of other pathogens (Neospora caninum, to 30 days old from a unit of rearing calves and heifers and Leptospira spp, Toxoplasma gondii, Ehrlichia canis and a family based dairy property (respectively, both located Brucella canis) or latent of herpervirus infection. The in the western region of the state of Paraná, Brazil).The stud dogs from kennels are constantly submitted to RV diagnosis was carried out in fecal samples by silver- stress, immunosuppression or pregnancy, which can stained polyacrylamide gel electrophoresis (SS-PAGE). In recidivate the virus. Moreover, the herpesvirus is not outsourced rearing units the RV-A was detected in 11.3% stable in the environment, with optimal replication at (17/150) of samples of all animals with clinical signs of temperatures less than 37ºC, and in tropical climate zone diarrhea, which was easily controlled. The family based the media annual temperature is high (mean 26ºC). This dairy property showed positivity of 47.3% (45/95) hypothesis has been raised in other studies, with a higher which 93% of it was with diarrhea and mortality of 20% incidence of cases in regions with milder temperatures. (9/45). The installation and the sanitary management adopted may explain the discrepancy in the frequency DNA in the blood of host, which may not have been of RV found in different systems. In outsourced rearing foundFurthermore, due to the we infection used the latency, qPCR that which identifies can be theresolved CHV- units, heifers were kept in individual masonry stalls with serological test. FINANCIAL SUPPORT: FAPESP - PROCESSO NO 13/06993-1. property, animals of different ages occupied stalls and collectiveand suspended plots with floor, poor while hygiene, in the familywhich basedfavored dairy the VV126 - ROTAVIRUS FREQUENCY IN DIFFERENT transmission cycle RV. Furthermore, the proposal of RAISING SYSTEMS OF DAIRY HEIFERS IN THE WEST a exclusive manpower in the work of creating heifers OF PARANA results in greater attention to the promotion of animal Gallego, J.1; Macedo, R.1; Ribeiro, A.S.1; Kunz, A.F.1; health, which is usually neglected in most of the dairy Mello, J.L.1; Grolli, P.1; Taffarel, L.E.2; Takiuchi, E.1 farms where lactating animals receive most attention 1. UFPR - Universidade Federal do Paraná, and investment in management. Already established in Rua XV de Novembro, 1299 - Centro, Curitiba - PR, 80060- the U.S. and EU, outsourced rearing units systems has 000 been performing promisingly in dairy farming of Paraná, 2. UNIOESTE - Universidade Estadual do and the expectation of control of neonatal diarrhea, Oeste do Paraná, R. Universitária, 1619 - Universitário, Cascavel - PR, 85819-110 particularlyVV130 - PARTIAL RV, is significant “M” PROTEIN advantage SEQUENCING to the producer. OF Currently, family dairy properties represent 38.8% CANINE CORONAVIRUS STRAINS CIRCULATING IN of the properties in the state of Parana and they are THE STATE OF RIO DE JANEIRO, BRAZIL responsible for 44% of the milk production. In order Bottino, F. de O.; Domingues, C.F.; Costa, E.M.; to reduce the production costs and improvement of Pimentel, F.B.; de Castro, T.X.; Garcia, R. de C.N.C. zootechnical and health rates, many familiar producers have run the raising of their heifers off-site the dairy UFF - Universidade Federal Fluminense, Rua Miguel farm, transferring them to outsourced rearing units. de Frias, 9, Icaraí, Niterói - RJ, 24220-900 The raising phase of heifers is the second largest cost Canine coronavirus (CCoV) is a single-stranded RNA in the dairy business and the occurrence of diarrhea virus belonging to the Coronaviridae family and is responsible for mild to severe gastroenteritis in dogs. To to producers. Rotavirus A (RV-A) is recognized as the during this period can cause significant economic losses major viral etiologic agent in cases of neonatal diarrhea. and CCoV-II according to the genetic identity observed date, CCoVs can be classified in two genotypes, CCoV-I September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

233 Veterinary Virology: VV between these viruses and Feline Coronavirus (FCoV) VV138 - HIGH FREQUENCY OF BOVINE VIRAL type I and II, respectively. The aim of this study was DIARRHEA VIRUS 2 IN RIO GRANDE DO SUL, BRAZIL to characterize the CCoV genotypes detected in fecal Weber, M.N.1; Silveira, S.1; Machado, G.1; Groff, F.H.S.2; samples from diarrheic puppies in Rio de Janeiro, by Mósena, A.C.S.1; Silva, M.S.1; Budaszewski, R.F.1; Duppont, P.M.1; Corbellini, L.G.1; Canal, C.W.1 41 fecal samples collected from diarrheic puppies (2006- 2014)partial that amplification tested positive of the for “M” CCoV protein by using gene. RT-PCR A total assay of 1. UFRGS - Universidade Federal do Rio Grande do Sul, Avenida Paulo Gama, 110 - Farropilhas, Porto for M gene (CCV1/CCV2) were used in this study. The Alegre - RS, 90040-060 2. SEAPA - ecretaria da Agricultura, Pecuária direct sequencing with the Big Dye Terminator® kit and 409bp amplicons were purified and then subjected to e Agronegócio, Avenida Getúlio Vargas, 1384. Menino Deus, an ABI Prism® 3730 DNA analyzer (Applied Biosystems Porto Alegre - RS, 90150-004 CA). Sequence editing and multiple alignments were performed with BioEdit Sequence Editor 7.0 software. Ruminant pestiviruses can infect cattle populations Nucleotide similarity with sequences deposited in the GenBank database was assessed using the BLAST tool. their impact on productivity and health. Knowledge of worldwide and cause significant economic losses due to Sequence analyses were performed with the MEGA 5.1 pestivirus diversity is important for control programs software program. For 27/41 strains, the sequences and vaccine development and for determining probable presented enough quality for analysis. Based on the AA sources of infection. In this work, we describe a search changes found in residues 127, 173, 193, 200 and 201 of for ruminant pestiviruses in the sera of 9,078 calves the M protein, eight strains were characterized as CCoV-I from six to 12 months of age that were sampled for foot while another 19 as CCoV-II. Alignment of deduced amino acid sequences showed 10 nonsynonymous analyzed in pools and the positive samples analyzed individually.and mouth diseaseThirty-three monitoring. RT-PCR Thepositive calves animals were were first detected (0.36%) from 6.64% (23) of the 346 herds substitutions. Among the 10 AA changes, five were found tested. Sequencing analysis of the 5’ non-coding region Thesein all CCoV-I AA changes strains: were 127 (Ile/Val→Ala),present in the 173 strains (Val→Thr), of this showed the presence of BVDV-1a (15 strains), -1b (3), study193 (Ile→Met), as well in the 200 reference (Asp→Glu) strains. and Moreover, 201 (Asn→His). six of the -1d (1) and -2b (14), with a higher frequency (42.4%) of Brazilian strains analyzed contained non-synonymous BVDV-2 in comparison with other countries. This study substitutions not previously described. AA changes at may also be the reference for future control programs in Southern Brazil because it reports the prevalence of in strain RJ1133/12. Another two strains (RJ915/08 and cattle with active infections and the genetic background RJ916/08)residues 119 contained (Ile→Thr) a non-synonymous and 121 (Gly→Cys) substitution were found at of the circulating strains. Financial support: CAPES, CNPq, FAPERGS and Propesq/UFRGS. residue 120 (Ala→Thr), while RJ999/10 was substituted 2011(RJ1084/11at residue 144 (Lys→Arg). and RJ1091/11). An AA In change this study, at residuethe AA changes137 (Ile→Phe) found in was residues found in127, two 173, CCoV-II 193, 200 strains and from201 of the M protein could distinguish between CCoV-I and CCoV-II strains. These non-synonymous substitutions had already been described in European samples, but the exact consequence of these mutations is not yet clear. FINANCIAL SUPPORT: FAPERJ, CNPQ, PROPPI-UFF, PROGRAD-UFF

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

234 Veterinary Virology: VV

VV143 - A QPCR PLATFORM FOR ZOONOTIC VIRAL AGENTS DIAGNOSTIC: APPLICATION ON RODENTS to 1. These initial results indicate that the platform can EMERGING VIRUSES DETECTION AT THE MINAS 96% (PCPV). The linearity coefficients (R2) were close GERAIS STATE, BRAZIL samples, which will be prepared and used as template be used for detection of the targeted viruses in field Alves, P.A.1; Borges, I.A.1; Amaral, C.D.1; Rezende, for our reactions in the near future. I.M.2; Drumond, B.P.2; Magalhães, C.L. de B.3; Almeida, VV145 - MUTATIONS IN THE CAPSID PROTEIN OF G.M.F.1; Ferreira, P.C.P.1; Abrahão, J.S.1; Kroon, E.G.1; PORCINE CIRCOVIRUS TYPE 2 INDICATE A VIRAL Trindade, G. de S.1 IMMUNE EVASION 1. UFMG - Universidade Federal de Minas Gava, D.1; Schaefer, R.1; Serrão, V.H.B.2; Cantão, M.E.1; Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, Silva, M.C.3; Zanella, J.R.C.1 Belo Horizonte - MG, 31270-901 2. UFJF - Universidade Federal de Juiz de Fora, 1. Embrapa Swine and Poultry Research Center, R. José Lourenço Kelmer - Martelos, Juiz de Fora - MG, 36036- Animal Health Laboratory, EMBRAPA 3. UFOP - Universidade Federal de Ouro Preto, 2. Institute of Physics of São Carlos – University Campos Morros do Cruzeiro, s/n - Bauxita, Ouro Preto - MG, of São Paulo-USP 35400-000 3. Diagnostic Center For Animal Health (CEDISA) The emerging infectious diseases play an important role in public health, and zoonoses have a great part Postweaning multisystemic wasting syndrome, the most in this scenario. In the context of emerging viruses, common clinical manifestation of porcine circovirus type it is believed that with environmental degradation, some species of small mammals are now approaching Canada and in 2000 in Brazil. PCVD has caused economic 2 (PCV2) diseases (PCVD), was first described in 1996 in anthropic disturbed areas, increasing contact with both losses with high mortality rate in Brazilian pig farms human populations with domestic animals and herds, until the introduction of PCV2 vaccine in 2008. During December of 2012, a suspect PCVD case was reported in natural and anthropic system. Among the viral agents associatedwhich could with increase these zoonoses, the flow ofthe zoonosesfollowing betweenfamilies in Southern Brazil. The affected pigs showed coughing, 57-67 days-old pigs in a vaccinated wean-to-finish farm should be highlighted: Bunyaviridae, Flaviviridae and dyspnea, enlargement of inguinal lymph nodes, wasting Poxviridae. The aim of this study is to develop and and diarrhea. The objective of this study was to diagnose validate a qPCR platform to detect potential zoonotic and characterize the PCV2 infection in a PCV2 vaccinated viral agents circulating in rodents distributed in the pig herd. Lymph node, kidney and lung tissue samples geographical extension of the state of Minas Gerais by were collected and processed according to conventional qPCR. This technique was chosen for its high sensitivity methods for histopathology (H&E), real-time PCR and and robustness considering the possibility of numerous immunohistochemistry (IHC). DNA sequencing was performed by Sanger method. The obtained sequences chosen and assembled in gene blocks (gBlocks™), cloned were analyzed and assembled with Phred/Phrap/Consed intosamples. pGEM-T For positiveeasy vector controls (Promega), specific and sequences used for were the softwares. Phylogenetic analyses of the whole genome and the ORF2 gene (capsid protein) were performed by were designed for all viruses of interest including Apeu Neighbor-Joining method in MEGA 5.2 software. Using virusvalidation (APEU), of the Juquitiba designed virus primer (JUQV), pairs. Yellow Specific Fever primers virus molecular modeling methodology (I-TASSER) a structural (YFV), St. Louis encephalitis virus (SLEV), Vaccinia virus model of the capsid protein was obtained. The structural (VACV) and Pseudocowpox virus (PCPV). Standard model was validated and the mutated residues were curves ranging from 1.0ng to 1.0fg were prepared, reactions were performed using the SYBR® Green PCR identified. PCV2 infection was demonstrated by H&E and Master Mix (Applied Biosystems), and the detected time PCR (5.67x1011 DNA copies/uL). Sequence analysis revealedconfirmed a PCV2-bby IHC. Agenotype. high viral The load comparison was detected among by real- the (JUQV), 103% (YFV), 99% (SLEV), 96% (VACV) and ORF2 amino acid sequence with other PCV2 sequences efficiency for each primer pair were: 94% (APEU), 108% September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

235 Veterinary Virology: VV revealed three amino acids substitutions, in domains VV166 - BETACORONAVIRUS LINEAGE 2A DETECTION F57I, N178S and A190T. The results of the structural IN YOUNG ALPACAS (VICUGNA PACOS) FROM FARMS analysis showed a substantial change on the physical- IN ANDEAN REGION OF SOUTHERN PERU chemical characteristics of one residue. Moreover, Luna, L.; Gregori, F. the analysis also indicates a possible disruption in the secondary structure of the capsid protein around the LABMAS/USP - Laboratório de Biologia Molecular Aplicada e Sorologia. Departamento de Medicina Veterinária 178 residue which is an important region for antibodies Preventiva e Saúde Animal da Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, São Paulo – SP, recognition. Therefore, modifications in the viral protein 05508-270 Thisconformation mechanism could could have explain caused the an recent inefficient vaccine antibody failures Coronavirus is a large family of viruses that may cause a detectedbinding that in swine was observed in Brazil. by However, the lack ofadditional vaccine efficacy. studies diversity of symptoms (e.g. respiratory, enteric, nervous), infecting many animal and human hosts. Considering the this hypothesis. high mortality rate in young alpacas due to occurrence using in vivo and in vitro models are needed to confirm of diarrhea and data regarding the etiology is scarce, the VV155 - PESTIVIRUS CONTAMINATION IN CELL objective of this work was to detect coronavirus in 50 CULTURES stool samples of young alpacas from 3 farms located at da Silva, M.S.; Mósena, A.C.S.; Weber, M.N.; Silveira, the Andean region of southern Peru by a RT-nested PCR S.; Torikachvili, M.; Dupont, P.M.; Budaszewski, R.F.; test targeting a fragment of RpRd gene (RNA dependent Streck, A.F.; Canal, C.W. RNA polymerase) followed by nucleotide sequencing. A total of 11 samples resulted positive and the nucleotide UFRGS - Universidade Federal do Rio Grande do Sul, phylogenetic analysis of sequences demonstrates that Avenida Paulo Gama, 110 - Farropilhas, Porto Alegre - RS, all were characterized as Betacoronavirus Lineage 90040-060 2a, a group frequently found in both domestic and The genus Pestivirus of the family Flaviviridae compriseS RNA viruses that often cause diseases in ruminants and circulation in the sampled area and may be implicated pigs. Pestiviruses contamination in cell cultures and withwild the ruminants. diarrhea in These young results alpacas. confirm coronavirus fetal calf serum (FCS), widely used as a nutrient in cell culture, is frequently reported in the literature. Viral VV167 - ANALYSIS OF ANTIBODY LIBRARY contaminations may negatively affect diagnostic tests, VARIABILITY FROM CHICKEN IMMUNIZED WITH vaccine production and research. In this study, 41 cell BOVINE HERPESVIUS TYPE 1 lines of different animal origins were analyzed by the Japolla, G.; Almeida, R.G.; Penido, B.A.F.; Brito, reverse transcription-polymerase chain reaction (RT- D.E.M.W.; Souza, L.R.G. UFG - Universidade Federal de Goiás, Av. Esperança, s/n - Setor Itatiaia, Goiânia - GO, 74001-970 productsPCR) targeting from the eight 5′ untranslated were sequenced. region (UTR). Phylogenetic Eighteen (43.9%) samples were positive and the amplification The bovine herpesvirus type 1 (BoHV-1) is a member BVDV-1, two to BVDV-2 and one to ‘HoBi’-like viruses. of the family Hesperviridae and is recognized as an analyses revealed that five strains are closely related to important agent of economic loss in cattle. The economic pestivirus control in FCS and cell cultures to keep the impact caused by the BoHV-1 is expressed, among biologicalThe current safety findings and correct reinforce diagnostic the need results. for a Financial constant other features, by embryonic death, abortion, birth of support: CAPES, CNPq, FAPERGS and Propesq/UFRGS. cows and bulls, poor feed conversion and growth delay indebilitated calves. In animals,order to produce reduced and reproductive analyze the efficiency variability of of antibody library, two chickens were immunized with BoHV1 (105.5 DICC/50mL). Then the chickens were euthanized and their spleens were quickly removed

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

236 Veterinary Virology: VV for total RNA extraction and cDNA synthesis. The light displaying similar clinical signs. However, recent studies in cats infected with CPV-2c reported marked clinical to produce the single chain Fv fragments (scFv). The signs of hemorrhagic gastroenteritis. The aim of the study wholeand heavy scFv chains repertoire of antibodies were cloned genes wereinto phagemid amplified was to detect and genotype parvoviruses in fecal samples of cats with distinct clinics symptoms. Stool samples were collected from 75 cats with and without diarrhea infection.vectors, expressed Plasmidial asDNA fusion from proteins15 clones in was filamentous extracted from different veterinary clinics in Porto Alegre, Brazil. andbacteriophages the product andwas amplifiedanalyzed byin agarose the E. coligel 2%. bacteria The The total extraction of DNA was performed using a silica DNA was digested with the restriction enzyme BstNI in based protocol and the PCR targeted a 583 bp fragment order to evaluate the variability of the library through the of viral protein 2 (VP2) gene. Eight out of 75 samples analysis of the restriction fragments. The results showed (10.16%) were positive in the PCR, and two (25%) of that only two of the 15 clones share the same fragment those eight were from animals presenting diarrhea. size characterizing the other 13 as unique clones with Four positive samples were selected for DNA sequencing to determine the species and the whole VP1/2 genes this variability of antibodies generated by chickens has were analyzed. Using BLAST analysis, two samples were specificities theoretically distinct. In another study, similar to FPV, and the other two were closer to CPV-2c. of some antigen. In this work, the chickens didn’t live The results showed the presence of CPV-2c in feces of innot sterile been verified,environment, probably getting due in to touch immunodominânce with a wide cats, reinforcing that canine parvovirus can also infect range of micro-organisms present in the environment, cats, and that there is the possibility of transmission food and water. This contact could generate an immune between dogs and cats. FINANCIAL SUPPORT: CAPES, response against these organisms and consequently CNPQ, FAPERGS AND PROPESQ/UFRGS.

VV180 - SEARCH FOR STUDY OF CORONAVIRUS IN usedamplification for a variety of gene of selection fragments and of screening antibodies strategies without WILD AND DOMESTIC BIRDS FROM ILHA DE MARAJÓ forspecificity other purposes,to BoHV-1. such In the as future diagnosis these or clones for vaccinewill be Barbosa, C.M.; Araújo, J.; Góes, L.G.; Ometto, T.; Seixas, production. FINANCIAL SUPPORT: CNPQ EDITAL: MCT/ M.; Thomazelli, L.; Durigon, E.L. CNPQ/FNDCT/FAPS/MEC/CAPES/PROCENTRO-OESTE Laboratory of Clinical and Molecular Virology, Instituto Nº 031/2010. de Ciências Biomédicas, USP VV176 - DETECTION AND CHARACTERIZATION OF Recently, new more virulent strains of human CoVs PARVOVIRUSES IN STOOL SAMPLES FROM DISEASED (HCoVs) emerged, including the betacoronavirus cause AND HEALTHY CATS of Severe Acute Respiratory Syndrome (SARS-CoV) Chiappetta, M.C.; Mósena, A.C.S.; Soares, M.S.; Weber, epidemic in Asia and a new CoV-MERS reported in the M.N.; Budaszewski, R.F.; Streck, A.F.; Canal, C.W. Middle East. Besides human infections, species of wild and domestic birds can also carry respiratory and enteric LABORATÓRIO DE VIROLOGIA, FACULDADE viruses, including coronaviruses (COVs). Recently, DE VETERINÁRIA/UFRGS - Universidade Federal do Rio Infectious Bronchitis Virus- like viruses and some new Grande do Sul, Avenida Paulo Gama, 110 - Farropilhas, Porto Alegre - RS, 90040-060 families of birds and mammals both wild and domestic. The genus (formerly known as DueCoVs to genetically their high distinct mutation from and IBV recombination were identified rate in Parvovirus), from the Parvoviridae family, consists of during replication, CoVs are able to generate extensive several species of viruses extremely resistant in the genotypic variation, which facilitates adaptation to environment that cause important diseases in domestic new host species so CoVs are considered not only animals. Cats can be infected with feline parvovirus pathogens of veterinary importance but also a threat (FPV) that causes high mortality in kittens, leukopenia, with zoonotic potential, enhancing the surveillance for gastroenteritis and nervous signs. Canine parvovirus possible animal reservoirs. Therefore, this study aims type 2 (CPV-2) infection of cats was already reported to identify the species of coronavirus that are affecting

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

237 Veterinary Virology: VV birds and establish its zoonotic potential . Initially, were dsRNA was extracted using a combination of phenol/ chloroform/isoamyl alcohol and silica/guanidinium the Laboratory of Clinical and Molecular Virology, in isothiocyanate methods and detected by silver-staining ananalyzed expedition 93 bird to Ilhasamples, de Marajó taken inby thethe statefield ofstaff Pará from in polyacrylamide gel electrophoresis (ss-PAGE) method. 2006. Those samples were collected from cloacal and Of the 40 animals evaluated, the presence of RVA in feces was demonstrated in 47.5% of the samples (19/40). of VTM and kept in ultra-freezer (-80°C).at the laboratory In addition, four animals died by dehydration and inorotracheal USP. Using swabs, Chu et placed al.(2011)’s in cryotubes methodology, containing the nucleic 500μL secondary infections, which two of it were attributed to acids were extracted using “Magmax ™ -96 Total RNA the RVA. Results indicate that maternal vaccination does Isolation Kit” and subjected to reverse transcription not prevent infection by RVA. Surprisingly, the morbidity reaction (RT). Samples were tested by conventional rate increased to 48% when compared to the population PCR (Polymerase Chain Reaction) using Coronavirus of calves before the introduction of vaccination. However, protocols followed by Nested-PCR. As positive controls, there was reductions of 9, 8 and 22% in mortality rates samples of bovine coronavirus and avian coronavirus were used. At the moment, two samples were detected by Nested -PCR to coronavirus, considered suspicious. 05).by diarrhea, Morbidity specific rate by mortalityRVA after byvaccination, RVA and have lethality, also beenrespectively, described but by with other no authors statistical and significancethe interpretation (p>0, must be cautious, because the co-infection by other sequencesThese samples of coronavirus. will be further FINANCIAL sequenced SUPPORT: to confirm CAPES the pathogens was not investigated, neither the possibility diagnosis and correlation of findings with the known of RVA infection with distinct genotypes of those VV186 - EPIDEMIOLOGICAL STUDY OF BOVINE contained in the vaccine. However, severe mistakes of ROTAVIRUS AFTER DAM VACCINATION IN AN health management of the dairy property, regarding ENDEMICALLY INFECTED DAIRY HERD IN cleaning and disinfection, animal stocking of different NORTHWEST PARANA ages in the same stalls and not separating the animals Mello, J.L.; Ribeiro, A.S.; Macedo, R.; Gallego, J.C.; with clinical signs of diarrhea, may have caused the Kunz, A.F.; Grolli, P.; Takiuchi, E. constant circulation and propagation of RV, suggesting CAMPUS PALOTINA/UFPR - Universidade Federal do Paraná - Campus Palotina, R. Pioneiro, 2153, Palotina - stipulated without the right management corrections in PR, 85950-000 athat endemically prophylactic infected vaccination dairy herd has by lowRVA. efficacy when Rotavirus A (RVA) is a major cause of diarrhea in young VV189 - CONJUGATION OF COLLOIDAL GOLD calves. A prophylactic strategy for RVA is based on PARTICLES WITH ANTI-BOVINE IGG FOR THE immunization of pregnant cows in order to increase the DETECTION OF ANTIBODIES AGAINST BOVINE concentration of neutralizing antibodies in colostrum and HERPESVIRUS TYPE 1 milk. The aim of this work was to evaluate RVA infection in Japolla, G.1; Almeida, R.G.1; Junior, C.P.J.2; Souza, newborn calves after dam vaccination in an endemically L.R.G.1 infected dairy herd in Mariluz, northwest region of Paraná state (March 2013 to February 2014). Before 1. UFG - Universidade Federal de Goiás, Av. introduction of dam vaccination (seven months prior the Esperança, s/n - Setor Itatiaia, Goiânia - GO, 74001-970 study) the selected farm presented recurrent episodes 2. UFU - Universidade Federal de Uberlândia, of neonatal diarrhea due RVA, showing morbidity and Av. João Naves de Ávila, 2121 - Santa Mônica, Uberlândia - MG, 38408-100 33% respectively. The dams were primo-vaccinated with The bovine herpesvirus type 1 (BoHV-1) is a member of aspecific commercial mortality inactivated and lethality vaccine rates containing of 39%, G6 13% and G10and the family Herpesviridae, subfamily RVA serotypes according to manufacturer’s instructions. and genus Varicellovirus. Viruses of this subfamily are After calving, 40 fecal samples were collected from characterized by a short replication cycle, relatively calves which ages ranged from seven to 21 days. The RVA large number of hosts and the ability to establish latent

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

238 Veterinary Virology: VV infections in neurons. The bovine species is primary VV194 - CELLULAR IMMUNE RESPONSE OF AVIAN host for this herpesvirus, what is widely acknowledged INFECTIOUS BRONCHITIS VARIANT BY FLOW due to the damage caused. BoHV-1 is associated with a CYTOMETRY variety of clinical disorders, including rhinotracheitis Mattos, G.L.M.1; Okino, C.H.2; Trevisol, I.M.2; Mores, (IBR), conjunctivitis, genital infections, miscarriages M.A.Z.2; Brentano, L.2; Esteves, P.A.2; Lopes, L. dos S.2; and neurological disorders. For an effective control Bastos, A.P. de A.2; Caron, L.F.3; Filho, T.F.3; Duarte, I.3; of diseases caused by this virus a correct diagnosis Montassier, H.J. of the same is necessary, and this, several diagnostic techniques are being used, but none of them allows the 1. Embrapa Suínos e Aves e Aluno de Doutorado no Programa de Pós Graduação em Medicina Veterinária. Área de Concentração Patologia Animal, da UNESP - conjugation of colloidal gold particles with bovine IgG Universidade Estadual Paulista, FCAV wasquick performed diagnosis as to a be precondition made in the for field. the developmentIn this study of a 2. Embrapa Suínos E Aves a rapid diagnostic test. For this, the antibody anti bovine 3. Imunova Análises Biológicas e Universidade trade done in goat was conjugated to colloidal gold using Federal do Paraná classical methods for reduction of gold ions through 4. UNESP - Universidade Estadual Paulista, the action of sodium citrate reducing agents, applying FCAV, Departamento de Patologia Veterinária, Programa de tetracloroauric acid as sources of gold ions. Next, the Pós Graduação em Medicina Veterinária slot-blot test was performed in duplicate coating a Avian infectious bronchitis virus (IBV) is an avian nitrocellulose membrane with different dilutions of coronavirus that causes a highly infectious disease BoHV-1 (107 to 102 DICC/50ml). As negative control characterized by respiratory, reproductive and renal reaction the membrane was coated with BSA, while signs, depending on the viral tropism and results serum of animals proved positive for BoHV-1. After the positive control was purified immunoglobulin of poultry industry. Failures of vaccine protection on the coating, the membranes were inoculated with a solution in a significant economic impact to the worldwide containing the same immunoglobulins used as positive between the vaccine strain and the variant strains. control (6.8mg/ml) overnigt at 19° C and washes with Thefield, present may be work related aimed to to incomplete evaluate, cross-protectionimmune-related PBST 0.05% were carried out shortly after inoculation markers (cluster of differentiation) in the peripheral of bovine IgG conjugated with colloidal gold. A strong mark was observed on the test with the lowest dilution (107) and in form of decreasing intensity, visualized dividedblood of into birds three challenged groups ofwith 9 animalsa Brazilian were field housed isolate in to the dilution of 104. At the negative control lines no positiveof IBV (F3735pressure strain), isolators. by At flow one cytometry. day old group SPF A birds was staining was observed, while in the positive control vaccinated with attenuated vaccine strain (H120) of IBV. lines a strong coloring was visualized, thus validating At 28 days of age, birds from both vaccinated group A the test performed. The results obtained demonstrate and unvaccinated group B were challenged with 10 4.0 the feasibility of developing a quick test for lateral EID50/bird of the variant strain F3735. Birds belonging to group C were not vaccinated nor challenged (negative of antigens or antibodies, aiding in the diagnosis of control group). Blood samples were collected from nine infectedflow diagnosis or vaccinated of infected animals. animals FINANCIAL for the SUPPORT: detection birds of each group on 5 days post-infection. Leukocyte CNPQ EDITAL: MCT/CNPQ/FNDCT/FAPS/MEC/CAPES/ PROCENTRO-OESTE Nº 031/2010. surface antigens were assessed with specific monoclonal Dataantibodies were analyzed combined by (CD4/TCRαβ-Vβ1/CD45; analysis of variance using Kul-1/ the MHCII/CD45; CD8α/CD28/CD45; and Bu-1a/CD45). of treatment. Comparison between means was made MIXED procedure of SAS (2008) by testing the fixed effects were observed between groups A and C, and group by Student’s t test (p ≤ 0.05). No significant differences September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

239 Veterinary Virology: VV

1.234 (99.4%) were not reagent, but 8 (0.6%) samples tested were positive for IgG antibodies for . B presented a significantly higher immune response Positive results were detected in the following rodent of, CD4+TCRvβ1+, CD4+TCRvβ1- and CD8α+CD28+ species: 3 Necromys lasiurus and 2 Calomys callosus cells. The subpopulations CD4-TCRvβ1+, CD8α- from Bodoquena county of Mato Grosso do Sul State, 1 differencesCD28+, CD8α+CD28-,Kul-1+MHCII-, between all treatments. As expected, Kul-1+MHCII+, these Necromys lasiurus from Campo Alegre de Goiás from resultsKul-1-MHCII+ showed and an Bu-1a+increased did population not present of significantnaïve and Goiás State and 2 Calomys tener from Espírito Santo do Pinhal in São Paulo State. Hemorragic fevers caused by arenaviruses are considered endemic diseases, occurring cellsactivated in blood T helper of unvaccinated (CD4+TCRvβ1- and IBV and challenged CD4+TCRvβ1+) birds, in restricted geographical areas, but the species of rodent whereascells and the naïve immune or memory cells dynamics T cytotoxic in the (CD8α+CD28+) vaccinated/ reservoirs may have wider geographic distribution. challenged group compared to the negative control Search for the circulation of these viruses in rodents group (not unvaccinated/not challenged) showed no captured in natural habitats is important for better understanding of the relationship between the virus against challenge conferred by vaccination, and a and its host, as well as to support the epidemiological systemicsignificant exacerbated differences, response suggesting mediated immune by both protection naïve surveillance and control of this zoonosis. and activated T helper cells and naive T cytotoxic cells. VV212 - ACTIVE AND PASSIVE IMMUNE RESPONSE VV205 - FREQUENCY OF ARENAVIRUS ANTIBODY INDUCED BY COMERCIAL VACCINE AGAISNT BOVINE IN WILD RODENTS CAPTURED IN MIDWEST, ROTAVIRUSES SOUTHEAST AND SOUTH REGIONS OF BRAZIL Godoy, H.P.1; Mazer, L.C.1; Gonçalves, A.C.S.1; Godoy, Oliveira, A.L.R.1; Bisordi, I.1; Souza, R.P.1; Levis, S.2; H.P.1; Sepulveda, L.2; Samara, S.I.1; Buzinaro, M.G.1 Pereira, L.E.1; Montoro, M.G.1; Barbosa, V.M.1; Suzuki, 1. FCAV/UNESP - Faculdade de Ciências 1 3 A. ; Timenetsky, M.C.S.T. Agrárias e Veterinárias da Universidade Estadual Paulista, 1. Center for Disease Vector Transmission, Via de Acesso Prof.Paulo Donato Castellane s/n, Jaboticabal, Adolfo Lutz Institute SP, 14884-900 2. Dr. Julio Maiztegui 2. Ouro Fino 3. Center Of Virology, Adolfo Lutz Institute The aim of this study was evaluated the active and passive Viral hemorrhagic fevers caused by arenavirus are immunity against rotavirus induced by vaccination emerging and severe illnesses. Humans are accidentally with an inactive commercial vaccine in cows and their infected mainly by mucosal exposure to excretions respective calves from a farm of Cravinhos, state of São from chronically infected wild rodents. The presence Paulo. The analysis of IgG, IgG1, IgG2 , IgM and IgA was of antibodies in reservoirs species may indicate viral done in serum 60 days before calving (before vaccination); 30 days before calving (before revaccination) and in the natural reservoir of the Sabia virus, the etiologic agent day of calving, with indirect ELISA. The levels of IgG, IgG1, ofcirculation Brazilian in hemorrhagic a population fever, of a specificremains region. unknown The IgG2, IgM and IgA in colostrum were evaluated in the day even after the occurrence of human cases in Cotia and of calving, with the same technique. The levels of calves’ Espírito Santo do Pinhal counties in São Paulo State. immunoglobulin were evaluated in the day of born and This study aims to search for wild rodents possibly 1, 7, 21 and 28 days of age. The excretion of rotavirus associated with arenavirus transmission. We randomly was evaluated in fecal samples of calves by SDS-PAGE. selected 1.242 rodents blood samples taken from a bank In this study, levels of IgM and IgA were increased in of biological samples formed during epidemiological cows that were not vaccinated, and their calves, fed their surveillance for hantavirus and analyzed by ELISA for the colostrums, have levels of IgM increased one day after born. Vaccinated cows have serum IgG1 and colostral selection was performed using a randomizing algorithm IgG2 higher than non vaccinated cows but statistics availabledetection ofat specificwww.randon.org. IgG antibodies The to results arenaviruses. show thatThe differences in levels of immunoglobulin were not found

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

240 Veterinary Virology: VV in calves. In both groups, was detected rotavirus in there could be an evidence of spatial autocorrelation feces of calves, and besides the low occurrence in this between positive cases and the geographic region. The study, one calf of vaccinated cow excreted rotavirus in disagreement between the statistical tests is probably Day 21, while two calves born of non vaccinated cows caused because of the lack of data from other more excreted rotavirus with 7 and 14 days. In conclusion, cities of the neighborhood, so further regional studies the vaccination of cows with a commercial inactivated are required. In conclusion, it was possible to show that vaccine did not prevent the rotaviruses in calves. BEL is present in bovine herds of São Paulo state, and affects mostly females. The central west area of the state VV227 - OCCURENCE AND GEOGRAPHIC is where the majority of positive herds are, which is very DISTRIBUTION OF BOVINE ENZOOTIC LEUKOSIS concerning once bovine breeding is one of the region’s (BEL) POSITIVE HERDS IN SÃO PAULO STATE main economical activity. FINANCIAL SUPPORT: CNPQ Oliveira, L.G.; Almeida, H.M. de S.; Borges, L.; Gatto, I.R.H.; Medeiros, A.S.R.; Oliveira, L.G.; Samara, S.I. VV230 - RABIES VIRUS PHOSPHOPROTEIN MRNA IS AN EFFECTIVE TARGET TO SIRNA-MEDIATED RNAI FCAV/UNESP - Faculdade de Ciências Agrárias e ANTIVIRAL TREATMENT IN VITRO Veterinárias da Universidade Estadual Paulista, Via de Acesso Prof.Paulo Donato Castellane s/n, Jaboticabal, SP, 14884-900 Durymanova Ono, E.A.; Brandão, P.E. Bovine Enzootic Leukosis (BEL) is an infecto-contagious FMVZ/VPS/USP - Faculdade de Medicina Veterinária disease caused by a virus of Retroviridae family. The e Zootecnia, Departamento de Medicina Veterinária disease, which has chronical evolution, causes malignant Preventiva e Saúde Anima da Universidade de São Paulo, Av. lymphoma in cattle and also the proliferation of lymphoid Duque de Caxias Norte, 225. Pirassununga - SP, 13635-900 cells. This study focused on analyzing the geographic Rabies is a zoonotic disease that affects all mammals distribution and prevalence of positive BEL cases in and leads to more than 55,000 human deaths every São Paulo state. A total of 5,916 bovine serum samples year caused by rabies virus (RABV) (: received between the years of 2011 and 2013 from 20 Rhabdoviridae: Lyssavirus: Rabies Virus). The search different municipalities of São Paulo state were tested for antivirals against rabies is one of the frontiers on the for BEL using the immunodiffusion in agar gel test at Universidade Estadual Paulista – UNESP Jaboticabal. The based on ketamin, ribavirin, midazolam and amantadin results were analyzed using the software Terraview® wasfield successful but, despite after a protocolthe treatment (the Milwaukee of a human Protocol) patient, version 4. 2. 2. Statistical analysis such as Moran index it was shown as not reproducible. RNA interference is and Local Moran Index (LISA) were used to determine if an alternative as an antiviral technology against RABV there was spatial autocorrelation between positive cases already shown as effective in vitro in cell cultures and and the region they occur. In the years of 2011, 2012 and in vivo in the mouse model. Most of the researchers on 2013 were detected 123 (7.48%) positive samples out of 1,645 tested samples, 107 (3.22%) out of 3,324 and glycoprotein or RNA polymerase (large) protein in 16 (1.69%) out of 947, respectively. In general, out mRNAs,this field but have from used until siRNAsnow there targeting are no reports nucleoprotein, on RNA of 5,916 tested samples, 246 were positive, showing a interference for knock down of RABV phosphoprotein prevalence of 4.16%. Only 62 or 1.05% of total samples P, which is a multifunctional protein in its interaction were considered suspect at the immunodifusion agar gel with N and a key component of the virion-associated test. All positive animals were female, except in one case, RNA polymerase complex as a regulatory protein in with age ranging from 12 months to 8 years; this occurs viral genome replication. The aim of this study was to mainly because female animals are kept for a longer assess the decrease in the titer of rabies virus in vitro period in the farm due to reproductive reasons. Living using stealth short-interfering RNAs against P mRNA. To longer increases the chances of chronic manifestation of this end, three siRNAs were used with antisense strands the disease. The Moran Index value of 0.17615 with p complementary to rabies virus P mRNA. BHK-21 cells value of 0.01 showed no spatial autocorrelation among monolayers were infected with 10 to 0.01 TCID50% of positives cases. However the LISA analysis showed that PV and AgV3 (Desmodus Rotundus, antigenic variant

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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3); after 2 hours of virus adsorption, the cells were Livestock and Food Supply (MAPA) to perform the transfected with each of tree stealth siRNAs (in separate serological diagnostic to AI and ILT. During 2013, 14.728 assays each) using Lipofectamine-2000™ and RABV serum samples from breeders, grandparent breeders and great grandparent breeders destined for exportation 24h of transfection. All three siRNAs reduced the titer of were forwarded to CAPTAA and submitted to the PVdirect and immunofluorescenceAgV3 strain, with the higher assay drop was in performed titer regarding after serological diagnosis to AI and ILT by IDGA tests. The siRNA 360 treatment (0,4log drop for Pasteur Virus and tests were perform according the OIE procedures. These 1,0log drop for AgV3). No cytotoxic effect was observed samples were from three Brazilian states: Sao Paulo, in the monolayers treated with Lipofectamine-2000™. Minas Gerais e Rio Grande do Sul, and corresponding to These results show that the phosphoprotein of rabies virus can be use like an effective target for antiviral strategy for rabies. 381 flocks: 57 flocks from great grandparent breeders breeders(11.61% of(27.91%), the total respectively. samples analyzed), There was 187 no flocks detection from VV246 - SEROLOGICAL SURVEILLANCE FOR AVIAN grandparent breeders (60.48%) and 137 flocks from INFLUENZA AND INFECTIOUS LARYNGOTRACHEITIS which contributes to attest that the health status of IN GENETIC MATERIAL FROM BRAZILIAN POULTRY theseof antibodies samples, againstintended AI to and exporting ILT in of any Brazilian of the poultry flocks, EXPORTS DURING 2013 genetic material was considerate free to AI and ILT. These Luciano, R.L.; Cardoso, A.L.S.P.; Tessari, E.N.C.; Stoppa, results are important to epidemiological point of view to G.F.Z.; Kanashiro, A.M.I.; Castro, A.G.M. demonstrate the high quality level in the Brazilian avian CAPTAA/INSTITUTO BIOLÓGICO - Centro genetic production. Avançado de Pesquisa Tecnológica do Agronegócio Avícola VV252 - STANDARDIZATION OF A CONVENTIONAL do Instituto Biologico, Avenida Conselheiro Rodrigues Alves, PCR FOR DETECCION OF BOHV-1 AND 5 IN SEMEN 1.252, São Paulo - SP, 04014-002 de Carvalho, I.L.Q.1; Gasparini, M.R.2; Leite, R.C.2; Brazilian poultry industry ranks important position Stancioli, E.F.B.1 in the international scene. Currently, the country kept its position as largest exporter and the third 1. ICB/UFMG - Instituto de Ciências Biológicas principal producer of poultry meat. In respect this, da Universidade Federal de Minas Gerais, Avenida Presidente Brazil has exported genetic material from poultry Antônio Carlos, 6627, Belo Horizonte - MG, 31270-901 (breeders, grandparent breeders and great grandparent 2. VET/UFMG - Escola de Veterinária da breeders) to many countries, so that is extremely Universidade Federal de Minas Gerais, Avenida Presidente Antônio Carlos, 6627, Belo Horizonte - MG, 31270-901 important to certify that these material obey the sanitary requirements ordered by the International Bovine herpesvirus 1 and 5 (BoHV-1 and BoHV-5) Zoo-sanitary Code (IZC). All exporting products must are important animal pathogens, causing sanitarian and economic losses in cattle herds. The BoHV-1 according with the establishment of requirements by the is responsible for various clinical manifestations, having the international zoosanitary certificate (IZC) including respiratory and reproductive tract diseases and generalized infection in young cattle. Beyond the theimporting World country.Organization Avian for influenza Animal (AI) Health and (OIE) Infectious and symptoms already mentioned, the BoHV-5 causes non havelaringotracheitis been causing virus great (ILT) losses are notifiableto industrial diseases poultry to supurative meningoencephalitis in bovines. These breeding in many countries around the world. One of

(AI) and infectious laryngotracheitis (ILT) is agar gel forviruses the epidemiologyare transmitted of viathese body pathogens. fluids like It semen, is estimated being immunodiffusiontechniques adopted test by OIE(AGID). to diagnosis The Centro of avian Avançado influenza de thatthe artificial in 2013 insemination about 14 million an important doses ofcontagion semen wereform Pesquisa Tecnológica do Agronegócio Avícola (CAPTAA), Instituto Biológico (IB), located in Descalvado, SP, Brazil Organization for Animal Health (OIE) recommends is a laboratory accredited by the Ministry of Agriculture, testingcommercialized the semen in productionBrazil, which for justifies the presence that the of World these

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

242 Veterinary Virology: VV viruses. This work aimed the development of a PCR from commercial herds in Rio de Janeiro State, Brazil. The clinical signs presented by the infected animals 5 in bovine semen samples. The assay was done using were also reported. A total of 201 pigs from 21 farrow to thetest geneticfor the rapidregions and which specific codify detection the glycoproteins of BoHV-1 and B (gB) and G (gG). The detection sensitivity was analyzed A Clinical examination and weighing were performed in culture of kidney bovine cells CRIB and in semen, beforefinish herds blood located collection. in Rio DNA de Janeirowas extracted State were from studied. serum both experimentally infected with the viruses. In the samples by High Pure Viral Nucleic Acid Kit, ROCHE® and submitted to polymerase chain reaction (PCR) and bp) the sensitivity of detection of the virus in cell culture Nested PCR using primer sets targeting the VP2 gene wasamplification 5,128 TCID50 of the for gene BoHV-1 which codifiesand 5,75 the TCID50 gG (~500 for of PCV-2 and non-coding region gene of TTSuV1 and BoHV-5. In experimentally infected semen the sensitivity TTSuV2. All the PCR amplicons were sequenced and was 51,28 TCID50 and 57,5 TCID50 for BoHV-1 and BoHV-5, respectively. The analytical sensitivity using in 28/201 serum samples; TTSuV-1 in 28/201; and recombinant plasmids was 300 ag for BoHV-1 and 3 fg for TTSuV2confirmed in using 25/201 the samples.BLAST tool. Co-infection PCV-2 DNA was was reporteddetected in 50/201 animals: TTSuV1 and TTSuV2 in 37/201 pigs; the gB (96 bp) the detection sensitivity of the virus in TTSuV1 and PCV-2 in 4/201 pigs; TTSuV2 and PCV-2 in cellBoHV-5. culture In thewas amplification 5,128 TCID50 of and the 0,575 gene TCID50,which codifies and in one pig and triple infection (TTSuV1, TTSuV2 and PCV- experimentally infected semen was 51,28 TCID50 and 2) in eight pigs. The majority of the co-infected animals 5,75 TCID50 for BoHV-1 and BoHV-5, respectively. The (27/50) did not showed any clinical signs. Eleven showed herpesvirus 1 (EHV-1), Equid herpesvirus 4 (EHV-4), showed enteric signs (diarrhea and presence of blood Suidreaction herpevirus specificity 2 (SuHV-2), standardized Ovine was herpesvirus tested using 2 (OvHV- Equid inrespiratory feces) and signs seven (sneezing showed and systemic respiratory signs sounds); (sneezing, five 2) e Human herpesvirus 1 (HHV-1) which shows its high blood in feces and arthritis). PCV-2 is the primary agente causing postweaning multisystemic wasting syndrome (PMWS). Nevertheless, the literature suggests that positivitiesspecificity and were efficiency. 54.4% After (gG) an and initial 92,6% standardization, (gB). The the co-infections with other vírus as TTSuVs can be developed54 clinical testssemen presented samples excellentwere tested performance, and the verified which associated with more severe clinical signs. Furthermore, shows its possible utilization for semen screening in TTSuV1 was recently associated with clinical porcine control programs. respiratory disease complex (PRDC), which suggests that this virus might play a role in the development of VV256 - MOLECULAR DIAGNOSIS OF PORCINE this complex. Our data indicates that PCV-2, TTSuV1 CIRCOVIRUS TYPE 2 (PCV-2) AND TORQUE TENO and TTSuV2 are circulating in swine herds from Rio de SUS VIRUS TYPE 1 AND 2 (TTSUV1 AND TTSUV2) IN Janeiro and cases of co-infection with PCV-2 and TTSuV SWINE HERDS FROM RIO DE JANEIRO STATE, BRAZIL may be common in clinically normal pigs, hindering the Castro, T.; Cruz, A.C.; Silveira, R.; Baez, C.; Silva, S.; adoption of biosecurity measures in such populations. Dias, J.; Varella, R.; Castro, T. FINANCIAL SUPPORT: FAPERJ UFF - Universidade Federal Fluminense, Rua Miguel VV257 - MOLECULAR CHARACTERIZATION OF de Frias, 9, Icaraí, Niterói - RJ, 24220-900 FELINE HERPESVIRUS TYPE 1 (FHV-1) AND FELINE Porcine Circovirus type 2 (PCV-2) and Torque teno sus CALICIVIRUS (FCV) INFECTING DOMESTIC CATS virus (TTSuV) are emergent in swine herds and endemic FROM RIO DE JANEIRO, BRAZIL in many swine-producing countries. These agents were Castro, T.; Baumworcel, N.; Soares, A.; Silva, S.; already described in Brazil, causing severe economic Almeida, N.; Castro, T. impact on swine production although the information concerning to these viruses circulation in Rio de Janeiro UFF - Universidade Federal Fluminense, Rua Miguel state is absent. The aim of this study was to perform the de Frias, 9, Icaraí, Niterói - RJ, 24220-900 molecular diagnosis of PCV-2, TTSuV-1 and TTSuV-2

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

243 Veterinary Virology: VV

Feline conjunctivitis can be caused by several pathogens, Av. Dr. Enéas de Carvalho Aguiar, 470, Jardim América, São leading to similar clinical signs of disease. Two viruses in Paulo - SP, 05403-000 particular, feline calicivirus (FCV) and feline herpesvirus The central nervous system (CNS) may be affected by 1 (FHV-1), are responsible for at least 76% of the several agents, including viral bacterial or fungal. CNS conjunctivitis cases. These agents are widespread in the infections can trigger severe symptoms and, according to domestic cat population, despite the use of the vaccines. the site of infection, maybe designated as encephalitis or Both viruses are related to persistent infections due meningitis. Viruses are the most common cause of these to their mechanism of evasion of the immunologic diseases, followed by bacteria and fungus. Agents such system (latency and high genetic variability). In this as polyomavirus, Herpesvirus (Simplex, 6 and varicella- study, 108 ocular discharge samples were obtained from unvaccinated kittens. The genome extraction tuberculosis and C. neoformans, are responsible for was performed immediately after collection using the mostzoster) of influenzathe CNS infections, A, enterovirus, and have mumps, a high flavivirus, incidence M. PureLink™ Spin Column-Based Kit (Invitrogen®). For worldwide. However, prevalence of different agents FHV-1 detection, a fragment of 287 bp of the thymidine varies according to population, individual’s immune status, age and region of study. In fact, the prevalence detection, reverse transcriptase PCR (RT-PCR) using of these infectious agents is well established in many kinase enzyme (TK) gene was amplified by PCR. For FCV random primer (Invitrogen®) was performed. These countries. However, there is a lack of information products were subjected to a Nested-PCR reaction, regarding the prevalence of these agents in the Brazilian using internal primers able to amplify a fragment of population. Although there are diagnostic methods available for identifying most of the etiologic agents Animal Health, USA), was used as a positive control. that cause encephalitis and meningitis (EM), to obtain 467 bp. A commercial vaccine Felocell CVR-C (Pfizer FHV-1 DNA was detected in 62/108 (57,4%) samples. results in short period is essential for targeting the FCV genome was detected in 40/108 (37,0%) samples. most appropriate treatment of the disease. Here we use Co-infection of FHV-1 and FCV infection was detected in next generation technology (Ion Torrent) to investigate 20/108 samples (18,5%). All the PCR amplicons were putative microbial agents in CNS infections through sequenced and nucleotide (nt) similarity with sequences metagenomic approaches in liquor. The advantage of this deposited in the GenBank database was assessed using technique over traditional ones is the capability to detect the BLAST tool. The phylogenetic analysis of TK FHV-1 not only known agents, but also identify pathogens not gene revealed an unique cluster comprising the FHV-1 commonly associated to these pathologies in a fast way. strains from this study with others FHV-1 from Brazil. Samples (n=4) from patients with suspected viral EM The phylogenetic analysis of RdRp FCV gene showed two is were spiked before extraction with baculovirus and clusters: one comprising only strains from this study bovine viral diarrhea viruses as a control for acid nucleic and another one grouping strains from this study with extraction. After a set of centrifugation steps to exclude human cells, genomic DNA/RNA was obtained using characterization of FCV and FHV-1 in specimens from Macherey Nagel commercial kit. Reverse transcription strains from around the world. This is the first molecular cats in the State of Rio de Janeiro and revealed that these was performed with random primers using High viruses are circulating in the domestic feline population. Capacity (Applied Biosystems) kit. Double-stranded DNA FINANCIAL SUPPORT: FAPERJ was synthesized using Klenow enzyme Fragment (3’>5 VV259 - IMPLEMENTATION OF NEXT GENERATION ‘exo-) (NEB), followed by NGS sequencing. Filtering, SEQUENCING TECHNIQUES FOR PATHOGEN trimming and assemble of the reads were performed in DISCOVERY IN CEREBROSPINAL FLUID OF PATIENTS CLC genomic workbench software. Our methods appear WITH ENCEPHALITIS AND MENINGITIS the samples as internal control were recovered during Nunes, C.F.; Soares, A.C.; Urbano, P.R.; Gerhardt, D.; analysis.to be efficient In one since sample small investigated, reads of viruses around used 150 to contigs spike Romano, C.M. of Burkolderia (bacterial agent) were assembled. In IMTSP - Instituto de Medicina Tropical de São Paulo, conclusion, we demonstrated that the metagenomic

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

244 Veterinary Virology: VV methods implemented in the present work are capable VV274 - EVALUATION OF AN INDIRECT ELISA to detect viral and bacterial agents affecting the CNS. FOR FELINE CORONAVIRUS (FCOV) USING A RECOMBINANT NUCLEOCAPSID (N) PROTEIN BY VV261 - ANTI-HCV ACTIVITY OF THE TOXIN PLA2-CB COMPARISON WITH A COMMERCIALLY AVAILABLE FROM VENOM OF CROTALUS DURISSUS TERRIFICUS IMMUNOASSAY Shimizu, J.F.; Russo, R.; Cintra, A.C.O.; Sampaio, S.V.; Almeida, A.C. da S.1; Filoni, C.1; Hora, A.S.2; Brandâo, Aquino, V.H.; Rahal, P.; Jardim, A.C.G. P.E.2; Araújo Jr., J.P.1 UNESP/IBILCE - Universidade Estadual Paulista 1. IBB/UNESP - Instituto de Biociências da - Instituto de Biociências, Letras e Ciências Exatas, Rua Universidade Estadual Paulista, Bairro: Distrito de Rubião Cristóvão Colombo, 2265 Bairro Jardim Nazareth, São José Junior S/N, Botucatu - SP, 18618-970 do Rio Preto - SP, 15054-000 2. USP - Universidade de São Paulo, Av. Prof. Hepatitis C is a disease caused by the Hepatitis C virus Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 (HCV) infection and represents a public health problem Feline coronavirus (FCoV) belongs to the Coronaviridae worldwide. There is no vaccine for HCV and the current family, Nidovirales order and seroprevalence range up therapy is not effective for all treated patients, is to 90% in domestic cats and their wild counterparts. expensive and presents many side effects. Thus, there Approximately 5 to 10% of FCoV exposed felids develop the feline infectious peritonitis, an immune- approaches. In this context, natural compounds can mediated, systemic, progressive, and fatal disease that is is an evident need to develop more efficient therapeutic considered one of the most important infectious diseases products with therapeutic potential. toxins extracted that affects felids. The aim of this study was to evaluate provide an alternative source for the identification of from animal venoms have shown antiviral activity for the indirect ELISA (previously standardized with the other viruses such as dengue virus, yellow fever, measles, use of recombinant FCoV nucleoprotein) based on the among others. Therefore, this study aims to evaluate comparison with the commercial ImmunoComb® FCoV the effects of PLA2-CB, a subunit of crotoxin complex Antibody Test Kit for anti-FCoV antibodies detection in sera from naturally infected cats. Serum samples from replication in vitro. Huh 7.5 cells line stably expressing 111 cats were tested by the commercial immunoassay extracted from Crotalus durissus terrificus, on HCV subgenomic replicon SGR-FEO of HCV were treated with and ELISA. The accuracy of the indirect ELISA relative different concentrations of each compound for 48 hours, to commercial kit was 94.7%, the relative sensitivity and viral replication and cell viability was measured by MTT and luciferase assays, respectively. The results calculated as two standard deviations higher than the showed that the treatment of the cells with PLA2-CB arithmetic85.9% and meanthe relative of the absorbancespecificity 75% of negative at the cut-off samples of concentrations of 6 µg / mL and 4 µg / mL showed a by the reference test (0.41 OD). The indirect ELISA using reduction of approximately 58% in replication, and the recombinant FCoV nucleoprotein is a new, safe, and presented an EC50 of 5.7 µg / mL. Western blot analysis rapid diagnostic quantitative method that can be used at also demonstrated a reduction of virus NS5A protein reasonable low costs for measuring the precise amounts expression in treated cells as a result of replication inhibition. Our data showed that the PLA2-CB presents promising results in inhibiting viral replication. However, ofVV275 specific - MOLECULAR antibodies against DIAGNOSIS FCoV. OF VIRAL AGENTS more studies are needed for a better understanding IN DIARRHEIC PUPPIES IN RIO DE JANEIRO, BRAZIL of how these compounds act in the machinery of HCV. Costa, E.M.; Pimentel, F.B.; Domingues, C.F.; Bottino, FINANCIAL SUPPORT: FINANCIAL SUPPORT: FAPESP F. de O.; Castro, T.X.; Garcia, R. de C.C. (2011/11753-4) E (2013/03897-1) UFF - Universidade Federal Fluminense, Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 Canine parvovirus (CPV) and canine coronavirus (CCoV) are considered to be the main pathogens responsible

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

245 Veterinary Virology: VV for acute gastroenteritis in young dogs. Recently, canine VV294 - CLINICAL SIGNS, TRACHEAL CILIOSTASIS calicivirus (CaCV), canine astroviruses (CaAstV) and AND DETECTION BY QRT-PCR OF BRAZILIAN IBV Group A-Rotavirus (RV-A) have also been reported as VARIANTS OF CHICKEN AND PIGEON ORIGIN IN potential viral pathogens. This study was carried out CHICKENS to identify the viral agents associated to gastroenteritis Martini, M.C.1; Caserta, L.C.1; Santos, M.M.A.B.2; in puppies in Rio de Janeiro,Brazil using molecular Barnabé, A.C.S.1; Padilla, M.A.1; Neto, D.F.L.1; Ferreira, methods. Fecal samples collected between January 2008 H.L.3; Arns, C.W.1 and March 2014 from 254 privately owned puppies with diarrhea were tested. Genomic RNA/DNA was extracted 1. UNICAMP - Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz - Barão from 10% fecal suspensions using the PureLink™ Spin Geraldo, Campinas - SP, 13083-970 Column-Based Kit (Invitrogen®). For CPV detection, 2. UFJF - Universidade Federal de Juiz de Fora, PCR was performed with primers 555F/555R (4003- R. José Lourenço Kelmer - Martelos, Juiz de Fora - MG, 36036- 4585) which amplify a 593bp fragment of the VP2 330 capsid gene. For RNA-viruses, the reverse transcription 3. USP - Universidade de São Paulo, Av. Prof. was performed with random primer (Invitrogen®) Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 and Superscript III enzyme (Invitrogen®). Differential In a previous study, a pigeon coronavirus grouped with primers were used in PCR assays for detection of CCoV the H120 (Massachusetts) IBV strain and a chicken (gene M), CaCV (RdRp gene), AstV (RdRp gene) and coronavirus characterized as BR-I IBV strain were RV-A (NSP-4 gene). Sequencing was performed in PCR- detected and characterized by our group. With the present positive samples for molecular characterization. A study we aimed to detect by qRT-PRC and analyzee the total of 91 samples were negative for all agents. Single clinical signs and tracheal ciliostasis lesions induced by infection was detected in 135 puppies (CPV=120, CCoV= these viruses in experimentally infected chickens. To 10, AstV=2, RV-A=2, CaCV=1). Mixed infections were do so, three groups named Corona-PigeonMass (67T), detected in 31 puppies: 20 cases of CPV/CCoV, three Corona-ChickenBR-I (801) and Corona-ChickenBR-I of CPV/RV-A, one of CCoV/CaCV, one of CCoV/AstV, (810), containing 25 animals per group, were inoculated one of CCoV/RV-A, four of CPV/CCoV/AstV and one of by intranasal route with 100mL containing 105.5 CPV/CCoV/RV-A. Among 166 positive puppies 52 had EID50 of the respective strain. A negative control group received at least one doses of polyvalent vaccines. Based inoculated with PBS solution was evaluated. The clinical on clinical reports, 76/135 puppies with single infection signs were observed during 21 days. Samples and and 10/31 puppies with mixed infection presented more severe clinical signs (vomiting, hemorrhagic diarrhea). per group at 4, 7, 9, 11, 14 and 21 days post inoculation For the remaining puppies the association of clinic (dpi)secretions at necropsy. were collected Viral RNA from was five investigated inoculated chickensby qRT- signs was variable (vomiting and/or nonhemorrhagic PCR assay for the UTR gene of IBV. The results showed that coronaviruses were detect at most around 5th dpi still the most prevalent type circulating in Rio de Janeiro. in sinus, trachea, ileum, kidney and tonsil; in cloaca and Baseddiarrhea). on partial Genotyping sequencing results of Mconfirmed gene, eight that strains CPV-2b were is testicle, coronaviruses were detected a bit earlier (2th characterized as CCoV-I while another 16 as CCoV-II. The and 4th dpi, respectively) and in pro-ventricle and lung a CaCV positive sample shared higher nucleotide identity bit latterly (7th and 11th dpi respectively) by qRT-PCR in with the genus. In conclusion, the results of this all inoculated groups. Clinical signs such as respiratory study suggest that puppies with acute gastroenteritis problems, sneezing, lethargy and nasal discharge were should be screened for AstV, CaCV and RV-A as well as the observed mainly at 5th dpi in all inoculated groups. A established pathogens CPV and CCoV. Financial support: complete ciliostasis was observed (0% of active cilia) at FAPERJ, CNPq, PROPPI-UFF, PROGRAD-UFF 4, 7 and 9 dpi in the 67T group; whereas 50% of the cilia activity was recovered at 14 dpi. As for the 801 and 810 groups a complete ciliostasis was observed slight later at 9 and 11 dpi; while the 50% of cilia activity was also

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

246 Veterinary Virology: VV recovery at 14 dpi. Our results suggests that Coronavirus of the Oswaldo Cruz Foundation (FIOCRUZ), Rio de strains of chicken and pigeon origins can induce a Janeiro. Sera from cynomolgus monkeys are being tested for the presence of cyCMV DNA by real time PCR (qPCR) for clinical signs and reiterate the pigeons’ importance and by nested PCR using primers for the presence of incomplete the dissemination ciliostasis in and chickens, evolution supporting of avian coronavirus. the findings cyCMV gB gene. Our results show that a large percentage Financial Support: CAPES, CNPq (475571/2012-6) and (77%) of the animals are positive for HCMV DNA. The FAPESP (Grant 2013/02058-6). determination of the presence and incidence of cyCMV infection in cynomolgous will lead to further studies VV308 - DETECTION OF CYNOMOLGUS of the molecular epidemiology, characterization of CYTOMEGALOVIRUS DNA IN BRAZILIAN MACACA CMV infection in vivo and virus evolution, as well for FASCICULARIS Stangherlin L.M.1; Santos, E.S.1; Pinto, M.A.2; Silva, separated from the rest of the animals and used in M.C.C.1 studiesthe identification of CMV infection, of CMV pathogenesis,free animals thatinteraction can be withkept 1. UFABC - Universidade Federal do ABC, Rua other pathogens and vaccine testing. Funding: UFABC Catequese, 242 - Jardim, Santo André - SP, 09090-400 VV312 - SUSCEPTIBILITY OF HAMSTERS TO 2. FIOCRUZ - Fundação Oswaldo Cruz, Av. EQUINE HERPESVIRUS TYPE 1 INFECTION CAUSING Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 ENCEPHALITIS AND RESPIRATORY DISEASE Arévalo, A.F.1; Mori, E.3; Mori, C.M.C.; Miyashiro, S.I.; members of the Betaherpesvirinae sub-family which Zanatto, D.A.; Cunha, E.M.S.; Lara, M. do C.C. de S.H.; haveCytomegaloviruses been isolated (CMV)from various are highly mammalian specie-specific species. Viillalobos, E.M.C.; Tonietti, P. de O.; Gamon, T.H.M.; The Human Cytomegalovirus (HCMV) is associated with Maiorka, P.C. serious diseases in immunocompromised individuals, such as transplant recipients and AIDS patients. HCMV 1. FMVZ/USP - Faculdade de Medicina is also a cause of severe neurologic abnormalities and Veterinária e Zootecnia da Universidade de São Paulo, Av. hearing loss in infected newborns. One of the limitations Duque de Caxias Norte, 225. Pirassununga - SP, 13635-900 of studying CMV is the fact that HCMV does not infect 2. INSTITUTO BIOLÓGICO, Av. Cons. Rodrigues Alves, 1252 - Vila Mariana, São Paulo - SP, 04014- 002 Beta-herpesviruses. Consequently, studies in vivo are 3. INSTITUTO PASTEUR, Av. Paulista, 393 - performedanimals, due in toCMVs the of characteristic the respective specie mammal specificity species. of Cerqueira César, São Paulo - SP, 01311-000 Due to their close relationship to humans, nonhuman primates are considered the ideal research model The mouse has proved to be an excellent model for for these studies. The Rhesus Cytomegalovirus CMV studying the pathogenesis of encephalitis caused (RhCMV) is being characterized regarding to its genomic by neuropathogenic strains of equine herpesvirus similarity to HCMV and pathogenicity in vivo. Thus, type 1 (EHV-1). However, recent studies showed that Rhesus macaques (Macaca mulatta), are primarily used intranasal infection in hamsters resulted in more acute in the CMV studies. However due to the high demand and severe symptoms than those observed in mice, and the short the supply of Rhesus monkeys other suggesting greater susceptibility of the specie to the monkey species are becoming of interest for these agent. To evaluate respiratory and neurological changes studies. Cynomolgus Monkeys (Macaca fascicularis) are resulted from EHV-1 infection in hamsters. Material and infected with cynomolgous cytomegalovirus (cyCMV), as Methods: Male Syrian hamsters, 3-wk-old, were infected shown by serological studies. Recently a cyCMV has been via intranasal route with EHV-1 strains obtained from isolated and completely sequenced, providing a novel infected aborted foal fetuses (A4/72, A9/92, A3/97 model for studies to address fundamental questions of and Iso/72). The animals were weighed and examined HCMV biology, pathogenesis and immunology. This study daily for clinical and neurological signs. According to aims to investigate the presence and natural prevalence of cyCMV infection in the cynomolgous monkey colony the symptoms, groups of five hamsters were euthanized September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersby - Veterinary overdose Virology: of isoflurane VV or intraperitoneal ketamine/ XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

247 Veterinary Virology: VV xylazine injection. During necropsy, CNS, lungs, liver, Rabies is a fatal disease that infects the Central Nervous spleen and thymus were collected for virus isolation in System (CNS) of mammals. Rabies virus (RABV) is a RNA E-dermal cells culture. In addition, bronchoalveolar lavage non-segmented and single-stranded virus, member of (BAL) was performed to determine total and differential the Lyssavirus genus within the Rhabdoviridae family. white blood cell (WBC) count. Results: Similar to the Despite the recognized stability of RABV, differences experiments with mice, hamsters challenged with A4/72 among isolates from different species have been found. and A9/92 strains showed severe clinical manifestations Members of the Chiroptera order are reservoirs of many in the 3rd d.p.i., such as weight loss, dyspnea, dehydration, lissaviruses and the vampire bat Desmodus rotundus recumbency and death. It was also observed neurological became the most important reservoirs of RABV in Latin signs such as hyperexcitability, spastic paralysis, loss of America. Rabies transmitted by bites from D. rotundus, proprioception, circling and seizures. However, unlike affects cattle and sporadically humans causing economic the mice, which did not developed the disease; hamsters losses in livestock and in public health. This work was inoculated with A3/97 and Iso/72 strains showed carried out with the aim to identify the stability of genetic clinical and neurological symptoms in the 4th d.p.i., in lineage of RABV from vampire bat D. rotundus. For this, which the most evident were respiratory changes, mainly a group of six Swiss Webster mice was inoculated with epistaxis. Virus isolation from the CNS was positive in all RABV strain isolated from vampire bat D. rotundus animals; however, lungs were positive only in the groups infected by strains A9/92 and A4/72. BAL showed that suspension was prepared and inoculated in another WBC count was higher in A4/72 infected hamster when group(first passage). of mice The(second CNS ofpassage) mice were this collected,procedure a newwas compared with A9/92, A3/97 and Iso/72 and control repeated by ten successive passages. The inoculation group. Activated macrophages with large vacuolated was made by intracerebral route and the animals were cytoplasm, some of them containing granules within, maintained with water and food at libidum. The CNS and a great amount of erythrocytes were observed in all inoculated animals’s smears unlike the control smears, to RNA extraction, RT-PCR and sequencing of the which had only inactivated cells and rare erythrocytes. nucleoproteinstrains of first, genefifth and(N) oftenth the passagesvirus. None were difference submitted in Conclusion: The results indicate that hamster is the most clinical signs of rabies after ten successive passages was susceptible specie to infection caused by EHV-1, serving as an experimental model for studies of respiratory and neurological disorders caused by this agent. Ouridentified results in inoculatedshow that mice.genetic In additionlineage ofwas RABV not foundfrom vampiresignificant bat differences D. rotundus in the is geneticstable aftersequence 10 successive of N gene. VV315 - GENETIC STABILITY OF RABIES VIRUS passages in mice. In conclusion high genetic stability ISOLATED FROM VAMPIRE BAT DESMODUS of RABV recovered from vampire bat D. rotundus, was ROTUNDUS Pereira, P.M.C.1; Fernandes, M.E.S.1; Achkar, S.M.1; this hypothesis. FINANCIAL SUPPORT: FAPESP Oliveira, R.N.1; Castilho, J.G.1; Roehe, P.M.2,3,4; Carnieli confirmed and other genes will be analyzed to confirm Jr, P.1; Batista, H.B.C.R.1 1. INSTITUTO PASTEUR, Av. Paulista, 393 - Cerqueira César, São Paulo - SP, 01311-000 2. IPVDF - Instituto de Pesquisas Veterinárias Desidério Finamor, Estrada Municipal do Conde, 6000. Eldorado do Sul - RS, 92990-000 3. FEPAGRO - Fundação Estadual de Pesquisa Agropecuária, Fundação Estadual de Pesquisa Agropecuária, R. Gonçalves Dias, 570, Porto Alegre - RS, 90130-060 4. UFRGS - Universidade Federal do Rio Grande do Sul, Avenida Paulo Gama, 110 - Farropilhas, Porto Alegre - RS, 90040-060

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248 Veterinary Virology: VV

VV317 - MOLECULAR ANALYSES OF VP6, VP7, VP4, understanding the viral phylogeny and epidemiology, as AND NSP4 GENES OF PORCINE ROTAVIRUS GROUP H well as the explanation of patterns of viral evolution and DETECTED IN BRAZIL biological properties of RVH. Financial Support: FINEP, Possatti, F.1; Molinari, B.L.D.1; Possatti, F.1; Lorenzetti, CAPES, CNPq, and Fundação Araucária/PR. E.1; Otonel, R.A.A.1; Leme, R.A.1; Freitas, L.A.1; VV318 - AN OLD VIRUS IN A NEW SPOT: DETECTION Bronkhorst, D.E.2; Alfieri, A.F.1; Alfieri, A.A.1 OF HANTAVIRUS IN WILD RODENTS FROM CENTRAL 1. UEL - Universidade Estadual de Londrina, REGION OF MINAS GERAIS STATE Rodovia Celso Garcia Cid, Km 380 - Campus Universitário, Alves, P.A.1; Amaral, C.D.1; Borges, I.A.1; Alves, P.A.1; Londrina - PR, 86057-970 Miranda, J.B.1; Sacchetto, L.2; Costa, G.B.1; Abrahão, 2. UNOPAR - Universidade Norte do Paraná, J.S.1; Paglia, A.1; Figueiredo, L.T.M.3; Drumond, B.P.2; Av. Paris, 675 - Jd. Piza, Londrina - Paraná, 86041-140 Trindade, G. de S.1 Rotaviruses (RV) are a common cause of viral 1. UFMG - Universidade Federal de Minas gastroenteritis in humans and animals. Despite the seven Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, groups/species of RV (A-G), recently it was proposed Belo Horizonte - MG, 31270-901 the creation of a new RV group/specie H (RVH) based 2. UFJF - Universidade Federal de Juiz de Fora, on VP6 sequence analysis. This species includes the R. José Lourenço Kelmer - Martelos, Juiz de Fora - MG, 36036- following: the novel adult diarrhea RV strain (ADRV-N) 330 isolated from specimens collected during an outbreak 3. USP - Universidade de São Paulo, Av. Prof. of gastroenteritis among adults during 1997 in China; Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070 Hantaviruses are important zoonotic pathogens related China in 1997; the human RV strain B219, detected in to rodents and small mammals. In nature, these viruses astrain sporadic J19, case identified of diarrhea during in Bangladesh the same during outbreak 2002; in cause asymptomatic and persistent infections, and may the porcine RV strain SKA-1 that was isolated from a pig be transmitted to humans via inhalations of aerosols with diarrhea in Japan; 3 Brazilian RVH-positive stool from urine, feces. Also, the virus can be transmitted specimens (BR59, BR60, and BR63), and 30 US RVH- through saliva or direct contact with skin lesions or positive stool specimens detected in swine herds in rat bites. The Hantavirus Cardiopulmonary syndrome 2014. In this study we determined the VP6, VP7, VP4, and (HPS) is considered one of de most important emerging NSP4 nucleotide and deduced amino acid sequences of 6 diseases in Brazil, with high mortality rate (~40%) (BR59, BR60, BR61, BR62, BR63, and BR64) RVH-positive in humans. The disease has been reported in several stool specimens obtained from piglets with diarrhea regions of the country, including the western part of in Mato Grosso do Sul, Central-West region of Brazil in Minas Gerais state with a high incidence. This work 2012, using RT-PCR assay. Based on the high sequence aimed to detect Hantavirus in small mammals trapped on rural areas of Serro city, central region, State of Minas among 5 of the studied fecal specimens (BR59 to BR63), Gerais. An effort of 4800 traps was performed between theyidentities are (˃considered 99%) of the the VP6, same VP4, local VP7, rotavirusand NSP4 straingenes 2012-2013 and 56 animals were caught (1,1%). Forest, denominated RVH/BRA-1. In contrast, since the fecal pasture and domiciliary areas were covered. A greater sample BR64 showed a relatively high difference (81.6% number of captures are seen in rain forest (52 %), nt identity and 83.4% aa identity) in the VP7 sequence followed by domiciliary areas (32 %) and pasture when compared to the other 5 specimens it was named (16 %), which demonstrates a high incidence of these RVH/BRA-2 strain. Comparative phylogenetic analysis rodents in disturbed areas. Sera from all rodents were showed that the 6 RVH strains do not cluster together tested by IgG-ELISA using the N recombinant protein with any available sequences of the members from the of Araraquara virus as antigen. Two positive samples established RV groups (RVA-RVG), however, it seems to were found. RNA was extracted from RNA Latter (Life Technologies) preserved lungs and Real time PCR was presence of RVH in Brazil, demonstrate their genetic also performed on 18 samples. One Olygorizomys sp diversity,be related and to RVBprovide and RVG.new Thesedata resultsthat will confirm assist thein September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

249 Veterinary Virology: VV was found positive for the hantavirus S segment. The performed with the same primers as those used for PCR Olygorizomys sp, was trapped in domiciliary area on an automated DNA sequencer (ABI3500). Nucleotide indicating a close contact among wild and domestic life. and amino acid sequence alignment was performed Indeed, this genera has already been associated to the with the Clustal method using the BioEdit software. Juquitiba virus, which is of the most widely distributed The CPV-2 was detected by PCR in 83.33% (20/24) of the samples tested. Of these, one animal had incomplete description of a Hantavirus circulating in this area of thehantavirus Minas Gerais found State. in southFINANCIAL Brazil. SUPPORT: This is FAPEMIG, the first two doses of vaccine, six were not vaccinated and the CAPES, CNPQ, MAPA vaccination history schedule of eight (3 doses), animals five was received unknown. one CPV- or 2 was detected in the feces of animals of different ages, VV320 - DIAGNOSIS AND MOLECULAR with higher incidence (80%) on the animals between CHARACTERIZATION OF CANINE PARVOVIRUS IN 1 and 12 months old. Surprisingly, CPV-2 was also DOGS TREATED AT THE VETERINARY HOSPITAL detected in the animalof 14 years old, which received OF FEDERAL UNIVERSITY OF PARANA - CAMPUS only one dose of vaccine when a puppy and whose death PALOTINA recorded. Of the 20 positive samples by PCR, seven Kunz, A.F.1; de Mello, J.L.1; Gallego, J.C.1; Macedo, R.1; (35%) were sequenced to determine the type of CPV-2. Steffens, R.1; Oyafuso, M.K.1; Possati, F.2; Alfieri, A.2; Takiuchi, E.1 revealing the presence of the codon GAA (Glu) at position In the sequence analysis, all were classified as CPV-2c, 1. UFPR - Universidade Federal do Paraná, 426 of the viral protein VP2. The wide detection of CPV- Rua XV de Novembro, 1299 - Centro, Curitiba - PR, 80060- 2c in the canine population of the state of Paraná, even 000 in adult animals with vaccination history, reinforces the 2. UEL - Universidade Estadual de Londrina, importance of investigations into the virulence of this Rodovia Celso Garcia Cid, Km 380 - Campus Universitário, variant and the need for its incorporation into vaccines Londrina - PR, 86057-970 for effective disease prevention.

Canine parvovirus is a highly contagious enteric disease VV322 - SEROLOGIC EVALUATION AND DETECTION caused by canine parvovirus type 2 (CPV2). Currently OF CANINE PARVOVIRUS TYPE 2C IN DOGS KEPT IN three antigenic variants of CPV-2 are circulating, called AN ANIMAL PROTECTION SHELTER IN THE CITY OF CPV 2a, 2b and 2c. The frequency distribution of these MEDIANEIRA, PARANA variants vary according to geographical location and the Kunz, A.F.1; Grolli, P.1; Gallego, J.C.1; Macedo, R.1; de time reference period of the studies. Recent studies have Mello, J.L.1; Possati, F.2; Alfieri, A.2; Takiuchi, E.1 reported the type 2c as the most prevalent in certain regions of Brazil. However, the types of CPV2 involved 1. UFPR - Universidade Federal do Paraná, in cases of canine gastroenteritis in Paraná state in Rua XV de Novembro, 1299 - Centro, Curitiba - PR, 80060- recent yearsare unknown. The objective of this study 000 was to detect and identify types of CPV-2 circulating 2. UEL - Universidade Estadual de Londrina, in the dog population residing in the city and around Rodovia Celso Garcia Cid, Km 380 - Campus Universitário, Palotina, Paraná between 2013 and 2014. Twenty-four Londrina - PR, 86057-970 fecal samples from dogs with gastroenteritis treated at Canine parvovirus type 2 (CPV-2) is the main agent of the Veterinary Hospital of UFPR campus Palotina were acute viral gastroenteritis, affecting predominantly evaluated. The animals were between one month and 14 young animals until six months old. Another clinical years old and had various historical of vaccination. The syndrome associated to CPV-2 is myocarditis in newborn viral DNA extraction was performedusing a combination of phenol/chloroform/isoamyl alcohol and silica/ weeks of life, culminating in sudden death. Currently guanidiniumisothiocyanatenucleic acid methods, and threedogs undergoingCPV-2 antigenic intrauterine variants infection are circulating or in the called first CPV-2a, 2b and 2c, which only 2c is not consisted in 4585) of the VP2 gene. The sequencing reactions were the vaccines available. The detection of antibodies (Ab) was amplified by PCR a 583 bp fragment (nt 4003- September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

250 Veterinary Virology: VV anti-CPV-2 in non-vaccinated animals indicates natural Neonatal diarrhea is a major health problem affecting exposure to the virus and determining the Ab title reveals the level of protection after natural infection or losses. Among the various agents involved in this vaccination. The aim of this study was to determine the syndrome,calves in the rotaviruses first weeks haveof life, beencausing reported huge economic as the title of Ab anti-CPV-2 and CPV-2c detection in dogs kept most frequently detected during disease outbreaks. in a shelter runned by a non-governmental organization Belonging to the family Reoviridae, rotavirus presents 75 nm in diameter, triple capsid protein, and has the from adult dogs with unknown vaccination history were genetic material as dsRNA, with 11 genomic segments. evaluated.in the city Toof Medianeira,measure Ab PR. title, Forty-five sera were serum subjected samples to The external capsid proteins, VP4 and VP7, induce the hemagglutination inhibition technique being considered production of neutralizing antibodies and are useful protective titles exceeding 80. In addition, samples were collected from feces of normal consistency of an to determine the frequency of rotavirus infection in adult bitch with a history of recent sudden death of calvesfor viral of classification.dairy and beef The herds objective between of the this years study 2006 was her litter. DNA was extracted using the combination of to 2010, on properties of Minas Gerais State, Brazil. the techniques phenol/chloroform/isoamyl alcohol For this, survey data of 19 municipalities with a total of 36 herds and 304 samples, of which 31 were obtained by PCR technique a fragment of 583 bp VP2 gene and from animals with diarrhea and 273 clinically normal and silica/guanidiniumisothiocyanate, being amplified animals were studied. Employing the PAGE technique tested, 43 (95.5%) had protective antibody titles against the observed frequency of infection was 11.1% (4/36) infectionsubsequent by CPV-2 sequencing. (above Forty-five80), titration of ranged the animal from sera128 and 2.6% (7/273) found in animals with and without to 2048. CPV-2 was detected by PCR in the fecal sample diarrhea, respectively. Genotyping of positive samples for rotavirus was performed by the method of reverse fragment revealed the CPV-2c variant. The presence of Ab transcription-polymerase chain reaction (RT-PCR) canof the be adult the result bitch of and the sequence exposure analysis to remnants of the of amplified the virus and highlighted the presence of infection by genotype or vaccine response resulting from previous exposure to G6P[5] and G10P[11] in dairy herds and the genotypes G6P[5] and G6P[5]P[11] in the beef herds. The results 2c in the fecal sample of the adult bitch can justify the suddenshelter life.death The of the confirmation litter from ofthe the infection. detection The of results CPV- beef herds. Hygiene measures, management and of this study highlight the importance of titration of Ab immunoprophylaxisindicated a significant are circulationimportant to of reduce rotavirus the rate in the of to evaluate the susceptibility of these animals to disease infection and the persistence of the agent in the herds. and consequently the need to institute prophylactic FINANCIAL SUPPORT: FAPESP vaccination. Also proves the circulation of CPV-2c in the canine population in our region, pointing the need for VV336 - DETECTION OF FOUR PUTATIVE NEW BOVINE further studies of its virulence and antigenicity, as well PAPILLOMAVIRUS TYPES FROM THE BRAZILIAN AMAZON protection against this variant. Chiappetta, C.M.; Daudt, C.; da Silva, F.R.C.; Chiappetta, as to define the effectiveness of vaccines in heterologous C.M.; Streck, A.F.; Canal, C.W. VV330 - OCCORRENCE OF BOVINE ROTAVIRUS IN LABORATÓRIO DE VIROLOGIA VETERINÁRIA/ DAIRY AND BEEF HERDS IN MINAS GERAIS STATE, UFRGS - Laboratório de Virologia Veterinária da Universidade BRAZIL BETWEEN 2006-2010 Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9090, Godoy, H.P.; Gonçalves, A.C.S.; Camargo, T.; Mazer, L.; Agronomia, Porto Alegre - RS, 91540-000 Samara, S.I.; Buzinaro, M. da G. Papillomaviruses (PV) are small and complex viruses FCAV/UNESP - Faculdade de Ciências Agrárias e that belong to the family, composed by Veterinárias da Universidade Estadual Paulista, Via de Acesso at least 29 genera. They have a circular double-stranded Prof.Paulo Donato Castellane s/n, Jaboticabal, SP, 14884-900 DNA containing approximately 8,000 base pairs (bp). The bovine papillomavirus (BPV) cause an infectious disease

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

251 Veterinary Virology: VV that is characterized by chronic benign proliferative negative RNA genome encoding eight proteins. Given tumors that affect cattle worldwide. Additionally, these the implications that recombination has for RNA viruses can also cause tumor and neoplasia at different virus evolution, it is clearly important to determine if body regions, such eyes, skin, urinary bladder, upper recombination plays a role in CDV evolution. In this study, gastrointestinal and genital tract. This report aimed to we analyzed 30 complete CDV strains isolated from dogs develop a prospective study of BPV infection in cattle and non-dog hosts using seven distinct algorithms to from Northern Brazil based on nucleotide sequences of detect genetic conversions and incongruent phylogenies. the L1 ORF that is a highly conserved region between Fourteen putative recombination events were detected, all papillomavirus. Twenty-one samples of skin warts being most of them potential multi-recombinants. clinically diagnosed as papillomatosis from Acre and Interestingly, most of the genome mosaics were produced by recombination events between strains of L1 gene was performed with the degenerated primer from different genotypes, including classical vaccine Rondônia States were analyzed. The partial amplification strains, and strains isolated from different host species, were sequenced. The phylogeny was estimated with a mainly dogs and raccoons. These results suggest that Bayesianpair FAP59/FAP64 MCMC method, and using the BEAST amplification version 1.7.2 products and recombination plays an important role in CDV evolution run using the GTR substitution model and the resulting and that attenuated vaccines can recombine with data were analyzed using Tracer software. The DNA circulating wild-type virus. FINANCIAL SUPPORT: CNPQ, FAPERGS, CAPES AND PROPESQ/UFRGS four BPVs types (BPV1, 2, 11 and 13) already reported, foursegment putative of the new BPV BPV L1 genetypes enabled (05RO10, the 09RO11, identification 02AC12 of VV349 - UNDERESTIMATION AND TEMPORAL and 06AC14) and three putative new BPV subtypes. To TREND OF INCREASING COMPLEXITY OF HIV-1 BF1 RECOMBINANTS IN RIO DE JANEIRO, BRAZIL Marques, B.C.L.; Morgado, M.G.; Guimarães, M.L. our knowledge, this is the first record of BPV from the genetic diversity of BPV types that can be present in this FIOCRUZ - Fundação Oswaldo Cruz, Av. Brasil, 4365 Brazilian Amazon and these findings point to the great region. Finally, the discovery and characterization of - Manguinhos, Rio de Janeiro - RJ, 21040-900 novel BPV types will increase the understanding of viral HIV has extreme genetic variability due to high evolution and provide support for further studies about mutation and recombination rates caused by the lack therapeutic treatment. FINANCIAL SUPPORT: CAPES, of proofreading activity of the reverse transcriptase, CNPQ, FAPERGS AND PROPESQ/UFRGS. associated to the high rate of viral replication. VV343 - IDENTIFICATION OF POTENTIAL MULTI- Notwithstanding, molecular epidemiological studies are RECOMBINANT STRAINS OF CANINE DISTEMPER mostly based in the characterization of just one genomic VIRUS region, resulting in an underestimation of recombinant Streck, A.F.; Budaszewski, R. da F.; Weber, M.N.; forms. Previous studies conducted in Rio de Janeiro Mósena, A.C.S.; da Silva, M.S.; Dupont, P.M.; Borchardt, depicted a prevalence of 5% to 28% of HIV-1 sub-subtype A.C.; Streck, A.F.; Canal, C.W. F1 and 2% to 15% of BF1 recombinants, depending on the genomic region and/or from the studied population LABVIR/UFGRS - Laboratório de Virologia do Instituto de Ciências Básicas da Saúde da Universidade Federal do genomic regions, including the most recombinogenic Rio Grande do Sul, Prédio do ICBS/UFRGS - Sala 208 Rua ones,group. to The estimate present the study real aimedprevalence to characterize of sub-subtype five HIV F1 Sarmento Leite, 500 - Bairro: Cidade Baixa, Porto Alegre - RS, and to understand the complexity of the recombination 90050-170 Canine distemper virus (CDV) is a highly contagious collected from 1998 to 2013 and previously characterized viral pathogen that can cause a lethal systemic disease profiles. From a total of 635 HIV-1 positive samples in dogs and other carnivores. CDV belongs to the From those, 56 had biological samples and were included genus Morbillivirus within the family Paramyxoviridae based on the C2-V3 env region, 74 were classified as F1. and possesses a non-segmented single-stranded and sequenced in gag, PR/RT, INT, env and nef regions. in this study. The DNA samples were extracted, amplified September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

252 Veterinary Virology: VV

The sequences were aligned with reference samples Medicina Veterinária em Aquicultura, Rua Vieira de Morais, by ClustalW. Phylogenetic analyzes were performed 1201, Campo Belo, São Paulo - SP, 04617-014 using Neighbor Joining with Tamura-Nei substitution Ranavirus (RV), members of genus Ranavirus, family method by MEGA 5.1. The recombination analysis , are emerging pathogens responsible for was performed by bootscan in Simplot 3.5.1 program. outbreaks of great ecological and economic impacts The prevalence of sub-subtype F1 sequences varied according to the analyzed genomic region from 46% to aquaculture around the world, including Brazil. Cyprinid 71%, as well as BF1 recombinants represented 14% to herpesviruson fish, amphibians 3 (CyHV-3), and also reptiles known as of Koi importance herpesvirus, in is a double-stranded DNA virus and belongs to genus and family . CyHV-3 is very 25% of the forms. All samples were amplified at least pathogenic and is responsible for a high rate of morbidity in three genomic regions and 39% of them confirmed and mortality in common carp (Cyprinus carpio carpio) the previous F1 classification, whereas 61% were the BF1 recombinants were unique recombinant forms and koi carp (Cyprinus carpio Koi) causing large-scale reclassified as BF1. Based in the studied regions most of and only two were a second generation of a Circulation losses in worldwide aquaculture. Additionally, it has been Recombinant Form (CRF_12BF and CRF_39BF). Based in our analysis, samples collected from 1998-2003 carriers of CyHV-3. The main goal of this study was to presented only intragenic recombination (14%), investigatesuggested that cause other of species death ofof fish tilapias can be asymptomatic(Oreochromis whereas samples from 2004-08 exhibited intergenic niloticus) from one nursery farm located in Paulo

of 30 to 50g body weight were collected. Fragments of (6%) and intragenic (21%) profile and in those from gill,Afonso, eye, Bahia.liver, intestine, For this kidneypurpose, and 14 spleen fishes were with sampled a range 2009-13 all profiles (intragenic-15%; intergenic-18%; inter/intragenic-26%) were verified. In conclusion, underestimated when just one genomic region was used signs included exophthalmia, ocular opacity, swimming our results confirmed that recombinant forms were inand whirling, fixed in skin buffered darkening formalin and solution apathy, 10%. resulting Clinical in complexity of the BF1 recombinant forms was observed mortality rate of 30%. Concurrent infections by different for classification and a temporal trend of increasing over time. bacterial agents were observed as Streptococcus VV358 - CONCURRENT IRIDOVIRAL AND agalactiae, Francisella sp. and Flavobacterium sp. PRESUMPTIVE HERPESVIRAL INFECTON Infestations by monogeneas and trichodinids were ASSOCIATED WITH MORTALITY IN TILÁPIA also detected. Histologically, the core of renal epithelial (OREOCHROMIS NILOTICUS) FROM PAULO AFONSO, cells exhibited chromatin margination, consistent with BAHIA, BRAZIL herpesvirus infection. Basophilic cytoplasmic inclusions were abundant in pancreas, in a pattern typical of Maganha, S.R. de L.1; Navarro, J. de O.1; Almeida infection. Aliquots of 50mg (pool of tissues) Queiroz, S.R. de1; de Pádua, S.B.2; de Menezes Filho, were submitted to DNA extraction followed by PCR R.N.2; Araújo, A.P.3; Alencar, A.L.F.1; Arruda, E. de P. 1; Rosa, M.P.1; Michelin, E.C.1; Fernandes, A.M.1; de of 409- and 348bp-fragments of the TK gene of CyHV- Sousa, R.L.M.1 3.(polymerase In addition, chain the samereaction) samples for sequential were subjected amplification to PCR 1. FZEA/USP - Faculdade de Zootecnia e Engenharia de Alimentos da Universidade de São Paulo, Av. followed by nucleotide sequencing of the correspondent Duque de Caxias Norte, 225 - Campus da USP, Pirassununga amplicons.amplification All withsamples primers were to positive the iridoviral for iridovirus MCP gene and - SP, 13635-900 the PCR products showed a high percentage of similarity 2. AQUIVET - Aquivet Saúde Aquática, Rua to the corresponding sequences deposited in GenBank. Antonio Ernesto Célico, número 359, quadra 8, lote 16, Although no amplicons of MCP gene could be obtained Residencial Marcia Damha III, São José do Rio Preto - SP, with the primers set used in this study, concomitant 15061810 infection of RV and CyHV-3 is not a rare event, indicating 3. ACQUAPISCIS - Acquapiscis Consultoria e that different molecular diagnostic approaches can be

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

253 Veterinary Virology: VV further used to better understanding the etiological in dogs from Brazil and suggests their role as a canine nature of these infections. FINANCIAL SUPPORT: FAPESP enteric pathogen. (PROC. Nº 2012/08846-3, 2014/04327-7) VV369 - FIRST DOCUMENTED EVIDENCE OF BOVINE VV364 - DETECTION AND CHARACTERIZATION OF VIRAL DIARRHEA VIRUS GENOTYPE 2 (BVDV 2) IN 5 IN DOGS FROM BRAZIL COLOMBIA Alves, C.D.B.T.; Souza, C.K.; Streck, A.F.; Budaszewki, Villamil, V.; Vera, V.; Ramirez, G.; Jaime, J. R. da F.; Granados, O.F.O.; Torikachvili, M.; Dupont, UNAL - Universidade Nacional da Colômbia, Avenida P.M.; Weber, M.N.; Canal, C.W. Carrera 30 # 45, Bogotá, Cundinamarca 111321, Colômbia UFRGS - Universidade Federal do Rio Grande do Sul, The Bovine Viral Diarrhea Virus (BVDV) has emerged Avenida Paulo Gama, 110 - Farropilhas, Porto Alegre - RS, as one of the economically important pathogens 90040-060 in cattle populations, generating different clinical (MamV) have been described in presentations that can range from asymptomatic to many animal species associated with diarrhea and respiratory disorders, reproductive failure, congenital digestive disorders. There are few studies about the abnormalities and general immunodepression. In epidemiology of MamVs in dogs; however, astrovirus- like particles and viral genomes have been sporadically in 1975 in a batch of heifers imported from Holland. The described. Currently, the canine astrovirus was accepted Colombia, the presence of the virus was first reported as a distinct species in the Astroviridae family, named and noncytopathic) and three genotypes (1, 2, 3) Mamastrovirus 5 (MamV5). MamV5 was detected accordingvirus has beento their classified nucleotide into twosequence. biotypes Genotype (cytopathic 1 is in symptomatic puppies occasionally suffering from widely distributed in Colombia causing reproductive diarrhea in association with other enteric viruses, such problems in cattle. Genotypes 2 and 3 have lower as rotavirus and coronavirus and/or parvo-like viruses. incidence and have not yet been detected in the country. In addition, recent studies reported that around 10% Both genotypes are differentiated based on the 5 ‘non- of puppies showing clinical signs of diarrhea have also coding region (5’NCR). Depending on the infecting the MamV genome. Therefore, the aim of the present strains, can be recognized at least three different clinical study was to verify the presence and characterize the syndromes associated with BVDV infection: an acute genome of MamV5 from dogs of different Brazilian disease in cattle, the mucosal disease and persistently regions. Rectal swab samples were collected from infected animals (PI). The purpose of this study was to determine the presence of BVDV genotype 2 in Colombia Brazil, with different ages and breeds and both genders. evaluating four representative areas of cattle production Total107 dogs RNA with was orextracted without anddiarrhea, RT-PCR from was five performed States of using RT-PCR on serum and ear notches. For this, 375 using two primer pairs: pan-Mamastrovirus primers prepartum cattle’s sera, 245 precolostral sera from calves born to these cows and 103 calve’s (25 post-birth days) sera were collected. Additionally, 178 ear notches Escherichiafor screening, coli and were specific used primersas an internal to identify control. MamV5. The of these calves were taken. Subsequently, extraction of resultsAdditionally, showed specific that primersthe pan-Mamastrovirus for 16S ribosomal primers RNA of viral RNA from samples was performed. RT-PCR was

to discriminate the presence of BVDV and those who thesedetected samples 39 positive were selected samples for and full-genome fifteen were sequencing positive testeddue using positive specific underwent primers newto amplify RT-PCR a using288 bp primers segment to andusing the the capsid specific protein primer phylogenetic pair for MamV5. analyses Three showed out of a high similarity between Brazilian samples and MamV5 2 on the 5’NCR region. 16 bovine females samples were sequences from other countries, such as China, Korea, positiveamplify afor 221 BVDV, bp specifictwo of which sequence corresponding for BVDV genotypeto BVDV- Italy and France. We hypothesize that one main MamV5 BVDV and two sera samples taken at 25 after birth days were2; five positive precolostral for BVDV. samples Finally, of serum 17 ear were notches positive were to clonal population was efficiently spread worldwide. TheseSeptember/October findings also2014 Volume indicate 19 – that Supplement MamV5 2 - Abstracts/Posters is present - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

254 Veterinary Virology: VV positive, of which 16 corresponded to BVDV-2. This study performed. After that, RNA was extracted from cell livestock production in our country and represents the fractions,vesicles, electronextracellular microscopy vesicle fraction of the finaland serum. pellet wasRT- confirms the presence of BVDV in the areas of major PCR was performed to amplify a fragment of 197bp, 2 BVDV in Colombia. This constitutes a new element to corresponding to ORF6 and ORF7 PRRSV genes. Vesicles befirst considered documented to controllingevidence of the the disease presence in theof genotype country. of different sizes, morphologically characterized as SOPORTE FINANCIERO: UNIVERSIDAD NACIONAL DE microvesicles or exosomes, were evidenced. All samples COLOMBIA, COLCIENCIAS, UNIDAD DE INVESTIGACIÓN were positive for PRRSV by RT-PCR. This study shows FMVZ, UNIVERSIDAD NACIONAL DE COLOMBIA present in porcine colostrum. The viral transmission by VV370 - DETECTION OF PRRSV RNA FROM PORCINE the identification of PRRSV in cell excretion vesicles COLOSTRUM – DERIVED MICROVESICLES Cano, L.; Vera, V.; Ramírez, G.; Jaime, J. stepcolostrum to demonstrate was also confirmedthat PRRSV by is PRRSV-RNAtransmitted detectionfrom sow in serum and cell fraction. This contribution is the first UNAL - Universidade Nacional da Colômbia, Avenida to suckling pigs using the Trojan Horse strategy and Carrera 30 # 45, Bogotá, Cundinamarca 111321, Colômbia contributes to understand the disease´s pathogenesis. FINANCIAL SUPPORT: UNIVERSIDAD NACIONAL DE Porcine Reproductive and Respiratory Syndrome Virus COLOMBIA, PROGRAMA NACIONAL DE SEMILLEROS (PRRSV) is an important disease for the swine industry DE INVESTIGACIÓN, CREACIÓN E INNOVACIÓN DE LA due to economic losses. In Colombia, the presence of UNIVERSIDAD NACIONAL DE COLOMBIA. UNIDAD DE the virus was reported in 1997 and produces recurring INVESTIGACION FMVZ, UNIVERSIDAD NACIONAL DE outbreaks. It has been determined that several viruses COLOMBIA. may be transmitted through released by cells. The extracellular vesicles, as microvesicles and exosomes, are VV371 - DETECTION OF PORCINE CIRCOVIRUS TYPE released by different types of cells and have been detected 1 (PCV1) IN PIGS FROM COMMERCIAL FARMS IN BRAZIL vesicles are a mechanism of intercellular communication Cruz, T.F.; Yamatogi, R.S.; Araujo Jr, J.P. employedin body fluids by proteins, including lipids milk and and RNAs colostrum. transportation. These The association between the extracellular vesicles with UNESP - Universidade Estadual Paulista, Rua Quirino molecules involved in viral cell entry, RNAs and proteins de Andrade, 215, São Paulo - SP, 01049-010 of viral envelope, suggested that these vesicles can act as Porcine circoviruses (PCV) are non-enveloped viruses, a possible transmission route and as well as mechanism with single-stranded DNA genome that belong to the of PRRSV immune evasion. This hypothesis is called family Circoviridae. PCV2 is associated with several mechanism of Trojan Horse, which proposes that the diseases, while PCV1 is considered a non-pathogenic virus virus or viral genome, using the exosome´s formation detected in vaccines, cell cultures or products used for and transportation processes is able to hide away from cell culture preparations. PCV1 is also found in domestic the immune system, can infect other cells and generate pigs but not much is known about its epidemiology and new virus particles. This mechanism represents an worldwide distribution. Thus, the aim of this study was important infection way and would have applications to detect PCV1 in blood samples collected from pigs in in antiviral therapies and vaccines. In this study, the possibility of PRRSV transmission from sow to piglets whole blood samples were collected from pigs (65- through colostrum extracellular vesicles was proposed 160several days-old) farms in the Brazil. period Eight from hundred March and 2013 fifty-eight to June 2014. These samples were collected for monitoring of milliliters of colostrum were collected from eight sows vaccination against PCV2, which were analyzed by PCR fromusing a the positive Trojan PRRSV Horse farm strategy. located A in hundred Valle del and Cauca, fifty quantitative (qPCR). Eighteen farms located in eight Colombia. Each colostrum was processed by differential states of Brazil were used in the study. The number of ultracentrifugation and sucrose density gradient to samples per state was: São Paulo (n=40), Minas Gerais purify extracellular vesicles. In order to identify the (n=30), Paraná (n=130), Santa Catarina (n=168), Rio

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Grande do Sul (n=170), Distrito Federal (n=200), Mato Fibropapillomatosis is a neoplastic disease of the skin and Grosso (n=90) and Ceará (n=30). Extracted DNAs were internal organs that affects green turtles, Chelonia mydas. analyzed by qPCR for PCV (ORF1, both PCV1 and PCV2). The etiology and pathogenesis is partly understood and One hundred and thirty-eight samples were positive for it has been associated with papillomavirus and mainly PCV by qPCR. Thus, seventy PCV positive samples (1 x herpesvirus, environmental and genetic factors. The 105 to 6.2 x 1010 copies of PCV DNA/ml) were selected aim of this study was to identify papillomavirus and to be tested for PCV1. Only eight samples were positive for PCV1. For the total of 858 samples, PCV1-positive the coast of Paraná, southern Brazil. Coastal (40 Km) and samples corresponded to 0.9%. For the 70 samples baysherpesvirus monitoring in skin of fibropapillomasParanaguá Estuary of green Complex turtles were on positive for PCV, PCV1-positive samples corresponded to performed from 2009 to 2012 and sea turtles carcass 11.4%. The eight samples positive for PCV1 were from the states of São Paulo (n=2, 160 days-old), Minas Gerais from six juveniles green turtles. The number of sampled (n=1, 150 days-old), Rio Grande do Sul (n=2, 90 and 145 were rescued. In total, 22 fibropapillomas were collected days-old), Santa Catarina (n=2) and Ceará (n=1). Five (average=3.7±3.01). DNA from all samples was extracted animals were vaccinated against PCV2, one animal was usingfibropapillomas commercial varied kit, accordingfrom one to to seven the manufacturer’s of each animal not vaccinated and two were not informed. The levels of instructions. Two consensus primers set were used to viremia were 9.6 x 103 to 7.8 x 104 copies of PCV1 DNA/ amplify the papillomavirus (PV) DNA, FAP59/FAP64 that ml. The results of this study suggest that PCV1 has a low target a fragment of 480 bp of PVs L1 ORF, and AR-E1F2/ prevalence in Brazil. Therefore, PCV1 infection is a rare E1R9 that target a fragment of E1 ORF. For herpesvirus event that can be associated with a lower transmission capacity. Moreover, as the frequency of PCV1 is very low, detection of PCV in commercial farms vaccinated against GTHV2DNA identification, (165 bp) and aGTHPR1/GTHPR2 nested-PCR was (110 performed bp). The PCV2 enables monitoring of the presence of PCV2 and using specific Chelonia mydas HV primers set GTHV1/ variants of the virus that may arise due to mutations in of approximately 580 bp in one sample, which was ORF2. The ORF2 is widely used to design primers for primers AR-E1F2/AR-E1R9 allowed the amplification PCV2, but this region has a high mutation rate. While low quality of the sequencing product failed to identify primers for PCV were designed to a more conserved negative in herpesvirus identification. However, the region (ORF1) and with lower mutation rate primers FAP59/FAP64. In the nested-PCR, the amplicons ofthe the PV expected type. No lengthsample of was herpesvirus amplified were by PV obtained detection in VV374 - IDENTIFICATION OF FIBROPAPILLOMA- 21 DNA samples. Five positive nested-PCR amplicons ASSOCIATED HERPESVIRUS IN GREEN TURTLE, CHELONIA MYDAS, ON THE COAST OF PARANÁ STATE, BRAZIL nucleotidewere sequenced sequence to confirm showed the presence100% ofidentity herpesvirus with de Alcantara, B.K.1; Domiciano, I.G.1; Domit, C.3; Cheloniaassociated Herpesvirus with fibropapilloma 5 (ChHV-5) of(GenBrank green turtle. accession The Claus, M.P.2; Saporiti, V.1; Alfieri, A.F.1; Bracarense, number AF299108.1). The present study represents the A.P.F.R.L.1; Alfieri, A.A.1 1. UEL - Universidade Estadual de Londrina, the coast of Paraná. The results reinforce the herpesvirus first DNA characterization of ChHV-5 in green turtles on Rodovia Celso Garcia Cid, Km 380 - Campus Universitário, Londrina - PR, 86057-970 and the participation of papillomavirus infection was 2. IFSC - Instituto Federal de Educação, notassociation excluded. with The ChHV-5 fibropapillomatosis variant is possibly in green widespread turtles, Ciência e Tecnologia de Santa Catarina, Rua 14 de Julho, 150 on southern Brazil, but more studies are necessary to - Coqueiros, Florianópolis - SC, 88075-010 understand the dissemination of the viral strain between 3. UFPR - Universidade Federal do Paraná, population stocks on the Brazilian coast, and the role of Rua XV de Novembro, 1299 - Centro, Curitiba - PR, 80060- 000 support: CNPq, FINEP, Fundação Araucária and CAPES. PV infection in fibropapilloma pathogenesis. Financial

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VV380 - RETROSPECTIVE STUDY SHOWED THAT in Brazil. Even though, the impact of this virus for the pig PPV4 IS CIRCULATING INTO THE BRAZILIAN SWINE industry remains unknown. FINANCIAL SUPPORT: SIGA HERDS SINCE 1999 NRP 4568/20013 Castro, A.M.M.G.2; Bersano, J.G.1; Ogata, R.A.1; Nara, VV381 - FIRST REPORT OF PPV4 DETECTION IN J.M.1 RODENTS CAPTURED IN BRAZILIAN SWINE HERDS 1. LABORATÓRIO DE DOENÇAS DE SUÍNOS Castro, A.M.M.G.2; Bersano, J.G.1; Silva, S.O.S.3; Ferrari, “WASHINGTON SUGAY/INSTITUTO BIOLÓGICO - K.L.3; Ogata, R.A.1; Nara, J.M.1; Richtzenhain, L.J.3 Laboratório de Doenças de Suínos “Washington Sugay” Instituto Biológico, Av. Cons. Rodrigues Alves, 1252 - Vila 1. LABORATÓRIO DE DOENÇAS DE SUÍNOS Mariana, São Paulo - SP, 04014-002 “WASHINGTON SUGAY/INSTITUTO BIOLÓGICO - 2. FMU - Faculdades Metropolitanas Unidas, Laboratório de Doenças de Suínos “Washington Sugay” Avenida Brigadeiro Luís Antônio, 1089 - Bela Vista, São Paulo Instituto Biológico, Av. Cons. Rodrigues Alves, 1252 - Vila - SP, 01317-002 Mariana, São Paulo - SP, 04014-002 3. FMVZ/USP - Faculdade de Medicina 2. FMU - Faculdades Metropolitanas Unidas, Veterinária e Zootecnia da Universidade de São Paulo, Av. Avenida Brigadeiro Luís Antônio, 1089 - Bela Vista, São Paulo Duque de Caxias Norte, 225. Pirassununga - SP, 13635-900 - SP, 01317-002 3. FMVZ/USP - Faculdade de Medicina Parvoviruses are small, non-enveloped viruses with a Veterinária e Zootecnia da Universidade de São Paulo, Av. linear, non-segmented single-stranded DNA of 4 to 6.3 Duque de Caxias Norte, 225. Pirassununga - SP, 13635-900 kb that infect a wide range of species of animals. Porcine parvovirus 1 (PPV1) is considered one of the major Porcine parvovirus 4 (PPV4) is a small, non-enveloped virus with a linear, non-segmented single-stranded different groups of porcine parvoviruses (PPV) have been DNA that was found in 2010 in lungs from a disease causes of reproductive failure in swine. Nowadays, five pig that was also infected with porcine circovirus 2 was to perform a PPV4 DNA retrospective analyses in (PCV2). Since then, the studies showed that PPV4 infect identified, being PPV4 one of them. The aim of this study pigs with different ages and that the overall prevalence over the period of 1999-2005. These were randomly in pigs herds varied from 1.5 % to 39.7 %. However, selectedfifty pig tissuefrom the samples archive collected of the Laboratório at maintained de Doençasat -80ºC the impact of this virus for the pig industry and the de Suínos “Washington Sugay”, Instituto Biológico and factors of the epidemiological chain remain unknown. tested for PPV4. DNA extraction was performed using the The aim of this study was to investigate the presence QIamp DNA/RNA Kit (Qiagen). PCR for PPV4 detection of PPV4 in rodents captured in Brazilian swine herds. was standardized in our laboratory using the pairs of Twelve samples of tissue pool of Rattus norvegicus primers PPV4a (5’ TCA TAG CAC TAT GGC GAG C 3’)/ were collected from a pig farm in the Sao Paulo State. The samples were homogenized using a StomacherH a fragment of 284 pb. The PCR products were analyzed 80 Biomaster (Seward) in 20% (v/w) TE buffer (pH onPPV4b 1.5 %(5’AGC agarose ATT gel CTG stained CGT TGG with ACA GelRed 3’) that (Uniscience). amplifies 8.0). DNA was extracted using phenol-chloroform A PCR control was performed by testing the extracted and proteinase K protocols and stored at 22 C. A PCR for PPV4 detection was performed using the⎕ pairs of negative PCR results. Positive and negative controls primers PPV4a (5’ TCA TAG CAC TAT GGC GAG C 3’)/ reconstitutedDNA in parallel with to sterilized β-actin amplification Milli Q water to were avoid used false- in a fragment of 284 pb. The PCR products were analyzed PPV4b (5’AGC ATT CTG CGT TGG ACA 3’) that amplifies and 9 of them (8 from São Paulo and 1 from Minas Gerais on 1.5 % agarose gel stained with GelRed (Uniscience). state)all reactions. corresponding All samples to 18 (50) % werewere positivepositive for for β-actin PPV4. submitted to bidirectional sequencing reactions that These positive samples were distributed into the years The amplified fragments from positive samples were of their collect as n=1 (1999); n=1 (2000); n=4 (2002); were performed using the BigDyeTM Terminator v3.1 n=2 (2003) and n=1 (2004). These results proved that (Applied Biosystems Life Technologies). Consensus PPV4 was circulating into the swine herds before 2000 sequences were assembled using the Phred/Phrap and

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CAP3 programs with an analysis quality point of 20. One between the 2nd and 5th dpi, in accordance with out twelve (8.3 %) of the tested Rattus norvegicus were observed clinical signs. Gross lesions were observed in kidney at 4th and 5th dpi; whereas high viral RNA levels were detected two days later by qRT-PCR. Quantities of sequenced.positive for PPV4.The fragment Because thisgenerated is the first was report submitted of PPV4 to viral RNA were detected in testis samples, but similarly in rodents, to confirm the positive result, this sample was to the proventriculus detection level, due to the absence These results proved the detection of PPV4 in rodents andBLAST/n further and phylogenetic confirmed thestudies PPV4 are identity necessary relatedness. to show that this quantity was possible to cause microscopic lesions.of macroscopic Histopathology lesions, itanalysis is still notwill possiblebe performed to affirm in FINANCIAL SUPPORT: SIGA NRP 4568/20013 the importance of these findings for the pig industry. tissues and clinical signs, and also, ELISA analysis will VV382 - BRAZILIAN VARIANT OF THE INFECTIOUS beorder performed to relate to microscopic evaluate the findings, presence viral of presence antibodies in BRONCHITIS VIRUS: RESPIRATORY AND RENAL against IBV. FINANCIAL SUPPORT: FAPESP – BIOTA PATHOGENICITY PROJECT 2911/50919-5 FAPESP 2013/02058-6, CNPQ Caserta, L.C.1; Martini, M.C.1; Barnabé, A.C.S.1; (475571/2012-6) AND FAPESP (GRANT 2013/02058- Ferreira, H.L.2; Arns, C.W.1 6). 1. IB/UNICAMP - Instituto de Biologia VV394 - PRELIMINARY STUDY ON MOLECULAR Universidade Estadual de Campinas, Cidade Universitária AND HISTOPATHOLOGICAL DETECTION OF Zeferino Vaz, Rua Monteiro Lobato, 255, Campinas - SP, IN FISH FROM SOUTHERN AND 13083-862 2. FZEA/USP - Faculdade de Zootecnia e NORTHEASTERN REGIONS OF BRAZIL Engenharia de Alimentos da Universidade de São Paulo, Av. Navarro, J. de O.1; Maganha, S.R. de L.1; Almeida Duque de Caxias Norte, 225 - Campus da USP, Pirassununga Queiroz, S.R. de.1; de Pádua, S.B.3; de Menezes Filho, - SP, 13635-900 R.N.3; Araújo, A.P.2; Alencar, A.L.F.1; Arruda, E. de P.1; 1 1 1 Infectious bronchitis virus (IBV) belongs to order Michelin, E.C. ; Munin, F.S. ; Fernandes, A.M. ; de 1 Nidovirales, family Coronaviridae, Gammacoronavirus Sousa, R.L.M. genus. A Brazilian variant (BR) of IBV is causing 1. FZEA/USP - Faculdade de Zootecnia e great economics losses in Brazilian poultry industry. Engenharia de Alimentos da Universidade de São Paulo, Av. Nonetheless, experimental studies to evaluate the clinical Duque de Caxias Norte, 225 - Campus da USP, Pirassununga signs and viral tropism to different organs are scarce. - SP, 13635-900 The present study aimed to evaluate clinical signs, gross 2. ACQUAPISCIS - Acquapiscis Consultoria e lesion, and viral RNA after an in-vivo experiment with IBV Medicina Veterinária em Aquicultura, Rua Vieira de Morais, Brazilian variant genotype BR-I in day-old chicks during 1201, Campo Belo, São Paulo - SP, 04617-014 42 days. Tissue samples (sinus swab, trachea, lungs, 3. AQUIVET - Aquivet Saúde Aquática, Rua proventriculus, ileum, cecal tonsil, cloacal swab, kidney Antonio Ernesto Célico, número 359, quadra 8, lote 16, Residencial Marcia Damha III, São José do Rio Preto - SP, 15061810 and used for real time RT-PCR (qRT-PCR) targeting the untranslatedand testis) were region collected (UTR) and gene. viral Clinical RNA wassigns purified were Betanodavirus (BNDV) is a non-enveloped, 25nm mainly respiratory and a growth reduce was noticed as icosahedral virus with a single-stranded positive-sense well. Macroscopic changes were hyperemia and mucous RNA of approximately 4.6 kilobases containing two exudate in trachea but the most remarkable lesion were segments (RNA1 and RNA2). BNDV is the etiologic agent of viral nervous necrosis (VNN) or viral encephalopathy viral levels were detected in organs of digestive system, exceptpale kidneys in proventriculus. and ureters filled In withthis system,urates. Higher viral RNA species, causing mass mortality worldwide. So far there and retinopathy (VER) that affects fish of different was detected until 42 days post inoculation (dpi). In are no reports of circulation of the BNDV in Brazil. The respiratory system, high viral RNA levels were detected aim of this study was to investigate the presence of BNDV

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Porcine group A rotavirus (RVA) is the main viral cause of pigs neonatal diarrhea in worldwide. The apparentlyin fish from healthy southern (n and = 63) northeastern specimens wereregions collected of Brazil. in most common combinations of G and P genotypes in A total of 150 fish samples from diseased (n = 87) and RVA strains in piglets are G5P[7] (OSU strain), G4P[6] and Mogi-Guaçu Rivers watersheds), Ceará (Jaguaribara (Gottfried strain), G3P[7] (CRW8 strain), and G11P[7] andfish farmsHorizonte and riverscities), in and the Bahiastates (Pauloof São PauloAfonso (Pardo city). (YM strain). However, several other combinations of G Fresh tissue fragments of the central nervous system, and P genotypes have been detected in piglets. The aim eye, liver and kidney were sampled and stored at -20ºC. genotype in wild-type porcine RVA strains from Brazil and Oreochromis niloticus (tilápia) specimens collected from itsof this combination study was with to describe the P[13] the genotype. identification Two of diarrheic the G26 Additionally, buffered formalin-fixed tissue samples from fecal samples (BRA381 and BRA382) from suckling (14 (Ceará and Bahia states). Aliquots of approximately 50 to 28-day-old) piglets collected in July 2012 from a pig mgfish farms(pool withof tissues) outbreaks were characterized subjected to by RNA mass extraction mortality farm located in the state of Rio Grande do Sul that were followed by reverse transcription-polymerase chain RVA-positive by polyacrylamide gel electrophoresis reaction (RT-PCR) and nestedPCR targeting a 420bp- (PAGE) were submitted for RT-PCR analyses of the VP7 (G fragment of the BNDV RNA2 segment. A tissue pool genotype) and VP4 (VP8* - P genotype) genes to amplify from a tilápia specimen collected in Jaguaribara (Ceará 1,062 bp and 876 bp products, respectively. The RT-PCR state) tested positive. When each tissue type was tested products were sequenced and submitted for nucleotide separately, liver was positive. Histopathological changes (nt) sequence analysis to identify the G and P genotypes. and pancreas of this specimen and others from the same high nt identity (94.6% to 94.7%) with the G26 porcine area,as basophilic suggesting inclusions a typical bodies BNDV were infection. identified Interesting, in liver strains.The VP7 Thegene VP4 of both (VP8*) porcine gene RVA of fieldone strainsstrain displayedexhibited no clinical signs consistent with VNN or VER were 83.5% to 96.3% nt identity with the P[13] porcine observed in these animals, indicating a subclinical strains, and the another porcine RVA strain carried an untypeable P genotype. Based on the sequence analysis in Jaguaribara as well as nucleotide sequencing and virusinfection. isolation A broader in cell prospection culture for for the BNDV positive in fish samples farms strains (BRA381 and BRA382) belong to the genotypes of the VP7 and VP4 (VP8*) genes, the two field porcine that BNDV infection has a worldwide distribution and are ongoing. These findings corroborate the assumption diarrheaG26P[13] in and the G26P[X],Miyazaki respectively.prefecture, Japan The G26(TJ4-1 genotype strain/ and BNDV other than mortality. Nevertheless, this study G26P[7])was recently and describedalso in a pig in afrom five-day-old Hebei Lulong, piglet China with pointscall attention out the presence for potential of a viral interactions agent associated between with fish porcine RVA was only detected in piglets from Asia and promising area in Brazilian animal production. (Z650 strain/G26P[X]). Thus far, the G26 genotype of significant economic losses in aquaculture, one the most description of the porcine G26 genotype in diarrheic VV400 - DIARRHEA IN PIGLETS BY AN UNCOMMON in combination with the P[7] genotype. This is the first GENOTYPE COMBINATION (G26P[13]) OF PORCINE uncommon combination G26P[13] genotype in porcine GROUP A ROTAVIRUS RVApiglets strains. outside FINANCIAL Asia and SUPPORT:is also the FINEP,first detection CAPES, CNPq,of the Lorenzetti, E.; Medeiros, T.N. da S.; Molinari, B.L.D.; and Fundação Araucária/PR. Ribeiro, J.; Possatti, F.; Leme, R. de A.; Pereira, F.L.; Crespo, S.E.I.; Alfieri, A.F.; Alfieri, A.A. LVA/DMVP/UEL - Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva da Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Pr 445 Km 380, Campus Universitário, Londrina - PR, 86057-97

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VV401 - MOLECULAR DETECTION AND 9 samples were grouped together in a separate clade CHARACTERIZATION OF BOVINE ASTROVIRUS IN from other BoAstV, a sample (BoAstV-155-BRA) formed CENTRAL-SOUTH REGION OF BRAZIL a paraphyletic branch to a clade that grouped both other de Almeida Queiroz, S.R.1; Candido, M.1; Alencar, BoAstV and deer astrovirus and the remaining sample A.L.F.1; Batinga, C.A.1; de Godoy, S.H.S.1; Munin, F.S.1; (BoAstV-267-BRA) formed a basal branch to bovine and de Almeida Queiroz, S.R.1; Buzzinaro, M. da G.2; porcine astrovirus. However, the positioning of the two Livonesi, M.C.3; Fernandes, A.M.1; de Sousa, R.L.M.1 different samples (BoAstV-155-BRA and BoAstV-267- BRA) did not derive from episodes of positive selection 1. FZEA/USP - Faculdade de Zootecnia e or recombination. In summary, the results indicate, in Engenharia de Alimentos da Universidade de São Paulo, Av. an unprecedented manner, the circulation of enteric Duque de Caxias Norte, 225 - Campus da USP, Pirassununga BoAstV variants in herds of cattle from states of the - SP, 13635-900 2. FCAV/UNESP - Faculdade de Ciências supported by FAPESP (Proc. nº 2012/18441-0) and Agrárias e Veterinárias da Universidade Estadual Paulista, South-Central region of Brazil. This work was financially Via de Acesso Prof.Paulo Donato Castellane s/n, Jaboticabal, CNPq (Proc. nº 472509/2010-1). SP, 14884-900 VV402 - IDENTIFICATION OF A NOVEL EQUINE 3. FCF/UNIFAL - Faculdade de Ciências INFECTIOUS ANEMIA VIRUS STRAIN BY MASSIVE Farmacêuticas da Universidade Federal de Alfenas, Rua SEQUENCING IN AN ENDEMIC ZONE IN PANTANAL, Gabriel Monteiro da Silva, 700. Alfenas - MG, 37130-000 BRAZIL Enteric diseases associated with diarrhea, dehydration Malossi, C.D.1; Cavalcante, R.V.2; Ullmann, L.S.2; and weight loss is one of the major problems of cattle Nogueira, M.F.T. de L.3; de Aguiar, D.M.5; Kroon, E.G.4; Araujo Jr, J.P.1 mortality, especially among newborns. In this context, demandsworldwide, for contributing improving tothe significant laboratory morbidity diagnosis and of 1. IBB/UNESP - Instituto de Biociências da infectious enteric diseases became constant, given Universidade Estadual Paulista, Bairro: Distrito de Rubião the economic losses involved. Among the major viral Junior S/N, Botucatu - SP, 18618-970 enteropathogens of worldwide distribution and recent 2. FMVZ/UNESP - Faculdade de Medicina history, highlight the bovine astrovirus (BoAstV), Veterinária e Zootecnia da Universidade Estadual Paulista, Distrito de Rubião Junior, s/n Caixa Postal 560, Botucatu - SP, 18618-970 induces diarrhea especially among newborns and 3. EMBRAPA, Rodovia SP 340 - Km 127,5 - immunocompromisedwhose first mention dates animals, from 1978.having Enterica prevalence BoAstV Tanquinho, Jaguariúna - SP, 13820-000 4. UFMG - Universidade Federal de Minas data concerning the occurrence of this virus in Brazil, Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, above 60% in the first 5 weeks of life. Due to lack of the present study was aimed at molecular detection Belo Horizonte - MG, 31270-901 and characterization of BoAstV strains in fecal samples 5. FAMEV/UFMT - Faculdade de Agronomia, from cattle with and without diarrhea of different ages. Medicina Veterinária e Zootecnia da Universidade Federal de The study was conducted on 272 animals from different Mato Grosso, Av. Fernando Corrêa da Costa, nº 2367 - Bairro states of Brazil, the fecal samples were tested by RT-PCR Boa Esperança. Cuiabá - MT, 78060-900 Equine infectious anemia (EIA) is a persistent lentiviral obtaining positivity of 14.3%. Eleven samples from thewith states primers of São specific Paulo, for Minas the pol Gerais gene and of astroviruses, Rio Grande do Sul were subjected to nucleotide sequencing and adisease limited withdiagnosis mandatory with false-negative notification. results Although and theno phylogenetic analysis. The similarity between the availabledisease was cure identified or vaccine. more Complete than 150 proviral years ago, genomic it has deduced amino acid sequences of the samples was greater than 86.8% when compared with each other and strains of equine infectious anemia virus (EIAV), EIAV was between 86.2 to 94.8% compared with sequences of Wyoming,sequences EIAV have onlyLiaoning been and obtained EIAV fromMiyazaki2011-A, three field other BoAstV described. In phylogenetic reconstructions, from USA, China, and Japan, respectively. There is no September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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sample. In Brazil, EIAV is endemic in Pantanal region. Thiscomplete study genomicaimed to sequence sequence EIAV’s of viral genomic RNA RNA from at field the knownThe bovine to infect immunodeficiency cattle worldwide. As virus in other (BIV) retrovirus is the infections,causative agenthosts develop of bovine a long immunodeficiency term infection and which most is horses from Corumba, MS, was collected. The sample infected animals remains asymptomatic. Some animals first time in naturally infected horses. Plasma of infected can present variable disease signs such as lymphocytosis, centrifuged at 16500 xg/30 minutes. Total RNA was lymphadenopathy, weight loss, weakness, decreased was clarified at 2000xg/10 minutes and supernatant milk production and some secondary infections. BIV synthesized with random primers or oligodT primer for is present both in dairy and cutting herds and can eachextracted sample of and pellet EIAV and was mRNAdetected purified. by PCR. The cDNA dsDNA was be transmitted vertically in utero via placenta and library was prepared using RNAse H, T4 polymerase colostrum, or horizontally through the exchange of and sequenced with the MiSeq System (Illumina Inc.). in several countries, the prevalence of this infection in Geneiousand, T4 ligase R6 was following used toby analyzeNextera-XT the kitsequences, (Illumina using Inc.) Brazilbodily is fluids still unknown. and blood. The Although aim of thisBIV studyhas been was reportedto detect map to reference with complete EIAV genome (accession BIV proviral DNA in blood samples of cattle and estimate no. AF247394). A coding sequence of 7528bp in length the occurrence of infection in the state of Minas Gerais, (without 5`and 3`UTR) presenting 96.7% of coverage Brazil. Blood samples from 391 cattle were collected showing 120 nucleotides of gaps and 30 nucleotides from two regions of the state, Zona da Mata and Central. Blood was centrifuged at 1609g to isolate the buffy coat just 81%, 81% and 80% nucleotide sequence identity which was subjected to DNA extraction. Proviral DNA withof insertions. the EIAV AmongLiaoning, the Wyoming, field strains, and Miyazaki2011-A this isolate has was detected by semi nested polymerase chain reaction strains, respectively. Furthermore, phylogenetic studies (SN-PCR) using primers to amplify the conserved using Corumba EIAV sequence against known viral region in the pol gene of BIV. The product of SN-PCR strains of EIAV strongly suggests this isolate comprise was subjected to electrophoresis on 1.5% agarose gel a separate monophyletic group. With these results it and stained with ethidium bromide. The SN-PCR results is possible to do a better characterization of the virus indicate a BIV occurrence of 12,5% in the state. The circulating in Brazil and may improve the molecular nucleotide sequencing. The similarity of the nucleotide strain.FINANCIAL SUPPORT: FAPESP, FUNDIBIO sequenceamplified of sequences the BIV from were Minas confirmed Gerais isolates by cloning with andthe diagnostic of AIE using primers specific for Brazilian VV403 - MOLECULAR DETECTION OF THE BOVINE reporting the presence of BIV in the Minas Gerais, Brazil. IMMUNODEFICIENCY VIRUS (BIV) IN CATTLE OF THE Thereference results strain indicate (R-29) the needwas 99%. to conduct This is a thedetailed first study MINAS GERAIS STATE, BRAZIL on the prevalence of BIV infection in Brazil, and more Rodrigues, A.P. de S.1; Fonseca Jr, A.A.2; Lima, G.K.1; particularly its association with various diseases that are Gasparini, M.R.1; Naves, J.H.F. de F.1; Bicalho, J.M.1; prevalent in cattle, which may contribute negatively to Wood,C.3; Leite, R. de C.1; dos Reis, J.K.P.1 1. UFMG - Universidade Federal de Minas virus, semi-nested PCR, occurrence, Minas Gerais. the cattle industry. Keywords: Bovine immunodeficiency Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, FINANCIAL SUPPORT: FAPEMIG, CNPq ; INCT-Pecuária. Belo Horizonte - MG, 31270-901 2. LANAGRO/MG - Laboratório Nacional Agropecuário de Minas Gerais, Av. Rômulo Joviano, s/nº Caixa Postal 35, 50, Pedro Leopoldo - MG, 33600-000 3. NEBRASKA CENTER FOR VIROLOGY UNIVERSITY OF NEBRASKA, 4240 Fair St, Lincoln, NE 68583, Estados Unidos

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VV410 - SEARCH FOR CANINE CIRCOVIRUS (CACV-1) VV411 - ANALYSES OF HUMAN HERPESVIRUS 1 IN DOGS ISOLATED FROM NATURALLY INFECTED MARMOSET Stefanello, F.1; Daros, F.1; Zanella, E.L.1; Costa, M.M.1; (CALLITHRIX SP) Zanella, J.R.C.2 Kurissio, J.K.; Ulmann, L.S.; Rodrigues, M.V.; Araújo Jr., J.P. 1. UPF - Universidade de Passo Fundo, BR 285, São José, Passo Fundo - RS, 99052-900 UNESP - Universidade Estadual Paulista, Rua Quirino 2. EMBRAPA SUÍNOS E AVES, Parque de Andrade, 215, São Paulo - SP, 01049-010 Estação Biológica - PqEB s/nº, Brasília, DF, 70770-901 Human Herpesvirus type 1 (HHV-1) is a virus Canine circovirus type 1 or CaCV-1 was recently transmissible from human to non-human primates. characterized in dogs and is associated with disease Primate from Callithrichidae family may be highly or death in these animals. The CaCV-1 belongs to the susceptible to infection by human herpesvirus with fatal Circoviridae family, which members have single strand cases.The use of molecular techniques may help the DNA, circular and non-enveloped. Circoviruses are detection of the agent in animals with clinical suspicion. known to infect animals and are associated with serious The present study aimed to conduct viral isolation diseases in swine and poultry, causing wasting in animals, and genomic analysis of herpesvirus from an infected reduction in production, malformations and tegument non-human primate.Thus, the detection of HHV-1 was necrosis, lymphoid depletion and immunosuppression. performed by nested PCR on samples of DNA extracted from brain fragments of 8 marmosets clinically suspected routine serological tests and was similar to the one found inThe swine, CaCV-1 but isolated not showing for the clinical first timesigns. inIn 2012this study, from 100 canine blood samples collected from February to wasafter performed death. Six by samples Sanger’s showed method amplification and the sequence of viral July 2014 at the Veterinary Hospital from the University analysisDNA that obtained were purified 100% and identity sequenced. to DNA The polymerasesequencing of Passo Fundo were tested for CaCV-1 at the Animal region of HHV-1 (GenBank KF498959, JQ352184 and Health and Genetics Laboratory from Embrapa Swine JQ780693).Viral isolation of HHV-1 was done in Vero and Poultry research center. Viral DNA was extracted cell culture, and the inoculum was a brain fragment from the positive on nested PCR. After 24 hours p.i. (post inoculation) cytopathic changes characteristic 1using were MagMAX used for detection (Ambiom) of isolationthe pathogen. kit, andThe qPCR real time test of herpesviral infection were observed on the infected detectedPCR (qPCR) sequences using specific for the primers capsid and and probes replicate for CaCV-genes cell monolayer. An aliquot of the culture supernatant of the CaCV-1, but all 100 samples tested were negative of infected cells 30 hours p.i. was collected and DNA for the presence of the CaCV-1. This work allowed was extracted for the whole genome sequencing. Deep implementing a real time diagnostic test for CaCV- sequencing with Miseq® (Illumina, Inc.) was used. 1, allowing an early diagnostic in dogs with different Genome analysis was performed with Geneious program clinical signs or even different species, like swine. This obtaining a sequence of 149,667pb.The sequence was diagnostic tool is also important to be accessible for assembled using as reference the genome of human studies of the pathogenesis and epidemiology of ssDNA herpesvirus type 1 (GenBank HM558507).The sequence viruses co-infections in economically important species showed 93.1% identity and 65.5% GC, with 62 divergent such as swine. Financial support: The authors thank amino acid from coding regions: UL (23) US (37), IRS Neide Simon for the technical support. F Daros and (1), RS1 (1), and inserting the sequence ACCGCC the JRCiacci Zanella is a fellow of the National Council for the UL26 region.The consensus sequence generated was submitted to phylogenetic analysis using MEGA 6.0 a fellow of FAPERGS. program with Tamura-Ney model and the 500 rounds of Scientific and Technological Development. F Stefanello is Bootstrap.In phylogenetic analysis, the clade marmoset herpesvirus was considering highly reliable (bootstrap 81%). The result showed that the phylogeny obtained from marmoset herpesvirus has the ancestral human September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

262 Veterinary Virology: VV herpesvirus type 1 (GenBank AF67648), but the samples in 2010 and 74 in 2011. A total of 141 samples as observed in the genomic characterization of molecular and 29 (39%) in 2011. outbreaks of rabies in the period sequencestructure ofanalysis. the assembly Therefore, showed the possible molecular modifications analyses received, 68 were confirmed positive, 39 (58% ) in 2010 them occurred in the middle region of the Wasteland, in isolated from the marmoset originated from human twoproved consecutive to be distributed years, probably in the five due meso to its State. topography Most of herpesvirusconfirmed that type infection 1. Nevertheless, with herpesvirus variations detected in relation and and because the milk basin State is located there. The to its ancestor can be attributed to interspecies jumps or spatial distribution shows that some municipalities changes or adaptive virus in this species that had outbreaks in 2010, showed no positive cases in

VV415 - SPATIAL DISTRIBUTION OF CASES OF measures more effective actions of the surveillance RABIES IN HERBIVORES OF PERNAMBUCO STATE IN system2011. This after decrease the focus. suggests It awas reflection observed of preventive that the THE PERIOD OF 2010 TO 2011 rabies virus is present in all mesoregions the State of Santos, G.R.1; - Silva, G.C.P.1; Santos, R.F.1; Godoy, H.P.1; Pernambuco, which emphasizes the need for effective Brandespim, D.F.3; Carvalho, A.A.B.1 and continuous actions of Agricultural Surveillance 1. FCAV/UNESP - Faculdade de Ciências to control the disease. Epidemiological studies are an Agrárias e Veterinárias da Universidade Estadual Paulista, important support for health surveillance in animal Via de Acesso Prof.Paulo Donato Castellane s/n, Jaboticabal, health. SP, 14884-900 2. UFRPE - Universidade Federal Rural de VV426 - FIRST DESCRIPTION OF PORCINE ENTERIC Pernambuco, Rua Dom Manoel de Medeiros, s/n, Dois Irmãos, FROM WILD BOARS IN BRAZIL Recife - PE, 52171-900 Leme, R. de A.1; Donin, D.G.1; Ratti, D.1; Molinari, 1 1 1 1 Rabie is considered one of the world’s largest B.L.D. ; Possati, F. ; Lorenzetti, E. ; Otonel, R.A.A. ; 1 1 2 1 antropozoonoses importance because it represents Pereira, F.L. ; Massi, R.P. ; Claus, M.P. ; Alfieri, A.F. ; 1 a strong economic impact of agribusiness and a high Alfieri, A.A. risk to public health, not only by the drastic and lethal 1. UEL - Universidade Estadual de Londrina, outcome, but also by the high social and economic Rodovia Celso Garcia Cid, Km 380 - Campus Universitário, cost. In Brazil, more than 23,000 suspected cases of Londrina - PR, 86057-970 rabies in herbivores have been reported in 10 years; 2. IFSC - Instituto Federal de Educação, however, due to sub-reports, it is almost impossible Ciência e Tecnologia de Santa Catarina, Rua 14 de Julho, 150 to determine the actual losses caused by the disease. - Coqueiros, Florianópolis - SC, 88075-010 In Pernambuco, the creations of large herbivores are Porcine (PTV), porcine (PSV), and on growth and employed at national ranking eighth in enterovirus G (EV-G) are porcine enteric picornaviruses milk production in the country. These data emphasize that circulate worldwide in asymptomatic domestic pigs. the importance of conducting epidemiological studies However, depending on the virus serotype and infection on the rabies situation in that State. The objective was conditions, these picornaviruses may be important to analyze the spatial distribution of positive cases of etiological agents of enteric, respiratory, reproductive, or rabies in herbivores in the years 2010 and 2011 through neurological disorders in young and adult animals. Wild boars (Sus scrofa scrofa, Suidae family) are susceptible areas through spatial and visualization of data. Mapping to infections by PTV, PSV, and EV-G. Pecari tajacu and ofthe cases GIS, reported a tool that in the allows state better of Pernambuco, identification Brazil of was risk Tayassu pecari are wild animals earlier believed to be

Agreste, Mata, São Francisco and Sertão), composed and Tayassu into the Tayassuidae family. Sus scrofa, ofconducted, 185 municipalities, comprising using the fiveMapInfo meso Professional (Metropolitana, 7.0 Pecarirelated tajacu,to the genus and Tayassu Sus and reclassifiedpecari belong as generato the Pecarisame software. The laboratory diagnosis of rabies LANAGRO order (Artiodactyla), are phenotypically and behaviorally Recife / PE received from the State of Pernambuco, 67 similar, and largely distributed in distinct geographical

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263 Veterinary Virology: VV regions of Latin America. The aim of this study was to 3. FMVZ/USP - Faculdade de Medicina evaluate the natural infection by these porcine enteric Veterinária e Zootecnia da Universidade de São Paulo, Av. picornaviruses from wild boars and peccaries in Brazil. Duque de Caxias Norte, 225. Pirassununga - SP, 13635-900 Fecal samples (n=36) from wild animals of Paraná state 4. FACULTY OF VETERINARY, UNIVERSITY were evaluated. Young (2 to 7-months-old, n=14) and OF GLASGOW, 464 Bearsden Road, Glasgow, G61 1QH, adult (2 to 4-years-old, n=8) asymptomatic wild boars Scotland living on a captive farm were sampled in June, 2013. The infection of domestic cats (Felis catus) by feline Pecari tajacu (n=5) and Tayassu pecari (n=9) aged 6 to 8-months-old were sampled on a zoo of Cascavel city in of the immune system involving mainly the depletion March, 2014. The animals had no contact with domestic immunodeficiency virus (FIV) results in the impairment pigs. Nucleic acid extraction was performed using a opportunistic infections and even death. The need to combination of phenol/chloroform/isoamyl alcohol and performof CD4+ diagnostic T cells, tests increasing and identify the susceptibilitypositive animals of silica/guanidinium isothiocyanate methods. Polymerase comes from the indigence to know the data of prevalence chain reaction (PCR) and nested-PCR (n-PCR) assays were performed using primers targeting the 5’-non- and control for infected animals. Veterinarians have few translated region of the porcine enteric picornavirus alternativesof diseases, tofor set the the detection profile prophylactic,of FIV infection therapeutic and the genomes. Porcine enteric picornaviruses were detected in 12 of the 22 wild boar fecal samples. Enterovirus G known face to Brazilian samples. Another relevant point was most frequently (11/22) detected, followed by PTV issensitivity the high andcost specificity of the tests, of thefactor diagnostic that often tests deters still notthe (10/22) and PSV (4/22). Young wild boars were more owners to request the exam to the vet. Analyzing two frequently (9/14) infected with PTV, PSV, and EV-G hundred and one (201) serum samples from domestic than adult animals (3/8). Sequencing analysis showed cats this study compared the ability to identify cats similarities varying from 97.7% to 100% for PTV, naturally infected by FIV of three different tests: ECOVET 92.4% to 96.2% for PSV, and 87.1% to 100% for EV-G. Fecal samples from Pecari tajacu and Tayassu pecari tested negative for the three viruses and these animals – FIV Ab + FeLV Ag test; SNAP COMBO® - Ab-FIV/Ag- did not represent infection reservoirs. To the authors’ showedFeLV, IDEXX agreement. and a recombinant p24 antigen ELISA. The sensitivity and specificity of the all tests carried out screening for PTV, PSV, and EV-G from wild boars of Latin VV431 - CHARACTERIZATION OF CIRCULATING knowledge, this study represents the first molecular STRAINS OF FELINE IMMUNODEFICIENCY VIRUS IN host species for porcine enteric picornavirus.FINANCIAL BRAZIL: PRELIMINARY DATA SUPPORT:America and FINEP, the first CAPES, to screen CNPQ, peccaries AND as FUNDAçãOalternative Teixeira, B.1; Ferreria, M.C.1; Heleno, N.1; Boni, T.2; ARAUCáRIA/PR. D’elia, M.1; Cruz, J.3; Miyashiro, S.2; Pereira, P.L.1; Reis, J.1; Hosie, M.4; Hagiwara, M.2; Heinemann, M.2 VV427 - COMPARISON OF DIFFERENT SOROLOGICAL TESTS FOR DIAGNOSTIC OF FELINE 1. DMVP/UFMG - Departamento de Medicina IMMUNODEFICIENCY VIRUS (FIV) INFECTION Veterinária Preventiva da Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, campus Pampulha, Belo 1 1 1 Ferreria, M.C. ; Heleno, N. ; Gonçalves, S. ; Martins, Horizonte - MG, 30161-970 2 3 1 3 4 N. ; Miyashiro, S. ; Reis, J. ; Hagiwara, M. ; Hosie, M. ; 2. FMVZ/USP - Faculdade de Medicina Heinemann, M.3; Teixeira, B.1 Veterinária e Zootecnia da Universidade de São Paulo, Av. 1. DMVP/UFMG - Departamento de Medicina Duque de Caxias Norte, 225. Pirassununga - SP, 13635-900 Veterinária Preventiva da Universidade Federal de Minas 3. INTA - Instituto Superior de Teologia Gerais, Av. Antônio Carlos 6627, campus Pampulha, Belo Aplicada, Rua Coronel Antônio Rodrigues Magalhães, 359, Horizonte - MG, 30161-970 Bairro Dom Expedito Lopes, Sobral - CE, 62050-100 2. UEMA - Universidade Estadual do 4. FACULTY OF VETERINARY, UNIVERSITY Maranhão, Tirirical - Cidade Universitária Paulo VI, São Luís OF GLASGOW, 464 Bearsden Road, Glasgow, G61 1QH, -MA, 65055-000 Scotland September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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The rapid emergence of AIDS in humans during the is completely contained within VP1 and it mainly period between 1980 and 2000 has led to extensive comprises the capsid of CPV; amino acid substitutions efforts to understand more fully similar etiologic agents in its sequence can alter biological characteristics of the virus. CPV-2 emerged as a new pathogen of dogs from a disease in several mammalian species. Lentiviruses variant of feline panleucopenia virus in the end of 1970s thatof chronic have andgene progressive sequence acquired homology immunodeficiency with human and spread rapidly. A few years later, CPV-2a replaced CPV-2. In 1984, a new type called CPV-2b emerged and different species (including sheep, goats, horses, cattle, co-circulated with CPV-2a. In 2000, a new variant was cats,immunodeficiency and several Old virus World (HIV) monkey have species). been found Feline in discovered, and at present it co-circulates with the other two types, but it seems to be replacing the other variants worldwide. Monitoring the circulation of CPV Timmunodeficiency cells, leaving the animals virus (FIV) susceptible causes into domesticopportunistic cats is important in order to establish control measures and infections,a progressive and decline sometimes of the numberdeath. Virusesof circulating related CD4+ to improve the quality of existing vaccines. The aim of this domestic cat FIV occur also in a variety of nondomestic study was to evaluate fecal samples of dogs from four felids. The objective of our study is to perform a better different geographical Brazilian regions to determine the characterization of circulating strains of this large and CPV subtypes currently circulating in dogs population. ancient group of viruses (FIVs) in the country. In this Forty-three stool samples of dogs aged 2 to 6 months work, we provide our preliminary data: the frequency were selected. All dogs presented clinical signs of of FIV antibodies in domestic cats was 17,2% (43/250 hemorrhagic gastroenteritis and their feces were sent to animals) and in wild felids was 0% (0/76 animals), by CPV detection. The samples were collected in the states ELISA. In addition, to date, all samples from this study of Paraná (n=13), Minas Gerais (n=3), Paraíba (n=24), and Mato Grosso (n=3). All samples were tested by B as the only subtype circulating in FIV positive domestic cats(isolates in Brazil. from Belo Anyway, Horizonte more and widespread São Paulo) surveys identified of a 583 bp fragment of CPV VP2 gene. The PCR products Brazilian isolates are required to determine whether wereconventional subjected PCR to assay RFLP using using a primer the restriction pair that amplifies enzyme a single subtype of FIV predominates in Brazil. This MboII, which recognizes GAAGA sites, present only in the preliminary data on FIV infection paves the way for residue 426 of CPV-2c, making it possible to differentiate future studies characterizing circulating strains of feline CPV-2c from other types. From the 43 samples analyzed, 14 (32.6%) were positive for CPV-2c and 24 (55.8%) for CPV-2/2a/2b. Five amplicons did not show digestion lentiviralimmunodeficiency infection invirus Brazilian in Brazil. felids. Such characterization in RFLP. All CPV-2c strains were from the states of will allow us to better understand the significance of Paraná and Mato Grosso. Two amplicons of each state VV441 - CPV-2B AND CPV-2C CO-CIRCULATION IN were selected and subjected to sequence analysis. The DOGS IN BRAZIL Silva, A.P.1; Freitas, L. de A.1; Spera, C.G.1; Miyabe, be CPV-2c. The amplicons from Minas Gerais and Paraíba F.M.1; Diniz, J.A.1; Crespo, S.E.I.1; Grecco, S.2; Barcellos, showedamplicons to be from CPV-2b. Paraná Although and Mato some Grosso reports confirmed claim that to M.2; Pérez, R.2; Alfieri, A.F.1; Alfieri, A.A.1 CPV-2c has been replacing other types of CPV, this study 1. UEL - Universidade Estadual de Londrina, demonstrates that there is still CPV-2b circulating in Rodovia Celso Garcia Cid, Km 380 - Campus Universitário, some Brazilian geographical regions. Financial support: Londrina - PR, 86057-970 CNPq, CAPES, and Fundação Araucária/PR 2. Universidade da República, Avenida 18 de Julio 1824, Montevideo 11100, Uruguai Canine parvovirus (CPV) is a pathogen responsible for hemorrhagic enteritis in pups. The single-stranded DNA genome has two ORFs that encode nonstructural (NS) and structural (VP) viral proteins. VP2 sequence

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265 Veterinary Virology: VV

VV443 - STANDARDIZATION AND VALIDATION OF literature studies of MCF performed with SYBR®Green MOLECULAR METHOD DIAGNOSTIC FOR MALIGNANT system, the results suggest that this is a reliable and safe CATARRHAL FEVER method for detecting OvHV-2, it showed good analytical Martins, M. de S.N.; Lima, M. dos S.; Silva, T.G.; Pinto, V. da S.C.; de Stefano, E.; Okuda, L.H.; Pituco, E.M. of the reaction. Therefore, it can and should be used as asensitivity tool for diagnosis and specificity, and effective reproducibility surveillance and in linearityorder to Instituto Biológico, Av. Cons. Rodrigues Alves, 1252 - avoid socioeconomic consequences. Vila Mariana, São Paulo - SP, 04014-002 Malignant catarrhal fever (MCF) is an acute viral disease, VV444 - COINFECTION OF VACCINIA BEEF AND DAIRY affects multiple systems, is reported worldwide, usually CATTLE PSEUDOVARIOLA IN PROPERTY OF A MATO fatal and affects mainly ruminants. Ten species of viruses GROSSO STATE, BRAZIL belonging to the MCF group are known, all Herpesvirus, Martins, M. de S.N.1; Silva, T.G.1; Martins, M. de S.N.1; Family Gammaherpesvirinae, genus . The Pinto, V. da S.C.1; Lima, M. dos S.1; Okuda, L.H.1; Santos, main in Brazil is sheep-associated MCF, induced by ovine S.P.2; Pituco, E.M.1 herpesvirus 2 (OvHV-2) strain .Due to the similarity of 1. Instituto Biológico, Av. Cons. Rodrigues lesions, with encephalitis/encephalopathy, foot and Alves, 1252 - Vila Mariana, São Paulo - SP, 04014-002 mouth disease bluetongue and vesicular stomatitis, it 2. Defesa Agropecuária must be considered as potential differential diagnoses Bovine vaccinia and pseudovaríola are vesicular disease where MCF is suspected. The use of molecular methods of cattle caused by two species of viral family Poxviridae: vaccinia virus (orthopoxvirus) and pseudocowpoxvirus infected animals and may also be useful for phylogenetic (gender ). They are enveloped viruses andallow epidemiological confirmation ofstudies the presence in both ofnatural MCF virusesand MCF- in and their genome consists of double-stranded DNA susceptible hosts. However, these are not yet routinely and its site of replication occurs in the cytoplasm. It available in laboratories. Therefore, the objective was is characterized by vesicular lesions cause teats of to standardize and validate a quantitative PCR (qPCR) cows and calves in the oral cavity, compromising the SYBR®Green system for diagnosis of OvHV-2. The production chain of milk, as it hampers milking and can primers used detect the region ORF75 that encodes also infect humans, becoming a public health problem. a protein highly conserved of the viral tegument. The Brazil in cases of coinfection caused by poxviruses of standard sample used was from both Laboratory of different genera have also been described in outbreaks Bovine Viral Diseases and Laboratory of Pathology of of vesicular disease in cattle and humans. This report the Biological Institute, São Paulo, with characteristic describes an outbreak of vesicular disease in dairy lesions for FCM. This sample was sequenced and in cattle in São José dos Quatro Marcos in the state of Mato the BLAST obtained 97-100% identity with various Grosso, where the animals had vesicular lesions on the OvHV-2 isolates from different parts of the world. To ceilings, with approximate travel 8-13 days and the determine the analytical sensitivity of the reaction, the serial dilutions were made from 105 to 100 copies of stomatitis,officers also bovine had vesicular epithelium lesions three on samples the hands. from After this DNA/uL.concentration Concentrations of the purified of primers viral DNA from was 200 checked to 1000nM and herdconfirmation were sent of negative to the Laboratory diagnosis for of FMD Viral and Diseases vesicular of were tested and the best concentration was 500nM, bovine Biological Institute for differential diagnosis. either for the reverse as the forward primers. The After extraction with Trizol ® DNA, the samples were threshold detection was 100 copies of DNA/uL, dilution subjected to semi-nested PCR reaction using primers at which all samples in triplicate in three reactions made that amplify, respectively, fragments of 890 bp for the HA on different days, showed positive results. The Melting gene of vaccinia virus and 256 bp for pseudocowpoxvirus B2L gene. All samples were positive in both semi-nested PCR and subjected to sequencing, which showed high thecurve threshold showed detectiona single peak was flowering satisfactory at approximatelyit is indicated identity between the Brazilian samples with vaccinia for86 °C,routine demonstrating laboratory. the Although specificity it was of notthe foundreaction. in the As September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

266 Veterinary Virology: VV virus (98%) and pseudocowpoxvirus (99%) deposited bovine of four months old, male, had incoordination, the literature has found that vaccinia virus coinfection and behavioral changes, and come to death in a few days. andin GenBank. pseudocowpoxvirus This study in corroborates cattle and demonstrates the findings the of Diagnosticspaddling movements, for Herpesvirus flaccid type paralysis 1 and of 5, the and hind Neospora limbs circulation of these viruses in the country. caninum were conducted with negative results. This is

VV447 - CASE REPORT: DETECTION OF BVDV-3 IN A symptom in Brazil being detected mainly in batches of BOVINE WITH ENCEPHALITIS FROM THE STATE OF FBS.the first It also report indicates of BVDV-3 the need in animal to mapping with neurologicalthe existing SÃO PAULO BVDV genotypes in Brazil and contribute to surveillance Pinto, V. da S.C.; Martins, M. de S.N.; Lima, M. dos S.; actions in cases of encephalitis. Silva, T.G.; Okuda, L.H.; Pituco, E.M. VV459 - A SEMI-NESTED PCR TO DETECT PROVIRAL Instituto Biológico, Av. Cons. Rodrigues Alves, 1252 - EQUINE INFECTIOUS ANEMIA VIRUS DNA IN Vila Mariana, São Paulo - SP, 04014-002 NATURALLY INFECTED HORSES The Bovine Viral Diarrhea Virus (BVDV) belongs to the Vilela, A.P.P.1; Cursino, A.E.1; Ferreira, P.C.P.1; de Lima, family Flaviviridae, genus Pestivirus. Its genome consists M.F.N.T.2; Kroon, E.G.1 of single-stranded RNA of positive polarity. Presents high level of antigenic variability and causes great losses 1. UFMG - Universidade Federal de Minas to the cattle, milk and meat industries. There are several Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, subtypes between genotypes of BVDV-1 and BVDV-2, Belo Horizonte - MG, 31270-901 2. EMBRAPA, Rodovia SP 340 - Km 127,5 - however, more recently it was reported the existence Tanquinho, Jaguariúna - SP, 13820-000 of what could become a new genotype, the BVDV-3, also called Hobi-like Virus, isolated from samples Fetal The Equine Infectious Anemia (EIA) is caused by Equine Bovine Serum (FBS) probable origin in South America, infectious anemia virus (EIAV), a retrovirus that infects especially the Brazil. The BVDV is responsible for a all members of the family Equidae, and clinical cases of wide variety of clinical manifestations varying from disease are reported in horses and ponies (Equus caballus) inapparent, acute and even fatal infections. The most and also in donkeys (Equus asinus). The major mode of important are gastroenteric, respiratory, and especially EIAV transmission is through mechanical transmission the reproductive system changes. Cases of encephalitis by biting insects or by contaminated fomites. The EIA is caused by BVDV are not very reported and for this reason a worldwide disseminated disease, and it is also broadly we describe this study. The Biological Institute of São disseminated in Brazil. It exhibits a high prevalence in Paulo received for differential diagnosis of encephalitis, Pantanal, affecting horses performance and, indirectly, a sample of central nervous system (CNS) from a the livestock farming. Given its burden, EIA is one of bovine with neurological signs belonging to a property of Patrocínio Paulista, São Paulo, Brazil, negative for (World Organization for Animal Health). Currently, EIA the eleven equine diseases listed as notifiable by OIE rabies. Nucleic acid extraction was performed using diagnosis is based on the serological assays, such as agar Trizol® according to manufacturer’s instructions. Then gel immunodiffusion (AGID), considered gold standard was subjected to qualitative RT-PCR using primers that test. However, there is a need for the development of amplify the gene 5´UTR, highly conserved, capable of detecting all genotypes of BVDV. The CNS sample since the serological tests show some limitations. This more sensivite and specific assays for EIA diagnosis, was positive for BVDV in comparison to the positive study aims to standardize a semi-nested PCR for EIAV. control, and subjected to sequencing to identify the DNA extracted from the peripheral blood leucocytes of genotype. The result revealed that it was BVDV-3 and naturally infected horses, from Corumbá, Mato Grosso showed high identity and genetic similarity to several do Sul, Brazil, were used for this study. For semi-nested sequences deposited in GenBank (HoBi_D32/00 access: PCR EIAVltr-reverse1 and EIAVltr-reverse2 primers AH013732.2; LV01/12 access: KC465388.1, Italy-68/13 were used (Dong et al., 2012), in combination with access: KJ627180.1, among others). The animal was a the EIAVltr-forward 4 primer (designed in this study),

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

267 Veterinary Virology: VV targeting the highly conserved 5’ end of the EIAV of the method of virus neutralization, the repeatability, genome, extending from the long terminal repeat (LTR) reproducibility and uncertainty of measurement were to the trans-activator (tat) gene. The PCR product was also evaluated. In addition, a comparison was made fractionated by polyacrylamide gel electrophoresis, and between the results obtained by ELISA and by virus neutralization for BVDV (two different strains) in 64 The sequences obtained showed nucleotide identity serum samples, before and after inactivation in a water withthe amplified EIAV sequences DNA was deposited excised, inpurified, the GenBank and sequenced. database. bath for 30 minutes at 56° C, besides the evaluation of the In summary, our preliminary results showed that the selectivity of the methods. In the results, the analytical semi-nested PCR was effective for EIAV detection in sensitivity of ELISA varied according to the sample, and alternative for EIA diagnosis. Experiments to determine eight samples tested the titer was 8. Repeatability and field samples from Pantanal, and may be useful as a the titers obtained varied from 2 to 16. In five of the assay are underway. FINANCIAL SUPPORT: CNPQ, CAPES,the sensivity EMBRAPA and specificity of this semi-nested PCR measurementreproducibility was of the 0.213. ELISA Furthermore, obtained a CVELISA (coefficient showed of variation) ≤ 10% and the expanded uncertainty of VV461 - VALIDATION OF THE SEROLOGICAL METHOD Veterinary Laboratory Agency. The virus neutralization OF DIAGNOSIS OF BOVINE VIRAL DIARRHEA (BVD) BY obtainedsatisfactory CV resultsvalues <2.3%in proficiency in samples tests of organized higher titer by andthe ELISA AND VERIFICATION OF THE NEUTRALIZATION around 13% in samples of lower titer and the expanded METHOD FOR DETECTION OF ANTIBODIES FOR uncertainty of measurement was 0.652. The results of BVDV the comparison between ELISA and virus neutralization Rodrigues, A.P. de S.1; Oliveira, T.F.P.2; Azevedo, I.C.2; (NADL strain) reached a concordance of 63.3% and using Rivetti Jr, A.V.2; Thomaz, M.M.2; Rodrigues, A.P. de S.2; Singer strain it was 86.7% and the results also indicate Oliveira, A.M.2; Camargos, M.F.2 that the methods are selective. Thus, the techniques of 1. UFMG - Universidade Federal de Minas viral neutralization and ELISA for BVDV are suitable for Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, Belo Horizonte - MG, 31270-901 Agriculture Department. Keywords: ELISA, virus 2. LANAGRO/MG - Laboratório Nacional neutralization,the routine use bovine of the officialviral diarrhea laboratories virus, of validation, Brazilian Agropecuário de Minas Gerais, Av. Rômulo Joviano, s/nº FINANCIAL SUPPORT: LANAGRO/MG Caixa Postal 35, 50, Pedro Leopoldo - MG, 33600-000 verification.VV462 - DETECTION OF PICOBIRNAVIRUS The methods of viral neutralization and ELISA are ASSOCIATED WITH CRYPTOSPORIDIUM IN THE commonly used in the diagnosis of viral diseases in DIARRHEAL FECES OF CALVES IN THE WESTERN animal laboratories, such as Bovine Viral Diarrhea (BVD), REGION OF PARANA which is caused by a virus from the family Flaviviridae, Macedo, R.1; Garcia, F.G.1; Snak, A.1; Ribeiro, A. de S.1; genus Pestivirus that affects cattle, sheep, goats, swine Gallego, J.1; Kunz, A.F.1; Lustosa, J.1; Otonel, R.A.A.2; Alfieri, A.A.2; Osaki, S.C.1; Takiuchi, E.1 laboratoriesand wild animals. from theCurrently, Brazilian validation Agriculture and Ministry verification as a 1. UFPR - Universidade Federal do Paraná, measureof analytical of quality methods of these are tests, usually besides required being to a criteria official Rua XV de Novembro, 1299 - Centro, Curitiba - PR, 80060- checked in national and international auditing. In this 000 way, the aim of this work was the validation of the ELlSA 2. UEL - Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Km 380 - Campus Universitário, detect antibodies against Bovine Viral Diarrhea Virus Londrina - PR, 86057-970 (BVDV).test and Theverification following of parameters viral neutralization were evaluated tests used in the to The Picobirnavirus (PBV) is an emerging group of non- validation of ELISA: analytical sensitivity, repeatability enveloped viruses with bisegmented RNA genome and reproducibility, uncertainty of measurement and whose detection has been reported in both samples of diarrheic feces and non-diarrheal feces in different hosts participationSeptember/October in 2014 proficiency Volume 19 tests. – Supplement For the 2 - verificationAbstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

268 Veterinary Virology: VV including man, birds and reptiles. Cryptosporidium are affected, but clinical signs are usually observed in are opportunistic protozoa that affect all animal sheep and include a high rate of mortality, reproductive problems, weight loss and indirect losses as a result of from self-limiting diarrhea episodes to death. The export restrictions. The aim of this study was to verify etiologyclasses, especiallyof diarrhea in outbreaks the first weeksattributed of life, to protozoa can lead the presence of BTV in sheep of different breed, from 14 Crypotosporidium, is well proven. However, the properties in State of São Paulo. A total of 538 animals, participation of the PBV as the causal agent of diarrhea is which age ranged from 37 months, were examined from still controversial. The intent of this study was to report 2007 to 2013. Were collected blood samples for direct the simultaneous occurrence of Cryptosporidium spp. detection of the virus by the methods of RT-PCR Real- and PBV in diarrheal feces of naturally infected calves. Time (RT-qPCR), isolation in embryonated chicken eggs 24 diarrheic fecal samples with less than 60 days old (ECE) and cell culture of BHK-21 strain, and sequencing were selected, collected between 2012 and 2013, in the of the positive samples. The sheep herds, 61.54% Western Region of Paraná. The samples were positive for (8/14) had viremic animals, which represented 12% PBV by electrophoresis on polyacrylamide gel (PAGE) (64/538) of occurrence of viremic animals, detected by RT-qPCR. Samples positive in RT-qPCR were subjected Genogroup I. For detection of Cryptosporidium spp. The to virus isolation in ECE and subsequently adapted in samplesstained with were silver analyzed nitrate using and confirmed Ziehl-Neelsen by RT-PCR coloring for cell culture BHK-21 resulted in 75% (48/64) adapted to sample this isolation system. The positive samples contained Cryptosporidium spp. These results are quitemodified. promising, The 28since positive few studies samples of detection PBV, (28.57%) of PBV by PAGE in cattle are reported in the literature, and avoidwere sequencedthe damage be caused confirmed by the strain spread of BTV-4. of the The disease. more frequency (0.69% to 3.67%) lower than that observed Also,sensitive allows and analysis specific, on early a large diagnosis scale in is a necessaryshort period to

Cryptosporidium spp. so far, has been described only international trade. Molecular studies for typing of in oursamples findings. of human Furthermore, feces, suggesting the detection the ofassociation PBV and BTVof time, isolates for certificationin Brazil are ofvery animals important for domesticto evaluate and of of these enteropathogens. Due to the lack of knowledge epidemiological situation thus contributing to sanitary about the epidemiology of PBV, mainly in cattle, and their measures of control; reducing the risk of introduction involvement in cases of diarrhea in natural or mixed of new BTV strains to minimize the negative impact of infections, more detailed studies are needed to complete the disease and to improve the exchange of products of the diagnosis and understanding of the association animal origin. between PBV and Cryptosporidium spp. of neonatal diarrhea episodes in calves. VV486 - SYNTHETIC NAPHTHOQUINONES INHIBIT IN VITRO REPLICATION OF BOVINE HERPESVIRUS-5 VV481 - DETECTION OF VIRUS BLUETONGUE IN Pinto, A.M.V.1; Leite J.G.P.2; Ferreira, V.F.1; Ferreira, SHEEP IN THE STATE OF SÃO PAULO S.B.1; Gonzaga, D.T.1; da Rocha, D.R.1; Paixão, I.C.N.P.1 Pinto, V. da S.C.; Lima, M. dos S.; Silva, T.G.; Martins, 1. UFF - Universidade Federal Fluminense, M. de S.N.; Pinto, V. da S.C.; Souza, S.F.; Venditti, L.L.R.; Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900 Okuda, L.H.; Pituco, E.M. 2. FIOCRUZ - Fundação Oswaldo Cruz, Av. INSTITUTO BIOLÓGICO, Av. Cons. Rodrigues Alves, Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900 1252 - Vila Mariana, São Paulo - SP, 04014-002 Bovine herpesvirus type 5 is an important agent of Bluetongue is an infectious, non-contagious disease transmitted by Culicoides sp. The bluetongue virus in outbreaks of neurological disease in bovine in (BTV) belongs to the family Reoviridae, genus Orbivirus. severalmeningoencephalitis Brazilian States. in Furthermore, cattle. It has BoHV-5 been identified has also been isolated from respiratory and genital tracts of subtropical and some temperate regions of the world. animals without neurological disease symptoms, from Sheep,So far 26 cattle, BTV goats serotypes and various have been species identified of wild inruminants tropical, cryopreserved semen, oocytes, embryos, and inside

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

269 Veterinary Virology: VV spermatozoids. These observations have stimulated Neonatal diarrhea is a major health problem affecting the search for compounds that can be used as means of controlling bovine herpesvirus infections or preventing losses. Among the various agents involved in this recrudescent. In order to investigate cytotoxicity and syndrome,calves in the rotaviruses first weeks have of life, been causing reported huge as economicthe most anti BoHV-5 activity three synthetic naphthoquinones frequently detected during disease outbreaks. Belonging (Dimero, Lapachol and Quins 28) obtained by chemical to the family Reoviridae, rotavirus present 75 nm in synthesis were assayed “in vitro” and showed antiviral diameter, triple capsid protein and has dsRNA as the activity on BoHV-5RJ42/01 replication. Acyclovir was genetic material with 11 genomic segments. The external assayed in all experiments as compound standard. capsid proteins, VP4 and VP7, induce the production Cytotoxic effect were measured in MDBK cells treated of neutralizing antibodies, setting the basis for viral with different compounds concentrations (25, 50, 250, 500 and 1000 µM) using the tetrazolium salt the frequency of rotavirus infection in calves of dairy and (3-(4,5-dimethylthiazol-2yl)-2,5diphenyl tetrazolium beefclassification. cattle, detected The objective between of thisthe yearsstudy 2006was to to determine 2010, on bromide and the concentration of compounds required properties of Minas Gerais state / Brazil. For this, survey to reduce the number of viable cells by 50 % were for data were included from 19 municipalities with a total Dimero (58 µM ± 3.5); Lapachol (49.7 µM ± 9.2); Quins of 36 herds and 304 samples, of which 31 were obtained 28 (396 µM ± 7.3) and acyclovir (989 ± 2). Antiviral from animals with diarrhea and 273 clinically normal analyses of compounds against BoHV-5RJ42/01 were animals. By the PAGE technique, frequencies of infection measured by inhibition of cytopathic effect on infected were 11.1% (4/36) and 2.6% (7/273) found in animals cells. The naphthoquinones derivatives EC50 (Dimero with and without diarrhea, respectively. Genotyping 2.3 µM ± 0.8; QUINS-28 = 2.8 µM ± 1.7); Lapachol = 10 µM of samples positive for rotavirus was performed by ± 1.2); and ACV = 166 µM ± 2). All compounds showed the method of reverse transcription-polymerase chain low virucidal ability but Dimero, Lapachol and Quins 28 reaction (RT-PCR) and highlighted the presence of blocked BoHV-5RJ42/01 attachment and penetration in infection by genotype G6P[5] and G10P[11] in dairy MDBK cell. For the time of addition studies monolayers herds and the genotypes G6P[5] and G6P[5]P[11] in were infected with 1x104 PFU BoHV-5RJ42/01 and treated with different compounds at time 0 h or at of rotavirus in the herds studied. Hygiene measures, intervals of 1, 2, 3, 4, 5, and 6 h post-infection. Time of managementbeef herds. The and results immunoprophylaxis indicated a significant are important circulation to addition studies revealed Dimero, Lapachol and Quins 28 reduce the rate of infection and the persistence of the compounds suppressed all stages of the BoHV-5RJ42/01 agent in herds. FINANCIAL SUPPORT: FAPESP replication cycle. On the other hand ACV showed slighter inhibitory activity on BoHV-5RJ42/01 replication before VV493 - AVIAN METAPNEUMOVIRUS (AMPV)) AND 3 h postinfection but reduction of 50% and 85% was INFECTIOUS BRONQUITES VIRUS (IBV) IN WILDLIFE observed after 3 and 6 h postinfection respectively. PSITTACIDAE Simas, P.V.M.1; Barnabé, A.C.S.1; Caserta, L.1; Martini, activity of these naphthoquinones are underway. Key M.C.1; Lima Neto, D.F.1; Durães Magalhães, R.1; words:Investigations BoHV-5, about naphthoquinones specific mechanisms and antiviral of the Financial antiviral Moraes, A.P.1; Nagel, N.E.2; Lierz, M.2; Hafez, H.M.2; support: CNPQ/UFF/ FIOCRUZ Felippe, P.A.N.1; Arns, C.W.1 1. UNICAMP - Universidade Estadual de VV489 - FREQUENCY OF BOVINE ROTAVIRUS IN Campinas, Cidade Universitária Zeferino Vaz - Barão DAIRY AND BEEF CATTLE IN MINAS GERAIS STATE Geraldo, Campinas - SP, 13083-970 BETWEEN THE YEARS 2006-2010 2. FREIE UNIVERSITÄT BERLIN, Godoy, H.P.; Gonçalves, A.C.S.; Samara, S.I.; Buzinaro, Kaiserswerther Straße 16-18, 14195 Berlin, Alemanha M.G. Psittacidae are some of the most intelligent birds. This FIOCRUZ - Fundação Oswaldo Cruz, Av. Brasil, 4365 family consists of one of the groups most affected by - Manguinhos, Rio de Janeiro - RJ, 21040-900

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posterstrafficking - Veterinary inVirology: wildlife, VV for its great diversity of colors and XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

270 Veterinary Virology: VV ability to imitate human speech arouses the interest of selected. In each farm, blood samples were randomly people around the world. Besides hunting for marketing, collected from 20 animals per category of the production suffer from the continued destruction of their habitat. cycle: breeding animals (sows and gilts), farrowing crate Long-lived animals, whose larger species can live over (2–3 weeks), nursery (4–7 weeks), grower pigs (8–14 50 years. Brazil is the country with the largest number of representatives of this family. The FAPESP-BIOTA Project of 100 samples per farm and a total of 3.000 animals has shown keen interest to know the real situation inweeks), the study. and Samples finishing were pigs tested (15–16 for weeks), hemagglutination for a total concerning these bird diseases in order to preserve these inhibition activity against H1N1 pandemic strain (A/ animals at Sao Paulo State. Viral diseases of psittacine birds are presently detected by PCR. For all these reason, the aim of this study is to identify IBV and aMPV using the animalsswine/Brazil/11/2009) with antibodies against and H3N2 H1N1pdm09 SIV (A ⁄ and swine H3N2 ⁄ RT-PCR technique. 289 samples were collected from 13 SIVIowa were ⁄ 8548-2 26.23% ⁄ 98) and reference 1.57%, strain.respectively, The percentages considering of birds’ species (119 oropharyngeal – O; 170 cloacal swabs all samples tested. The percentages of seropositive herds – C), of which 2.77% (2 O and 6 C) were positive for IBV for both viruses were 96.6% and 13.2%, respectively. and 2.77% (6 O and 2 C), for aMPV. Interesting evidence Only four farms had antibodies anti-H3N2, suggesting was the fact that there is a greater positivity for aMPV in lower circulation of this virus subtype in the State. Means samples from the cloaca. Thus, we can conclude that the of antibodies titers for H1N1pdm09 were higher in sows epidemiological surveillance in birds is necessary since and gilts and decreased along the other categories. they can serve as a reservoir for viral evolution and may In general, serology followed a characterized pattern cause outbreaks in wild birds, animals of commercial interest and even in humans, given its proximity to the higher in females group, decreasing through eight to 14 humans and also for the degradation of their natural weeksof antibodies of age when profile, seroconversion in which antibodies by natural titers infection are habitat. normally occurs. However, farms from South/South-

VV498 - DISTRIBUTION OF ANTIBODIES AGAINST seroconversion normally occurring earlier (4-7 weeks) INFLUENZA VIRUS IN PIGS FROM FARROW-TO-FINISH inwest the area production showed very system. variable In serologicalthis area, profiles,some farms with FARMS IN MINAS GERAIS STATE, BRAZIL kept detectable antibodies levels throughout the ages Dias, A.S.1; Costa, É.A.1; Guedes, M.I.M.C.1; Rajão, D.S.1; and means of antibodies titers did not range negative Guedes, R.M.C.1; Zanella, J.R.C.2; Lobato, Z.I.P.1 values, suggesting virus circulation in all groups. In 1. UFMG - Universidade Federal de Minas the other hand, one farm showed means of antibodies Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, titers reasonably constant throughout the ages in the Belo Horizonte - MG, 31270-901 production cycle, suggesting virus circulation in all 2. EMBRAPA SUÍNOS E AVES, Parque categories. FINANCIAL SUPPORT: CNPQ AND FAPEMIG Estação Biológica - PqEB s/nº, Brasília, DF, 70770-901 VV504 - SCREENNING AND DETECTION OF ALPHA- AND BETACORONAVIRUS IN BATS FROM SÃO PAULO respiratory disease affecting swine worldwide. In Brazil, AND PARANA STATE Swine influenza virus (SIV) is the cause of an acute Goes, L.G.B.1; Campos, A.C.1; Ambar, G.2; Carvalho, C.3; emergence of pandemic H1N1 in 2009 (H1N1pdm09), 3 1 3 2 SIV has been identified in pigs since 1978 and after Favaro, A.B. ; Crispin, L.A. ; Queiroz, L.H. ; Neto, A.C. ; Durigon, E.L.3 in Brazilian herds. The objective of this study was to few studies reported the presence of influenza virus 1. ICB/USP - Instituto de Ciências Biomédicas da Universidade de São Paulo, Edifício III USP - Administração Thirtyevaluate farms the serological with no SIV profile vaccination for influenza history virus from in pigs the - Av. Prof. Lineu Prestes, 2415 - Butantã, São Paulo - SP, fourfrom largerfarrow-to-finish pig production farms areasin Minas in GeraisMinas state,Gerais Brazil. state 05508-900 (Zona da Mata, Triângulo Mineiro/Alto Paranaíba, South/ 2. DEPARTAMENTO DE ZOOLOGIA/IB/ South- west and Belo Horizonte metropolitan area) were UNESP - Departamento de Zoologia do Instituto de Biociências

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

271 Veterinary Virology: VV da Universidade Estadual Paulista, Bairro: Distrito de Rubião Junior S/N, Botucatu - SP, 18618-970 BatCoV detected in same genera bat from Panama and 3. DEPARTAMENTO DE APOIO PRODUÇÃO α-CoV detected in A. lituratus was genetic related with E SAÚDE ANIMAL/FMVA/UNESP - Departamento de Apoio Produção e Saúde Animal da Faculdade de Medicina ContinuedCosta Rica. surveillance To our knowledge, of New Worldthis is bats the forfirst novel report CoVs of Veterinária da Universidade Estadual de São Paulo, R. Clóvis andthe possiblefurther research detection efforts of a to β-CoV comprehend lineage Cthe in genetic Brazil. Pestana, 793 - Dona Amelia, Araçatuba - SP, 16050-680 diversity of CoV in different bat species and biomes from Coronaviruses are enveloped positive-sense single Brazil are needed. FINANCIAL SUPPORT: FAPESP strand RNA viruses capable to infect birds and mammals including humans and associated with respiratory, VV506 - AVIAN CORONAVIRUSES SCREENING AND enteric, neurological or hepatic disease in a great DETECTION IN ANTARCTIC SEABIRDS diversity of species. The Chiroptera order harbor the Goes, L.G.B.1; Seixas, M.1; Campos, A.C.1; Araujo, J.1; biggest number and genetic diversity of Alpha- and Ometto, T.1; Hurtado, R.1; Thomazelli, L.1; Kruger, L.2; Betacoronavirus including virus phylogenetically Petry, M.V.3; Durigon, E.L.1 1. ICB/USP - Instituto de Ciências Biomédicas SARS (Severe Acute Respiratory Syndrome) and the da Universidade de São Paulo, Edifício III USP - Administração CoV-MERSrelated to emergent (Middle East coronavirus Respiratory afflicting Syndrome). humans, Despite CoV- - Av. Prof. Lineu Prestes, 2415 - Butantã, São Paulo - SP, the large number of bat species in Brazil, and the great 05508-900 diversity of CoV in bats, studies about the occurrence 2. CENTRO DE CIÊNCIAS DA SAÚDE/ and diversity of CoV in Brazilian bats are scarce. We UNISINOS - Universidade do Vale do Rio dos Sinos, Avenida analyzed the presence of CoV RNA in intestinal samples Unisinos, 950 - Cristo Rei, São Leopoldo - RS, 93022-000 of 189 bats specimens of 10 different bat species 3. Laboratório de Ornitologia e Animais (Artibeus lituratus, Artibeus planirostris, Carollia Marinhos/UNISINOS - Laboratório de Ornitologia e Animais perspicillata, Eptesicus furinalis, Eumops glaucinus, Marinhos da Universidade do Vale do Rio dos Sinos, Avenida Lasiurus cinereus, Molossus molossus, Molossus rufus, Unisinos, 950 - Cristo Rei, São Leopoldo - RS, 93022-000 Myotis nigricans and Sturnira lilum) captured in cities Coronaviruses are RNA single-stranded positive polarity from northwest region of São Paulo and two different virus capable to infect different species of birds and sites from Parana State, between 2010 and 2013. Total RNA was extracted from 30mg of tissue in NucliSENS® into four different genera: Alpha- and Betacoronavirus easyMAG® automatic extractor (Biomérieux) and the presentmammals in mammals including and man. Gamma- Coronaviruses and Deltacoronavirus are classified complementary DNA were obtained by RT-PCR using mainly present in different species of birds. Infections by random primers. The screening of coronavirus were coronavirus (CoV) are associated to pathologies of the performed as described by Chu et al., 2011, a Nested PCR digestive, respiratory, hepatic, renal tract and the central assay that amplify 440bp of the gene RNA dependent nervous system depending on the host. Over the past RNA polymerase. Amplicons were sequenced for 10 years, two new highly pathogenic CoV were detected in humans, the SARS-CoV (Severe Acute Respiratory detected in four samples obtained from bats captured in Syndrome) and the newly described CoV-MERS (Middle BatCoV genotype identification. Coronavirus RNA were East Respiratory Syndrome), both with a high mortality rate and probably originated from “spill-over” events Parana State, two Alphacoronavirus (α-CoV) in Carollia from bats and camels to humans respectively. In contrast wasperspicillata, most closely one related Betacoronavirus to coronaviruses (β-CoV) detected and one in of the great number of studies and the discovery of a Carolliaα-CoV in perspicillata specimens from of Artibeus Costa Rica, lituratus. and grouped The β-CoV with others Betacoronavirus lineage C, same lineage from little is known about the biology of avian coronaviruses in wildlife,significant and number even less of novel in Antarctic coronavirus seabirds. in bats, The relatively diversity was most related to the BatCoV detected in same species of coronaviruses in avian hosts, the potential of mutation ofMERS bat from CoV. BahiaThe two state α-CoV in a previously detected instudy. C. perspicillata Finally, the and recombination of these viruses and the detection

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

272 Veterinary Virology: VV of Gamma- and Deltacoronavirus in few mammals make essential the knowledge about the diversity of of the agent is desirable for treatment and control of coronavirus in different avian species. The present theTherefore, infection, as thea clinical clinical diagnosis diagnosis should and identification always be study aims to analyze the presence and molecularly been developed for the laboratory diagnosis of CPV. Region including Southern giant petrel, Chinstrap Thisconfirmed study byaimed laboratory to identify tests. CPV Several in fecal methods samples have of penguin,characterize Gentoo CoV inpenguin, five avian Cape species petrel from and theBrown Antarctic skua dogs examined at the Veterinary Teaching Hospital that birds. Endotracheal and cloacal swabs samples were had hemorrhagic enteritis using hemagglutination test collected from animals captured at the Elephant Island (HA), immunochromatography assay, and PCR, and in Subantarctic region, between December of 2010 compare the use of different techniques to detect CPV. and February of 2011. Total RNA was extracted from Twenty samples were selected; all of them were positive the samples with MagMaxTM RNA Isolation Kit and for CPV in a PCR assay that target a 583 pb fragment the randomic complementary DNA generated by RT- of the VP2 gene. Eight (40%) samples obtained titles PCR with Superscript Reverse Transcriptase enzyme. The detection of CoV was performed by Nested PCR samples were considered positive in a fast test based onconsidered immunochromatography. positive (≥ 512) Five in HA, positive and twelvePCR products (60%) RNA-polymerase, enabling the detection and genotype assay for amplification of 440pb of the RNA-dependent showing that this variant is circulating in Brazil. Methods eight samples were screened and one Gammacoronavirus basedwere sequenced, on detection all of of CPV them by PCRwere have identified been shown as CPV-2c, to be RNAidentification was detected after sequencing.in a Southern One giant hundred petrel and sample. eighty- highly sensitive. However, immunochromatography is an The phylogenetic analysis demonstrate a close relation optimal diagnostic tool when taking into consideration with a CoV detected previously in a Glaucous-winged that it has low cost and is more practical in routine gull (Charadriiforme Order) from Commander Island clinical medicine, assisting veterinarian in situations where needs to make decisions and take immediate of coronavirus in migratory seabirds of the Antarctic measures safely, given that it has a higher sensitivity region.(Russia). FINANCIAL This is the SUPPORT: first report FAPESP about (12/14255-8) the circulation than HA. Also, it is easier to perform and has shown better results than the HA. FINANCIAL SUPPORT: CNPq, VV514 - COMPARISON OF PCR, HEMAGGLUTINATION CAPES, and Fundação Araucária/PR TEST (HA), AND IMMUNOCHROMATOGRAPHY ASSAY (IC) TO DETECT CANINE PARVOVIRUS IN STOOL VV516 - IDENTIFICATION OF PERSISTENTLY SAMPLES OF DOGS INFECTED CALVES WITH BOVINE VIRAL DIARRHEA Balbo, L. de C.; Silvia, A.P.; Beuttemuller, E.A.; VIRUS BY RT-PCR Freitas,L. de A.; Spera, C.G.; Massi, R.P.; Miyabe, F.M.; Finoketti, F.; Firpo, R.; Campos, F.S.; Immig, J.; Torres, Alfieri, A.F.; Alfieri, A.A. F.D.; Franco, A.C.; Roehe, P.M. UEL - Universidade Estadual de Londrina, Rodovia UFRGS - Universidade Federal do Rio Grande do Sul, Celso Garcia Cid, Km 380 - Campus Universitário, Londrina Avenida Paulo Gama, 110 - Farropilhas, Porto Alegre - RS, - PR, 86057-970 90040-060 Canine parvovirus (CPV) is one of the most common Bovine Viral diarrhea Virus (BVDV) is a member of the viruses responsible for acute hemorrhagic enteritis genus Pestivirus within the Flaviviridae family. The in dogs and is an important cause of morbidity and virus can be transmitted horizontally to embryos and mortality in 6 to 12 week-old pups. Clinical signs in fetuses, what may result in fetal absorption, abortion, puppies include hemorrhagic or mucoid diarrhea, cerebellar hypoplasia, teratogenicity or the birth of vomiting, and dehydration, and secondary infections can persistently infected animals (PI) Persistent infections develop rapidly. The clinical diagnosis of CPV infection play a key role on virus perpetuation, since PI calves is inconclusive, since clinical signs found in CPV are may continually shed the virus in secretions and very similar to symptoms caused by other pathogens. excretions and are major disseminators of the infection.

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is the most physiological phenomenon associated of the main objectives in attempting to control BVDV with corticosteroid increased levels, especially during infectionsConsequently, in endemically the identification infected of cattle.the PI animalsIn the present is one parturition. There are still no reports comparing cortisol levels in cows during parturition with shedding of BoHV- reverse transcription-polymerase chain reaction (RT- 1 and antibody response to that virus. Thus, the aim of PCR)study, is a described. method for Ear identification tissue fragments of PI of animals 560 animals using this study was to compare levels of cortisol with viral of different ages were collected in a farm on Bacia de shedding in nasal secretions and milk in two different Castro, Paraná, Brazil. The samples were screening phases of the dairy cows naturally infected: four months using a commercial enzyme-linked immunossorbent of gestation and parturition day. For this purpose, 35 pregnant dairy cows naturally infected by the virus were Ten samples were ELISA positive and 21 samples - subjected to collect of blood serum, nasal secretions and positiveassay (HerdChek animals and BVDV their Ag/Serum –progenitors Plus Test were Kit, submitted IDEXX). milk and the cows were divided into two groups according to RT-PCR. RNA from ear tissues was extracted with to the production phase as previously mentioned. Doses a commercial kit (Purelink, Invitrogen®). The RNA of the hormone cortisol by chemiluminescence assay, was submitted to reverse transcription using random serum levels of immunoglobulin M and G by indirect primers and to PCR with primers OPES 13A and OPES ELISA and assessment of viral load in nasal secretions 14A (Elvander et al., 1998), expected to amplify a 296 bp and milk by real time PCR assays were performed. We region on the non-coding 5’ portion of the viral genome had observed increased levels of cortisol and viral load . Out of 21 samples, 10 (47,6%) were positive to RT- in milk postpartum (P <0,05). In addition, in the same PCR and then submitted to molecular characterization period, there was a positive correlation between blood through nucleotide sequencing. As reported by the IgG levels and viral load in milk (P = 0.0088, r = 0.7939), and between blood IgM levels and viral load in nasal ELISA and the RT-PCR was 100%. Therefore, the RT-PCR secretions (P = 0.0249 , r = 0,5750). From these results, result, we may affirm that the correlation between the it is concluded that, in naturally infected dairy cows, animals. FINANCIAL SUPPORT: CAPES; CNPq parturition is a favorable situation to increase viral was effective on the identification and confirmation of PI shedding and antibody response against BoHV-1. High VV517 - CORTISOL PRODUCED IN PARTURITION levels of cortisol in the postpartum period are related PERIOD OF DAIRY COWS INFECTED NATURALLY to the reactivation of productive infection by BoHV-1, BY BOVINE HERPESVIRUS 1 STIMULATE VIRAL promoting its largest shedding in secretions. The viral SHEDDING AND ANTIBODY RESPONSE load in the milk is directly related to the blood IgG level Junior, A.S.1; Ferreira, H.C.C.1; Granda, M.C.1; Silva, as well as IgM response is also proportional to shedding A.A.B.2; Ribeiro, M.G.2; Stancioli, E.F.B.2; Rebouças, viral load in nasal secretions. FINANCIAL SUPPORT: M.S.1; Valério, O.C.1; Almeida, M.R.1 CAPES, FAPEMIG, CNPq 1. UFV - Universidade Federal de Viçosa, VV518 - NEW HEPACIVIRUS IDENTIFIED IN CAPTIVE Avenida Peter Henry Rolfs, s/n - Campus Universitário, Viçosa NON-HUMAN PRIMATES FROM SOUTHERN BRAZIL - MG, 36570-000 1 2 3 3 2. UFMG - Universidade Federal de Minas Ullmann, L.S. ; Li, L. ; Cubas, Z.S. ; Morais, W. ; Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, Fantinatti, B.1; Deng, X.2; Pusterla, N.4; Leutenegger, Belo Horizonte - MG, 31270-901 C.5; Biondo, A.W.6; Delmart, E.2; Araujo Jr, J.P.1 Bovine herpesvirus 1 (BoHV-1) are related to respiratory 1. UNESP - Universidade Estadual Paulista, and reproductive diseases in young and adults animals. In Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010 dairy cattle, the impact is extremely harmful. The ability 2. BSRISF - BLOOD SYSTEMS RESEARCH of bovine herpesvirus to establish latent infection makes INSTITUTE, 270 Masonic Ave, San Francisco, CA 94118, them perpetually attached to their hosts. So, they may Estados Unidos return to the infection productive phase in the presence 3. RBBV of high levels of corticosteroid hormones. Pregnancy 4. UC-DAVIS - University of California, Davis, 7551 Madison Ave, Citrus Heights, CA 95610, Estados Unidos September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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5. IDEXX LABORATORIES - Idexx with GBV-B. Results suggest a new species of Hepacivirus Laboratories Innovative Diagnostics and Technologies, cp similar to GBV-V. FINANCIAL SUPPORT: FAPESP, CAPES, 132. Ponte Preta - Louveira - SP, 13.290-000 6. UFPR - Universidade Federal do Paraná, Rua XV de Novembro, 1299 - Centro, Curitiba - PR, 80060- ANDVV519 IDEXX - DETECTION LABORATORIES. OF INFLUENZA A VIRUS IN 000 MIGRATORY BIRDS 1 1,2 1 The genera Flavivirus, Pestivirus, Hepacivirus, and Santos, M.C. ; Ferreira, D. de L. ; Lima, S.T. ; Santos, 1 1 1,2 Pegivirus belong to the Flaviviridae family. Hepatitis M.C. ; Mello, W.A. ; Medeiros, R. C virus and Hepatitis G virus (GBV-B) are within 1. IEC - Instituto Evandro Chagas, Rodovia BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000 non-human primates, wild rodents, dogs, and bats. 2. NÚCLEO DE MEDICINA TROPICAL/ the Hepacivirus genus, with variants identified in UFPA - Núcleo de Medicina Tropical da Universidade Federal important diseases such as dengue and yellow fever do Pará, Rua Augusto Corrêa, 01 - Guamá, Belém - Pará, occurBrazil throughout is considered the country. a sanctuary As non-human of flaviviruses primates and 66075-110 contribute to important human diseases, the study of the viruses present in these animals represents variety of animals, such as mammals (humans, pigs, an important tool for detection of possible emergent horses),Influenza poultry virus is (chicken, known forgoose, its abilityturkey) to and infect even a widewild viruses. Thus, viral metagenomics was performed with birds of Anseriformes (duck, wild goose and swan) and plasma samples from 10 non-human primates (Alouatta guariba and Cebus apella) from Southern Brazil. Viral virus,Charadriiformes favoring possibility (gulls, terns, of spreading waterfowl to and other kingfishers) birds and (0.45 µm membranes), and treatment with nucleases. aquaticorders. Wildanimals birds when are the considering natural hosts the of migration avian Influenza factor. enrichment was made by centrifugation, filtration The Brazilian territory is crossed by distinct migratory with random primers and the SuperScript III Reverse routes of wild birds from the hemispheres north and Viral nucleic acid was purified, cDNA was obtained Transcriptase kit, and dsDNA was obtained with the south of the continent. Therefore, it is acknowledged the need for monitoring this virus in these birds considering with 5 µL of dsDNA, random primer and AmpliTaq its implications for the health of the human population. Klenow enzyme. An amplification step was performed magnetic beads, dsDNA was used for library preparation in migratory birds captured in the coastal regions of Gold DNA Polymerase. After two purifications with with TruSeqTM Sample Preparation v2 LT kit. Library Bahia,To investigate Pernambuco the and occurrence Pará states, of theBrazil. Influenza The samples virus quality was checked with 2100 Bioanalyzer and the were analyzed using techniques of molecular biology, comprising two main steps: a) extraction of DNA / Kit. Libraries were pooled and sequencing with MiSeq® quantification was estimated with KAPA Library Quant System with 2x250 cycles reagent kit. Geneious R7 gene encoding manipulator of citocromooxidase by and Mega 6.0 were used for analysis. Out of 2,181,312 theRNA technical from biological control chain specimen; reaction b) amplification mediated by of(PCR) the generated reads, 3,819 had hit to GBV-B (NC001655) and were used for de novo assembly, resulting in 10 contigs. transcription followed by real-time PCR (RT-qPCR). The One contig had 8,885 bp and was used as reference for resultsamplification showed and that, RNAv from usingthe total technique number of samples reverse concatenation with the whole raw data from the pool sequenced, resulting in a consensus sequence of 9,708 by RT-qPCR. There was a difference of positivity for the bp (HQ 99.1%). Amino acid analysis resulted on the virustested, among 7.2% the(n = analyzed 158) were bird positive species, for being Influenza 35.4% A to virus the viral polyprotein with a CDS of 2,830 aa. The sequence Charadriiformes order, 44.9% of the Anseriformes order, showed 63% identity with 39 gaps compared with the 1.2% for the birds belonging to the Pelecaniformes order deposited sequence (BAK24073). Phylogenetic analysis and 18.3% for those of the Suliformes order. Between with neighbor-joining tree (1,000 rounds of bootstrap) samples of the Passeriformes and Columbiformes resulted in a monophyletic group that shares ancestry

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Postersorders, - Veterinary no Virology: sample VV was positive for Influenza virus. XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

275 Veterinary Virology: VV

The data suggest variation between sampling sites, as do so, A RT-PCR test targeting polymerase gene previously Pará state showing the lowest percentage of positivity standardized was tested in our lab with different vaccine (11.3%), Bahia with the higher rate (65.8%), followed by viruses. Then, 100 samples (50 oropharyngeal and 50 Pernambuco with the second higher value of positivity cloacal swabs) from wild birds were collected and tested (22.7%). This study shows that although the presence of by the test. RT-PCR test was able to detect different rare investigations in Brazilian territory, there has been viruses representing four different genera of the viral family. Nonetheless, this test was up to 1000 times less migratory birds using the Pará, Bahia and Pernambuco statescirculating as stopping Influenza andA viruses reproducing among severalplaces speciesfor their of virus. Two out of tested samples were detected by this species. These reports justify further investigations to assay.sensitive This when test seemscompared to be to a a potential specific test tool targeting to detect eachnew viruses despite its low sensitivity. Therefore, we expect the population of circulating wild birds in Brazil, and its to increase the knowledge about the epidemiology of roleunderstand as a potential the dynamics source ofof infection avian Influenza for other viruses animals, in avian paramyxoviruses in Brazil. FINANCIAL SUPPORT: including man. FINANCIAL SUPPORT: CNPQ, SVS/MS FAPESP (NUMBER: 2011/09019-0).

VV527 - IDENTIFICATION OF AVIAN VV528 - CHALLENGES IN MONITORING AVIAN PARAMYXOVIRUSES BY A CONVENTIONAL RT-PCR INFLUENZA VIRUS IN OROPHARYNGEAL AND CLOACAL SWABS FROM Ferreira, H.L. WILD BIRDS DEPARTAMENTO DE MEDICINA VETERINÁRIA/ 1 2 2 Scagion, G.P. ; Silva, R.K. ; Sebastiani, M.C. ; Ciconello, FZEA/USP - Departamento de Medicina Veterinária da 2 1 1 3 F.N. ; Caserta, L.C. ; Martini, M.C. ; Felippe, P.A.N. ; Faculdade de Zootecnia e Engenharia de Alimentos da 1 2 Arns, C.W. ; Ferreira, H.L. Universidade de São Paulo, Av. Duque de Caxias Norte, 225 - 1. DGEB/IB/UNICAMP - Departamento de Campus da USP, Pirassununga - SP, 13635-900 Genética, Evolução e Bioagentes do Instituto de Biologia da Wild waterbirds and particularly Anatidae are the Universidade Estadual de Campinas, Rua Bertrand Russel, s/n Caixa Postal 6109, Campinas - SP, 13083-970 family Orthomyxoviridae) for most of the known 16 2. DEPARTAMENTO DE MEDICINA hemagglutininmain reservoir (HA) of and avian 9 neuraminidase influenza (NA) viruses subtypes (AIV, VETERINÁRIA/FZEA/USP - Departamento de Medicina in birds. Active surveillance in wild birds has increased Veterinária da Faculdade de Zootecnia e Engenharia de worldwide after the emergence of the H5N1 subtype; Alimentos da Universidade de São Paulo, Av. Duque de Caxias nonetheless, nucleotide sequence data are still limited Norte, 225 - Campus da USP, Pirassununga - SP, 13635-900 in comparison to other regions of the world. This 3. ICS/UNIP - Instituto de Ciências da Saúde da Universidade Paulista, Av. Paulista, 900 - Cerqueira César, information remains important to better understand the São Paulo - SP, 01310-100 pose threats to human health. In Brazil, AIV surveillance Avian paramyxoviruses belong to genus Avulavirus of isAIV realized ecology byas influenzaRRT-PCR, virusesvirus isolation,circulating and in animalsgenetic characterization of cleavage site in HA after a suspicion 12 serotypes (AMPV-1 to APMV-12). They have already Paramyxoviridae family. These viruses are classified into based on clinical signs and mortality or routinely. Village chickens are also sampled for AIV surveillance. economic losses in poultry industry. AMPV-1 was already The importance, challenges in monitoring AIV will detectedbeen identified in different in wild regions birds of andBrazil. are Nonetheless, responsible only for be discussed. FINANCIAL SUPPORT: EC (NUMBER: recently other serotypes, such as APMV-2 and APMV-10, 2011/09019-0). were detected in samples from wild birds on the coast of Espirito Santo and Rio de Janeiro States by virological tests. Studies in other regions of nearby Brazilian poultry industry are, thus, still scarce. The present study aimed to evaluate the circulation of those viruses in wild birds by RT-PCR targeting a conserved gene of this viral family. To September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV INDEX XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

278 Index

A Alves, P.A. 64, 225, 227, 233, 248 Assis, F.L. 12, 21, 108 Alves, P.T. 15, 41 Assis, M. 124 Abdelhaleem, K.R. 136 Alves, R. 86, 122, 124 Augusto, L.T.S. 149 Abrahão, J. 122, 123 Alves, T.M. de A. 86 Azevedo, I.C. 266 Abrahao, J.A. 225 Alvim, L.B. 69 Azevedo, R. 172 Abrahão, J.S. 10, 12, 13, 17, 18, 21, 54, 66, Amaral, C.D. 64, 227, 233, 248 Azevedo, S.S.D. 154 68, 69, 74, 101, 102, 103, 108, 149, Amarilla, A. 134 233, 248 Amarilla, A.A. 78 B Abrão, E. 184 Ambar, G. 270 Baccarin, A.M. 14, 57 Abrão, E.P. 183 Ambrosini, V.A. 197 Abreu, C.M. 15, 43 Badial, R.M. 169 Ambrósio, L.L.D. 227 Badra, S.J. 82, 134, 145, 156 Achkar, S.M. 13, 55, 247 Amorim, J.H. 137 Acosta, P.O.A. 180, 184 Badr, K. 65, 120 Amorim, L. 12, 28, 107 Badr, K.R.A. 145 Acrani, G.O. 12, 21, 88, 91 Amorim, L. dos S.C. 90 Affonso, M.Z. 229 Baez, C. 242 Amorim, L.M.F. 110, 111 Baffini, V.R. 173, 194 Afonso, L.A. 118 Amorim, N.A. 14, 27 Aguiar, A. 162 Baggio, M.L. 154 Amorim, R. 10, 14, 18, 24, 80 Baggio, M.P.D. 210, 211 Aguiar, R.W. de S. 213 Andrade, A.C. dos S.P. 68, 69 Aguirre, A.A. 14, 60 Balani, D. 215 Andrade, A. de S. 146, 202 Balbo, L. de C. 272 Aita, C. 103 Andrade, G.P. 222 Akinaga, M.M. 72, 73 Baldi, C. 158 Andrade, J. 124 Bandeira, R. da S. 112, 161 Albanese, J.M. 12, 30 Andrade, J.S.R. 133 Albuquerque, A.C.C. 143 Barardi, C.R.M. 12, 28, 104, 105, 109 Andrade, K.R. 102, 103 Barbagelata, L. 195 Albuquerque, N.R.M. 229 Andrade, L.O. 116, 117 Alencar, A.L.F. 252, 257, 258 Barbagelata, L.S. 171, 174 Andrade, M. de S. 218 Barbosa, C.M. 236 Alessandra, V.B.T.G. 12, 22 Andrade, S.T.Q. de 83 Alfieri, A. 248 , 249 Barbosa, E. de C. 86 Andriguetti, N.B. 12, 29, 111 Barbosa, J.E.F. 111 Alfieri, A.A. 14 , 57, 229, 247, 255, 258, Antão, K.L. 95, 140 262, 264, 267, 272 Barbosa,M.L. 198 Anthony, S.J. 14, 60 Barbosa, M.R. 112 Alfieri, A.F. 14 , 57, 247, 255, 258, 262, Apolinário, I.B. 230 264, 272 Barbosa, M.RF. 12, 29 Aquino, V.H. 78, 134, 186, 243 Barbosa, V.M. 239 Alfonso, H.L. 78, 145 Arantes, A. de M. 11, 34, 134 Almeida, A.C. da S. 244 Barboza, J.D.B. 224 Arantes, T. 12, 31 Barca Junior, F.A. 229 Almeida, G. 122, 123 Arantes, T.S. 66, 102, 103 Almeida, G.G. 13, 54 Barcellos, M. 264 Araújo, A.P. 252, 257 Barnabé, A.C. 207 Almeida, G.M. de F. 74 Araújo, A.P. de 14, 58 Almeida, G.M.F. 10, 18, 102, 233 Barnabé, A.C.S. 11, 51, 245, 257, 269 Araujo, B.R. 178 Barrela, K.M. 112 Almeida, H.M. de S. 239 Araújo Coutinho, C.J.P. da C. 210 Almeida, J.F. 80 Barreto, D.M.V.S. 129 Araujo, J. 224, 271 Barreto, E.S. 167 Almeida, L.L. 13, 55 Araújo, J. 236 Almeida, M.G.B. 170 Barriolli, C.M.C. 220 Araújo, J.M.G. 127 Barros, C. 71 Almeida, M.R. 273 Araujo Jr, J.P. 13, 14, 56, 57, 227, 231, 254, Almeida, M.T.R. 147 Barros, C.A. 85 259, 273 Barros, C.S. 64, 77 Almeida, N. 242 Araújo Jr, J.P. 120, 137, 226 Almeida Queiroz, S.R. de 252, 257 Barros, H.L.S. 62 Araújo Jr., J.P. 244, 261 Barros, I.C. 150, 177 Almeida, R.G. 235, 237 Araujo, L. 150 Almeida, S.E. de M. 11, 12, 29, 50, 111 Barros, J. de A. 180 Araújo, S.B. 70 Barth, O.M. 170 Almeida, S.M.V.S. 129 Ardisson, A.D.M.P. 11, 46 Almeida, T.N.V. 125, 134, 136 Barth, O.M.S. 193 Arévalo, A.F. 246 Bastos, A.P. de A. 238 Alvarenga, P. 122 Arns, C.W. 11, 51, 98, 207, 245, 257, 269, Alves, A. de A. 147 Batinga, C.A. 258 275 Batista H.B.C.R. 55 Alves, C.D.B.T. 52, 252 Arruda, E. 11, 14, 33, 38, 88, 89, 91, 175, Alves, F.A.G. dos S. 173, 194 Batista, H.B.C.R. 247 181 Batista, I.C.A. 137 Alves, M.R. de M. 142 Arruda, E. de P. 252, 257 Alves, M.R.M. 159 Batista, M.N. 72, 73, 83 September/October 2014 Volume 19 – Supplement 2 - Index XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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Batista, T.N. 14, 57 Borges, I.A. 64, 227, 233, 248 Calmon, M. de F. 75, 169 Battisti, M.A. 149 Borges, L. 239 Calzavara Silva, C.E. 138 Baumworcel, N. 242 Borges, L.G. dos A. 99 Calzavara-Silva, C.E. 86 Bellei, N. 14, 38, 94, 179, 188 Botelho, J. 121 Camargo, C. 188 Belley, N. 204 Botelho, J.A. 186 Camargo, C.N. 179 Bello, G. 168, 182, 189 Botelho, L. 10, 18, 102 Camargos, M.F. 266 Belo, C.A.D. 14, 59 Botosso, V.F. 203 Camargo, T. 250 Bélot, J.L. 11, 44 Bottino, F. de O. 232, 244 Camilo, H.P. 169 Benites, H.G. 210 Brabo, M.V.V. 171 Campbell, C.M.P. 154 Bergmann, M.R. 11, 46 Bracarense, A.P.F.R.L. 255 Campos, A.C. 270, 271 Bergo, E.S. 152 Braga, A.C.S. 72, 73, 83 Campos, A.D. 176 Bergter, E.B. 11, 44 Braga, R.M. 170 Campos, D. 14, 39 Bernardino, J. de S.T. 86, 122, 210 Brancalhão, R.M.C. 210, 211 Campos, D.A. 179, 197 Bersano, J.G. 255, 256 Brandão, C. 202 Campos, F.C. 12, 31 Bertani, G.R. 15, 42, 192, 228 Brandão, C.J. de F. 146 Campos, F.S. 11, 50, 75, 99, 229, 272 Bertol, J.W. 176 Brandâo, P.E. 244 Campos, G.R.F. 12, 23, 82 Bertran, A.G.M. 215 Brandão, P.E. 225, 240 Campos, G.S. 146, 202 Beuttemuller, E.A. 272 Brandespim, D.F. 261 Campos, M. 123 Bezerra, D.A.M. 12, 53, 182 Brasileiro, A.C.M. 218 Campos, R.K. 10, 17, 101 Bhatt, N. 175 Brentano, L. 12, 52, 231, 238 Campos, R.M. 73, 84, 143 Bicalho, J.M. 260 Brioso, P.S.T. 216 Canal, C.W. 13, 52, 56, 233, 235, 236, 250, Bila, D. 175 Brito, C.J.B. 90 251, 252 Biondo, A.W. 273 Brito, D.E.M.W. 235 Candeias, J.M.G. 163 Biselli, J. 135 Brito, M.E.D. 229 Candiani, T. 122 Biselli, J.M. 137 Brito, V. de L. 87 Candido, M. 258 Bisordi, I. 11, 31, 239 Bronkhorst, D.E. 14, 57, 229, 247 Cano, L. 253 Bittar, C. 12, 13, 23, 36, 82 Bronzoni, R.V. de M. 162, 167 Cantão, M.E. 13, 53, 234 Bittencourt, L.H.F.B. 166 Brown, D.T. 84 Cardoso, A.L.S.P. 241 Bittetti, M. 64 Budaszewki, R. da F. 252 Cardoso, B.F. 130, 132, 166 Bloom, D.C. 98 Budaszewski, R. da F. 251 Cardoso, D. 65, 120 Boas, L.V. 87 Budaszewski, R.F. 52, 233, 235, 236 Cardoso, D. das D. de P. 11, 34, 125, 126, Boff, L. 81 , 147 Bueno, F.C. 208 127, 131, 134, 136, 163 Bogado, A.L.G. 229 Bueno, V.D.C. 157, 158 Cardoso, D.D.P.C. 145 Boiteux, L.S. 218, 220 Burbano, R.M.R. 150 Cardoso, E.S. de C. 129 Boldrini, N.A.T. 139, 153 Burlandy, F.M. 12, 29 Cardoso Jr, C.A.M. 14, 26 Bolpetti, A. 163 Burth, P. 87 Cardoso, R.S. 91 Bomfim, W.A. 67 Burutarán, L. 101 Carenzi, L. 175 Bonafé, C.F.S. 207 Bustamante, F.C. 198 Carenzi, L.R. 181 Bonanno, V.M. dos S. 112 Buzinaro, M. da G. 250 Carestiato, F.N. 118, 159 Bonanno, V.M.S. 12, 29 Buzinaro, M.G. 191, 239, 269 Carlos, L.Z. 86 Bonfim, C.M. do 79 Buzzato, G.P. 181 Carmona, R. de C.C. 142, 159 Boni, T. 263 Buzzinaro, M. da G. 258 Carneiro, A.C.A. 69 Bonjardim, C.A. 13, 54, 74, 149, 230 Carneiro, A.P. 14, 26 Bonon, S. 186 C Carneiro, B.M. 72, 73, 83 Bonon, S.H.A. 139, 158, 193 Cabello, M. 155 Carneiro, R. dos S. 131, 163 Boratto, P.V. de M. 66 Cabrera, E. 135 Carnieli Jr, P. 247 Boratto, P.V.M. 10, 12, 18, 21, 101, 102, Caetano, D.G. 182, 189 Carnieli, Jr. P. 55 103 Cahu, G.G. de O.M. 143, 180 Caron, L.F. 238 Borchardt, A. 13, 56 Cahú, G.G.O.M. 156 Carraro, E. 176, 179, 197 Borchardt, A.C. 251 Calastri, L.P. 116 Caruzo, T.A.R. 176 Bordignon, J. 202 Caldas, L.A. 73 Carvalho, A.A.B. 261 Bordin, L.C. 228 Caldas, S. 72, 84 Carvalho, B.K.S. 14, 40 Borges, A.A. 93, 94, 185, 186 Calegari, L.P. 78 Carvalho, C. 270 Borges, A.S. 80 Calixto, R.S. 68, 69, 149 Carvalho, C.A.M. 85, 92 September/October 2014 Volume 19 – Supplement 2 - Index XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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Carvalho, D.G. 87 Colina, R. 101 Cruz, J. 263 Carvalho-Filho, J.R. 86 Colmanetti, T. 224 Cruz, O.G. 178 Carvalho, L.D. 63 Colombo, A.C.F. 133 Cruz, T.F. 14, 57, 226, 227, 254 Carvalho, L.R. 167 Colombo, T.E. 135, 137, 196 Cubas, Z.S. 273 Carvalho, M.O. de O. 193 Coltro, V.P. 77 Cunha, A.C.C. 90 Carvalho, M.O.O. 170 Comone, P. 203 Cunha, E.M.S. 246 Carvalho, R.C.P. 222 Conceição, A.O. 70 Cunha, M. dos P. 125, 127, 131, 163 Carvalho, V.L. 213 Corbellini, L.G. 233 Cunha, M.S. 11, 31 Carvalho, W.J. 14, 26 Cordeiro, T.I. 159 Cursino, A.E. 266 Caserta, L. 11, 51, 269 Correa, A. 113 Caserta, L.C. 207, 245, 257, 275 Correa, A. de A. 113 D Castellanos, L. 95 Correa, R.A. 12, 31, 99, 113 Dabelic, R. 14, 25 Castilho, J.G. 13, 55, 247 Correa, R.F.T. 207 da Costa, L.C.F. 15, 40 Castrignano, S.B. 112 Corrêa, R.L. 221 da Costa, L.J. 10, 14, 18, 27 Castro, A.G.M. 241 Correa, T. dos S. 11, 34 da Costa, S.M. 10, 18 Castro, A.M.M.G. 226, 255, 256 Corrêa, T. dos S. 136 da Costa, V.G. 125 Castro, A.R. 187 Corrêa, V.M. 123 da Fonseca, F.G. 13, 35 Castro, F. 122 Côrtes, F.H. 182, 189 Dalla Vecchia, A. 105, 106 Castro, F.C. 213 Cortines, J. 12, 21 Damaso, C. 70 Castro, Í. de A. 126, 127 Costa, A.G. 14, 58 Damaso, C.R.A. 107 Castro, K. da S. 213 Costa, A.O. 10, 18, 102 Damazo, N.A. 109 Castro, L.F.F. 194 Costa, A.P.F. 127, 150, 152, 201 Dantas, G. 210 Castro,L.F.F. 164 Costa, C.S. 75 da Paixão, C.G.S. 133 Castro, M.E.B. 217 Costa, D.T.M. 147 da Rocha, D.R. 268 Castro, T. 242 Costa, É.A. 270 da Rosa, R.D. 77 Castro, T.X. 244 Costa, E.M. 232, 244 Daros, F. 260 Catroxo, M.H.B. 225 Costa, E.P. 13, 15, 34, 41 da Silva, A.A.S. 186 Cavalcante, M. 64, 71, 77 Costa, E.V. 12, 29 da Silva, D. 21 Cavalcante, R.V. 259 Costa, G.B. 13, 54, 129, 149, 248 da Silva, D.J.F. 167 Cavalcanti, S.M.B. 118, 159 Costa, I.B. 150, 177 da Silva, D.M. 180 Cavaleiro, N. 10, 18 Costa, L.C.P. das N. 160 da Silva, E.E. 10, 12, 18, 29 Cecílio, A.B. 72, 84 Costa, L.D.C. 126 da Silva, É.E. 173 Chagas, C. 64 Costa, L.F. de M. 146 da Silva, E.M.L. 14, 27 Chanasit, J.S. 143 Costa, L.F.M. 202 da Silva, E.R.M. 164 Chávez, J.H. 62 Costa, L.J. 97 da Silva, F.C. 80 Chiappetta, C.M. 250 Costa, L.J. da 14, 24, 80 da Silva, F.E.R. 147 Chiappetta, M.C. 236 Costa, M.A. 150 da Silva, F.G. 11, 31 Cibulski, S.P. 113 Costa, M.C.M. 150 da Silva, F.R.C. 250 Ciconello, F.N. 275 Costa, M.M. 260 da Silva, G.P.D. 97 Cilli, A. 142 Costa, P.M.G. 222 da Silva, J.L. 143 Cintra, A.C.O.; 243 Costa, S. 186 da Silva Jr, H.C. 201 Cirne-Santos, C.C. 95 Costa, S.B.F.G. 212 da Silva, L.D. 161 Cirqueira, C. dos S. 162 Costa, S.C.B. 139, 158, 193 da Silva, L.L.P. 14, 27, 91 Claus, M.P. 255, 262 Costenaro, J.G. 11, 50, 75 da Silva, L.P. 11, 34 Clemente, W.T. 103 Covas, D.T. 10, 13, 19, 36, 172 da Silva, L.S. 191, 196 Coelho, A. 14, 39 Crapez, M.A. 108, 110, 111 da Silva, M.C.C. 88 Coelho, L.F.L. 12, 15, 22, 40, 69, 119, 128, Craveiro, S.R. 217 da Silva, M.G. 189, 190 130, 131 Crespo, S.E.I. 14, 57, 258, 264 da Silva, M.R.S. 73, 84 Coelho, M.R.C.D. 143 Criado, M. 175 da Silva, M.S. 52, 235, 251 Coêlho, M.R.C.D. 156, 180 Criado, M.F. 14, 38, 88, 89, 91 da Silva, M.V. 159 Coimbra, R. 122 Crispin, L.A. 270 da Silva, T.R. 11, 50 Coimbra, T.L.M. 11, 31 Cruz, A.C. 242 da Silva, V.A.G.G. 98 Colares, J.K.B. 146 Cruz, C.A. 191 da Silveira, N.J.F. 131 Coldebella, A. 12, 52, 231 Cruz, F. da S.P. 15, 42, 192 Daszak, P. 14, 60 September/October 2014 Volume 19 – Supplement 2 - Index XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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Daudt, C. 13, 56, 250 De Oliveira, F. 62 dos Santos, F.A.L. 132, 166 de Aguiar, D.M. 224, 259 de Oliveira, J.G. 86 dos Santos-Junior, N.N. 78 de Albuquerque, A.C.C. 180 de Oliveira, J.M. 178 dos Santos, M.A.M. 130 de Albuquerque, N.R.M. 12, 31 de Oliveira, P.S.L. 94 dos Santos, M.C. da S.G. 15, 40 de Alcantara, B.K. 255 de Oliveira, T.S. 163 dos Santos, R.N. 12, 31 de Alencar, A.L.F. 14, 58 de Pádua, S.B. 252, 257 dos Santos, T.A. 198 de Almeida, D.V. 185 de Paula, F.E. 14, 38 dos Santos, V.R. 106 de Almeida, G.M. 10, 17 de Paula, S.O. 12, 30, 78, 117 Drummond, B.P. 131 de Almeida Jr, R.F. 92 de Pinho, R.A.P. 171 Drumond, B. 135 de Almeida, M. da G.B. 193 Dergovics, F.L. 112 Drumond, B.P. 64, 119, 120, 121, 123, de Almeida Queiroz, S.R. 258 de Sá, J.P.O. 95, 140 137, 196, 227, 233, 248 de Amorim, L.M. da F. 98 de Sena, A.A.S. 80 Duarte, I. 238 de Andrade, M.A.A.M. 89 de Siqueira, T.R. 227 Duarte, P. de S.G. 13, 47, 212, 214 de Araujo, J. 11, 50 de Sousa, D.D. 180 Duarte, P.S.G. 13, 47, 215 de Araújo, J.M.G. 92 de Sousa, E. 89 Dupont, P.M. 235, 251, 252 de Azevedo, K.M.L. 11, 32 de Sousa, M.S. 196 Duppont, P.M. 52, 233 de Azevedo, M.L.B. 201 de Sousa, R.L.M. 14, 58, 252, 257, 258 Durães Magalhães, R. 269 de Barros, L.H.R. 140 de Souza, E.M.A. 174 Durigon, E.L. 11, 14, 50, 60, 198, 224, 236, de Campos, A.M. 149 de Souza, F.F. 231 270, 271 de Carvalho, I.L.Q. 241 de Souza, K.M.C. 126, 127 Durymanova Ono, E.A. 240 de Carvalho, I.M.V.G. 86, 122, 210 de Souza, M.C.B.V. 90 Dutra, A.G.S. 84 de Castro, C.A.V. 168 de Souza, M.R 14, 58 Dutra, K.R. 123 de Castro, F.L. 11, 50, 227 de Souza, R.P. 11, 31 de Castro, I.R.D. 86 de Souza, V.C. 180 E de Castro, R.O. 14, 27 de Souza, W.M. 135 Ebani, E.C. 116 de Castro, T.X. 232 de Stefano, E. 225, 264 Edreira, N. 215 de Faccin-Galhardi, L.C. 66, 67 Dias, A.S. 270 Eduardo, M.B.P. 12, 29 de Fatima, A. 81 Dias, C.M.G. 143 Eiras, A.E. 155 de Figueiredo, M.L.G. 134 Dias, J. 242 Éleouët, J.F. 12, 24 de Freitas, G.V. 117 Dias, M.C. 169 Eleutério Jr, J. 201 de Godoy, S.H.S. 258 Dias, R.S. 12, 30, 117 Elias, T.C. 131 de Jesus, B.L.S. 14, 38, 89 Dibo, M.R. 155 Ellott, R.M. 12, 21 Delatorre, E. 168 Di Felice, E. 63 Elmahdy, E.M. 104 D’elia, M. 263 Diminitrova, Z.E. 13, 36 Escremim, F. 175 de Lima, L.M.P. 87, 172 Diniz, J.A. 264 Espada, S.F. 66, 67 de Lima, M.C. 95, 140 Dobao, E. 118 Esposito, D.L.A. 14, 25, 183 de Lima, M.F.N.T. 266 do Carmo, A.P. 13, 35 Esteves, P.A. 12, 52, 231, 238 de Lima, M.M. 130 Dolabella, S.S. 189, 190 Evers, F. 166 de Lima, P.C. de S. 180 Domiciano, I.G. 255 Dellariva, T.C. 139 Domingues, C.F. 232, 244 F Delmart, E. 273 Domit, C. 255 de Lucena, W.A.T. 180 Domitrovic, T. 12, 22 Fabres, R.B. 12, 29 de Macedo, M.D. 10, 19 Donin, D.G. 262 Faccin-Galhardi, L.C 118 de Magalhães, D.A.R. 10, 19 Dorea, A. 146 Fagotti, A. 153 de Medeiros, R.M. 11, 50 Dornas, F.P. 10, 12, 17, 21, 66, 101, 102, Fagundes, E.M.S. 63, 81 de Mello, J.L. 248, 249 103 Fagundes, J.M. 76 de Melo, W.A. 174 do Rosario, J.S.C. 198 Fagundes, L.G. 131 de Meneses, M.D.F. 84 dos Anjos, K. 76 Fantinatti, B. 273 de Menezes Filho, R.N. 252, 257 dos Reis, A.S. 143 Faria, M.G. 13, 15, 34, 41 de Moraes, A.P. 98 dos Reis, J.K.P. 260 Faria, N.A. 111 de Morais, V.M.S. 180 dos Santos, A. de O. 173, 197 Farias, K.J.S. 92, 127, 150, 152 Denani, C.B. 85 dos Santos, A.O. 194 Farias, L.M. 76 Deng, X. 273 dos Santos, C.N.D. 202 Farman, M. 13, 47 de Oliveira, A. do S.L. 182 dos Santos, C.P. 12, 31 Fausto, A.K. da S. 11, 44

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Favarin, M. do C. 13, 36 Figueiredo, C. 71 Gaidet, N. 11, 50 Favaro, A.B. 270 Figueiredo, L.T. 78 Galan, L. 154 Favaro, E.A. 120, 155 Figueiredo, L.T.M. 82, 120, 134, 135, 145, Galbieri, R. 11, 44 Favero, L.M. 14, 57 156, 162, 248 Galindo, D.B. 11, 50 Fecury, P.C.M.S. 182 Figueiredo, M.L.G. 135 Gallego, J. 166, 232, 267 Feldner, A.C. de C.A. 86, 122 Figueiredo, P. de O. 13, 54 Gallego, J.C. 237, 248, 249 Felippe, P.A.N. 11, 51, 269, 275 Filho, A.A. da S. 84 Galler, R. 201 Fernandes, A.M. 14, 58, 225, 252, 257, Filho, E.F. de O. 63 Gama, L. 15, 43 258 Filho, F.A.X.M. 146 Gamon, T.H.M. 246 Fernandes, A.T.G. 170, 193 Filho, J.R.C. 122 Ganime, A.C. 113 Fernandes, C.D. 216 Filho, R.N. de M. 14, 58 Garboggini, F.F. 98 Fernandes, G.C. 119, 121 Filho, S.L. de M. 128 Garcia, D.G. 87 Fernandes, J.E.F. 13, 48 Filho, T.F. 238 Garcia, F.G. 267 Fernandes Jr, P.C. 15, 41 Filizzola, E.M.A. 171 Garcia, L.A.T. 109, 114 Fernandes, M.E.S. 13, 55, 247 Filoni, C. 244 Garcia, L.A.T.; Nascimento, M.A.; Barardi, Fernandes, M.J.B. 70 Finoketti, F. 229, 272 C.R.M. 114 Fernandes, O.A. 13, 49 Firpo, R. 272 García, M. 101 Fernandez, J.C. 154 Firpo, R.M. 229 Garcia, P. 205 Fernandez, J.C.C. 175 Florencio, C.M.G.D. 147 Garcia, R. de C.C. 244 Ferrari, K.L. 256 Florêncio, C.M.G.D. 148 Garcia, R. de C.N.C. 232 Ferraz, A. de B.F. 81 Floriano, V.G. 125 Garcia, S.C. 12, 29, 112 Ferraz, M.L. 165 Fongaro, G. 12, 28, 104, 105 Garrafa, P. 112 Ferraz, M.L.C.G. 86, 122 Fonseca, B.A.L. 183, 184 Garrido, V. 64, 71, 77 Ferreira, A.C. 126, 151, 152, 153 Fonseca Jr, A.A. 260 Gasparini, M.R. 241, 260 Ferreira, C.G.M. 119 Fonseca, L.A.B.V. 12, 30 Gatti, M.S.V. 176 Ferreira, D. de L. 274 Fonseca, L.P. 116 Gatto, I.R.H. 239 Ferreira, D.F. 84, 92, 107, 110, 111, 143 Fraiha, A.L.S. 14, 58 Gauger, C.P. 15, 41 Ferreira, F.D. 73 Francisco, F.L. de M. 86 Gava, D. 13, 53, 234 Ferreira, H.C.C. 273 Franco, A.C. 11, 12, 31, 50, 75, 99, 229, Gerhardt, D. 14, 37, 243 Ferreira, H.L. 245, 257, 275 272 Gheit, T. 154 Ferreira, J. 195 Franco, G.M. 81 Gibrail, M.M. 145 Ferreira, J.C. 13, 55 Franco, I.R. 15, 40, 131 Giehl, I. 105 Ferreira, J. de A. 171, 174 Franco, O.L. 87 Giehl, I.C. 96, 106 Ferreira, J.G.G. 138 Fregolente, M.C.D. 176 Gilio, A.E. 142 Ferreira, J.M.S. 12, 22, 68, 123, 128 Freitas, D.M.T.A. 217, 220 Gil, L.H.V. 228 Ferreira, L.C. de S. 137 Freitas, G.M. 12, 21 Gil, L.H.V.G. 15, 42, 192 Ferreira, L.S.S. 133, 141 Freitas, G.R.O. 14, 26, 65, 68, 135 Giongo, A. 99 Ferreira, M.U. 13, 37 Freitas, J.C. de O.C. 127, 152, 201 Giongo, V. 12, 28, 71, 77, 107, 108 Ferreira P.C.P. 138 Freitas, L.A. 247 Giongo, V.A. 110, 111 Ferreira, P.C.P. 10, 13, 17, 18, 54, 74, 149, Freitas, L.B. 139, 153 Giraldo, P.C. 201 230, 233, 266 Freitas, L. de A. 264 Giuliano, A.R. 154 Ferreira, P.G.A. 222 Freitas,L. de A. 272 Godinho, M.T. 11, 43, 207, 209 Ferreira, S. 154, 163 Freixo, H. de O. 178 Godoi, A.M. 66, 118 Ferreira, S.B. 268 Froelich, L. 65, 135 Godoi, A.M. de 67 Ferreira, V.F. 80, 268 Fujimura, P.T. 14, 26 Godoy, H.P. 191, 239, 250, 261, 269 Ferreira, V.F.F. 90 Fumagalli, M.J. 15, 40, 131 Godoy, I.J. 222 Ferreira, V.N. dos S. 92 Fumian, T.M. 112, 124, 133 Góes, L.G. 236 Ferreria, M.C. 263 Fusaro, A. 220 Goes, L.G.B. 270, 271 Ferro, C.G. 209, 212 Goes, T. 93, 94 Fiaccadori, F.S. 11, 34, 125, 126, 127, 131, G Gomes, A.C.C. 116 134, 136, 145, 163 Gabbay, Y.B. 112, 138, 144, 160, 161, 191 Gomes, A.M. de O. 92 Figueira, A. 215 Gadelha, S.R. 129 Gomes, A.M.O. 85 Figueira, A. dos R. 13, 47, 212, 214 Gagliardi, T. 175, 181 Gomes, A.V.B.T. 69, 128, 130 Figueira, A.R. 13, 47 Gagliardi, T.B. 14, 25 Gomes, E. 195 September/October 2014 Volume 19 – Supplement 2 - Index XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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Gomes, E.R. 174 Hachich, E.M. 12, 29 Júnior, W.P.L. 180 Gomes, É.R. 171 Hafez, H.M. 11, 51, 269 Junqueira, B.R.T. 210 Gomes, L.G. da S. 129 Hagiwara, M. 263 Junqueira, D.M. 11, 50 Gomes, M. 147 Harakava, R. 208 Junqueira, R.M. 185 Gomes, R.A. 63 Harsi, C.M. 148, 204 Justino, M.C.A. 182 Gomes, Y.M. 141 Hassan, R. 152 Gonçalves, A.C.S. 239, 250, 269 Headley, S.A. 229 K Gonçalves, A.K. da S. 201 Heck, T.M. da S. 12, 29, 111 Kamikawa, J. 188 Gonçalves, G. dos S. 15, 43 Heinemann, M. 263 Kanamura, C.T. 162 Gonçalves, M.C. 208 Heinen, L.B. da S. 130, 132, 166 Kanashiro, A.M.I. 241 Gonçalves, M. dos S. 171, 174 Heleno, N. 263 Kashima, S. 10, 13, 19, 36, 172 Gonçalves, R.B. 85 Henzel, A. 12, 29, 96, 103, 105, 106, 111, Katto, S. 229 Gonçalves, S. 263 113, 228 Khan, M.J. 145 Gonzaga, D.T. 268 Hernandez, J. das M. 161 Khudyakov, Y. 13, 36 Gonzalo, B. 155 Hora, A.S. 244 Kimura, L.M. 194 Goodin, M.M. 13, 47 Hosie, M. 263 Kisielius, J.J. 11, 31, 112 Gotardi, A.H.B. 188 Howard, J.C. 230 Klein, T.M. 184 Goulart, L.R. 13, 14, 15, 26, 34, 41, 80 Huatuco, E.M.M. 198, 205 Kluge, M. 12, 31 Gouvea, M.N. 203 Hurtado, R. 14, 60, 271 Knak, M.B. 11, 50, 75 Gouvea, T.D. 118 Hurtado, R.F. 11, 50 Kobayashi, M.T. 169 Gradiz, J.J. 14, 57 I Kohn, L.K. 98 Granados, O.F.O. 252 Kramer.B. 228 Granato, C. 94, 157, 158, 165, 179, 188 Ibidi, S.M. 142 Kratz, J.M. 81, 147 Granato, C.F.H. 164, 204 Iglezias, S.D.A. 162 Kroon, E. 122, 123 Granda, M.C. 273 Igreja, R.R. 146 Kroon, E.G. 10, 12, 13, 15, 17, 18, 21, 40, Granja, F. 180, 184 Immig, J. 272 54, 66, 68, 69, 74, 86, 101, 102, 103, Grecco, S. 264 Inglis, P.W.; 217 108, 121, 149, 155, 225, 230, 233, Gregianini, T.S. 99 Ismael, N. 175 259, 266 Gregori, F. 235 ivonesi, M.C. 258 Kruger, L. 271 Gregório, D. de S. 142 Kunz, A. 12, 28, 105 Grinsztejn, B. 182, 189 J Kunz, A.F. 166, 232, 237, 248, 249, 267 Groff, F.H.S. 233 Kurissio, J.K. 13, 14, 56, 57, 163, 231, 261 Grolli, P. 232, 237, 249 Jaime, J. 253 Jain, S.A. 189, 190 Kury, C.M.H. 178 Grotto, R.M.T. 167 Kuster, R.M. 73 Grynberg, P. 217 Jani, I. 175 Gudo, E. S. 175 Januário, M.E. da S. 14, 27 L Guedes, M.I.M.C. 14, 58, 270 Japolla, G. 235, 237 Guedes, R.M.C. 270 Jardim, A.C.G. 13, 36, 243 Lacerda, A.L. 218 Guerra, J.M. 194 Jesus, L.F. de 96 Lacorte, C. 218, 220 Guerra, O.G. 188 Johnson, J.E. 12, 22 Lamar, D.C. 207 Guerra, S. de F. 191, 196 Joventino, K.M. de S. 127, 150, 152 Lamas, N.S. 217 Guerra, S. de F. dos S. 144, 182 Julião, J.A.S. 13, 34 Landell, M.G.A. 208 Guimarães, M. 14, 39, 187 Junior, A.S. 273 La Scola, B. 10, 12, 17, 18, 21, 66, 101, Guimarães, M.L. 185, 251 Junior, C.P.J. 237 102, 108 Guimarães, T.E. 73 Júnior, E.C.S. 171 Lau, D. 209, 212 Guimarães, V.N. 125, 127, 131, 163 Junior, J.C.R. 14, 57 Laurindo, M.F.L. 157 Guioti, F. 126, 152, 153 Junior., J.P.A. 196 Lazari, C. dos S. 157, 158 Guissoni, A.C. 65, 120 Júnior, J.W.R. 213 Lazari, C.S. 164 Guissoni, A.C.P. 145 Junior, L,B.D. 133 Leastro, M.O. 10, 13, 17, 49 Gurjão, T.C.M. 112 Junior, L.B.D. 141 Lei, G. 11, 46 Gusmao, A.F. 152 Junior, M.C.R. 172 Leite, F. de S. 215 Gusmão, A.F. 126 Junior, P.C.F. 13, 34 Leite J.G.P. 268 Junior, R.B.M. 175 Leite, J.P.G. 14, 59, 101, 124, 141 H Júnior, S.M. de A. 11, 50 Leite, J.P.V. 76

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Leite, R.C. 241 Lopes, N. 66, 67, 118 Marin, L.J. 129 Leite, R. de C. 260 Lopes, T.R.R. 143 Marques, B.C.L. 251 Leite, T. 14, 39, 187 Lopes,T.R.R. 156 Mar, T.B. 209, 212 Leme, R.A. 247 Lorenzetti, E. 14, 57, 247, 258, 262 Martinelli, A. de L.C. 172 Leme, R. de A. 258, 262 Loureiro, E.C.B. 138 Martines, R.B. 162 Lemes, L.N. 11, 34 Loving, L.C. 15, 41 Martinez, R.T. 219 Lemos, T.M.A.M. 152 Lucas, F. 96 Martini, M.C. 11, 51, 98, 207, 245, 257, Leutenegger, C. 273 Lucas, F.M. 219 269, 275 Levis, S. 239 Lucas, M.A. 212 Martins, C.P.S. 63 Lewis, M. 15, 43 Lucena, M.S.S. de 161 Martins, D. de L. 87 Lierz, M. 11, 51, 269 Lucena, W.A.T. 143 Martins, F. dos S. 68, 69 Li, L. 273 Luchs, A. 142 Martins, M. de S.N. 264, 265, 268 Lima, A. 10, 18, 102 Luciano, R.L. 241 Martins, M.L. 13, 35, 63, 81 Lima, A.S.N. 86 Lucinda, N. 76 Martins, N. 263 Lima, A.T.M. 11, 43, 207, 209 Luiz, A.P.F. 122 Mascarenhas, D.O.J.D’A.P. 196 Lima, D.M. 146 Luiz, A.P.M.F. 225 Mascarenhas, J.D’A.P. 161, 182, 191 Lima, G.A. 178 Luna, L. 235 Mascarenhas, J.D.P. 12, 53, 112, 138, 144 Lima, G.K. 260 Luna, L.K. de S. 11, 33 Mascitti, A.K. 228 Lima, G.M. 87 Lunel, N.L.T. 12, 21 Massi, R.P. 262, 272 Lima, M. dos S. 264, 265, 268 Lunge, V.R. 228 Mathijs, E. 63 Lima, M.F. 220 Lustosa, J. 267 Matias, B.F. 13, 15, 34, 41 Lima, M.I.S. 15, 41 Lustosa, J.M. 166 Matos, A.C.D. 14, 58 Lima, M.P. 14, 58 Luz, C.R.N.E. da 160 Matos, N.B. 194 Lima, M.T. 68, 69 Luz, I. 113 Matos, R.P.A. de 75 Lima, N.C. da S. 194 Luz, R.B. 12, 29 Matsui, T. 12, 22 Lima Neto, D.F. 11, 51, 207, 269 Lyra, J.S.P.O. 90 Mattos, B.B.B. 11, 44 Lima, P.C. de S. 143 Mattos, G.L.M. 12, 52, 231, 238 Lima, R.G. 139 M Mauroy, A. 63 Lima, R.N. 96 Mabunda, N. 175 Mazer, L. 250 Lima, S,F. 141 Macedo, M.D. de 13, 36 Mazer, L.C. 239 Lima, S.F. 133 Macedo, R. 166, 232, 237, 248, 249, 267 Mazzarotto, G.A.C.A. 202 Lima, S.T. 274 Machado, A. de M.V. 15, 42, 192 M.B. de L.D. 131 Lima, T.L.C. 92 Machado, A.M. 188 Medeiros, A.S.R. 239 Lima, W.A. 175 Machado, A.M.V. 76 Medeiros, M.A. 201 Lima, W.T.A. 11, 14, 33, 38, 181 Machado, A.R.S.R. 188 Medeiros, N.P.T. 185 Linhares, A. da C. 144, 160, 182, 191, 196 Machado, B.C. 142, 159 Medeiros, R. 171, 174, 195, 274 Linhares, A.G. 13, 56 Machado, D. 93 Medeiros, R.S.S. 194 Linhares, L.S.A. 13, 56 Machado, G. 123, 233 Medeiros, T.N. da S. 14, 57, 258 Linhares, R.E.C. 66, 67, 118 Machado, P.R.L. 92, 127, 150, 152, 201 Mees, L. 75 Lira, E.L. 186 Maeda, A.Y. 11, 31 Mehnert, D.U. 112 Livonesi, M.C. 15, 40 Magalhães, C.L. de B. 233 Meira, G.L.S. 73 Lizasoain, A. 101 Maganha, S.R. de L. 14, 58, 252, 257 Meire, G.L.S. 143 Lobato, Z.I.P. 14, 58, 270 Magri, M.E. 12, 28, 105 Melchiades, V. 64, 71, 77 Lobo, P. dos S. 144 Maiorka, P.C. 246 Melgaço, F.G. 113 Lo, D.S. 142 Malaquias, L.C.C. 12, 15, 22, 40, 69, 128, Mello, F.C. do A. 159 Loh, E. 14, 60 130, 131 Mello, I.M.V.G.C. 13, 36 Lopes, A.P. de M. 215 Malossi, C.D. 259 Mello, I.O. 141 Lopes, B.P.T. 119 Mamani, P.R. 82, 156 Mello, J.L. 232, 237 Lopes, D.O. 123 Marcio, M.S. 11, 45 Mello, W. 195 Lopes, E. 165 Marcolino, L.D. 189, 190 Mello, W.A. 119, 133, 141, 171, 274 Lopes, K.G.S. 228 Maria, F.H. de O.S. 95, 140 Melo, D.M. 94 Lopes, L. dos S. 238 Mariano, A.P.M. 129 Melo, D.M. de 185, 186 Lopes, L.V.A. 116 Marinheiro, J.C. 148 Melo F.L. 216 Lopes, L.V. de A. 116, 117 Marinho, R.S.S. 14, 59 Melo, F.L. 13, 48, 49, 213, 217, 218 September/October 2014 Volume 19 – Supplement 2 - Index XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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Mendes, G. 87 Moreira, L.P. 179 Nepomuceno, A.M. 110, 111 Mendonça, L.A. 119, 121 Moreira, M.S.A. 93 Neto, A.C. 270 Mendonça, L.M. 97 Moreira-Vieira, F. de F. 68 Neto, D.B. 229 Mendoza, Y.Y. 155 Moreli, M.L. 125, 167 Neto, D.F.L. 245 Meneguin, C. 224 Moreno, E.C. 149 Neto, E.A. 91 Meneses, M.D.F. 73 Moresco, V. 109 Neto, E.D. 11, 32 Menezes, E. de F.C. 138 Mores, M.A.Z. 12, 52, 231, 238 Neto, F.C. 151, 155 Menezes, I. de Q. 178 Morgado, L.N. 173 Neto, J.P.N. 213 Menezes, W. da R. 118 Morgado, M. 175 Neto, L.L.S. 146 Menoni, S. 186 Morgado, M.G. 14, 39, 154, 168, 182, 189, Neto, M.M. 157 Menoni, S.M.F. 193 251 Neurauter, A.L. de A. 90 Merlone, M.P. 13, 37, 178 Mori, C.M.C. 246 Neves, A.P. 64 Miagostovich, M. 101 Mori, E. 246 Neves, M.A.O. 138 Miagostovich, M.P. 113, 124, 133, 141 Morillo, S.G. 142 Nguyen, J.B. 14, 25 Michelin, E.C. 252, 257 Moriones, E. 212 Nicolini, C. 216 Migliolo, L. 87 Mósena, A.C.S. 233, 235, 236, 251 Nogueira, A.H.C. 225 Milanelo, L. 13, 56 Mota, B.E.F. 74 Nogueira, G.B. 157 Minnicelli, C. 152 Mota, G.C. 86 Nogueira, J.S. 11, 31 Miquelitto, F. 184 Mota, M.T. de O. 73 Nogueira, L.M. 123 Miranda, A.E. 139, 153 Mota, T.Q. 152 Nogueira, M.F.T. de L. 259 Miranda, E.A. 189, 190 Moura, F. de B.P. 93 Nogueira, M.L. 73, 120, 131, 135, 137, Miranda, F.J.A. 150 Moura, F.E.A. 147, 148 155, 167, 196 Miranda, J.B. 13, 54, 64, 227, 230, 248 Moura, M. 11, 46 Nogueira, R. 172 Mirandola, P.H. 188 Moura, M.E.G. 157, 158 Nonogaki, S. 194 Miura, V.C. 167 Muller, E.C.A. 191, 196 Nozawa, C. 66, 67, 118 Miyabe, F.M. 264, 272 Müller, U.B. 230 Nunes, C.F. 14, 37, 243 Miyaki, C. 203 Muller, V. 78 Nunes, E.M. 154 Miyashiro, S. 263 Muller, V.D.M. 93, 94, 186 Nunes, M.R.T. 12, 21 Miyashiro, S.I. 246 Munin, F.S. 14, 58, 258 Nunes, M.S. 13, 37 Miyazaki, R.D. 166 Myiazaki, R.D. 132 Modena, J.L.P. 14, 38, 181 O N Modis, Y. 14, 25 Ocadaque, C.J. 147, 148 Molinari, B.L.D. 247, 258, 262 Nagasse-Sugahara, T.K. 112 Oda, J.M.M. 188 Mondini, A. 126, 151, 152, 153, 155 Nagata, T. 76, 133, 172, 210, 216 Ogata, R.A. 255, 256 Monezi, T.A. 112 Nagel, N.E. 11, 51, 269 Ogawa, J. 12, 24 Monta, M.J. 146 Nali, L.H. da S. 11, 32, 33 Okano, W. 229 Montassier, H.J. 12, 52, 231, 238 Nara, J.M. 255, 256 Okino, C.H. 12, 52, 231, 238 Montebianco, C.B. 11, 44 Nardi, M.S. 14, 60 Okuda, L.H. 225, 264, 265, 268 Monteiro, D.C.S. 14, 40 Nascimento, B.L.S. 213 Olival, G.S. do 11, 33 Monteiro, H.A. de O. 213 Nascimento, I. 215 Oliveira, A. 12, 24 Monteiro, J.M. de C. 78, 117 Nascimento, M.A. 104, 114 Oliveira, A.C. 85 Monteiro, T.A.F. 150, 177 Nascimento, V.A. 14, 27, 40 Oliveira, A.C.R. 125, 126 Montoro, M.G. 239 Nascimento, Y.M. 92 Oliveira, A. do S. 191, 196 Moraes, A.P. 269 Nassar, A.F.C. 225 Oliveira, A.L.R. 11, 31, 239 Moraes, E.M.S. 229 Nava, A. 14, 60 Oliveira, A.M. 266 Moraes, F.M. 183 Navarro, I.T. 166 Oliveira, A.S. 78, 88, 91 Morais, L.D. da S. 80 Navarro, J.A.S. 10, 13, 17, 49 Oliveira, C.P. 157 Morais, L.L.C. de S. 112 Navarro, J. de O. 14, 58, 252, 257 Oliveira, D. 122, 123 Morais, S.M. de S. 12, 22, 128 Navarro, J.S. 13 Oliveira, D.B. 10, 17, 191, 198 Morais, V.M.S. 143, 156 Naveca, F.G. 14, 27, 40, 180, 184 Oliveira, D.C.A. 203 Morais, W. 273 Naves, J.H.F. de F. 260 Oliveira, F.M.S. 147 Morandi, B.C. 98 Negri Filho, L.C. 229 Oliveira, G.P. 68, 69 Moreira, C.M. 158 Nelson, M. 13, 53 Oliveira, I.B. 95 Moreira, L. 94, 188 Nepomuceno, A. 108 Oliveira, I.B.R. 210 September/October 2014 Volume 19 – Supplement 2 - Index XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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Oliveira, J.G. 138 Pallás, V. 10, 13, 17, 49 Pilotto, J.H. 168 Oliveira, J.M. 13, 37 Pandolfi, J.R. 228 Pilotto, M.R. 109 Oliveira, L. do H. dos S. 11, 32 Pannuti, C.S. 11, 32 Pimenta, S.T.S. 191 Oliveira, L.G. 239 Pantoja, M.B. 222 Pimentel, F.B. 232, 244 Oliveira, L.M. 133 Pardo-Vargas, A. 95 Pinhatti, A.K. 73 Oliveira, M.D. 117 Parente, A.F. 120 Pinheiro, M. da S. 95, 140 Oliveira, M.M. 65, 135 Parente, A.M.B. 146 Pinheiro, T.M. 137, 196 Oliveira, R. 219 Parente, J. 65 Pinheiro, T.M.L. 95, 140 Oliveira, R.A. 70 Parra, M.C.P. 155 Pinho, J.B. 224 Oliveira, R. das N. 203 Pascoal, J. de O. 160 Pinto, A.M. 71 Oliveira, R.G. de O. 90 Pascoli, G.V.T. 160 Pinto, A.M.V. 14, 59, 268 Oliveira, R.N. 247 Passetti, F. 119 Pinto, A.R. 77 Oliveira, R.N. 13, 55 Passos, A.M. 165 Pinto, L.B. 224 Oliveira, R.S. 139 Paterson, A. 11, 46 Pinto, L.R. 208 Oliveira, S. 210 Paula, D.F. 96 Pinto, M.A. 13, 37, 173, 178, 245 Oliveira, T.F. de M.S. 62, 160 Paula, F.L. 146 Pinto, M.T. 10, 19 Oliveira, T.F.M.S. 65 Paula, J.E.F.B. 12, 28, 107, 108, 110 Pinto, V.B. 209 Oliveira, T.F.P. 266 Paulini, I.J. 204 Pinto, V. da S.C. 264, 265, 268 Oliveira, T.S. 131 Paulo, W.M. 229 Pires, L.M. 196 Oliveira, V.M. 12, 30 Pedrosa, C.F. 176 Pires, M.A. 74 Ometto, T. 11, 14, 50, 60, 224, 236, 271 Pedrosa, F.C. 179, 197 Pituco, E.M. 225, 264, 265, 268 Ometto,T. 224 Pedrosa, L.G. 12, 28 Piva, M.R. 190 Ordonez, F.J.P. 226 Pedrosa, L.G.M. 111 Polaro, A.A. 150, 177 Ordoñez, F.J.P. 227 Pedrosa-Macena, L.G. 107, 108, 110 Polloni, L.C. 62 Orellana, M.D. 10, 13, 19, 36 Peiró, A. 13, 49 Ponce, E.L. 154 Orílio, A.F. 213, 215 Peña, B.P. 205 Pontes, C.M.L. 146 Orsi, M.A. 13, 54 Penalva de Oliveira, A.C. 11, 33 Portal, T.M. 160 Ortamnn, C.F. 149 Penido, B.A.F. 235 Portari, E.A. 193 Osaki, S.C. 267 Perecin, D. 208 Possati, F. 248, 249, 262 Otaguiri, K.K. 13, 36 Pereira, A.B. 14, 59 Possatti, F. 247, 258 Otonel, R.A.A. 247, 262, 267 Pereira, A.F. 225 Prado, A.A.O. 69, 130 Oyafuso, M.K. 248 Pereira, A.V.C. 194, 197 Prates, M. 178, 181 Pereira, F.L. 258, 262 Prates, M.C. 14, 38 P Pereira, H. de S.P 90 Price, S.L. 15, 43 Pacca, C.C. 73 Pereira, H. de S.P. 90 Primo, F.L. 75 Pacheco, S.M. 52 Pereira, L.E. 239 Provazzi, P.J.S. 167, 169 Pacheco, T. dos 166 Pereira, M.G. 127, 152 Pujol, S. 103 Pacheco, T.J.A. 218 Pereira, O.M.D. 11, 32 Pusterla, N. 273 Pereira, P.L. 263 Padilla, M.A. 98, 245 Q Pádua, R.M. 76 Pereira, P.M.C. 13, 55, 247 Paglia, A. 248 Pereira, P.S. 110 Queiroz, A. 93 Paglia, A.P. 64, 227 Pereira, S.A.R. 147, 148 Queiroz, A.T.L. 210 Paixão, C.G.S da 141 Pereira, U.P. 15, 41 Queiroz, L.H. 270 Paixão, I. 71 Pereira, W.V.E.G. 126, 153 Queiroz, S.R. de A. 14, 58 Paixão, I.C.N. de P 87, 90 Pérez, R. 264 Quintana, S.M. 169 Paixão, I.C.N. de P. 90 Périco, J.M.B. 196 Quintana, V.H.A. 127, 145, 152 Paixão, I.C.N.de P. 98 Perissini, L.H. 83 Quirino, M.S. 213 Paixão, I.C.N.P. 12, 28, 77, 95, 107, 108, Perosa, A.H. 14, 38 268 Pessoa, C.R. 78 R Paixão, IC.N.P. 14, 59 Pestana, N.F. 14, 38 Petersen, E. 224 Rabelo, A. 207 Paixão, I.C.P. 110, 111 Rabelo, D.C.C. 143, 180 Paixão, I.C.P.N. 64 Petry, M.V. 224, 271 Pianowski, L.F. 15, 43 Racaniello, V.R. 14, 25 Paixão, L.C.F. 150 Raeva, L.M.G. 13, 36 Paixão, S. 133 Pierrotti, L.C. 11, 32 September/October 2014 Volume 19 – Supplement 2 - Index XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

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Rafael, E. 120 Rigotto, C. 12, 29, 96, 103, 105, 106, 111, Sabino, U. 224 Rahal, P. 12, 13, 23, 36, 72, 73, 75, 79, 82, 113 Sacchetto, L. 64, 227, 248 83, 120, 167, 169, 243 Rios, W.M. 78 Sacchetto, L.P. 121 Raissa, L. 177 Ritterbusch, G.A. 12, 52 Sacramento, P.R. 158 Rajao, D. 15, 41 Rivetti Jr, A.V. 266 Sakata, S.T. 13, 54 Rajão, D.S. 270 Rocco, I.M. 11, 31 Salcedo, J.M.V. 173, 194, 197 Ramalho, T.O. 13, 47 Rocha,B.A. 203 Sales, C. de B.P.M. 93 Ramirez, G. 253 Rocha, D.R. 80 Salla, P. de F. 228 Ramírez, G. 253 Rocha, E.S. de O. 15, 40 Salles, S.T. 73 Ramos, A. da C. 162 Rocha Junior, M.C. 10, 19 Salles, T.S. 84 Ramos, C.J.B. 14, 59 Rocha, L.B. 204 Salustiano, S.G. 172 Ratti, D. 262 Rocha, M.A.L. 93, 94 Salvador, F.S. 137 Real-Hohn, A. 85 Rocha, N. 170 Sá, M. 103 Rebouças, A.S.J. 70 Rocha, N. de S.P.D. 150 Samara, S.I. 239, 250, 269 Rebouças, M.S. 273 Rocha, R.P. 15, 40, 131 Sampaio, M.L. da S. 146, 202 Rechenchoski, D.Z. 66, 67, 118 Rocha, S. 111 Sampaio, S.V. 243 Reginatto, F.H. 81, 147, 149 Rodrigues, A.P. de S. 260, 266 Sanches, M.M. 217 Rehfeld, I.S. 14, 58 Rodrigues, C.M. 80 Sanchez, E.F. 72 Reinaldo, M.R. 165 Rodrigues, E.A.M. 161 Sangrillo, F.S.S. 90 Reis, C.F. 14, 26 Rodrigues, E.S. 10, 13, 19, 36, 172 Santana-Pereira, P. 12, 28, 107, 108, 111 Reischak, D. 13, 54 Rodrigues, F.C. das C. 193 Santiago, J.C.L. 222 Reis, J. 263 Rodrigues, I.C. 138 Santos, B.A. 214 Reis, M.B.A. 217 Rodrigues, J.S.V. 132, 166 Santos, C. 12, 31 Rendon, D.S.C. 13, 36 Rodrigues, M.C. 141 Santos, C.J. 89 Resende, R. 216 Rodrigues, M.V. 261 Santos, D.A. 10, 18, 102 Resende, R. de O. 13, 215 Rodrigues, N.F. 15, 40, 69, 130 Santos, D.F. 172 Resende, R.O. 10, 13, 17, 49, 96 Rodrigues, N.F.S. 74 Santos, D.I. 220 Resque, H.R. 160, 161 Rodrigues, R.A. 12, 21 Santos, D.M. da S. 13, 35 Reys, C. 158 Rodrigues, R.A.L. 66, 102, 103 Santos, E.S. 88, 245 Rezende, B. 70 Rodrigues, S.V. 171 Santos, F.M. 117, 185 Rezende, I.M. 121, 233 Roehe P.M. 55 Santos, G.R. 261 Rezende, I.M.de 64 Roehe, P.M. 11, 12, 31, 50, 75, 99, 113, Santos, H.C.P. 134, 136 Ribeiro, A. de S. 267 247, 272 Santos, H.F. 113 Ribeiro, A.L.M. 132, 166 Rohrmann, G.A. 11, 46 Santos, I.G. de A. 189, 190 Ribeiro, A.S. 232, 237 Rollie, J.C. 11, 46 Santos, I.M. 77 Ribeiro, B.M. 13, 48, 207, 213, 218 Roma, E.H. 170 Santos, J. das V. 194 Ribeiro, C. 64 Romanel, E. 11, 44, 46, 221 Santos, J.R. 203 Ribeiro, C.P. 98, 225 Romano, C.M. 11, 14, 32, 33, 37, 137, 243 Santos Jr, C.D. 14, 26 Ribeiro, G.P. 222 Romano, E. 217 Santos Jr, J.A. dos 185 Ribeiro, G.S. 13, 48 Romeiro, F. 134 Santos, L.F.P. 150 Ribeiro, J. 258 Romeiro, M.F. 135, 162 Santos, L.L. 123 Ribeiro, L.F.C. 210, 211 Rosa, J.C.A. 13, 52, 55 Santos, L.L.R. 143 Ribeiro, M. 71, 77 Rosales, C.A.R. 13, 54 Santos, L.S. 11, 32 Ribeiro, M. de S.R 90 Rosa, L.H. 86 Santos, M. 195 Ribeiro, M. de S.R. 90 Rosa, M.P. 252 Santos, M.C. 171, 174, 274 Ribeiro, M.G. 273 Rosa, P.C.R. 167 Santos, M.M.A.B. 245 Ribeiro, M.G.M. 189, 190 Rostal, M. 14, 60 Santos, R. 85 Ribeiro, M.R. 73 Roxo, T. 85 Santos, R.F. 191, 261 Ribeiro, M.S. 143 Rribeiro, J. 14, 57 Santos, S.P. 265 Ribeiro, S.G. 217, 218, 220 Russomano, F.B. 170, 193 Santos, T.C. 149 Ribeiro, Z.M.A. 217 Russo, R. 243 Santos, T.F.Q. 98 Ricardo, N.M.P.S. 66, 67, 118 Russo, R.R. 78 Santos, T.V. 178 Ricci, E. 157 Santos, W.K. da S. 127, 150, 152 Richtzenhain, L.J. 226, 227, 256 S Saporiti, V. 255

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Saraiva, H.A.C. 213 Silva, J.L. 85, 92 Siqueira, C.E.H. 162, 167 Sarda, F.N. 147 Silva, J.L.A. 156 Siqueira, J.A.M. 144, 160 Sardi, S.I. 146, 202 Silva, J.P. 13, 37, 209, 212 Siqueira, T.R. 119, 121 Sato, M.I.Z. 12, 29 Silva Jr, J.V.J. 15, 42, 192 Skums, P. 13, 36 Saturno, T. 14, 38 Silva, J.S. 204 Slavov, S.N. 13, 36, 172 Saturno, T.H. 181 Silva Júniro, J.V.J. 228 Slhessarenko, R.D. 130, 132, 166 Sbegue, A. 193 Silva, K.N. 216 Snak, A. 267 Scagion, G.P. 275 Silva, L.A. 13, 49 Soares, A. 242 Schaefer, R. 13, 53, 234 Silva, L.C. 229 Soares, A.C. 14, 37, 243 Schenkel, E.P. 147 Silva, L.C.F. 10, 12, 17, 21, 30, 101, 102, Soares, A.M. 78 Schissi, C.D. 12, 28, 105 103 Soares, C. 65, 120 Schissi; C.D. 104 Silva, L.D. 138 Soares, L. da S. 112, 138, 144, 161, 182 Schrago, C.G. 11, 44 Silva, L.K. dos S. 66, 102, 103 Soares, L.S. 12, 53 Sciani, J.M. 203 Silva, L.M. 170 Soares, M.S. 236 Sebastiani, M.C. 275 Silva, L.P. 134 Sobrinho, J.S. 79 Segura, M. de N.O. 213 Silva, M. 175 Soliman ,M. 113 Seixas, M. 224, 236, 271 Silva, M.A.N. 170 Soliman, M.C. 12, 29, 103, 105, 106, 111 Seixas, M.M.M. 11, 50 Silva, M.C. 234 Solorio, M.R. 14, 60 Semaan, L.M. 197 Silva, M.C.C. 89, 164, 194, 245 Sotero, A. de J. 13, 47 Sena, A. 157, 165 Silva, M.C. de M. 138 Sotero, A.J. 13, 47 Senem, J.V. 211 Silva, M.F. 208 Sousa, C.A. 142 Sepulveda, L. 239 Silva, M.G. 163 Sousa, D.D. 184 Serra, O.P. 130, 132, 166 Silva, M.H.R. 188 Sousa, J.G.A. 217 Serrão, V.H.B. 234 Silva, M.J.M. 12, 53 Sousa Jr, E.C. 119 Shimizu, J.F. 243 Silva, M.L. 14, 38, 88, 91 Sousa, M.P. 12, 30 Shirata, N.K. 194 Silva, M.S. 216, 233 Sousa, M.S. de 112 Sichero, L. 79, 154, 163 Silva, M.X. 117 Souza, A.C.S. 89 Sihler, W. 11, 45 Silva, P.A. 87, 172 Souza, C. 203 Silva, A. 64 Silva, R. 107 Souza, C.K. 252 Silva, A.A.B. 273 Silva, R.J.C. 154 Souza, E. 195 Silva, A.C.M. 125 Silva, R.K. 275 Souza, F.G. de 12, 29, 105, 106, 111 Silva, A.E.B. 86, 122 Silva, R.R. 12, 53 Souza, J.G. 63, 81 Silva, A.K. 133, 141 Silva, R.U. 138 Souza, K. 120 Silva, A.K.F. 219 Silva, S. 242 Souza, K.C. 15, 41 Silva, A.L. de S. 157 Silva, S.O.S. 256 Souza, K.F.C.S. 87 Silva, A.P. 264 Silva, T.C. 87 Souza, K.M. 136 Silva, B.F.G. 164 Silva, T. da F. 11, 44 Souza, K.M.C. 11, 34 Silva, B.M. 69 Silva,T.F. 221 Souza, K.S. 71 Silva, C.C. 12, 30 Silva, T.G. 264, 265, 268 Souza, L.B.F.C. 150, 201 Silva, C.E.C. 137 Silva, V.S. 228 Souza, L.F.B. 173, 194, 197 Silva, C.L. 78 Silveira, A.C. 163 Souza, L.M.S. 127, 150, 152 Silva, C.O. 11, 32 Silveira, F.A. 159 Souza, L.R.G. 235, 237 Silva, D.C.P.S. 219, 221 Silveira, R. 242 Souza, M. 11, 34, 134, 136 Silva, D.F. 11, 33 Silveira, S. 233, 235 Souza, M.B. de L.D. 125, 126, 127, 163 Silva, D.M. 143, 156 Silveira, V.R. 11, 31 Souza, M.C.P. 14, 60 Silva, E. 188 Silvestre, R.V.D. 119, 133, 141 Souza, M.L. 11, 45 Silva, F. de O. 72, 84 Silvia, A.P. 272 Souza, M.M. 91 Silva, G.A.V. 184 Simabuco, F. 12, 24 Souza, N.V. 146 Silva, G.C.D. 196 Simas, P.V.M. 11, 51, 269 Souza, R.P. 239 Silva, G.C.P. 261 Simões, C.M.O. 76, 81, 147, 149 Souza, S.F. 225, 268 Silva, G.F. 167 Simões, M. 124 Souza, V.C. 14, 27, 40 Silva, I.T. 76, 81, 149 Simões, R.S. de Q. 193 Souza, W.M. 82, 156, 162 Silva, Í.T.S.S. 70 Simoes, R.S.Q.S. 170 Spada, P.K. de P. 112 Silva, J. de M. 186 Siqueira, A.B. 191 Spano, L.C. 139, 141, 153

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Spera, C.G. 264, 272 Togawa, R.C. 217 Veloso, V. 14, 39 Sperança, M.A. 88 Tolardo, A.L. 135, 162 Veloso, V.G. 182, 189 Spiegel, M. 12, 21 Tommasino, M. 154 Venditti, L.L.R. 268 Spilki, F.R. 12, 29, 96, 103, 105, 106, 111, Tonietti, P. de O. 246 Ventura, A. 12, 24 113, 228 Toniollo, G.H. 231 Veras, R.M. de A. 93, 94 Staggemeier, R. 12, 29, 96, 103, 105, 106, Toppa, N.H. 116 Vera, V. 253 111, 113 Torikachvili, M. 235, 252 Vergueiro, M. 195 Stancioli, E.F.B. 13, 35, 63, 81, 241, 273 Torquato, E.B 211 Vessaro Silva, S.A. 210 Stangherlin L.M. 245 Torrelio, C.M.Z. 14, 60 Vicentini, F. 141 Stangherlin, L.M. 164, 194 Torres, A.P.R. 12, 30 Victoria, M. 101 Stefanello, F. 260 Torres, F.D. 272 Vieira, C.J. da S.P. 167 Steffens, R. 248 Torres, S. 186 Vieira, D.S. 173, 194, 197 Steindel, M. 147 Torres, S.V.S. 193 Vieira, E.M. 14, 57 Stephens, P.R.S. 90 Tort, L.F.L. 101 Vieira, F.N. 64, 227 Stoppa, G.F.Z. 241 Tozato, C.C. 14, 57 Vieira, H.R. 142, 159 Streck, A.F. 13, 52, 56, 235, 236, 250, 251, Trabuco, A.C. 78, 145 Vieira, R. de S. 160 252 Trento, C.L. 189, 190 Viillalobos, E.M.C. 246 Suhette, R. 12, 30 Trevisol, I.M. 12, 52, 231, 238 Vilela, A.P.P. 266 Suzuki, A. 11, 31, 239 Trindade, A.C.G. 157 Villa, L.L. 75, 79, 154, 163 Szabo, M.P.J. 160 Trindade, G. de S. 13, 54, 64, 74, 149, 227, Villamil, V. 253 230, 233, 248 Villani, F.N.A. 138 T Trindade, G.S. 12, 21 Villeth, G.R.C. 218 Taffarel, L.E. 232 Trindade, G.T. 225 Vincent, L.A. 15, 41 Takayanagui, O.M. 10, 13, 19, 36 Tunin, A. 225 Vitral, C.L. 13, 37, 173, 178 Vogel, V.A.R. 110, 111 Takiuchi, E. 166, 232, 237, 248, 249, 267 U Tamashiro, E. 11, 14, 33, 38, 175, 181 Voguel, V.R. 108 Tanaka, T. 187 Ueira,C.U.V. 14, 26 Volpini, L.P.B. 139, 153 Tanaka, Y. 10, 19 Ullmann, L.S. 13, 56, 120, 137, 196, 259, Vorsatz, C. 182, 189 Tanuri, A. 15, 43 273 Vubil, A. 175 Tarboda, R.L.M. 173 Ulmann, L.S. 261 W Tavares, D.C. 231 Urbano, P.R. 11, 14, 32, 37, 243 Tedesco, A.C. 75 Urbano, P.R.P. 11, 33 Walker, D. 11, 50 Teixeira, B. 263 Wang, R. 13, 47 Teixeira, D.M. 112 V Watanabe, A.S.A. 94, 179, 188 Teixeira, G. 64, 77 Vago, A.R. 116, 117 Webby, R.J. 11, 50 Teixeira, M.M. 120 Valera, F. 175, 181 Weber, M.N. 52, 233, 235, 236, 251, 252 Teixeira, S. 14, 39 Valera, F.C.P. 11, 14, 33, 38 Webster, R.G. 11, 50 Teixeira, T.F. 113 Valério, O.C. 273 Weidmann, D.M.D.S. 12, 21 Teixeira, V. 71 Vancini, R. 84 Wood,C. 260 Teixeira, V.L. 14, 59, 77, 87, 90, 95 Varella, R. 242 X Tenório, E.C.N. 203 Vargas, M.D. 64 Terzian, A.C.B. 120 Vasconcelos, K. de O. 210 Xavier, A.A. 76 Tesh, R.B. 213 Vasconcelos, P.F.C. 145 Xavier, C.A.D. 11, 43, 207 Tessari, E.N.C. 241 Vasconcelos, P.F. da C. 213 Xavier, M.A. 208 Thiry, E. 63 Vasli, M.F.S. 11, 44 Thomazelli, L. 236 , 271 Vaslin, M.F.S. 11, 44, 219, 221 Y Thomazelli, L.M. 11 , 50, 224 Vaughan, G. 13, 36 Thomaz, L. 204 Yamamoto, A.Y. 169 Vaz, E.R. 14, 26 Yamamoto, K.A. 84 Thomaz, M.M. 266 Vecchi, L. 14, 26 Tigre, D.M. 146 Yamamoto, K.I. 73 Vedovello, D. 120, 135, 137, 196 Yamasaki, L.H.T. 13, 36 Timenetsky, M.C.S.T. 239 Vedovello,D. 73 Timenetsky, M. do C.S.T. 142 Yamatogi, R.S. 254 Veiga, A.B.G. 99 Yokosawa, J. 14, 26, 62, 65, 68, 135, 160 Tinôco, R.S. 13, 49 Veiga, R. 119 Toffoli, B. 63 September/October 2014 Volume 19 – Supplement 2 - Index XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology September/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

290 Index

Z Zacalusni, S. 187 Zaguini, J. 12, 28, 105 Zanatto, D.A. 246 Zanella, E.L. 260 Zanella, J.R.C. 13, 53, 234, 260, 270 Zanluca, C. 202 Zapana, E.M. 205 Zerbini, F.M. 11, 43, 207, 209, 212 Zeredo, A.C.B. 12, 28 Ziber, G. 103 Zirbes, G. 12, 29 Zonta, W. 63 Zubiaur, Y.M. 212 Zuchi, N. 130, 132, 166

September/October 2014 Volume 19 – Supplement 2 - Index