SPECIAL THANKS

EMPRESAS COLABORADORAS SEV

VI R O L O G Í A Publicación Oficial de la Sociedad Española de Virología

13th Spanish National Congress Of

Madrid 2015

CONGRESO NACIONAL DE

Volumen 18 Número 1/2015 EXTRAORDINARIO

VIROLOGÍA . PUBLICACIÓN OFICIAL DE LA SOCIEDAD ESPAÑOLA DE VIROLOGÍA

13th Spanish National Congress Of Virology

Madrid 2015

Del 7 al 10 de junio de 2015 Auditorio de la Fábrica Nacional de Moneda y Timbre Real Casa de la Moneda de Madrid Volumen 18 Madrid 2015 Número 1/2015 EXTRAORDINARIO

Edición y Coordinación: M Angeles Muñoz-Fernández &M. Dolores García-Alonso

Diseño y Maquetación: DM&VCH.events.S.L.

Diseño Portada: M. Dolores García-Alonso

Impresión: Fragma: Servicios de impresión digital en Madrid

ISSN (versión digital): 2172-6523

SEV – Sociedad Española de Virología Centro de Biología Molecular “Severo Ochoa” C/ Nicolás Cabrera, 1 28049 Cantoblanco – Madrid [email protected]

Página web del congreso: www.congresonacionalvirologia2015.com

La responsabilidad del contenido de las colaboraciones publicadas corresponderá a sus autores, quienes autorizan la reproducción de sus artículos a la SEV exclusivamente para esta edición. La SEV no hace necesariamente suyas las opiniones o los criterios expresados por sus colaboradores.

Virología. Publicación Oficial de la Sociedad Española de Virología

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B I E N V E N I D A

El Comité Organizador del XIII Congreso Nacional de Virología (XIII CNV) tiene el placer de comunicaros que éste se celebrará en Madrid, del 7 al 10 de junio de 2015. En el XIII CNV participan conjuntamente la Sociedad Española de Virología (SEV) y la Sociedad Italiana de Virología (SIV), y estará abierto a la participación de virólogos de Latinoamérica. La SEV, la SIV y el Comité Organizador os da la bienvenida. El Acto Inaugural del Congreso se celebrará en el Ateneo de Madrid el día 7 de Junio y la sede del Congreso será el Ayre Gran Hotel Colón de Madrid y la Fábrica Nacional de Moneda y Timbre - Real Casa de la Moneda en las Sesiones de los días 8 al 10 de Junio. El Ayre Gran Hotel Colón y la Fábrica Nacional de Moneda y Timbre - Real Casa de la Moneda se ubican junto al famoso Parque del Retiro y el Palacio de Deportes. Los miembros del Comité Organizador y Científico hemos diseñado un Programa Científico atractivo, contando con la presencia de virólogos españoles y extranjeros de reconocido prestigio, cubriendo los diversos campos de la virología, básicos, aplicados, traslacional y clínicos en sus vertientes humana, animal y vegetal. También se muestra la interacción de la virología con otras áreas de la biología, la medicina, la nanotecnología, la informática, la química o la física. El programa científico ha buscado que estén representadas las distintas áreas de la virología y os sintáis identificados, de tal forma que participéis de forma activa y enviéis comunicaciones. Todos los asistentes podremos discutir sobre los avances, logros y retos en el estudio de los , para generar nuevas ideas y promover la investigación entre la comunidad de virólogos, en un Congreso que esperamos sea fructífero y distendido. Para que los jóvenes tengan una mayor presencia, se ha destinado una Sesión a presentaciones rápidas, no mas de tres minutos, las denominadas "flash presentations" en las que en breve tiempo, como si de un anuncio se tratara, deberéis defender y difundir el gran trabajo que estáis desarrollando. Agradecemos a los miembros del Comité Organizador y del Comité Científico del XIII Congreso Nacional de Virología su esfuerzo e ilusión en la preparación de este importante Evento, especialmente al personal del Hospital General Universitario Gregorio Marañón y de otras instituciones de Madrid por su ayuda con los temas locales. Hacemos extensivo nuestro agradecimiento al Presidente y a los miembros de la Junta Directiva de la Sociedad Española de Virología por el apoyo que nos han prestado desde el inicio de los preparativos. También agradecemos a todas las instituciones participantes y empresas colaboradoras en este XIII Congreso Nacional de la Sociedad Española de Virología como patrocinadores, ya que sin su apoyo hubiera sido imposible realizar este Evento tan importante para la virología. Finalmente, Madrid ofrece los atractivos turísticos de una moderna capital europea. Por lo tanto, sería una gran satisfacción para nosotros si marcarais estas fechas en vuestras agendas. Deseamos que todos disfrutéis de este Congreso.

Un cordial saludo en nombre del Comité Organizador, Mª Angeles Muñoz-Fernández, Presidenta del Comité Organizador del XIII CNV

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W E L C O M E

The Organizing Committee of the 13th National Virology Congress (CNV) are pleased to announce that the Congress will be held from 7th to 10th June, 2015 at the Ateneo de Madrid where the Opening Session will take place. Whereas the Scientific Sessions from 8th to 10th June will be held at the Fábrica Nacional de Moneda y Timbre - Real Casa de la Moneda, the building of which is located next to the famous Retiro Park and Palacio de los Deportes. The 13th CNV will have great pleasure to host the Italian Society for Virology (SIV) together with the Spanish Society for Virology (SEV). The participation of virologists from Latin America will be especially welcom. The SEV, the SIV and the Organizing Committee hope that you will enjoy the Congress in Madrid and exploit the opportunity to interact with your colleagues.

The members of the Organizing and Scientific Committees have planned an attractive Scientific Programme with the presence of Spanish and foreign virologists of recognized prestige that will cover various fields of basic, applied, translational and clinical aspects of human, veterinary and plant virology. The programme will also cover the fruitful interactions of virology with other areas of biology, medicine, nanotechnology, computing, chemistry and physics. We hope that basic and clinical researchers working in any branch of virology will be interested in submitting abstracts and actively participating in this Congress. All together, we will be able to discuss current progress, achievements and challenges in the study of with the aim of generating new ideas and promoting research among the community of virologists, in a Congress that we hope will be both a fruitful and relaxed. To make the participation for young researchers in the scientific sessions easy, short (3 minutes) "flash-presentation" sessions have been scheduled in the Scientific Programme.

We would like to thank the Scientific and Organizing Committees of this 13th National Congress of Virology for the enthusiasm and effort in the preparation of this important event. We are especially grateful to the staff of the Hospital General Universitario Gregorio Marañón and other institutions of Madrid for their help with the local issues. We extend our thanks to the President and the Executive Board of the Spanish Society of Virology for the support to this Congress. Moreover, we gratefully acknowledge the sponsorship of several institutions and companies: the 13th CNV would have not been possible without their support. Moreover, we believe that Madrid will offer you all the tourist attractions of a modern European capital. Therefore, it would be great pleasure for us if you could mark these dates in your agenda

With my best regards on behalf of the Organizing Committee,

Mª Angeles Muñoz-Fernández Chairwoman of the Organizing Committee of the 13th CNV.

Virología. Publicación Oficial de la Sociedad Española de Virología

13th Spanish National XIII CONGRESO NACIONAL DE Congress Of Virology Madrid 2015 VIROLOGÍA ABREVIATURAS TOPICS

PS: SESIÓN PLENARIA (Invited plenary lecture) PS: PLENARY SESSION (Invited plenary lecture) P: CONFERENCIAS PLENARIAS P: PLENARY LECTURE OP: PRESENTACIONES ORALES (Invited paper) OP: ORAL PRESENTATIONS (Invited paper) CO: PRESENTACIONES ORALES CO: ORAL PRESENTATIONS PO: SESIÓN POSTERS PO: POSTERS SESSION

ÍNDICE TABLE OF CONTENTS

Páginas Pages COMITES 5 COMMITTEES 5 PROGRAMA-VERSIÓN REDUCIDA 13 PROGRAMME-REDUCED VERSION 13 PROGRAMA 19 PROGRAMME 19 ABSTRACT SESIONES PLENARIAS 31 ABSTRACT PLENARY SESSION 31 SESIÓN PLENARIA I: FRONTIERS IN VIROLOGY 33 (PI): FRONTIERS IN VIROLOGY 33 SESIÓN PLENARIA II: EMERGING VIRUSES 36 (PII): EMERGING VIRUSES 36 PRESENTACIÓN ORAL I: EMERGING VIRUSES 38 (OP I): EMERGING VIRUSES 38 SESIÓN PLENARIA III: HEPATITIS A, B AND C: BASIC, 40 (P III): HEPATITIS A, B AND C: BASIC, TRANSLATIONAL 40 TRANSLATIONAL AND CLINICAL RESEARCH AND CLINICAL RESEARCH

SESIÓN PLENARIA IV: INNATE IMMUNITY 44 (P IV): INNATE IMMUNITY 44 PRESENTACIÓN ORAL II: INNATE IMMUNITY 44 (OP II): INNATE IMMUNITY 44 SESIÓN PLENARIA V: STRUCTURAL ANALYSIS OF VIRUS 47 (P V): STRUCTURAL ANALYSIS OF VIRUS AND 47 AND BIOTECHNOLOGY BIOTECHNOLOGY PRESENTACIÓN ORAL III: STRUCTURAL ANALYSIS OF 49 (OP III): STRUCTURAL ANALYSIS OF VIRUS AND 49 VIRUS AND BIOTECHNOLOGY BIOTECHNOLOGY SESIÓN PLENARIA VI: 51 (P VI): PLANT VIRUS 51 SESIONES PARALELAS 55 PARALLEL SESSION 55 SESIÓN PARALELA I: EMERGING VIRUSES AND 55 PARALLEL SESSION I: EMERGING VIRUSES AND 55 VETERINARY (CO 9 – CO 16) VETERINARY (CO 9 – CO 16) SESIÓN PARALELA II: HIV (CO 17 – CO 24) 62 PARALLEL SESSION II: HIV (CO 17 – CO 24) 62 SESIÓN PARALELA III: PLANT VIRUS 70 PARALLEL SESSION III: PLANT VIRUS 70 (CO 25 – CO 32) (CO 25 – CO 32) SESIÓN PARALELA IV: ANIMAL 77 PARALLEL SESSION IV: ANIMAL VACCINES 77 (CO 33 – CO 40) (CO 33 – CO 40) SESIÓN PARALELA V: CELL-VIRUS INTERACTION 85 PARALLEL SESSION V: CELL-VIRUS INTERACTION 85 (CO 41 – CO 48) (CO 41 – CO 48) SESIÓN PARALELA VI: ROLE OF VIRUS IN PEDIATRIC 92 PARALLEL SESSION VI: ROLE OF VIRUS IN PEDIATRIC 92 DISEASES (SEV-SEIP) (CO 49 – CO 52) DISEASES (SEV-SEIP) (CO 49 – CO 52) SESIÓN PARALELA VII: MECHANISMS 95 PARALLEL SESSION VII: VIRAL ENTRY MECHANISMS 95 (CO 53 – CO 60) (CO 53 – CO 60) SESIÓN PARALELA VIII: SPECIFIC IMMUNITY 101 PARALLEL SESSION VIII: SPECIFIC IMMUNITY 101 (CO 61 – CO 68) (CO 61 – CO 68)

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ÍNDICE TABLE OF CONTENTS Páginas Pages SESIÓN PARALELA IX: MICROBIOME AND HEALTH 108 PARALLEL SESSION IX: MICROBIOME AND HEALTH 108 (CO 69 – CO 72) (CO 69 – CO 72) SESIÓN PARALELA X: TEACHING AND DISSEMINATION 111 PARALLEL SESSION X: TEACHING AND DISSEMINATION 111 OF THE VIROLOGY (CO 73 – CO 78) OF THE VIROLOGY (CO 73 – CO 78) SESIÓN PARALELA XI: ANTIVIRAL DRUGS 116 PARALLEL SESSION XI: ANTIVIRAL DRUGS 116 (CO 79 – CO 86) (CO 79 – CO 86) SESIÓN PARALELA XII: REPLICATION MECHANISMS 124 PARALLEL SESSION XII: REPLICATION MECHANISMS 124 (CO 87 – CO 94) (CO 87 – CO 94) POSTERS 131 POSTERS 131 POSTERS FLASH PRESENTATION 131 POSTERS FLASH PRESENTATION 131 POSTERS 153 POSTERS 153 ÍNDICE DE AUTORES 279 AUTHOR/SPEAKER INDEX 279

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PRESIDENTE /CHAIRWOMAN 13th Spanish National Congress Of Virology Mª Angeles Muñoz Fernández Hospital General Universitario Gregorio Marañón, Madrid Madrid 2015

COMITÉ CIENTÍFICO / SCIENTIFIC COMMITTEE

Antonio Alcamí, Centro de Biología Molecular, Severo Ochoa, Madrid

Jose María Almendral, Universidad Autónoma de Madrid, Madrid

Alfredo Berzal-Herranz, Instituto de Parasitología y Biomedicina "López-Neyra", Granada

Albert Bosch, Universitad de Barcelona, Barcelona

Carlos Briones Llorente, Centro de Astrobiología, CSIC-INTA, Madrid

Javier Buesa, Universidad de Valencia

Angel L. Corbí, Centro de Investigaciones Biológicas, CSIC, Madrid

Julia del Amo, Instituto de Salud Carlos III, Madrid

Ana Maria Doménech, Universidad Complutense de Madrid, Madrid

Esteban Domingo, Universidad Autónoma de Madrid, Madrid

Luis Enjuanes, Centro Nacional de Biotecnología, Madrid

José A. Esté, Irsi Caixa, Barcelona

Ricardo Flores Pedauyé, Instituto de Biología Molecular y Celular de Plantas, Valencia

Felipe García, Hospital Clinic, Barcelona

Pablo Gastaminza, Centro Nacional de Biotecnología, Madrid

José María Gatell, Hospitla Clinic, Barcelona

Esperanza Gómez-Lucia Duato, Universidad Complutense de Madrid

Ana Grande Pérez, Universidad de Málaga, Málaga

Josep Gregori, Vall d'Hebron Research Institute, Barcelona

Manuel Leal, Hospital Virgen del Rocío, Madrid

José Antonio López Guerrero, Universidad Autónoma de Madrid, Madrid

Javier Martínez-Picado, ICREA ;amp irsiCaixa, Barcelona

Jose Antonio Melero, Instituto de Salud Carlos III, Madrid

Luis Menéndez Arias, Centro de Biología Molecular Severo Ochoa, Madrid

Andreas Meyerhans , Universitat Pompeu Fabra, Barcelona

Amelia Nieto, Centro Nacional de Biotecnología, Madrid

Marisa Navarro, Hospital General Universitario Gregorio Marañón, Madrid

Juan Ortín, Centro Nacional de Biotecnología, Madrid

Josep Quer, Hospital Universitari Vall d'Hebron, Barcelona

Francisco Rodríguez-Frías, Hospital Universitari Vall d'Hebron, Barcelona

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13th Spanish National Congress Of Virology COMITÉ CIENTÍFICO / SCIENTIFIC COMMITTEE Madrid 2015

Fernando Rodríguez González, Universitat Autonoma de Barcelona, Barcelona

Javier Romero, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid

Franco Ruggeri, Presidente Sociedad Italiana de Virología

José Ruíz Castón, Centro Nacional de Biotecnología, Madrid

Noemí Sevilla, Centro de Investigación en Sanidad Animal, Madrid

Héctor Tejero, Universidad Complutense de Madrid, Madrid

Nuria Verdaguer, Institut de Biología Molecular, Barcelona

Enrique Villar Ledesma, Universidad de Salamanca, Salamanca

Eloisa Yuste, Hospital Clinic, Barcelona

COMITÉ ORGANIZADOR / ORGANIZING COMMITTEE

Secretarios / Secretary:

Josep Quer Sivila, Hospital Universitari Vall d'Hebron, Barcelona

Dolores García Alonso, Hospital General Universitario Gregorio Marañón, Madrid

Vocales / Members:

José Luis Jiménez Fuentes, Hospital General Universitario Gregorio Marañón, Madrid

Susana Álvarez Losada, Hospital General Universitario Gregorio Marañón, Madrid

Pedro Majano, Hospital de la Princesa, Madrid

Esperanza Gómez-Lucia Duato, Universidad Complutense de Madrid, Madrid

Ana Maria Doménech, Universidad Complutense de Madrid, Madrid

Covadonga Alonso, Inst. Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid

José Alcamí, Instituto de Salud Carlos III, Madrid

Juliá Blanco, Fundación Irsi Caixa, Barcelona

Mª Isabel González Tomé, Hospital 12 de Octubre, Madrid

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JUNTA DIRECTIVA DE LA SEV ------SEV EXECUTIVE BOARD

Presidente / Chairman: Ana María Doménech Gómez (Universidad Complutense de Madrid, Madrid) Albert Bosch Navarro (Universidad de Barcelona, Barcelona) José Antonio López Guerrero (Universidad Autónoma de Madrid, Madrid) Vicepresidente / Vice Chairman: Fernando de Ory Manchón Jesús Navas Castillo (Universidad de Málaga -(IHSM-UMA-CSIC), Málaga) (Centro Nacional de Microbiología, ISCIII Madrid) Juan García Costa Secretario / Secretary: (Complejo Hospitalario Cristal Piñor, Ourense) Fernando Rodríguez González Dolores García Villalón (Centre de Recerca en Sanitat Animal, UAB-IRTA, (Secretaría Técnica de la SEV) Barcelona) Esperanza Gómez-Lucía y Duato Vicesecretario / Vice Secretary: (Universidad Complutense de Madrid, Madrid)

Inmaculada Casas Flecha Mª Ángeles Muñoz Fernández (Centro Nacional de Microbiología, ISCIII Madrid) (Hospital General Universitario Gregorio Marañón, Madrid) Tesorero / Treasurer: Josep Quer Sivila Amelia Nieto Martín (Hospital Universitari Vall d'Hebron, Barcelona) (Centro Nacional de Biotecnología, CSIC, Madrid)

Vicente Pallás Benet Vocales / Members: (Instituto de Biología Molecular y Celular de Plantas Esteban Domingo Solans (IBMCP) (UPV-CSIC), Valencia) (Centro de Biología Molecular “Severo Ochoa”,CSIC- UAM, Madrid) Enrique Villar Ledesma (Universidad de Salamanca, Salamanca) Covadonga Alonso Martí (I. N. de Investigación y Tecnología Agraria y Alimentaria, Madrid)

Javier Buesa Gómez (Universidad de Valencia, Valencia)

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DAILY PROGRAMME - REDUCED VERSION

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DAILY PROGRAMME - REDUCED VERSION

Fábrica Nacional de Moneda y Timbre - Real Casa de la Moneda

C/ del Doctor Esquerdo, 36, 28028 Madrid

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ATENEO DE MADRID: Salón de Actos C/Calle del Prado, 21, 28014 Madrid

Teléfono: 914 29 17 50

SundaySunday June june 7, 20157, 2015

AUDITORIUM ATENEO DE MADRID

17:00-17:15 WELCOME CEREMONY

17:15-19:45 Plenary Session I: Frontiers in virology

Welcome Lectures

Chairpersons: JUAN ORTÍN - RICARDO FLORES

17:15-17:50 *RALF BARTENSCHLAGER

17:50-18:25 *ESTEBAN DOMINGO

18:25-19:00 *NOBUHIRO SUZUKI

The Virologist Conference Senior Award

19:10-19:45 *ANTONIO TENORIO

*Invited Speakers 20:00-22:00 WELCOME COCKTAIL (Restaurant Ateneo de Madrid)

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Mo nday june 8, 2015 Fábrica Nacional de Moneda y Timbre - Real Casa de la Moneda Monday, June 8 2015

8:00 -9:00 REGISTRATION AUDITORIUM REAL CASA DE LA MONEDA WHITE ROOM AUDIOVISUAL ROOM 9:00-10:00 Plenary Session II: EMERGING VIRUSES

Chairpersons: JAVIER BUESA - ANTONIO ALCAMÍ 9:00-9:30 *LUIS ENJUANES 9:30-10:00 *FRANCO RUGGERI

10:00-11:00 Oral Presentations Chairperson: RAFAEL DELGADO 10:00-10:15 *Mª PAZ SÁNCHEZ 10:15-10:30 *ANA NEGREDO 10:30 -11:00 COFFEE BREAK // VISIT POSTERS [ GOYA ROOM - EXHIBITION AREA] 11:00-12:20 Plenary Session III: HEPATITIS A, B AND C: BASIC, TRANSLATIONAL AND CLINICAL RESEARCH Chairpersons: JOSEP QUER - PABLO GASTAMINZA 11:00-11:20 *ROSA M. PINTÓ 11:20-11:40 *MANUEL ROMERO 11:40-12:00 *FRANCISCO RODRÍGUEZ-FRÍAS

12:00-12:20 *SOFIA PEREZ DEL PULGAR 12:30-14:00 LUNCH– [TRUSS MADRID ]

14:15-15:00 AUTHOR WORKSHOP: HOW TO GET PUBLISHED Sponsored by the Journal. Virus Research FRENSDORFF BRENRING AND ALINA HELSLOOT

15:00-17:00 Parallel Session I: EMERGING VIRUSES AND VETERINARY Parallel Session II: HIV Parallel Session III: PLANT VIRUS Chairpersons: ANA M. DOMENECH - JAVIER ORTEGO Chairpersons: MANUEL LEAL - JOSÉ ALCAMÍ Chairpersons: JESÚS NAVAS - VICENTE PALLÁS

15:00-15:15 MIGUEL ANGEL JIMÉNEZ-CLAVERO 15:00-15:15 MARJORIE PION 15:00-15:15 VERÓNICA TRUNIGER 15:15-15:30 COVADONGA ALONSO 15:15-15:30 MAURO DI PILATO 15:15-15:30 ARES MINGOT 15:30-15:45 ANA BELÉN BLAZQUEZ 15:30-15:45 ESTHER BALLANA 15:30-15:45 MIKEL VALLE 15:45-16:00 CARLOS CASTAÑO-RODRIGUEZ 15:45-16:00 BEATRIZ PACHECO 15:45-16:00 SUSANA RUIZ-RUIZ 16:00-16:15 LETICIA FRANCO 16:00-16:15 CRISTINA LORCA-ORÓ 16:00-16:15 ELVIRA FIALLO-OLIVÉ 16-15-16:30 ELENA PASCUAL 16-15-16:30 JOSE LUIS JIMENEZ 16-15-16:30 ENZA MARIA TORCHETTI 16:30-16:45 LILIANNE GANGES 16:30-16:45 JUAN GARCÍA-ARRIAZA 16:30-16:45 INMACULADA FERRIOL 16:45-17:00 NATALIA BARREIRO PIÑEIRO 16:45-17:00 YOLANDA M. PACHECO 16:45-17:00 VIJI VIJAYAN 17.00-17:30 COFFEE BREAK // VISIT POSTERS [GOYA ROOM - EXHIBITION AREA]

17:30-19:30 Parallel Session VI: Parallel Session IV: ANIMAL VACCINES Parallel Session V: CELL-VIRUS INTERACTION ROLE OF VIRUS IN PEDIATRIC DISEASES (SEV-SEIP) [ Joint Session SEV-SEIP ] Chairpersons: FERNANDO RODRIGUEZ – ALEJANDRO BRUN Chairpersons: COVADONGA ALONSO - ENRIQUE VILLAR Chairpersons: M. ISABEL GONZÁLEZ - MARISA NAVARRO 17:30-17:45 ALEJANDRO MARÍN-LÓPEZ 17:30-17:45 GRACIELA ALONSO 17:30-17:45 *LUIS PRIETO 17:45-18:00 MANUEL DURAN FERRER 17:45-18:00 JORDI ARGILAGUET 17:45-18:00 *ROSA RODRIGUEZ-FERNÁNDEZ 18:00-18:15 NADIA INGLESE 18:00-18:15 LUCÍA BARRADO GIL 18:00-18:15 *CRISTINA CALVO 18:15-18:30 ANA MARIA FALCON 18:15-18:30 LAURA SANZ-SÁNCHEZ 18:15-18:30 *DANIEL BLAZQUEZ-GAMERO 18:30-18:45 JOSÉ MIGUEL AVIA 18:30-18:45 DANIEL PÉREZ-NUÑEZ 18:45-19:00 EVA CALVO-PINILLA 18:45-19:00 JOSE MARÍA ALMENDRAL 19:00-19:15 PAULA LÓPEZ-MONTEAGUDO 19:00-19:15 PILAR GARCÍA-BRONCANO 19:15-19:30 JAVIER ORTEGO 19:15-19:30 PABLO RÍOS-MARCO *Invited Speakers 19:00-21:00 SEV GENERAL MEETING [BOARDROOM] // POSTERS SESSION

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Tuesday june 9, 2015 Tuesday, June 9 2015 Fábrica Nacional de Moneda y Timbre - Real Casa de la Moneda AUDITORIUM REAL CASA DE LA MONEDA WHITE ROOM AUDIOVISUAL ROOM

9:00-9:40 Plenary Session IV: INNATE IMMUNITY

Chairperson: MARIANO ESTEBAN 9:00-9:40 *VOLKER THIEL

9:40-10:40 Oral Presentations Chairperson: ANGEL CORBÍ – JAVIER MARTÍNEZ-PICADO 9:40-10:00 *JUAN JOSÉ BERLANGA 10:00-10:20 *MARIA TERESA COIRA 10:20-10:40 *EZEQUIEL RUIZ MATEOS 10:40-11:15 COFFEE BREAK // POSTERS [ GOYA ROOM - EXHIBITION AREA] 11:15-12:45 Plenary Session V: STRUCTURAL ANALYSIS OF VIRUS AND BIOTECHNOLOGY Chairpersons: JOSÉ ESTÉ - MARJORIE PION 11:15-11:45 *BEN BERKHOUT

11:45-12:15 *MARCO VIGNUZZI

12:15-12:45 *GIORGIO PALÚ

12:45-13:30 Oral Presentations Chairperson: FRANCISCO SOBRINO 12:45-13:00 *MARGARITA SALAS 13:00-13:15 *CARLOS BRIONES 13:15-13:30 *JOSÉ ÁNGEL MARTÍNEZ ESCRIBANO 13:30-15:00 LUNCH – [TRUSS MADRID ] 15:00-17:00 Parallel Session IX: Parallel Session VII: VIRAL ENTRY MECHANISMS Parallel Session VIII: SPECIFIC IMMUNITY MICROBIOME AND HEALTH [Joint Session SEV-SEM] Chairpersons: JOSÉ A. MELERO - JOSÉ MARÍA ALMENDRAL Chairpersons: MARGARITA DEL VAL - YOLANDA PACHECO Chairpersons: ALBERT BOSCH AND ROSA DEL CAMPO 15:00-15:15 COVADONGA ALONSO 15:00-15:15 JOSÉ ANTONIO MELERO 15:00-15:30 *ROSA DEL CAMPO 15:15-15:30 RAFAEL CEÑA-DIEZ 15:15-15:30 SARA MARTIN DELGADO 15:30-16:00 *ESTHER JIMÉNEZ QUINTANA 15:30-15:45 VIRGINA GONDAR 15:30-15:45 SILVIA LÓPEZ-ARGÜELLO 16:00-16:30 *ALBERTO LÓPEZ BUENO 15:45-16:00 FLAVIA CARIDI 15:45-16:00 MARTA LOPEZ DE DIEGO 16:30-17:00 *TUIJA KEKARAINEN 16:00-16:15 CRISTIAN SMERDOU 16:00-16:15 F. XAVIER LÓPEZ-LABRADOR 16:15-16:30 SUSANA GUIX 16:15-16:30 MAURO DI PILATO

16:30-16:45 FERNANDO MÉNDEZ 16:30-16:45 SILVIA GÓMEZ-SEBASTIAN 16:45-17:00 MONTSERRAT DE CASTELLARNAU 16:45-17:00 MARIA TERESA SÁNCHEZ-APARICIO 17.00-17:30 COFFEE BREAK // VISIT POSTERS [GOYA ROOM - EXHIBITION AREA] 17:30-19:30 Parallel Session X: TEACHING AND DISSEMINATION OF THE VIROLOGY Parallel Session XI: ANTIVIRAL DRUGS Parallel Session XII: REPLICATION MECHANISMS Chairpersons: Chairperson s: JULIÁ BLANCO - RAFII MOHAMED Chairpersons: AMELIA NIETO - LUIS MENÉNDEZ ESPERANZA GÓMEZ-LUCÍA - JOSE A. LÓPEZ-GUERRERO 17:30-17:33 *JOSE ANTONIO LOPEZ-GUERRERO 17:30-17:45 AMELIA NIETO 17:30-17:45 CARMEN RIVAS 17:33-17:41 *JAVIER MEDINA 17:45-18:00 GLORIA LOZANO 17:45-18:00 LAURA LERMA 17:41-17:49 *MANUEL SEARA 18:00-18:15 DANIEL SEPÚLVEDA-CRESPO 18:00-18:15 BERNAT BLASCO-MORENO 17:49-17:57 *ANA DOMÉNECH 18:15-18:30 PALOMA RODRÍGUEZ-RODRÍGUEZ 18:15-18:30 LILIANA CUBAS 17:57-18:05 *RAFAEL AÑEZ 18:30-18:45 JOSÉ A. DEL CAMPO 18:30-18:45 GINÉS ÁVILA 18:05-18:13 *MIGUEL ÁNGEL JIMÉNEZ-CLAVERO 18:45-19:00 ANA J PÉREZ-BERNÁ 18:45-19:00 ROCIO COLOMA 18:13-18:21 *ESPERANZA GÓMEZ-LUCÍA 19:00-19:15 SANDRA FRANCO 19:00-19:15 JENNIFER S. JUNGFLEISCH 18:21-18:30 *JOSE ANTONIO LOPEZ-GUERRERO 19:15-19:30 ESTER LÁZARO 19:15-19:30 JASMINA VASILIJEVIC 18:30-19:00 DISCUSIÓN 19:00-19:30 GRAN CONCURSO DE EPIDEMIA VIRTUAL *Invited Speakers 20:30 CONGRESS DINNER

16 Virología. Publicación Oficial de la Sociedad Española de Virología

XIII CONGRESO NACIONAL DE 13th Spanish National Congress Of Virology

Madrid 2015 VIROLOGÍA

Wednesday june 10, 2015 Wednesday June 10 2015 Fábrica Nacional de Moneda y Timbre - Real Casa de la Moneda AUDITORIUM REAL CASA DE LA MONEDA WHITE ROOM AUDIOVISUAL ROOM 9:00-10:20 Plenary Session VI: PLANT VIRUS

Chairperson: JUAN ANTONIO GARCÍA *Invited plenary lecture 9:00-9:40 NEW CONCEPTS IN THE BIOLOGY OF MULTIPARTITE VIRUSES *STÉPHANE BLANC INRA, UMR BGPI, Montpellier, France 9:40-10:20 MEMBRANE REARRANGEMENTS IN PLANT VIRUS RNA REPLICATION *LUISA RUBINO Institute for Sustainable Plant Protection, CNR, Bari, Italy 10:20-11:30 FLASH PRESENTATIONS Chairperson: SUSANA ALVAREZ AND JOSÉ LUIS JIMÉNEZ

11:30-12:00 COFFEE BREAK // VISIT POSTERS [ GOYA ROOM - EXHIBITION AREA ] 12:00-12:30 THE VIROLOGIST CONFERENCE YOUNG AWARD Chairperson: PEDRO MAJANO

*Invited plenary lecture TEN YEARS OF HEPATITIS C VIRUS CELL CULTURE INFECTION MODELS *PABLO GASTAMINZA Centro Nacional de Biotecnologiá -Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid 12:30-13:15 CLOSING LECTURE Chairperson: MIGUEL ÁNGEL JIMÉNEZ-CLAVERO *Invited plenary lecture MECHANISMS OF VIRAL PERSISTENCE IN INSECTS *CARLA SALEH Institut Pasteur, Viruses and RNA interference Unit, Paris, France 13:15-13:30 CLOSING CEREMONY Chairperson ALBERT BOSCH AND MANUEL RODRÍGUEZ

13:15-13:30 AWARD GIVING CEREMONY

13:30-13:45 ANNOUNCEMENT OF THE UPCOMING “XIV CONGRESO NACIONAL DE VIROLOGÍA”

CLOSING OF THE MEETING

LUNCH-MAP

TRUSS MADRID | PREMIUM EVENTS VENUE (PALACIO DE LOS DEPORTES)

Volumen 18 – Número 1/2015 - EXTRAORDINARIO 17

18 Virología. Publicación Oficial de la Sociedad Española de Virología

13th Spanish National XIII CONGRESO NACIONAL DE Congress Of Virology

Madrid 2015 VIROLOGÍA

Volumen 18 – Número 1/2015 - EXTRAORDINARIO 19

20 Virología. Publicación Oficial de la Sociedad Española de Virología

13th Spanish National XIII CONGRESO NACIONAL DE Congress Of Virology

Madrid 2015 VIROLOGÍA

SUNDAY JUNE 7, 2015

ATENEO DE MADRID: Salón de Actos C/Calle del Prado, 21, 28014 Madrid Teléfono: 914 29 17 50

17:00-17:15 WELCOME CEREMONY

17:15-19:45 Plenary Session (PL1): Frontiers in virology

Welcome Lectures Chairpersons: JUAN ORTÍN - RICARDO FLORES

*Invited plenary lecture 17:15-17:50 CELL BIOLOGY OF CYCLES: A COMPARISON OF HEPATITIS C VIRUS AND DENGUE VIRUS *RALF BARTENSCHLAGER Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany Division Virus-associated carcinogenesis, German Cancer Research Center, Heidelberg, Germany *Invited plenary lecture 17:50-18:25 VIRUS BEHAVIOR AT EXTREME FITNESS VALUES

*ESTEBAN DOMINGO Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona 18:25-19:00 *Invited plenary lecture A NEW VIRUS LIFE STYLE: A CAPSIDLESS SSRNA HOSTED BY AN UNRELATED NOVEL DSRNA VIRUS *NOBUHIRO SUZUKI Institute of Plant Science and Resources, Okayama University, Kurashiki, Okayama 710-0046, Japan 2NARO Institute of Fruit Tree Science, 92 Shimokuriyagawa, Morioka, Iwate 020-0123, Japan THE VIROLOGIST CONFERENCE SENIOR AWARD 19:10-19:45 *Invited plenary lecture FRONTIERS IN PUBLIC HEALTH : A CONTINUING CHALLENGE *ANTONIO TENORIO Arbovirus and Imported Viral Diseases, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid

20:00-22:00 WELCOME COCKTAIL (Restaurant Ateneo de Madrid)

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MONDAY JUNE 8, 2015

AUDITORIUM WHITE ROOM AUDIOVISUAL ROOM

08:00-09:00

RECEPTION CASA DE LA MONEDA REGISTRATION 9:00-10:15 Plenary Session (PL2): Emerging Viruses

Chairperson: Javier Buesa - Antonio Alcamí

9:00-9:35 *Invited plenary lecture CORONAVIRUS PATHOGENESIS AND PROTECTION *Luis Enjuanes (CNB-CSIC), Madrid 9:35-10:15 *Invited plenary lecture ZOONOTIC, FOODBORNE AND ENVIRONMENTAL TRANSMISSION IN AND HEPATITIS E VIRUS INFECTION *Franco Ruggeri

Dept. of Veterinary public health & food safety, Istituto Superiore di Sanità, Rome, Italy 10:15-11:00 Oral Presentations (OP1) Chairperson: Rafael Delgado 10:15-10:30 *Invited paper THE ROLE OF THE NATIONAL REFERENCE LABORATORY REGARDING ARBOVIRUSES *Mª Paz Sánchez Centro Nacional de Microbiología. Instituto de

Salud “Carlos III”, Madrid 10:30-10:45 *Invited paper LABORATORY DIAGNOSIS OF ÉBOLA VIRUS DISEASE IN SPAIN. DIAGNOSTICS OF ÉBOLA VIRUS DISEASE SUSPETED CASES

*Ana Negredo Centro Nacional de Microbiología. Instituto de Salud “Carlos III”, Madrid

10:30-11:00 VISIT POSTERS AND COFFEE BREAK [GOYA ROOM - EXHIBITION AREA]

11:00-12:20 Plenary Session III: Hepatitis A, B and C: Basic, Translational and Clinical Research

Chairperson: Josep Quer - Pablo Gastaminza 11:00-11:20 *Invited plenary lecture HEPATITIS A VIRUS: NEW PARADIGMS OF AN OLD PATHOGEN *Rosa M. Pintó University of Barcelona, Barcelona 11:20-11:40 *Invited plenary lecture CONTINUUM OF CARE IN HEPATITIS C: FROM DETECTION TO CURE AND ERADICATION *Manuel Romero Valme University Hospital, University of Seville, Sevilla

22 Virología. Publicación Oficial de la Sociedad Española de Virología

MONDAY JUNE 8, 2015

AUDITORIUM WHITE ROOM AUDIOVISUAL ROOM

11:40-12:00 *Invited plenary lecture

HEPATITIS B AND D: WHEN EVERY SINGLE DETAIL HAS A HIDDEN MEANING *Francisco Rodríguez-Frías Hospital Universitari Vall d'Hebron, Barcelona(VHIO), CIBERehd

12:00-12:20 *Invited plenary lecture

VIROLOGY OF HEPATITIS C VIRUS INFECTION AFTER LIVER TRANSPLANTATION

*Sofia Perez Del Pulgar

Hospital Clínic, IDIBAPS, CIBERehd, Barcelona

12:30-14:00 LUNCH [Truss Madrid - c/Jorge Juan, 99]

14:15-15:00 *Invited paper

AUTHOR WORKSHOP: HOW TO GET PUBLISHED

Sponsored by the Journal.Virus Research

*Frensdorff Brenring and *Alina Helsloot

ELS – HJ (ELS-AMS) 15:00-17:00 15:30-17:00 15:30-17:00 Parallel Session (PS1): Parallel Session PS2): HIV Parallel Session (PS3): Plant Virus Emerging Viruses And Veterinary Chairpersons: Manuel Leal - José Alcamí Chairpersons: Jesús Navas - Vicente Pallás Chairpersons: Ana M. Doménech - Javier Ortego 15:00-15:15 15:00-15:15 15:00-15:15 STUDY OF THE PROCESSIGN-BODIES (P-BODIES) ROLE 2003-2015: 12 YEARS OF RESEARCH ON MOSQUITO- IN THE RESTRICTION AGAINST HIV-1 IN PRIMARY STRUCTURAL AND FUNCTIONAL DIVERSITY OF PLANT BORNE EPORNITIC IN SPAIN. WEST NILE, HUMAN T CELLS VIRUS 3´-CAP-INDEPENDENT TRANSLATIONAL USUTU, BAGAZA…AND BEYOND ENHANCERS Marjorie Pion Miguel Angel Jiménez-Clavero Verónica Truniger H. G. U. Gregorio Marañón, Madrid INIA-CISA, Valdeolmos, Madrid CEBAS-CSIC, Murcia 15:15-15:30 15:15-15:30 15:15-15:30 NEUTROPHIL MIGRATION VIA NFκB ACTIVATION BY THE ANTIVIARL EFFECT OF INTERFERON INDUCED MODIFIED VACCINIA VIRUS AS A NOVEL EXPRESSION AND FUNCTION OF THE TRANS-FRAME TRANSMEMBRANE PROTEINS (IFITMS) IN AFRICAN MECHANISM TO ENHANCE HIV-SPECIFIC T CELL P1N-PISPO GENE PRODUCT OF THE SWINE FEVER VIRUS INFECTION RESPONSES SWEET POTATO FEATHERY MOTTLE VIRUS (SPFMV) Covadonga Alonso Mauro Di Pilato Ares Mingot I. N. de Investigación y Tecnología Agraria y Centro Nacional de Biotecnología (CNB-CSIC), Centro de Investigación Agrigenómica (CRAG), Alimentaria, Madrid Madrid Barcelona 15:30-15:45 15:30-15:45 15:30-15:45 AMINO ACID SUBSTITUTIONS IN THE NON- IDENTIFICATION AND CHARACTERIZATION OF THE STRUCTURE OF FLEXIBLE FILAMENTOUS PEPINO STRUCTURAL PROTEINS 4A OR 4B MODULATE THE MOLECULAR PATHWAY LEADING TO SAMHD1- MOSAIC VIRUS BY HIGH RESOLUTION CRYOEM INDUCTION OF AUTOPHAGY IN WEST NILE VIRUS MEDIATED VIRAL RESTRICTION Mikel Valle INFECTED CELLS Esther Ballana Structural Biology Unit, CIC bioGUNE, Derio, Vizcaya Ana Belén Blazquez IrsiCaixa, Barcelona 15:45-16:00 Departamento de Biotecnología. INIA, Madrid 15:45-16:00 GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE 15:45-16:00 INTRACELLULAR FACTORS BLOCKING EARLY STEPS IS CO-OPTED FOR REPLICATION OF CITRUS TRISTEZA ROLE OF SARS-CoV VIROPORINS E, 3a AND 8a IN VIRUS OF THE HIV-1 REPLICATIVE CYCLE IN COMMON VIRUS VIA INTERACTION WITH THE VIRAL-ENCODED REPLICATION AND VIRULENCE MARMOSET LYMPHOCYTES PROTEIN p23 Carlos Castaño-Rodriguez Beatriz Pacheco Susana Ruiz-Ruiz Centro Nacional de Biotecnologia (CNB-CSIC), Madrid Centro de Biologia Molecular Severo Ochoa. Madrid IBMCP (UPV-CSIC), Valencia 16:00-16:15 16:00-16:15 16:00-16:15 VIROLOGICAL AND EPIDEMIOLOGICAL FEATURES OF CHARACTERIZATION AND INHIBITION OF THE HIV-1 BIOLOGICAL CHARACTERIZATION OF NON-CODING CHIKUNGUNYA VIRUS INFECTION AMONGST NUCLEOCAPSID MATURATION-CONDENSATION DNA SATELLITES ASSOCIATED TO NEW WORLD TRAVELERS RETURNING TO SPAIN, 2008-2014 STEP Leticia Franco Cristina Lorca-Oró Elvira Fiallo-Olivé Instituto de Salud Carlos III, Madrid IDIBAPS, Barcelona Universidad de Málaga - Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), 16-15-16:30 16-15-16:30 Málaga HEMAGLUTININ PROTEIN OF PESTE DES PETITS PREVENTION OF HIV-1 VAGINAL TRANSMISSION

RUMINANTS VIRUS ACTIVATES THE INNATE IMMUNE AND MODE OF ANTIVIRAL ACTION BY TOPICAL RESPONSE VIA TOLL-LIKE RECEPTOR 2 SIGNALING POLYANIONIC CARBOSILANE DENDRIMER G2-S16 IN Elena Pascual HUMANIZED BLT MICE INIA-CISA, Valdeolmos, Madrid Jose Luis Jiménez

H.G.U. Gregorio Marañón, Madrid

Volumen 18 – Número 1/2015 - EXTRAORDINARIO 23

MONDAY JUNE 8, 2015

AUDITORIUM WHITE ROOM AUDIOVISUALROOM

16:30-16:45 16:30-16:45 16:30-16:45 POSTNATAL PERSISTENT INFECTION WITH CLASSICAL HEAD-TO-HEAD COMPARISON OF POXVIRUS NYVAC UNRAVELLING THE RNA2-ENCODED POLYPROTEIN SWINE FEVER VIRUS IN DOMESTIC PIGS AND WILD AND ALVAC VECTORS EXPRESSING IDENTICAL HIV-1 CLEAVAGE SITES OF TOMATO-INFECTING BOARS: OPENING PANDORA'S BOX CLADE C IMMUNOGENS IN PRIME/BOOST TORRADOVIRUSES USING N-TERMINAL PROTEIN Lilianne Ganges COMBINATION WITH ENV PROTEIN IN NON- SEQUENCING AND A REVERSE GENETICS SYSTEM HUMAN PRIMATES (CReSA)-(IRTA), Campus de la UAB, Barcelona Inmaculada Ferriol Juan García-Arriaza 16:45-17:00 University of California Davis –USA Centro Nacional de Biotecnología (CSIC), Madrid GETTING IC-TAGGING TO WORK INTO THE 16:45-17:00 16:45-17:00 ENDOPLASMIC RETICULUM ANALYSIS OF TOLERANCE MECHANISMS AND Natalia Barreiro Piñeiro IMMUNOVIROLOGICAL TRAITS OF HIV SUBJECTS TRADE-OFFS IN PLANT-VIRUS INTERACTIONS WITH DELAYED INITIATION OF CART AND Viji Vijayan Centro Singular de Investigación en Química Biológica y SUBSEQUENT POOR CD4 RESTORATION. STUDY ON Materiales Moleculares, Santiago de Compostela PRE-TREATMENT SAMPLES Centro de Biotecnologia y Genomica de Plantas, Madrid Yolanda Pacheco

Hospital Virgen del Rocío, Sevilla

17:00-17:30 VISIT POSTERS AND COFFEE BREAK [GOYA ROOM - EXHIBITION AREA]

17:30-19:30 17:30-19:30 17:30-19:30 Parallel Session (PS4): Parallel Session (PS5): Parallel Session (PS6): Role of Virus Animal Vaccines Cell-Virus Interaction in Pediatric Diseases (SEV-SEIP) [ Joint Session SEV-SEIP ] Chairpersons: Fernando Rodriguez Chairpersons: Covadonga Alonso Alejandro Brun Enrique Villar Mª Isabel González-Tomé Chairpersons: Marisa Navarro 17:30-17:45 17:30-17:45

A NOVEL STRATEGY FOR MULTISEROTYPE PROTECTION RNA-SEQ BASED TRANSCRIPTOME ANALYSIS OF THE 17:30-17:45 *Invited paper AGAINST BLUETONGE VIRUS USING MUNS-MI INTERFERON HOST RESPONSE UPON VACCINIA IMPLICATIONS OF CONTROL OF MICROSPHERES AND RECOMBINANT MVA EXPRESSING VIRUS INFECTION DURING PREGNANCY AND RISK OF HIV-1 MOTHER- VP2, VP7 AND NS1 PROTEINS Graciela Alonso TO-CHIELD TRANSMISSION Alejandro Marín-López Centro de Biología Molecular Severo Ochoa-CSIC, *Luis Prieto INIA-CISA, Valdeolmos, Madrid Madrid Hospital Universitario de Getafe, Madrid 17:45-18:00 17:45-18:00 17:45-18:00 *Invited paper TRIAL FOR CHECKING THE PROTECTIVE IMMUNITY OF A SYSTEM BIOLOGY APPROACH REVEALS GENES RSV BRONCHIOLITIS: CHALLEGES IN 2015 A COMMERCIAL AGAINST THE “NEW AND PATHWAYS INVOLVED IN T-CELL EXHAUSTION VARIANT” OF THE RABBIT HAEMORRHAGIC DISEASE AT TISSUE LEVEL *Rosa Rodriguez-Fernández VIRUS Jordi Argilaguet Hospital Materno-Infantil Gregorio Marañón, Madrid Manuel Duran Ferrer Universitat Pompeu Fabra, Barcelona 18:00-18:15 *Invited paper Laboratorio Central de Veterinaria (MAGRAMA), 18:00-18:15 Madrid NEUROLOGICAL AND SYSTEMIC AFRICAN SWINE FEVER VIRUS REPLICATION IS INFECTIONS IN CHILDREN 18:00-18:15 AFFECTED BY THE INHIBITION OF THE PROTEASOME SYLVATIC RABIES IN THE NORTH-EAST OF ITALY: SYSTEM *Cristina Calvo MONITORING AND EVALUATION OF THE Lucía Barrado Gil Hospital Severo Ochoa, Leganés – Madrid EFFECTIVENESS OF PROPHYLAXIS IN WORKERS AT RISK AND TRAVELERS Instituto Nacional de Investigacion y Tecnología 18:15-18:30 *Invited paper Agraria y Alimentaria (INIA), Madrid Nadia Inglese CONGENITAL (cCMV) 18:15-18:30 INFECTION: HOW, WHEN, WHERE Dept. Molecular Medicine, University of Padua CORRELATIVE LIGHT AND ELECTRON MICROSCOPY *Daniel Blazquez-Gamero 18:15-18:30 TO STUDY VIRAL MORPHOGENESIS AND EGRESS Hospital 12 Octubre, Madrid ROLE OF VIRUS SMALL RNAs CONTROLLING Laura Sanz-Sánchez PATHOGENICITY IN VIVO Centro Nacional de Biotecnologia CNB-CSIC, Madrid Ana Maria Falcon 18:30-18:45 Centro Nacional de Biotecnología (CNB-CSIC), Madrid 18:30-18:45 CD2v INTERACTS WITH ADAPTOR PROTEIN AP-1 DURING AFRICAN SWINE FEVER INFECTION BLOCKING OF TYPE I IFN PATHWAY BY PESTE DES PETITS RUMINANTS VIRUS (PPRV) Daniel Pérez-Núñez José Miguel Avia Centro Biología Molecular Severo Ochoa (CBMSO), Madrid Centro de Investigación en Sanidad Animal (CISA)-INIA, Madrid INIA-CISA, Valdeolmos, Madrid

24 Virología. Publicación Oficial de la Sociedad Española de Virología

MONDAY JUNE 8, 2015

AUDITORIUM WHITEROOM AUDIOVISUAL ROOM

18:45-19:00 18:45-19:00 ADMINISTRATION OF ANTISERUM FROM MICE THE MAMMALIAN CELL CYCLE REGULATES VACCINATED WITH MODIFIED VACCINIA ANKARA PARVOVIRUS NUCLEAR ASSEMBLY VIRUS EXPRESSING AFRICAN HORSE SICKNESS VIRUS Jose María Almendral (AHSV) VP2 PROTEIN CONFERS PROTECTION WHEN ADMINISTERED BEFORE OR AFTER CHALLENGE Centro de Biología Molecular Severo Ochoa, Madrid Eva Calvo-Pinilla 19:00-19:15 POLYANIONIC CARBOSILANE DENDRIMERS The Pirbright Institute, Pirbright-United Kingdom PREVENT VAGINAL/RECTAL HSV-2ENTRYIN VIVO 19:00-19:15 Pilar García-Broncano BA71ΔfX: A LIVE ATTENUATED VACCINE THAT Hospital General Universitario Gregorio Marañón, CONFERS PROTECTION AGAINST HOMOLOGOUS AND HETEROLOGOUS AFRICAN SWINE FEVER VIRUSES Madrid

19:15-19:30 Paula López-Monteagudo RECRUITMENT OF HOST FACTORS BY THE CRE IRTA-CReSA, Barcelona ELEMENT OF THE HEPATITIS C VIRUS 19:15-19:30 Pablo Ríos-Marco EXPERIMENTAL BLUETONGUE VIRUS 4 SUBUNIT VACCINE DELIVERED TO ANTIGEN PRESENTING CELLS Consejo Superior de Investigaciones Científicas, Madrid Javier Ortego

INIA-CISA, Valdeolmos, Madrid

19:00-21:00 SEV GENERAL MEETING [BOARDROOM] // POSTERS SESSION

Virología. Publicación Oficial de la Sociedad Española de Virología 25

TUESDAY JUNE 25, 2013

TUESDAY JUNE 9, 2015

AUDITORIUM WHITE ROOM AUDIOVISUAL ROOM

09:00-9:45

Plenary Session (PL4):

Innate Immunity

Chairperson: MARIANO ESTEBAN

9:00-9:45 *Invited plenary lecture

TO SENSE OR NOT TO SENSE VIRAL RNA - ESSENTIALS OF CORONAVIRUS INNATE IMMUNE

EVASION *Volker Thiel

University of Bern, Switzerland

9:45-10:40

Oral Presentations (OP2):

Innate Immunity

Chairperson: ANGEL CORBÍ

JAVIER MARTÍNEZ-PICADO

9:45-10:00 *Invited paper

FUNCTIONAL INTERACTION BETWEEN THE EIF2Α KINASE GCN2 AND THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1

*Juan José Berlanga

Centro de Biología Molecular Severo Ochoa,

Madrid 10:00-10:20 *Invited paper

SAMHD1 ANTIVIRAL FUNCTION, INTERFERING WITH HIV-1 REPLICATION

*Maria Teresa Coira

Instituto de Salud “Carlos III”, Madrid

10:20-10:40 *Invited paper ROLE OF MONOCYTES AND PLASMACYTOID

DENDRITIC CELLS IN THE CONTROL AND IMMUNOPATHOGENESIS OF HIV AND HCV INFECTION

*Ezequiel Ruiz Mateos

Instituto de Biomedicina de Sevilla (IBiS)/Hospital Universitario Virgen del Rocío. Seville

10:40-11:15 COFFEE BREAK // VISIT POSTERS [ GOYA ROOM - EXHIBITION AREA]

26 Virología. Publicación Oficial de la Sociedad Española de Virología

TUESDAY JUNE 9, 2015

AUDITORIUM WHITE ROOM AUDIOVISUAL ROOM

11:15-12:45 Plenary Session (PL5):

Structural Analysis Of Virus And Biotechnology

Chairpersons: JOSÉ ESTÉ - MARJORIE PION

11:15-11:45 *Invited plenary lecture HIV-1 EVOLUTION: DRUG-RESISTANCE AND NUCLEOTIDE COMPOSITION *Ben Berkhout Academic Medical Center of the University of Amsterdam-(CINIMA),Holland 11:45-12:15 *Invited plenary lecture RNA VIRUS POPULATION DYNAMICS IN SEQUENCE SPACE AND FITNESS LANDSCAPES *Marco Vignuzzi

Institut Pasteur, Paris, France 12:15-12:45 *Invited plenary lecture VIRUSES AS TOOLS FOR THERAPEUTIC INTERVENTIONS IN CANCER AND INFECTIOUS DISEASES *Giorgio Palú Department of Molecular Medicine, University of Padova, Padova, Italy

12:45-13:30 Oral Presentations (OC3): Structural Analysis Of Virus And

Biotechnology

Chairperson: FRANCISCO SOBRINO

12:45-13:00 *Invited paper Ø29: FROM MOLECULAR BIOLOGY TO BIOTECNOLOGY *Margarita Salas Centro de Biología Molecular “Severo Ochoa” (CSIC–UAM) Madrid 13:00-13:15 *Invited paper STRUCTURAL ANALYIS OF VIRAL AND VIROIDAL RNA BY ATOMIC FORCE MICROSCOPY *Carlos Briones Department of Molecular Evolution, Centro de Astrobiología (CSIC-INTA), Madrid 13:15-13:30 *Invited paper BREAKING THE BARRIERS TO INCREASE THE PRODUCTIVITY OF BACULOVIRUS-BASED TECHNOLOGIES *José Ángel Martínez Escribano Departamento de Biotecnología, INIA, Madrid

13:30-15:00 LUNCH [Truss Madrid - c/Jorge Juan, 99]

Volumen 18 – Número 1/2015 - EXTRAORDINARIO 27

TUESDAY JUNE 9, 2015

AUDITORIUM WHITE ROOM AUDIOVISUAL ROOM

15:00-17:00 15:00-17:00 15:00-17:00 Parallel Session VII: Parallel Session VIII: Parallel Session IX Viral Entry Mechanisms (PL7) Specific Immunity (PL8) Microbiome And Health (PL9) [Joint Session SEV-SEM] Chairpersons: JOSÉ A. MELERO Chairpersons: MARGARITA DEL VAL JOSÉ MARÍA ALMENDRAL YOLANDA PACHECO Chairpersons: ALBERT BOSCH 15:00-15:15 15:00-15:15 ROSA DEL CAMPO LIPID COMPONENTS IN AFRICAN SWINE FEVER THE STRUCTURALLY RELATED FUSION PROTEINS OF 15:00-15:15 *Invited paper VIRUS ENTRY AND REPLICATION HUMAN RESPIRATORY SYNCYTIAL VIRUS AND Covadonga Alonso METAPNEUMOVIRUS ARE ANTIGENICALLY AND SCIENTIFIC EVIDENCES OF GUT MICROBIOTA I. N. de Investigación y Tecnología Agraria y IMMUNOGENICALLY DISSIMILAR IMPLICATIONS IN HUMAN DISEASES Alimentaria, Madrid José Antonio Melero *Rosa Del Campo 15:15-15:30 Instituto de Salud Carlos III, Madrid Microbiology Department, University Hospital Ramón y Cajal, Madrid POLYANIONIC CARBOSILANE DENDRIMERS AS 15:15-15:30 PROMISING MICROBICIDE CANDIDATES AGAINST 15:15-15:30 *Invited paper HSV-2 INFECTION: BROAD-SPECTRUM ACTIVITY VIH-1 INDUCE A DEREGULATION IN B-CELL INITIAL BACTERIAL COLONIZATION: IMPACT AND ACTION MECHANISM POPULTIONS THROUGH A PARTIAL REGULATORY B- CELL PHENOTYPE IN VITRO ON HUMAN HEALTH Rafael Ceña-Diez Sara Martin Delgado *Esther Jiménez Quintana Hospital General Universitario Gregorio Marañón, Departamento de Nutrición, Bromatología y Madrid. Hospital General Universitario Gregorio Marañón, Madrid Tecnología de los Alimentos. Universidad 15:30-15:45 Complutense de Madrid 15:30-15:45 ROLE OF CLATHRIN AND CLATHRIN ADAPTOR 15:30-15:45 *Invited paper PROTEIN-1 IN HEPATITIS C VIRUS EGRESS ENGINEERED THERMOSTABLE EMPTY OF FMDV FOR IMPROVED VACCINES METAGENOMIC ANALYSIS OF VIRUSES IN THE Virgina Gondar HUMAN ORAL CAVITY Silvia López-Argüello Instituto de Investigación Sanitaria Hospital La *Alberto López Bueno Centro de Biologia Molecular Severo Ochoa, Madrid Princesa, Madrid Centro de Biología Molecular Severo Ochoa 15:45-16:00 15:45-16:00 (C.S.I.C.-U.A.M.), Madrid ROLE OF INTERFERON STIMULATED GENES IN THE pH STABILITY OF FOOT-AND-MOUTH DISEASE 15:45-16:00 *Invited paper VIRUS PARTICLES IS MODULATED BY RESIDUES INFLUENZA VIRUS PRODUCTION AND ANTIVIRAL VACCINE DRIVEN EVOLUTION OF PORCINE LOCATED AT THE PENTAMERIC INTERFACE AND IN SIGNALLING THE N TERMINUS OF VP1 Marta Lopez De Diego *Tuija Kekarainen Flavia Caridi University of Rochester, Rochester, NY, USA Centre de Recerca en Sanitat Animal (CReSA) - Centro de Biología Molecular “Severo Ochoa” 16:00-16:15 Institut de Recerca i Tecnologia (CSIC), Madrid EPIDEMIOLOGÍA MOLECULAR DE GRIPE A Y B EN Agroalimentàries (IRTA), Bellaterra 16:00-16:15 ENFERMEDAD RESPIRATORIA GRAVE Y ESTADO ANALYSIS OF THE ORIGIN AND INFECTIVITY OF VACUNAL INFECTIOUS MICROVESICLES DERIVED FROM F. Xavier López-Labrador SEMLIKI FOREST VIRUS DEVOID OF CAPSID (FISABIO-Salud Pública/Universtat de València), Cristian Smerdou (CIBER-ESP), Instituto de Salud Carlos III, Madrid FIMA, Zaragoza 16:15-16:30 16:15-16:30 MODIFICATION OF PROMOTER SPACER LENGTH IN TYPE I INTERFERON RESPONSE IS DELAYED IN VACCINIA VIRUS AS A STRATEGY TO CONTROL THE HUMAN INFECTED CELLS ANTIGEN EXPRESSION Susana Guix Mauro Di Pilato University of Barcelona, (INSA-UB), Barcelona, Centro Nacional de Biotecnología (CNB-CSIC), Spain Madrid 16:30-16:45 16:30-16:45 LA PROTEÍNA VP5 JUEGA UN PAPEL ESENCIAL EN HIGHLY EFFICIENT INSECT CELL-BASED PLATFORM LA DISEMINACIÓN DEL VIRUS DE LA BURSITIS FOR VIRUS-LIKE PARTICLE VACCINES PRODUCTION INFECCIOSA USING AN IMPROVED BACULOVIRUS VECTOR Fernando Méndez Silvia Gómez-Sebastian Centro Nacional de Biotecnología (CSIC), Madrid Alternative Gene Expression S.L (ALGENEX), Madrid 16:45-17:00 16:45-17:00 A MODEL FOR HEPATITIS A VIRUS TRANSCYTOSIS VIRAL PROTEINS TARGET COMPLEXES IN THE RIG-I IN HEPATOCYTES LIKE RECEPTOR Montserrat De Castellarnau Maria Teresa Sánchez-Aparicio Universidad de Barcelona, Barcelona ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI, NY-USA

17:00-17:30 VISIT POSTERS AND COFFEE BREAK [GOYA ROOM - EXHIBITION AREA]

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TUESDAY JUNE 9, 2015

AUDITORIUM WHITE ROOM AUDIOVISUAL ROOM

17:30-19:30 17:30-19:30 17:30-19:30 Parallel Session X: Parallel Session XI: Parallel Session XII: Teaching and Dissemination of the Antiviral Drugs (PL11) Replication Mechanisms (PL12) Virology (PL10) Chairpersons: JULIÁ BLANCO Chairpersons: AMELIA NIETO RAFII MOHAMED LUIS MENÉNDEZ Chairpersons: ESPERANZA GÓMEZ-LUCÍA JOSE A. LÓPEZ-GUERRERO 17:30-17:45 17:30-17:45 MODULATION OF P85β ACTIVITY BY SUMO 17:30-17:33 *Invited paper CHD1 CHROMATIN REMODELER IS A POSITIVE MODULATOR OF INFLUENZA VIRUS REPLICATION Carmen Rivas INTRODUCTION THAT PARALLELS RNAP II DEGRADATION IN THE *Jose Antonio Lopez-Guerrero INFECTED CELLS Universidade de Santiago de Compostela (CIMUS- IDIS), Santiago de Compostela Universidad Autónoma de Madrid, Madrid Amelia Nieto 17:45-18:00 17:33-17:41 *Invited paper Centro Nacional de Biotecnologia (CNB-CSIC), Madrid EXPRESSION OF PSEUDORABIES VIRUS IE180 PROTEIN TEACHING VIROLOGY AT A PREUNIVERSITARY 17:45-18:00 UNDER THE CONTROL OF HUMAN TUMOR-SPECIFIC LEVEL LOCAL RNA FLEXIBILITY PERTURBATION OF THE IRES PROMOTERS (hTERT AND CEA): I.- APPLICATION TO *Javier Medina ELEMENT INDUCED BY A NOVEL LIGAND INHIBITS OBTAIN CITOLYTIC VECTORS IN TUMOR CELLS VIRAL RNA TRANSLATION Departamento de Ciencias Naturales, IES ALPAJÉS Laura Lerma (Consejería de Educación, Comunidad de Madrid), Gloria Lozano Universidad Autónoma de Madrid, Madrid Aranjuez, Madrid Centro de Biologia Molecular Severo Ochoa, Madrid 18:00-18:15 17:41-17:49 *Invited paper 18:00-18:15 THE EXONUCLEASE XRN1P IS SPECIFICALLY REQUIRED THE RADIO AS A VIROLOGY INFORMATION ANTIVIRAL ACTIVITY OF POLYANIONIC CARBOSILANE FOR THE TRANSLATION OF BROME MOSAIC VIRUS DIFFUSOR DENDRIMERS AGAINST HEPATITIS C VIRUS IN CELL Bernat Blasco-Moreno *Manuel Seara CULTURE Universitat Pompeu Fabra, Barcelona Radio Nacional de España. Prado del Rey, Madrid Daniel Sepúlveda-Crespo 18:15-18:30 17:49-17:57 *Invited paper H. G.U. Gregorio Marañón, Madrid IFN-α TREATMENT CAUSES A MASSIVE APOPTOSIS VIROLOGY FOR ALL IN FREE AND ONLINE 18:15-18:30 IN IBDV INFECTED CELLS DIVULGATION JOURNALS APTAMERS DESIGN AS ANTIVIRAL AGENTS AGAINST Liliana Cubas *Ana M. Doménech INFLUENZA VIRUS Centro Nacional de Biotecnología (CSIC), Madrid Universidad Complutense de Madrid, Madrid Paloma Rodríguez-Rodríguez 18:30-18:45 17:57-18:05 *Invited paper Centro Nacional de Biotecnologia (CNB-CSIC), Madrid BIOGENESIS AND DYNAMICS OF YOUNG VIROLOGISTS TO RECEIVE THE BATON 18:30-18:45 REPLICATIVE STRUCTURES SIMVASTATIN AND METFORMIN INHIBIT CELL *Rafael Añez Ginés Ávila PROLIFERATION AND HEPATITIS C REPLICATION IN Universidad17.00-17:30 ComplutenseCOFFEE BREAK de Madrid, // VISIT Madrid POSTERS [GOYA ROOM - EXHIBITION AREA] VITRO, BY DOWNREGULATING TCTP AND Centro Nacional de Biotecnología (CSIC), Madrid 18:05-18:13 *Invited paper INCREASING PTEN 18:45-19:00 DISSEMINATION OF VIROLOGY THROUGH BLOGS José A. Del Campo STRUCTURAL BASIS OF INFLUENZA VIRUS RNP AND OTHER SOCIAL MEDIA Valme University Hospital. Sevilla ACTIVITY *Miguel Ángel Jiménez-Clavero 18:45-19:00 Rocio Coloma INIA-CISA, Valdeolmos, Madrid HEPATITIS C VIRUS REPLICATION FACTORY STUDIED Centro Nacional de Biotecnología (CSIC), Madrid 18:13-18:21 *Invited paper BY CRYO SOFT-X-RAY TOMOGRAPHY: PLATFORM FOR 19:00-19:15 GAMES AS TOOLS FOR TEACHING VIROLOGY PHARMACEUTICAL TRIALS OF NEW ANTIVIRAL DRUGS AT CELLULAR LEVEL THE DEAD-box HELICASE DHH1 PROMOTES *Esperanza Gómez-Lucía TRANSLATION OF HIGHLY STRUCTURED mRNAS Ana J. Pérez-Berná Universidad Complutense, Madrid, Madrid Jennifer S. Jungfleisch Sincrotrón ALBA, Barcelona 18:21-18:30 *Invited paper Universitat Pompeu Fabra, Barcelona 19:00-19:15 CONCLUSIONS 19:15-19:30 DETECTION OF A SEXUALLY TRANSMITTED HEPATITIS Jose Antonio Lopez-Guerrero C VIRUS PROTEASE INHIBITOR-RESISTANCE VARIANT INCREASED PATHOGENESIS OF INFLUENZA A H1N1 VIRUS LED BY A PA RESIDUE DETECTED IN A FATAL Universidad Autónoma de Madrid, Madrid IN A HUMAN IMMUNODEFICIENCY VIRUS-INFECTED CASE HOMOSEXUAL MAN 18:30-19:00 Jasmina Vasilijevic DISCUSSION Sandra Franco Centro Nacional de Biotecnologia (CNB-CSIC), Madrid 19:00-19:30 Función irsiCAIXA, Barcelona 19:15-19:30 QUIZ VIRTUAL EPIDEMIC TRANSIENT INCREASES IN THE ERROR RATE CAN OPEN NEW ADAPTIVE PATHWAYS IN AN RNA VIRUS Ester Lázaro Centro de Astrobiología (INTA-CSIC), Madrid

20:30 CONGRESS DINNER

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WEDNESDAY JUNE 10, 2015

AUDITORIUM

9:00-10:20 Plenary Session VI: Plant Virus (PL6)

Chairperson: JUAN ANTONIO GARCÍA

9:00-9:40 *Invited plenary lecture NEW CONCEPTS IN THE BIOLOGY OF MULTIPARTITE VIRUSES *Stéphane Blanc

INRA, UMR BGPI, Montpellier, France 9:40-10:20 *Invited plenary lecture MEMBRANE REARRANGEMENTS IN PLANT VIRUS RNA REPLICATION *Luisa Rubino Institute for Sustainable Plant Protection, CNR, Bari, Italy

10:20-11:30 FLASH PRESENTATIONS

Chairpersons: SUSANA ALVAREZ AND JOSÉ LUIS JIMÉNEZ

11:30-12:00 COFFEE BREAK // VISIT POSTERS [ GOYA ROOM - EXHIBITION AREA

]

12:00-12:30 THE VIROLOGIST CONFERENCE YOUNG AWARD

Chairpersons: PEDRO MAJANO

*Invited plenary lecture TEN YEARS OF HEPATITIS C VIRUS CELL CULTURE INFECTION MODELS *Pablo Gastaminza Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid

12:30-13:15 CLOSING LECTURE

Chairpersons: MIGUEL ÁNGEL JIMÉNEZ-CLAVERO

*Invited plenary lecture MECHANISMS OF VIRAL PERSISTENCE IN INSECTS *Carla Saleh Institut Pasteur, Viruses and RNA interference Unit, Paris, France

13:15-13:30 CLOSING CEREMONY

Chairpersons: ALBERT BOSCH AND MANUEL RODRÍGUEZ

13:15-13:30 AWARD GIVING CEREMONY

13:30-13:45 ANNOUNCEMENT OF THE UPCOMING “XIV CONGRESO NACIONAL DE VIROLOGÍA”

CLOSING OF THE MEETING

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32 Virología. Publicación Oficial de la Sociedad Española de Virología

PS: P L E N A R Y S E S S I O N

PLENARY SESSION I (PS I): remodel intracellular membranes in order FRONTIERS IN VIROLOGY to build up their replication factories. In my Chairpersons: presentation, I will summarize our current JUAN ORTÍN AND RICARDO FLORES state of knowledge how these viruses Sunday June 7, 2015 usurp host cell pathway to achieve LECTURE ROOM ATENEO DE MADRID efficient replication and virus production and describe an example how this *Invited plenary lecture knowledge has been used to develop a 17:15-17:50h (P1) highly potent antiviral therapy. CELL BIOLOGY OF VIRAL REPLICATION CYCLES: A COMPARISON OF HEPATITIS C *Invited plenary lecture VIRUS AND DENGUE VIRUS 17:50-18:25h (P2) RALF BARTENSCHLAGER 1,2 VIRUS BEHAVIOR AT EXTREME FITNESS 1Department of Infectious Diseases, Molecular VALUES Virology, Heidelberg University, Heidelberg, ESTEBAN DOMINGO1,2, NM BEACH1, E Germany 1 1,2,3 2 MORENO , C PERALES Division Virus-associated carcinogenesis, German 1 Cancer Research Center, Heidelberg, Germany Centro de Biología Molecular Severo Ochoa (CSIC- UAM), Cantoblanco, Madrid, Spain, 2Centro de Investigación Biomédica en Red de Positive strand RNA viruses replicate in the Enfermedades Hepáticas y Digestivas (CIBERehd), cytoplasm in distinct membranous Barcelona, Spain replication factories. Thus, a hallmark of 3Liver Unit, Internal Medicine, Laboratory of this virus class is the induction of profound Malalties Hepàtiques, Vall d’Hebron Institut de Recerca-Hospital Universitari Vall d’Hebron, (VHIR- membrane alterations that fall into two HUVH), Universitat Autònoma de Barcelona, morphological groups: the Barcelona, Spain invagination/spherule type and the double membrane vesicle (DMV) type. These Fitness is a parameter that captures the morphotypes are represented by the overall replicative efficacy of a virus in a Dengue virus (DENV) and the hepatitis C given environment. A recent study with virus (HCV), respectively. These viruses hepatitis C virus (HCV) replicating in human belong to the same family, the , hepatoma Huh-7.5 cells has documented because they share a similar genome that high fitness correlates with resistance organization and overall replication to several anti- HCV agents in the absence strategy. Yet, the interaction between of recognized resistance mutations in the these viruses and their host cell is HCV mutant spectra (Sheldon et al. J. Virol. fundamentally different. While DENV 88: 12098, 2014). Previous studies have induces an acute lytic infection, HCV shown that fitness tends to increase when replicates persistently with little large viral populations are allowed to cytopathogenicity. Moreover, both viruses multiply in a constant environment, and appear to utilize different cellular tends to decrease when the virus is pathways and host cell machineries to subjected to repeated bottleneck

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passages. Work with vesicular stomatitis increasingly perceived as relevant for the virus and foot-and-mouth disease virus management of HCV infections. Some documented stochastic fluctuations when recent clinical data point to low inhibitor very high or very low fitness values are sensitivity of HCV, independent of the approached (Novella et al. J. Virol. 73: presence of resistance mutations. 1668, 1999; Lázaro et al. PNAS 100: 10830, Selection of specific mutations is one 2003). In the case of HCV, viral fitness and among other mechanisms of drug multidrug resistance reach a plateau (and resistance that are still largely unexplored. even decrease slightly) when the number Implications for current antiviral therapies of serial passages in hepatoma cells will be discussed. approaches 200. Interestingly, a pattern of increasing fluctuations in virus titer has *Invited plenary lecture been observed between passages 100 and 200, with a trend towards a decrease of 18:25-19:00h (P3) specific infectivity (ratio of infectivity to DENDRITIC EFFECTS: THE ROLE OF amount of viral RNA). Two non-mutually DENDRIMERS AS SOFT SUPER-ATOMS IN exclusive mechanisms may contribute to NANOPERIODIC PROPERTY PATTERNS fitness fluctuations: (i) fitness dependence R. ZHANG1, S. HISANO1, A. TANI1, H. of mutation deleteriousness, and (ii) KONDO1, S. KANEMATSU2, NOBUHIRO participation of defective genomes in SUZUKI1 modulation of fitness levels. Experiments 1 Institute of Plant Science and Resources, Okayama are now in progress to identify the University, Kurashiki, Okayama 710-0046, Japan mechanism involved, because fitness 2NARO Institute of Fruit Tree Science, 92 variations may impact efficacy not only of Shimokuriyagawa, Morioka, Iwate 020-0123, Japan monotherapy but also of combination treatments. In particular, in lethal A rapidly growing number of viruses of mutagenesis-based antiviral protocols, the lower eukaryotes have been reported in advantage of a sequential inhibitor- the past few decades. These have mutagen administration over the contributed to further enhance our corresponding combination (Perales et al. understanding of virus evolution and TIM 20: 595, 2012) was largely lost when diversity. Simultaneously, some unusual applied to high fitness HCV. Recent results viruses have challenged the “common suggest that the doses of inhibitor rules” of viruses in sizes and concepts. One (telaprevir) required to control viral load such virus group includes the so called for an effective ribavirin mutagenesis- dsDNA megaviruses that viruses exceed driven lethality are higher for high fitness those of bacterial parasites such as than low fitness HCV. Also, high fitness mycoplasmas and in coding capacity (>1.2 HCV displays increased resistance to both the inhibitory and mutagenic activity of discovery of giant viruses was followed by ribavirin, as compared with low fitness HCV the identification of DNA viruses (Perales et al. in preparation). Fitness and termed “virophage” co-infecting amoeba its connection with viral load are with helper giant dsDNA viruses.

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Virophages have much smaller particles of (+) RNA virus, YkV1, which highjacks CP of 50 nm in diameter and dsDNA genomes the dsRNA virus, YnV1, for the trans- (18 kb) which encode no DNA or RNA encapsidation of its genome and RNA polymerases. Other unusual virus polymerase at the replication site. examples include “naked” or “capsidless” RNA viruses that are unable to form virus THE VIROLOGIST CONFERENCE SENIOR particles, exemplified by hypoviruses, AWARD which were the first to be reported as such

a virus from the chestnut blight . *Invited plenary lecture Rosellinia necatrix is a filamentous ascomycete that causes white root rot in 12:00-13:00h (P4) diverse perennial crops worldwide. This FRONTIERS IN PUBLIC HEALTH fungus is one of versatile fungal hosts to MICROBIOLOGY: A CONTINUING many viruses and suitable for studying CHALLENGE virus/virus and virus/host interactions. ANTONIO TENORIO Herewith, we show unique interactions in Arbovirus and Imported Viral Diseases, Centro R. necatrix between an as-yet-undescribed Nacional de Microbiología, Instituto de Salud Carlos positive-strand (+) RNA virus, with III, Madrid, España properties similar to but distinct from a virophage, and a novel, hosting double- My first contact with public health virology stranded (ds) RNA virus. We found a mixed was in 1963, when I received the oral viral infection in a hypovirulent strain of R. poliovirus vaccine. Twenty years later I was necatrix. Co-infecting viruses are in charge of the last outbreak due to tentatively termed yado-kari virus 1 (YkV1) autochthonous transmission of wild with a (+) RNA genome of approximately 6 poliovirus in Spain. It affected a social kb and yado-nushi virus 1 (YnV1) with a group rejecting vaccinations, like dsRNA genome of approximately 9 kb. everywhere with this and other vaccines. YkV1 possesses one single ORF encoding In the meantime, efforts for controlling RNA-dependent RNA polymerase (RdRp), the circulation of eradicable viruses are while YnV1 has two ORFs, each encoding continuous and have been including new capsid protein (CP) and RdRp. threats and methodologies. Immunological and molecular analyses In the nineties, our priority was improving revealed trans-encapsidation of not only the capability of virus diagnostics in the YkV1 RNA but also RdRp by the major CP of Spanish hospitals, developing innovative the other virus, YnV1. Virion transfection molecular tools and transferring methods assay and previous epidemiological data and knowledge to the National Health strongly suggest that YkV1 depends on System. Now, the majority of them are YnV1 for viability, although it probably able to diagnose most of the common viral encodes functional RdRp. This hypothesis infections in both immunocompetent and was confirmed by establishing infectious immunodeficient patients. full-length cDNA of YkV1. We propose the term “RNA virophage” for the capsidless

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With the new millennium, new threats country, impoverished health systems and suddenly exploded, directly derived from improved communication ways. the globalization: clusters of imported viral To address this new challenge, we need a hemorrhagic fevers in different European clear leadership and funding, joining the Countries, old viruses causing epidemics in efforts of social sciences, epidemiology, naïve continents, wild viruses producing ecology and virology. Otherwise, the new new infections in humans, new viral threat could be a virus adapted by serial diseases transmitted by imported vectors passage to human species. or reservoirs.

The only way to control these imported PLENARY SESSION (PS II): infections (including Dengue and Chikunguna in areas with competent EMERGING VIRUSES vectors) was the construction of national Chairpersons: and international networks, integrating JAVIER BUESA - ANTONIO ALCAMÍ tropical medicine and virology groups. Tuesday June 8, 2015 On the other hand, Spain could not be the AUDITORIUM REAL CASA DE LA MONEDA exception in Europe, and wild, zoonotic viruses were also circulating and causing disease in humans. The assembly of *Invited plenary lecture different research groups in a 9:00-9:30h (P5) multidisciplinary network allowed us to CORONAVIRUS PATHOGENESIS AND detect dozens of viruses in wild life in PROTECTION Spain, some of them infecting humans LUIS ENJUANES,J. L. NIETO-TORRES, M. (West Nile, Lymphocytic Coriomeningitis, DEDIEGO, J. M. JIMENEZ-GUARDEÑO, J. A. or Toscana, but also some new viruses), or REGLA-NAVA, C. CASTAÑO-RODRIGUEZ, R. potentially infecting humans (Crimean- FERNANDEZ-DELGADO, JAVIER CANTON- Congo Hemorrhagic Fever or the new BAILON, JAVIER GUTIERREZ-ALVAREZ, S. filovirus Lloviu). Moreover, we had a ZUNIGA, AND I. SOLA. qualitative change when we started to Department of Molecular and Cell Biology.National work with the viruses as ecological entities, Center of Biotechnology (CNB-CSIC), Madrid, Spain. fighting for their survival with other viruses or environmental frontiers. The identification of the genes involved in The current Ebola outbreak in West Africa coronavirus (CoV) virulence and in is the last example of a virus emerging signaling pathways contributing to from several globalization effects: pathogenesis has been addressed using migration of reservoirs from their original SARS- and MERS-CoVs. SARS-CoV non- habitats, rapid spread of infected essential genes have been deleted using a individuals to other countries –mainly war reverse genetics system. Among them, refugees resident in the first infected area– deletion of E gene led to an attenuated , or to the main cities of each infected phenotype (SARS-CoV-ΔE). The expression of proinflammatory cytokines was reduced

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in lungs of mice infected with a mouse or modified are promising vaccine adapted SARS-CoV-MA15-ΔE compared to candidates. lungs infected with the wild type virus. In infections by SARS-CoV with and without E *Invited plenary lecture protein, NF-κB was the only proinflammatory pathway differentially 9:30-10:00h (P6) activated. Addition of an inhibitor of NF-κB ZOONOTIC, FOODBORNE AND led to a reduced inflammatory response ENVIRONMENTAL TRANSMISSION IN after SARS-CoV infection and to an ROTAVIRUS AND HEPATITIS E VIRUS increase in mice survival. Therefore, these INFECTION inhibitors could serve as antivirals. A FRANCO M. RUGGERI 1 reduction in neutrophil migration to lung- 1 Dept. of Veterinary public health & food safety, infected areas was observed in mice Istituto Superiore di Sanità, Rome, Italy infected with SARS-CoV-MA15-ΔE, probably contributing to the lower degree Viruses harbored in the intestinal tract can of inflammation detected and to SARS- persist in a harsh environment for long CoV-ΔE attenuation. SARS-CoV E protein is time and are normally shed with feces of a viroporin with three domains: amino infected hosts in large amount. In addition terminus, transmembrane and carboxy- to direct passage between individuals, they terminus. The role of the different domains can therefore be transmitted via a of E protein in SARS-CoV virulence, contaminated environment, including including its ion channel activity and a PDZ surface waters and foodstuff. Moreover, binding domain (PBM) mapping at the viruses such as rotavirus and the hepatitis most carboxy-terminus of this protein was E virus can infect a wide range of animal evaluated. Alteration of the three domains species, and animal strains can be attenuated the virus, and the mechanisms efficiently transmitted zoonotically of attenuation have been studied. The adapting replication in human hosts. attenuated mutants provided long-term Rotavirus is the major cause of acute protection both in young and elderly mice gastroenteritis in children worldwide and against the challenge with pathogenic vaccination is showing high efficacy against SARS-CoVs. We showed that E protein PBM common human strains in an increasing binds syntenin, activating p38 MAPK and number of countries. Nonetheless, the causing lung inflammation and edema. large genetic variation of rotavirus makes it Inhibition of p38 MAPK protected 80% of unclear if protection is similar for all viral the mice infected with virulent SARS-CoV. genotypes of animal origin. During 8 years Deletion of E gene in MERS-CoV using a of molecular surveillance of rotavirus in reverse genetics system, led to a Europe, several thousands of rotavirus replication-competent propagation- strains from infected children were defective virus that is a safe vaccine genotyped to investigate shifts in the candidate. These data indicated that SARS- distribution of common strains and the CoV and MERS-CoV with E protein deleted possible emergence of rare, animal or exotic genotypes. Animal strains were also

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investigated from domestic animal species, ORAL PRESENTATIONS (OP I): confirming that different hosts are mostly EMERGING VIRUSES infected with species-specific genotypes. Chairperson: RAFAEL DELGADO However, in sporadic cases close genetic relatedness was identified between several Wednesday June 8, 2015 genes of human and animal strains, AUDITORIUM REAL CASA DE LA MONEDA suggesting human infection with animal- derived reassortant . Rotavirus *Invited paper infection of adults was also confirmed, 10:00-10:15h OP I(CO1) which may be related to strains acquired during travelling or from animal hosts. THE ROLE OF THE NATIONAL REFERENCE Animal and human strains were shown to LABORATORY REGARDING ARBOVIRUSES 1 1 be present in sewage samples highlighting MP SÁNCHEZ-SECO, , A. VÁZQUEZ , A. 1 1 1 possible risks of dual infection and inter- NEGREDO , L. FRANCO ,F. DE ORY , A. 1 species gene . TENORIO The hepatitis E virus (HEV) is the cause of 1. Centro Nacional de Microbiología. Instituto de Salud “Carlos III”. Most of the authors (MPSS, AV, sporadic cases of acute human hepatitis in AN, LF and FO) are members of ViroRed and (MPSS, industrialized countries and of large AV, AN and LF) RICET networks. waterborne outbreaks of disease particularly in Asia and Africa. Of the four The European Centre for Disease main genotypes, genotype g3 is largely Prevention and Control defines five spread among domestic swine bred activities to carry out by a National throughout Europe, and small outbreaks Reference Laboratory (NRL): Reference and an increasing rate of human infections diagnostics, Reference resource materials, are being reported in several countries in Monitoring, alert and response, Scientific association with raw pork consumption. In advice and Collaboration and research. addition to farmed swine, also wild boar, Low prevalence diseases are of special deer and other wild animal species are importance for NRLs since their susceptible to HEV infection, and genetic requirement of a high degree of characterization of human and animal g3 specialization. The National Centre of and g4 strains are in support of actual risks Microbiology in the Institute of Health of zoonotic transmission of infection. The “Carlos III” is the NRL for zoonoses. finding of HEV genome in swine feces, liver and meat of pigs at slaughter age, and the Many zoonoses are caused by arthropod- large import-export of animals and pork borne viruses (arboviruses) and most of products highlights needs of controlling them are clear examples of emerging the spread of infection in Europe. viruses. The emergency and / or re-emergency of virus diseases may cause the eruption in

our environment of very low or zero prevalence diseases. This fact will be, obviously, associated with the emergence

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of a new, "weird" or exotic virus. Beyond *Invited paper their actual involvement in Public Health, 10:15-10:30h OP I(CO2) these situations imply the need for swift LABORATORY DIAGNOSIS OF EBOLA action whose first step is the detection of VIRUS DISEASE IN SPAIN DIAGNOSTICS OF the agent. EBOLA VIRUS DISEASE SUSPECTED CASES In our country, except for Toscana virus, a A NEGREDO1, A VÁZQUEZ1, L FRANCO1, JM phlebovirus transmitted by sandflies that RUBIO2, I JADO2, G FEDELE2, P ANDA2,, MP produces cases of neurological infection SÁNCHEZ-SECO1. every year, the circulation of arboviruses in 1. Laboratory of Arboviruses and Viral Imported man has been highly limited. However, this Diseases, Centro Nacional de Microbiología. situation can be reversed at any time. And Instituto de Salud “Carlos III”, Majadahonda, this possibility should not be forgotten as Madrid, Spain. less probable events as the appearance of 2. Health Alert Team, Centro Nacional de the first case of Ebola virus infection out of Microbiología. Instituto de Salud “Carlos III”, Africa occurred recently (October 2014) Majadahonda, Madrid, Spain. due to the ongoing crisis of West Africa caused by this virus. The Spanish National Center of In recent years, there have been four Microbiology (CNM) is the National situations that deserve special Reference Laboratory for zoonosis in Spain. consideration: the first outbreak of West In the current Ebola virus disease (EVD) Nile virus in Spain (2010), the first outbreak epidemic in West Africa, the CNM has been of Chikungunya virus in a European assigned as the laboratory in charge of country (Italy, 2007), the arrival of the analyzing suspicious sample from EVD virus to the Americas (2013- 2014) with a patient under investigation. large increase in the number of viremic In the current EVD epidemic, CNM as patients in our environment coming from National Ebola Reference Laboratory have that continent and the appearance in received samples from 45 EVD suspicious Extremadura of ticks carrying the virus patients during 2014. In this group are causing Crimean-Congo haemorrhagic included travellers from African hotspot fever. area (34 suspicious cases), repatriated Previous work on preparedness is required suspicious cases from Liberia, Sierra Leone to be able to respond properly to these and Mali (2 confirmed cases and 2 challenging situations. Previous suspicious cases), and high risk local development and maintenance of contacts (7 suspicious cases). We followed techniques and protocols and specialized the European Centers for Disease Control personnel capable of dealing with these and Prevention´s (eCDC) diagnostic very rare situations is part of the duties of algorithm indicating ebolavirus nucleic acid a NRL. detection on a blood sample as diagnostic procedure. In our case we used a BSL-3 facility to inactivate the clinical samples

and after that, RNA extraction was carried

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out in BSL-2 conditions. Positive results PLENARY SESSION III (PSIII): HEPATITIS A, were confirmed by using a second PCR B AND C: BASIC, TRANSLATIONAL AND assay on a different genomic target and we CLINICAL RESEARCH sent the positive samples to a BSL-4 WHO Chairpersons: collaborative laboratory (Bernhard Nocht JOSEP QUER AND PABLO GASTAMINZA Institute for Tropical Medicine, Hamburg and Germany) for the confirmation of the Thursday June 8, 2015 results. Differential diagnosis was made for AUDITORIUM REAL CASA DE LA MONEDA malaria infection. With the exception of the 2 EVD *Invited plenary lecture repatriated patients all travellers from 11:00-11:20h (P7) Africa were negative (36/38). 22 out of 38 HEPATITIS A VIRUS: NEW PARADIGMS OF suspected cases were malaria positive AN OLD PATHOGEN patients. We confirm Ebola virus (EBOV) R.M. PINTÓ, F.J. PÉREZ, M. DE positive cases in samples from 2 CASTELLARNAU, L. D’ANDREA, S. GUIX, A. repatriated patients and we detected the BOSCH first case of infection outside Africa ever Enteric Virus Laboratory, Departament of reported. This case was detected in a nurse Microbiology, University of Barcelona, Barcelona, assistant who had been in contact with the Spain second repatriated person that came from Sierra Leone in September. In addition, The Hepatitis is still the most common acute EBOV RNA concentration in plasma was hepatitis worldwide, in spite of efficient measured daily from the 3 EBOV positive available vaccines. In developed countries, patients. The 2 EBOV positive repatriated the men-having-sex-with-men (MSM) patients died during the critical period of group is particularly susceptible to large EVD but the third EBOV patients attended outbreaks and foodborne outbreaks also in Spain was discharged when RNA was not occur frequently due to the global food detected in any biological fluid. trade. The outbreak of EVD in West Africa has hit The hepatitis A virus (HAV) is the causative our Public Health Systems and agent of the infection and although coordination among different actors belongs to the Picornaviridae family, it is a involved in the response has been very unique at the genomic, established. In relation with the Laboratory structural, antigenic and biological levels. diagnosis in CNM it was necessary to Its capsid crystal structure has recently create an alert team to be able to attend been described, revealing a much health alert situation the 24 hours/7 days. smoother particle compared to other Laboratory diagnostic have been carried , with higher thermal out by this alert team and by Arbovirus and stability at acid than neutral pH. By the Imported laboratory. The time way, HAV has an extremely stable capsid at of response is less than 24 hours after an acid pH and at high biliary salt alert is received. concentration, features required for its

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biological cycle. These particularities may would help in the ultimate fate of an be related to its very special codon usage, otherwise low replicative virus. shaped by fine tuning translation selection and resulting in a highly regulated *Invited plenary lecture ribosome traffic pace. Additionally, there is also a very fine tuning between capsid 11:20-11:40h (P8) codon usage and IRES efficiency or in other CONTINUUM OF CARE IN HEPATITIS C: words HAV has a very inefficient IRES FROM DETECTION TO CURE AND which correlates with an overall slow ERADICATION speed of translation. MANUEL ROMERO-GÓMEZ The requirement of stability for a virus UCM Digestive Diseases and ciberehd.Valme with long periods in the environment, out University Hospital, University of Seville, Sevilla, of the host body, is ensured by negative Spain. selection of mutations affecting capsid rare codons. Many of these rare codons encode Hepatitis C virus infection is a major health for surface residues located in the epitope problem affecting to millions of people sites avoiding the emergence of new infected during the second part of the XXth serotypes. Antigenic variants may, century. Blood transfusions and drug users however, arise in particular situations of are the two main risk factors for hepatitis immune pressure. Overall, codon usage C. A selection of just one clone is usual may be considered a genomic constraint to during the infection process. Butyrophilin antigenic variability. family genes are crucial in genotype Additionally, HAV has adopted several selection and female gender, IL28B-CC strategies to avoid its elimination from genotype and HCV-genotype 1 are crucial blood. On the one hand, its capsid to promote spontaneous viral clearance. structure at neutral pH is unable to Humans are the only one reservoir for the interact with glycophorin A, a decoy factor virus and HCV does not integrate on the present at the erythrocyte membrane, human genome giving the opportunity for avoiding its clearing from the blood. On cure of the disease and effective the other, it has recently described that eradication of the outbreak using directed HAV exists in a double phenotype: as free antiviral drugs. Viral genotype and fibrosis particles and enveloped in exosomes. A stage are two key aspects making decisions hypothesis has been proposed regarding on hepatitis C care. UDPS has HAV exit from hepatocytes. Exocytosis demonstrated to be useful detecting through the basolateral membrane would genotype and subtype o even mixed mainly give rise to enveloped particles in infections in patients non correctly typed blood, and exit through the apical by second generation Lipa®. Transient membrane into the biliar canaliculi would elastography allow a close monitorization give rise to free particles in feces. The of fibrosis progression. Changes on liver advantage of the occurrence of enveloped stiffness one year apart classify patients at particles in blood is the capacity to escape risk for liver diseases progression and the antibody interaction. These strategies lower survival. Combination of Sofosbuvir

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+ Ledipasvir or Parataprevir/r + Ombitasvir disease, recognized as "incurable" HBV has + Dasabuvir with or without ribavirin for 8, infected a third of the world population 12 or 24 weeks reach sustained virological from whom, 300 million are chronic response rate higher than 95% in patients carriers, and responsible for more than half with compensated liver disease genotype 1 of the liver cancers worldwide. Besides, or genotype 4. Sofosbuvir + Daclatasvir HDV, which infectivity depends on the HBV could be useful in patients infected by envelope, infects about 15 million HBV genotype 3a but failed in cirrhotics carriers ,being responsible for the most previously non-responders. In patients severe form of hepatitis). The molecular with previous fail to first-generation biology of these two agents is absolutely protease inhibitor triple therapy or amazing. HBV viral cycle, despite being a decompensated cirrhosis the combination DNA virus, includes a reverse transcription of sofosbuvir + ledipasvir with ribavirin step and the viral genome remains in the could achieve SVR in more than 90% and infected cell nucleus as a "mini- improved liver dysfunction in a large chromosome" (known as cccDNA). In fact proportion of patients. Eradication of the in an HBV infected hepatocyte four virus means blocking transmission of the different forms of the viral genome can virus and cure of the disease in non- coexist: as a mini-chromosome; as inserted cirrhotics. In patients with cirrhosis into the host genome; as "incomplete" sustained virological response is associated DNA molecule in secreted virions and as with decreased risk of liver dysfunction RNA pre-genome in "intracellular and improved survival for all causes. A previrions". Like a coin with four faces?. residual risk of liver cancer remained and The HBV-DNA is completely coding and surveillance is required. almost 70% of it has overlapping genes, a “Guinness record of genome *Invited plenary lecture compactness”. Among all coded regions, the HBV X protein (154 aa) is associated to 11:40-12:00h(P9) an incredible variety of interactions with HEPATITIS B AND D: WHEN EVERY SINGLE hepatocyte mechanisms and being DETAIL HAS A HIDDEN MEANING considered oncogenic. Despite the FRANCISCO RODRIGUEZ FRÍAS availability of highly efficient antiviral Unitat Patologia Hepàtica. Serveis de Bioquímica i treatments inhibiting viral replication, Microbiologia (Unitat Virologia). Hospital none of them is able to resolve the Universitari Vall d'Hebron, Barcelona, Spain. Institut infection, therefore any individual who has Recerca Vall d’Hebron (VHIO), CIBERehd been infected with HBV infected cannot be

considered as cured, 2000 million The hepatitis B (HBV) and delta (HDV) people¡¡¡. His occasional and small viruses are surprising mainly for the “roommate”, the HDV, is even more extraordinary compactness of their small surprising, its tiny 1.7 kb circular RNA genomes: HBV 3.2 kb DNA, HDV 1.7 kb genome has a high rate of self- RNA, the smallest known animal viruses, complementarity (74%) and a single coding both are responsible for chronic liver region, inhibiting HBV production despite

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needing HBV activity to produce envelope to study the pathogenesis of HCV infection particles. The HDV genome has a single for several reasons: (1) tissue and serum coding region, however, two proteins samples can be obtained before and after (Delta antigens) are detected as a result of HCV infection; (2) infection can be a multicodification mechanism (RNA monitored from the beginning and thus, it editing). The question is: are these two is possible to obtain data on HCV kinetics proteins enough to control and enslave and host factors; (3) hepatitis C recurrence HBV infection?. Multiple posttranslational after LT is a rapidly progressive disease and modification mechanisms of the delta patients with mild or very severe hepatitis antigen (isoprenylation, methylation, recurrence can be well characterized. acetylation and phosphorylation) could Studies on early kinetics have shown that give them a huge multifunctionality similar HCV replication starts a few hours after to the multiple social functions that can reperfusion of the graft and that HCV-RNA have a person who dresses in different concentrations increase within the first ways. "the suit defines the function". The days after LT, despite the presence of anti- new in-depth technologies to study viral HCV antibodies. The main source of HCV quasispecies by massive sequencing are infection is circulating virions. reporting surprises that represent real Nevertheless, some data suggest that HCV challenges on both viruses biology. present in extrahepatic compartments may contribute to hepatitis C recurrence. *Invited plenary lecture Escape from neutralizing antibodies and efficient entry into hepatocytes play a 12:00-12:20h (P10) major role in reinfection of the liver graft. VIROLOGY OF HEPATITIS C VIRUS Indeed, HCV receptor levels at the time of INFECTION AFTER LIVER LT may modulate early HCV kinetics and TRANSPLANTATION hepatitis C recurrence is associated with SOFÍA PÉREZ-DEL-PULGAR increased levels of claudin-1 and occludin Liver Unit, Hospital Clínic, IDIBAPS, CIBERehd, at the tight-junctions. Recent estimates Barcelona, Spain. show that the proportion of infected hepatocytes ranges from 1 to 54% and End-stage liver disease due to chronic correlates with viral load. Some studies hepatitis C virus (HCV) infection is the have shown that infection is not random. leading indication for liver transplantation Clustering of HCV-positive hepatocytes (LT) in the Americas, Europe, and Japan. suggest a localized mechanism of Unfortunately, infection of the graft is intrahepatic propagation and control (cell- universal in patients with detectable viral to-cell transmission and innate immune load at the time of LT. Furthermore, the responses, respectively) and may have course of recurrent HCV infection is implications for HCV therapy. On the other accelerated after LT, with approximately hand, the quasispecies population changes 30% of patients developing chronic significantly after LT, most likely because of hepatitis and liver cirrhosis within 5 years the strong immunosuppression and the after LT. The LT setting is a unique model need to adapt to a new environment.

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However, there are no conclusive data sensors or actively, by encoding viral supporting the role of HCV quasispecies antagonists to counteract the effects of composition and disease outcomes. interferons. Since many cytoplasmic Treatment of recurrent HCV infection after viruses exploit similar mechanisms of LT is often compromised by adverse effects innate immune evasion, mechanistic and limited efficacy of interferon-based insight into the direct interplay between therapies. The advent of direct-acting viral RNA, viral RNA-processing enzymes, antivirals, particularly in interferon-free cellular sensors and antiviral proteins will regimens, is very likely to improve the be highly relevant to develop novel prognosis and outcome of patients with antiviral targets and to restrict important severe hepatitis C recurrence. animal and human infections.

PLENARY SESSION IV (PSIV): ORAL PRESENTATIONS (OP II): INNATE IMMUNITY INNATE IMMUNITY Chairperson: MARIANO ESTEBAN Chairperson: ANGEL CORBÍ AND Tuesday June 9, 2015 JAVIER MARTÍNEZ-PICADO AUDITORIUM REAL CASA DE LA MONEDA Tuesday June 9, 2015 AUDITORIUM REAL CASA DE LA MONEDA *Invited plenary lecture 9:00-9:45h (P11) *Invited paper TO SENSE OR NOT TO SENSE VIRAL RNA – 9:45-10:00h OP II(CO3) ESSENTIALS OF CORONAVIRUS INNATE FUNCTIONAL INTERACTION BETWEEN THE IMMUNE EVASION eIF2α KINASE GCN2 AND THE HUMAN 1,2 V OLKER THIEL IMMUNODEFICIENCY VIRUS TYPE 1 1 Institute of Virology and Immunology, Federal J. DEL PINO, J.J. BERLANGA Department of Home Affairs, Bern, Switzerland Centro de Biología Molecular Severo Ochoa (CSIC- 2Department of Infectious Diseases and UAM), Madrid, Spain Pathobiology, Vetsuisse Faculty, University of Bern, Switzerland The reversible phosphorylation of the α- An essential function of innate immunity is subunit of eukaryotic translation initiation to distinguish self from non-self and factor 2 (eIF2α) is a well-characterized receptors have evolved to specifically mechanism of translational control in recognize viral components and initiate the response to a wide variety of cellular expression of antiviral proteins to restrict stresses, including viral infection. In viral replication. Coronaviruses are RNA mammalian cells there are four eIF2α viruses that replicate in the host cytoplasm kinases which specifically phosphorylate and evade innate immune sensing in most this factor at serine 51 in response to cell types, either passively by hiding their different forms of stress: PKR, GCN2, HRI viral signatures and limiting exposure to and PERK. Beside PKR, GCN2 participates in

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the cellular response against viral infection *Invited paper by RNA viruses with central nervous 10:00-10:20h OP II(CO4) system tropism, such as Sindbis virus, p56Lck AND PKC INHIBITORS PRESERVE through its activation by viral RNA. PKR has SAMHD1 ANTIVIRAL FUNCTION, been involved in the innate antiviral INTERFERING WITH HIV-1 REPLICATION response against HIV-1, although this M.R. LÓPEZ-HUERTAS1, M. BERMEJO1, S. antiviral effect is very limited due to the RODRÍGUEZ-MORA1, E. MATEOS1, JOE distinct mechanisms evolved by the virus HEDGPETH2, JOHN SWINDLE2, J. ALCAMÍ1, to counteract PKR action. Here we report M. COIRAS1 that infection of human cells with HIV-1 1. Inmunopatología del SIDA, Instituto de Salud conveys the proteolytic cleavage of GCN2 Carlos III, Madrid, Spain and that purified HIV-1 and HIV-2 2 . Complegen, Inc., Seattle, WA proteases produce direct proteolysis of

GCN2 in vitro, abrogating the activation of GCN2 by HIV-1 RNA. Moreover, the HIV-1 BACKGROUND. HIV-1 cannot presently be protein Tat interacts with PKR and inhibits eradicated due to the existence of latently its in vitro activity by acting as a infected cells that persist even with competitive inhibitor due to binding to the antiretroviral therapy (ART). HIV-1 may same site of eIF2α on the kinase. We have infect resting and activated CD4+ T cells assayed in vitro eIF2α kinase activity of but only replicates in activated cells. GCN2 and the other two eIF2α kinases, HRI Among other mechanisms, the inactivation and PERK, in the presence of Tat, and we of SAMHD1, an antiviral factor linked to have observed a reduction of eIF2α innate immunity, is essential to overcome phosphorylation in all cases. On the other HIV-1 restriction. SAMHD1 activity is hand, although the limited sequence greatly dependent on cell cycle similarity between eIF2α and the HIV-1 progression, as cyclin A2/CDK1 are integrase, we have found that this viral responsible for SAMHD1 phosphorylation protein is able to inhibit GCN2 eIF2α kinase at T592 (pSAMHD1) and subsequent activity in vitro. These significant findings inactivation. CD4+ T cell activation strongly Lck suggest that cleavage of GCN2 by HIV-1 relies on kinases such as p56 and PKC protease, and competitive inhibition by Tat theta (). PKC is selectively expressed on and the viral integrase, could represent CD4+ lymphocytes and its activation is distinct mechanisms of HIV-1 to counteract mediated by the lymphocyte-specific Lck GCN2 antiviral activity. tyrosine kinase p56 , which is required for PKC translocation to the plasma membrane, initiating a cascade of events that culminates in the activation of essential factors for HIV-1 replication such as AP-1, NFAT, and NF-B, as well as in SAMHD1 inactivation. We hypothesized Lck that p56 and PKC inhibitors could thwart HIV-1 replication using as

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mechanism the preservation of SAMHD1 is the first compound currently used in antiviral function. clinic that has been described to preserve MATERIAL & METHODS. PBMCs were the antiviral function of an innate factor isolated from healthy donors. pSAMHD1 at such as SAMHD1. T592 was determined by immunoblotting. Retrotranscription and proviral integration *Invited paper was analyzed by qPCR. PKC inhibitors 10:20-10:40h OP II(CO5) CGX1079 and CGX0471 were provided by ROLE OF MONOCYTES AND Complegen. Dasatinib (BMS-354825, PLASMACYTOID DENDRITIC CELLS IN THE Sprycel), an inhibitor of tyrosine kinases CONTROL AND IMMUNOPATHOGENESIS such as p56Lck, was provided by Bristol- OF HIV AND HEPATITIS C VIRUS Meyers Squibb. INFECTION RESULTS: 1) PHA/IL-2 induced pSAMHD1 in E. RUIZ-MATEOS1 PBMCs and full HIV-1 replication. 2) 1Instituto de Biomedicina de Sevilla (IBiS)/Hospital CGX1079, CGX0471 and Dasatinib Universitario Virgen del Rocío. Seville. Spain. interfered with HIV-1 retrotranscription and proviral integration. This interference Plasmacytoid dendritic cells (pDCs) are was not related to viral entry. 3) CGX1079 able to sense viral and bacterial infections and CGX0471 slightly reduced T-cell through Toll Like Receptors (TLRs)-7 and 9, proliferation, partially protecting SAMHD1 respectively. pDCs activation produces vast from T592 phosphorylation. These amounts of type I interferon (IFN-I). This inhibitors also interfered with NF-B, activation also induces pDC maturation, NFAT, and AP-1 activity. 4) Dasatinib expressing co-stimulatory molecules and completely abrogated T-cell proliferation, TRAIL (TNF-Related Apoptosis Inducing impeding SAMHD1 phosphorylation. This Ligand) that mediates CD4+ T-cell was the major mechanism of action of apoptosis. In the HIV-infection scenario we Dasatinib as it showed no effect on viral have shown that these functions are transcription. 5) Activation ex vivo of associated with the spontaneous control of PBMCs from patients with chronic myeloid the virus. This occur in less than 1% of HIV- leukemia on treatment with Dasatinib for infected subjects, the so called HIV elite several years showed that in vivo controllers (EC), who are able to maintain treatment with Dasatinib prevented undetectable viral loads during a long SAMHD1 phosphorylation. period of time in the absence of CONCLUSIONS: SAMHD1 plays a central antiretroviral treatment. These role in HIV-1 replication and in the mechanisms also account for hepatitis C establishment of viral reservoirs, thereby virus (HCV) infection. We have shown representing a major target for therapeutic lower HCV viral loads and others, superior intervention. PKC and p56Lck inhibitors, rates of HCV virus spontaneous clearance able to preserve SAMHD1 antiviral in HIV elite controllers. The understanding function, could be promising as adjuvant of of the mechanisms and characteristics of ART to reduce the reservoir size. Dasatinib the pDCs in this extraordinary subjects who

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are able to control these two infections, immunotherapeutic strategies for the may be very interesting for the treatment of chronic viral infections. development of immunotherapeutic strategies for HIV and HCV infection. PLENARY SESSION V (PS V): STRUCTURAL On the other hand, in most of the HIV- ANALYSIS OF VIRUS AND infected subjects the control of viremia is BIOTECHNOLOGY possible thanks to combined antiretroviral Chairpersons: treatment (cART). However, despite viral load remains undetectable, survival of JOSÉ ESTÉ AND MARJORIE PION patients on cART is ten year lower than the Tuesday June 9, 2015 general population. Most of the patients AUDITORIUM REAL CASA DE LA MONEDA died because of non AIDS events (NAEs), such as, hepatic complications in HCV co- *Invited plenary lecture infected patients, cardiovascular disease, 11:15-11:45h (P12) tumours and other age-related diseases, caused by low grade systemic chronic HIV-1 EVOLUTION: DRUG-RESISTANCE inflammation due to the activation of the AND NUCLEOTIDE COMPOSITION innate immune system. Using B. BERKHOUT multiparametric flow cytometry we are Laboratory of Experimental Virology, Department of observing that after TLR-2, TLR-4 and TLR- Medical Microbiology, Center for Infection and 7-specific stimulation the polyfunctionality Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the of monocytes, i.e.: production of cytokines Netherlands at the same time per cell, of HIV-infected patients on suppressive cART is higher than both, elderly (85 years old) and healthy Evolution of the human immunodeficiency subjects with the same age, pointing out to virus type 1 (HIV-1) was studied in diverse settings. First, we will discuss how HIV-1 specific HIV-related dysregulation of the st nd rd monocyte function contributing to the gains resistance to the 1 /2 /3 long-term development of NAEs. In the generation of peptidic entry inhibitors. We same way, aberrant IFN-I production of describe for the first time the evolution of pDCs also occurs on cART which drug-dependent virus variant in a patient. contributes to immune system We discuss the cascade of more and more dysregulation and has been associated difficult molecular resistance mechanisms with cardiovascular diseases development. against the three generations of inhibitor. We also demonstrate that resistance The double-edged sword of pDCs and against the optimized peptides is monocytes/macrophages in the control extremely difficult to achieve and that it and pathogenesis of HIV and HCV-infection comes at a prize: a significant loss of HIV-1 will help to both: the better understanding fitness. Although the therapeutic power of of the physiological function of these these improved peptides is enormous, also components of the innate immune system for non-HIV viruses that use similar and to the development of entry/fusion strategies, the need for drug

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injection remains a significant vivomodels of virus evolution, and disadvantage for this drug class. mathematical modeling to examine several HIV-1 evolution was also studied from a aspects of RNA virus biology. We provide quite different perspective: the profoundly evidence that evolutionary trajectories of biased nucleotide composition of the RNA viruses may be inherently predictable single-stranded viral RNA genome. HIV-1 and we show that phenotype is RNA is A-rich (more than 36%) and C-poor (less than 17%). Other (retro)viruses have determined by the group contribution of found other particular niches in sequence minority variants. Finally, we show how space. For instance, the HTLV-I multi-dimensional reduction of deep RNA is C-rich and G-poor and the sequence data may be used to generate coronavirus RNA is U-rich and C-poor. We maps of sequence space, with which we will discuss the evolutionary pressures that can generate empirical fitness landscapes may have shaped these genomes, but also the functional consequences, e.g. in codon to better monitor virus populations during usage. Using a novel phylogeny-instructed adaptive walks. mutagenesis (PIM) strategy, we recently generated HIV-1 constructs with an *Invited plenary lecture increased and decreased A-count in a small genome segment. The properties of these 12:15-12:45h (P14) virus variants will be presented. VIRUSES AS TOOLS FOR THERAPEUTIC INTERVENTIONS IN CANCER AND *Invited plenary lecture INFECTIOUS DISEASES 11:45-12:15h (P13) G. PALÙ RNA VIRUS POPULATION DYNAMICS IN Department of Molecular Medicine, University of Padova, Padova, Italy SEQUENCE SPACE AND FITNESS

LANDSCAPES After a short overview of the field, a few M. VIGNUZZI approaches will be described of preclinical Institut Pasteur, Paris, France and clinical deployment of viral vectors for new advanced molecular therapies of cancer, AIDS and allied genetic disorders. RNA viruses form highly diverse Details will be reported on: i) appropriate populations or quasispecies. Recent design, ii) suitable strategies of advances in sequencing technologies now HIV gene knock-down, iii) metabolic and makes it feasible to characterize the immune-inflammatory treatment of cancer genetic structure of these populations and and iv) gene editing of stem cells derived monitor their evolution in space and time from somatic lines. as they adapt to a host environment. We use NGS approaches, in vitro and in

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ORAL PRESENTATIONS (OP III): *Invited paper STRUCTURAL ANALYSIS OF VIRUS AND 13:00-13:15h OPIII(CO7) BIOTECHNOLOGY STRUCTURAL ANALYSIS OF VIRAL AND Chairperson: FRANCISCO SOBRINO VIROIDAL RNA USING ATOMIC FORCE Tuesday June 9, 2015 MICROSCOPY AUDITORIUM REAL CASA DE LA MONEDA CARLOS BRIONES

Department of Molecular Evolution, Centro de *Invited paper Astrobiología (CSIC-INTA), Torrejón de Ardoz, 12:45-13:00h OPIII (CO6) Madrid, Spain. BACTERIOPHAGEø29. FROM MOLECULAR Centro de Investigación Biomédica en Red de enfermedades hepáticas y digestivas (CIBERehd), BIOLOGY TO BIOTECHNOLOGY Spain. MARGARITA SALAS Centro de Biología Molecular “Severo Ochoa” (CSIC– RNA is a macromolecule of paramount UAM) Madrid. importance in biology. Apart from storing heritable information in RNA viruses and The Bacillus subtilis phage ø29 has a linear, viroids, RNA plays key roles in the flow of double-stranded DNA (19.275 bp) with a genetic information, including the control protein, named terminal protein (TP), of gene expression, the initiation of cap- covalently linked to the 5’ DNA ends. In the independent translation by Internal presence of the TP-DNA template a Ribosome Entry Site (IRES) elements, and molecule of TP primes the initiation of ø29 the catalysis of the peptidyl transferase DNA replication by formation of the TP- reaction within the ribosome. Additionally, dAMP initiation complex catalyzed by the different RNA molecules function as phage DNA polymerase. Then, the same efficient catalysts (ribozymes), natural polymerase catalyzes chain elongation in a RNAs (riboswitches) as well as in vitro highly processive way coupling selected ones (aptamers) specifically polymerization to strand displacement. recognise molecular targets, and certain These two properties of the ø29 DNA RNAs provide a structural scaffold in polymerase, high processivity and strand ribonucleoprotein aggregates. displacement capacity, together with a RNA molecules adopt specific three- high fidelity, make of this enzyme an dimensional structures critical to their outstanding polymerase to amplify DNA. In function, and different physicochemical fact, ø29 DNA polymerase has been techniques are currently used to analyse commercialized to amplify both circular RNA structure. Among them, Atomic Force and linear genomic DNA. In addition, we Microscopy (AFM) is a nanotechnology- have improved the ø29 DNA polymerase based technique belonging to the group of performance by fusion of DNA binding Scanning Probe Microscopies, whose motifs. nanometre resolution in air and liquid environments is optimal for the visualisation of RNA molecules of different

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lengths, as well as RNA-RNA or RNA- 5. Flores et al. (2014). Annu. Rev. Microbiol. 68: protein complexes adsorbed on flat 395. surfaces (1). AFM does not require any staining or coating of the imaged molecule, *Invited paper thus minimising its structural disruption. 13:15-13:30h OPIII (CO8) Currently, AFM is used in different fields of BREAKING THE BARRIERS OF virology (2). BACULOVIRUS-BASED PRODUCTION We have set up AFM technique to analyse TECHNOLOGIES the native structure of viral and viroidal J.M. ESCRIBANO RNA molecules. First, the Mg2+-dependent folding of the Hepatitis C Virus (HCV) IRES Departamento de Biotecnología, INIA, Madrid, element has been investigated. A sharp Spain structural switch was monitored in a HCV IRES-containing, 574 nt-long RNA molecule Baculoviruses are widely used in different 2+ (3) when Mg concentration increased biotechnology applications including pest from 2 to 4 mM. This conformational control pests and production of adeno- rearrangement was hindered by the associated viruses or recombinant presence of the microRNA miR-122. The proteins. The use of baculovirus vectors in 2+ competing effect of Mg and mir122 research about protein function and allowed envisaging a model for the long- structure or to industrial production of range RNA-RNA interaction within HCV different recombinant proteins has grown IRES in its natural sequence context (4). in recent decades. Since the development We are also analysing the 3D structure of of the baculovirus vector expression genomic viroid RNAs belonging to the system (BEVS) in the ’80s, thousands of families Pospiviroidae (PSTVd, 359 nt-long) recombinant proteins, ranging from and Avsunviroidae (ELVd and PLMVd, 332- cytosolic enzymes to membrane-bound 351 nt-long) in different ionic conditions. proteins, have been successfully produced Our AFM images confirm the main features in baculovirus-infected insect cells. During of their previously known rod-like and more than 30 years of continuous multibranched secondary structures, improvements in the BEVS, only a modest respectively (5), and provide information 30-40% increase in productivity was on viroid tertiary structure. This talk will achieved. Two bottle necks limited the summarise our main results and challenges industrial use of the BEVS, the maximum ahead. production yields reached in comparison to 1. Hansma et al. (2004). Curr. Opin. Struct. Biol. the most optimized systems (milligrams vs 14, 380. grams per liter), and the frequent partial 2. Kuznetsov et al. (2010). Nucleic Acids Res. 38, proteolysis found as a consequence of the 8284. damages induced in insect cells during 3. Beguiristain et al. (2005). Nucleic Acids Res. infection by the vector (apoptosis). 33, 5250. Recently (Gómez-Sebastian et al., 2014), a 4. García-Sacristán et al. (2015). Nucleic Acids baculovirus vector expression cassette Res. 43, 565. containing rearranged baculovirus-derived

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genetic regulatory elements has been productive method to develop the next described. This newly designed expression generation vaccines. cassette reduces the above mentioned limitations in production of the BEVS PLENARY SESSION VI (PS VI): PLANT VIRUS (400% increase), and also improved protein integrity by prolonging cell viability Chairperson: JUAN ANTONIO GARCÍA as a consequence of a virus-induced Wednesday June 10, 2015 apoptosis delay with respect to a standard AUDITORIUM REAL CASA DE LA MONEDA baculovirus vector. Baculoviruses modified with this expression cassette have been *Invited plenary lecture used for efficient production of vaccines 9:00-9:40h (P15) based on virus-like particles and surface glycoproteins. In addition, during the last NEW CONCEPTS IN THE BIOLOGY OF decade, several research groups and MULTIPARTITE VIRUSES companies have been using insects as A SICARD1, J-L ZEDDAM1,2, M YVON1, Y living biofactories in combination with MICHALAKIS3, S GUTIERREZ1 AND S baculovirus vectors for recombinant BLANC1#. protein production. The most widely used 1. INRA, UMR BGPI, Montpellier, France. insects are Bombyx mori and Trichoplusia 2. IRD, UMR RPB, Montpellier, France niLepidoptera. Insects offer unique 3. CNRS, UMR MIVEGEC 5290, Montpellier, France. advantages of productivity, scalability and success with difficult-to-express proteins. A Multipartite viruses are characterized by a considerable number of recombinant genome composed of two or more nucleic proteins have been produced in insects as acid segments, each encapsidated living bioreactors and in most cases those individually. A classical view in virology were produced at levels never reached in assumes that the viral replication cycle insect cells, obtaining yields of grams per occurs within individual cells, where the liter of extract, comparable to the most whole viral genome information is productive systems based on bacteria, replicated, and is then reiterated in yeast or mammalian cells. These insect- successively infected cells during host based production methods are currently invasion. In the context of multipartite used for production of diagnostic reagents viruses, this view implies that at least one and several subunit vaccines for animal copy of each of the genome segments health, produced in these biofactories, are must repeatedly enter together in under development and will be marketed individual cells for successful infection. in the near future. In conclusion, Because one or more genome segments baculovirus vectors are excellent allies for may be missing in numerous susceptible the biotechnological and pharmaceutical cells, thus aborting infection, these viral companies in the production of biologics systems are believed to bear an enormous and recent advances configured the BEVS cost, which drastically increases with the as one of the most cost-efficient and number of segments constituting the viral

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genome. It has even been concluded that *Invited plenary lecture multipartite viruses with an elevated 9:40-10:20h (P16) number of segments (as for example MEMBRANE REARRANGEMENTS IN PLANT member species of the family ) VIRUS RNA REPLICATION appear so costly that they should not have evolved and should thus not exist! L RUBINO Institute for Sustainable Plant Protection, CNR, Bari, To address this apparent paradox, we have Italy experimentally tested the thus far undisputed assumption that the segments of a multipartite virus must be together Positive-strand RNA viruses constitute a within individual cells for the system to be large group of infectious agents causing functional. For this, we used the major plant, animal and human diseases. Faba bean necrotic stunt virus, which Genome replication occurs in association genome is composed of 8 ssDNA segments, with host cell membrane structures each encapsidated individually. Our results derived from the endoplasmic reticulum indicate that the various segments are not (ER) (picornaviruses, , always together within individual cells and , and yet, that the system scattered over several bromoviruses) or from the limiting distinct cells appears functional. This membrane of organelles such as lysosomes observation has several important or endosomes (), vacuoles implications. First, it questions the cost (cucumoviruses), mitochondria that has always been attributed to (nodaviruses, some , multipartite viral systems, where gathering carmoviruses, ampeloviruses and a copy of each segments in single cells was maculaviruses), peroxisomes (several though to be mandatory. Second, it tombusviruses) and chloroplasts demonstrates that the replication cycle of (tymoviruses and some marafiviruses). a virus is not necessarily “cell- Viral proteins are involved in targeting the autonomous” and that the spatial unit of a replication complex to the specific virus replication cycle can be, in some intracellular membranes. Intracellular cases, an ensemble of interconnected cells membranes are normally modified to form within which the various part of viral vesicular structures with a narrow neck genetic information are obviously through which the interior of the vesicles communicating. communicates with the cytosol. A variety of observations indicates that, indeed,

virus replication takes place in the closed environment of the vesicles, including co- localization of virus replicase and virus RNA progeny with cell membranes and strong dependance of viral synthesis on lipid metabolism. Confinement of the virus

replication complexes in closed environments represents an advantage for

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the viral RNA, which is protected from THE VIROLOGIST CONFERENCE SENIOR degradation of host ribonucleases and AWARD recognition of host defence reactions. Chairperson: JUAN ANTONIO GARCÍA Vesiculation of the target cellular Wednesday June 10, 2015 membrane in natural hosts and in the yeast Saccharomyces cerevisisae, an AUDITORIUM REAL CASA DE LA MONEDA alternative model host for studying virus replication, is well documented for *Invited plenary lecture nodaviruses (animal viruses) and bromo- 12:00-12:30h (P17) and tombusviruses (plant viruses). Flock TEN YEARS OF HEPATITIS C VIRUS CELL house virus (FHV, genus Nodavirus, family CULTURE INFECTION MODELS ) protein A is a transmembrane PABLO GASTAMINZA LANDART protein that contains N-terminal signals Laboratorio de Infección por el Virus de la Hepatitis targeting the outer membrane of C mitochondria and elicits the formation of Centro Nacional de Biotecnología-Consejo Superior vesicular structures. Brome mosaic virus de Investigaciones Científicas (CNB-CSIC)-Madrid (BMV, genus , family (SPAIN) ) replication occurs on the ER membranes, in spherules containing Since its identification as the etiologic genomic RNA, the 2a replicase protein and agent for the non-A, non-B hepatitis in the 1a virus RNA replication factor. The 1a 1989, hepatitis C virus (HCV) could not be multifunctional protein has RNA capping robustly cultured in vitro. In fact, to date and helicase functions, and directs and despite enormous efforts from the targeting and assembly of the replication scientific community, it is not possible to complex on the ER membranes. The efficiently culture primary virus isolates replication of members of the genus from HCV-infected patients. In 2005, (family ) has several laboratories reported the been studied in plant and yeast cells. possibility of producing infectious hepatitis Carnation Italian ringspot virus p36 protein C virus from cloned cDNA. This contains the determinants for targeting the recombinant genotype 2a strain was replication complex to the outer generated by Dr. Takaji Wakita from a membrane of mitochondria; the p33 of consensus sequence of the viruses several other tombusviruses contains circulating in a Japanese patient with sequences necessary to localize virus Fulminant Hepatitis (JFH-1). Transfection of replication on the limiting membrane of in vitro transcribed, full-length genomic peroxisomes. RNA, produced, for the first time, infectious HCV virions with relatively high efficacy in the supernatants of the transfected cells.Ever since, new infectious molecular clones capable of producing infectious virions from different HCV genotypes have been developed. By

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recapitulating every aspect of the viral CLOSING LECTURE lifecycle, these unique tools have been Chairperson: instrumental for the study of basic aspects MIGUEL ÁNGEL JIMÉNEZ-CLAVERO of HCV infection in cell culture, especially those that were not recapitulated in other Wednesday June 10, 2015 experimental systems such as replicons AUDITORIUM REAL CASA DE LA MONEDA and HCV glycoprotein-pseudotyped retroviral vectors. In this sense, our *Invited plenary lecture contribution to the field throughout these 12:30-13:15h (P18) years involves the description of MECHANISMS OF VIRAL PERSISTENCE IN biophysical and ultrastructural features of INSECTS infectious HCV virions as well as the identification of cellular determinants that MARIA CARLA SALEH mediate infectious HCV assembly and Institut Pasteur, Viruses and RNA interference Unit, Paris, France. secretion. In recent years, we have focused our attention in the study of host-virus interactions to better understand the The establishment and maintenance of molecular and cellular mechanisms persistent viral infections is widely debated underlying different aspects of HCV and remains largely misunderstood. We infection. In addition, our group has taken show that in Drosophila, persistence is advantage of cell-based screening assays achieved through a mechanism involving to identify novel molecules with antiviral reverse transcription of non-retroviral RNA potential, with the aim of developing new virus and the RNA interference pathway. tools to dissect the virus life cycle and with Fragments of diverse RNA viruses are the hope of identifying new ways of reverse transcribed early during infection, tackling this important pathogen. resulting in DNA forms embedded within LTR-retrotransposon sequences. Inhibition of reverse transcription hinders the

appearance of these DNA forms and is accompanied by increased viral titers and cell death. These viral/retrotransposon- DNA chimeras produce transcripts that are processed by the RNAi machinery. Knocking down RNAi components in

persistently infected cells shifts the equilibrium from persistent to acute infection. Our results reveal an unanticipated physiological function for retrotransposons and propose a role in RNAi-mediated immune protection for

parasitic viral insertions into host genomes.

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PL: P A R A L L E L S E S S I O N

Parallel Session I: had been observed since the 1950’s, EMERGING VIRUSES AND VETERINARY although ceased in the 1970’s and 80’s, to Chairpersons: re-emerge in the 90’s. Since then the virus increased its incidence and geographic ANA M. DOMENECH AND JAVIER ORTEGO spread steadily, up to 2008 where a Monday June 8, 2015 recrudescence of the epidemiological AUDITORIUM REAL CASA DE LA MONEDA situation was observed in most of Europe, still lingering. 15:00-15:15h (CO 9) In Spain, little was known about WNV until 2003. That year, the EVITAR (Vector and 2003-2015: 12 YEARS OF RESEARCH ON Rodent-Borne Viral Diseases) network MOSQUITO-BORNE EPORNITIC started its activity. Since then a systematic FLAVIVIRUSES IN SPAIN. WEST NILE, collaborative work conducted in an USUTU, BAGAZA…AND BEYOND interdisciplinary way (the “One Health M. A.JIMÉNEZ-CLAVERO1, E. PEREZ- 1 1 approach”) produced an outstanding RAMÍREZ , F. LLORENTE , J. FERNÁNDEZ- advance in the knowledge of WNV and PINERO1, A. VÁZQUEZ2, M. P. SÁNCHEZ- 2 3 3 other epornitic flaviviruses in our country. SECO , J. FIGUEROLA , R. C. SORIGUER , S. Serosurveys in wild birds, horses and RUIZ4, R. VILLALBA5, C. GÓMEZ-TEJEDOR5, 5 2 1 humans in Southern Spain evidenced WNV M. AGÜERO , A. TENORIO . . sylvatic circulation for the first time in 1 2 INIA-CISA, Valdeolmos, Spain; CNM-ISCIII, 2004, while monitoring of mosquito Majadahonda, Spain; 3Doñana Biological Station, 4 populations in that area identified WNV CSIC, Seville, Spain; Servicio de Control de Mosquitos, Diputación de Huelva, Spain; and many other flaviviruses, some new to 5Laboratorio Central de Veterinaria, Algete, Spain. science, including a new WNV genetic lineage, while others, (e.g. Usutu virus), had known zoonotic potential. The group In 1999 an African virus, West Nile virus, developed new technologies to detect the unexpectedly emerged in New York, and virus and antibodies to it, and toinvestigate spread relentlessly throughout America, the feeding preferences of mosquito causing tens of thousands of encephalitis species found relevant for cases in humans and horses, and transmission, thus unveiling transmission uncountable (probably millions) deaths in pathways potentially leading to WNV wild birds, in one of the most remarkable spillover to humans and horses in the area, episodes of virus emergence one can eventually occurring in 2010 in Cádiz. mention. The virus still persists and has Members of the network isolated WNV for become endemic in wide areas in the the first time in Spain in 2007 from Americas. affected golden eagles, while in 2010 they Meanwhile in the Old World the virus isolated another flavivirus, Bagaza virus, produced sporadic, self-limited outbreaks new to Europe, in an encephalitis outbreak with little or no human affection. WNV was affecting wild birds in Cádiz. Since then, long known in Europe and the dozens of flavivirus isolates, from Spain Mediterranean where sporadic outbreaks and elsewhere, were characterized by full

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genome sequencing, establishing new effect in double stranded DNA viruses. We phylogenetic relationships, and have analyzed a possible antiviral function determined their virulence and host of IFITMs against a double stranded DNA competence using newly established or virus: The African swine fever virus (ASFV). optimised bird and mammal models of This virus has been shown to be inhibited flavivirus infection. by other IFN-response gene such as MxA The “core” of the EVITAR network, (Netherton et al, 2009). Infection by ASFV represented by those signing this work, still is IFN (interferon)-sensitive and induces pursues high quality research on vector- IFITMs expression in vitro. Then, we borne zoonotic diseases of public health investigated whether IFITMs expression importance in our country. could impair viral infection. Expression of IFTM1, 2 and 3 reduced virus replication,

with IFITM2 and 3 having an impact on 15:15-15:30h (CO 10) viral entry/uncoating. We will discuss the THE ANTIVIRAL EFFECT OF INTERFERON potential mechanisms to inhibit virus INDUCED TRANSMEMBRANE PROTEINS infection induced by IFITMs. (IFITMs) IN AFRICAN SWINE FEVER VIRUS *Animal Disease Notification System: INFECTION Outbreaks per Disease. European MUÑOZ-MORENO, R.1,2, C. MARTÍNEZ- Comission,pp. 8. 2 1 1 ROMERO , I. GALINDO , L. BARRADO-GIL M. A. CUESTA-GEIJO1, M. TAMAYO1, A. 15:30-15:45h (CO 11) GARCÍA-SASTRE2 AND C. ALONSO1. AMINO ACID SUBSTITUTIONS IN THE 1 Department of Biotechnology, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria NON-STRUCTURAL PROTEINS 4A OR 4B (INIA), Madrid, Spain MODULATE THE INDUCTION OF 2 Present address: Department of Microbiology, AUTOPHAGY IN WEST NILE VIRUS Icahn School of Medicine at Mount Sinai, New York, INFECTED CELLS USA A.B. BLÁZQUEZ1, M.A. MARTÍN-ACEBES1,2, 1 J.C. SAIZ Rapid spread of African swine fever virus 1Departamento de Biotecnología. INIA, Madrid, across Eastern Europe in the recent years Spain resulted in emergence of the disease in a 2 Centro de Biología Molecular Severo Ochoa (CSIC- number of EU countries including the Baltic UAM), Cantoblanco, Madrid,Spain Republics and Poland*. The interferon- induced transmembrane (IFITM) protein West Nile virus (WNV) is a neurotropic family is a group of antiviral restriction mosquito-borne flavivirus responsible for factors that impair flexibility and inhibit outbreaks of meningitis and encephalitis, membrane fusion restricting viral for which no vaccines or antivirals for progression at early infection. While human use are available. Autophagy is a IFITMs are widely known to inhibit several catabolic mechanism that sequesters single-stranded RNA viruses, there are cytoplasmatic components for limited reports available regarding their degradation. The autophagic pathway can

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be upregulated to cope with diverse forms the ability to induce the hallmarks of of cellular stress, including viral infections. autophagy such as LC3 modification and In the case of flaviviruses, the autophagic aggregation. Even more, the differences on pathway can play multifaceted roles during the induction of an autophagic response the infection of these pathogens that observed among WNV variants in infected include rearrangements of cellular lipid cells did not correlate with alterations on metabolism, contribution to viral the activation of the unfolded protein maturation, or involvement in the early response (UPR), suggesting an uncoupling steps of the infection. Whereas the of UPR and autophagy during flavivirus activation of autophagy in cells infected infection. The findings here reported could with other flaviviruses is well known, the help to improve the knowledge of the interaction of WNV with the autophagic cellular processes involved on flavivirus- pathway still remains unclear and there are host cell interactions and contribute to the reports describing opposite findings design of effective strategies to combat obtained even analysing viral strains with a these pathogens. common origin. To clarify this controversy, we first analysed the induction of 15:45-16:00h (CO 12) autophagic features in cells infected with a panel of WNV strains. WNV was ROLE OF SARS-CoV VIROPORINS E, 3a AND determined to induce autophagy in a strain 8a IN VIRUS REPLICATION AND dependent manner. We observed that all VIRULENCE 1 WNV strains or isolates analyzed, except C. CASTAÑO-RODRIGUEZ , JL. NIETO- 1 1 one (NY99), upregulated the autophagic TORRES , ML. DEDIEGO , C. VERDIÁ- 2 1 pathway in infected cells. Even more, BÁGUENA , JM. JIMENEZ-GUARDEÑO , JA. 1 1 interestingly, a variant derived from this REGLA-NAVA , R. FERNANDEZ-DELGADO , 2 1 NY99 isolated from a persistently infected VM. AGUILELLA VM , L. ENJUANES mouse (B13) also increased LC3 1Department of Molecular and Cell Biology, modification and aggregation. Complete National Biotechnology Centre (CNB-CSIC), Darwin genome sequencing of this B13 variant 3, Universidad Autonoma de Madrid, 28049 Madrid, Spain. revealed only two non-synonymous 2 Department of Physics, Laboratory of Molecular nucleotide substitutions when compared Biophysics. Universitat Jaume I, 12071 Castellón, to parental NY99 strain. These nucleotide Spain. substitutions introduced one amino acid replacement in NS4A and other in NS4B. SARS-CoV has three viroporins: 3a, E and Using genetically engineered viruses we 8a. We have engineered recombinant showed that introduction of any one of SARS-CoV (rSARS-CoV) variants missing these replacements, alone or in each of these proteins. Their analysis has combination, was sufficient to upregulate shown that none of them are essential for the autophagic pathway. Thus, in this work virus replication and that proteins E and 3a we have shown that naturally occurring are relevant in virulence. Interestingly, a point mutations in the viral non-structural virus lacking both E and 3a genes could not proteins NS4A and NS4B confer WNV with

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be rescued suggesting that a for the complementation. Experiments to complementation between proteins E and define the basis of this complementation 3a is required for virus viability. This are being performed. hypothesis is supported by the fact that the two proteins co-localize in the cell. 16:00-16:15h (CO 13) These two viroporins share at least two activities: PDZ binding motif (PBM) and ion VIROLOGICAL AND EPIDEMIOLOGICAL channel (IC), which are virulence factors in FEATURES OF CHIKUNGUNYA VIRUS SARS-CoV. In the present work we studied INFECTION AMONGST TRAVELERS whether 3a and E protein IC activities are RETURNING TO SPAIN, 2008-2014 1,2,3 †1,4 responsible for this complementation. To L FRANCO , MD FERNÁNDEZ , M † 1, 5 1,2 1 this end, rSARS-CoV in which IC activities of BANGERT , F DE ORY , A POTENTE ,L 1 1 1 E protein or 3a protein are knocked out, HERNANDEZ , F LASALA , L HERRERO , F 1 1,3 were firstly engineered. In order to MOLERO , A I NEGREDO , MP SÁNCHEZ- 1,2,3 generate rSARS-CoV without E or 3a SECO AND SNS HOSPITALS. protein IC activity (rSARS-CoV-EIC-, rSARS- † Equally contribution CoV-3aIC-), the amino acids involved in 1-Centro Nacional de Microbiología, Instituto de their IC activity were determined. E protein Salud Carlos III, Madrid-Spain 2-VIRORED,CYTED has a single transmembrane domain (TMD) 3-Red de Investigación Cooperativa en while 3a protein has three TMDs. The Enfermedades Tropicales (RICET),RETICS conductance of synthetic peptides of each 4- Pasteur Institute, Dakar-Senegal of the TMDs and full-length proteins with 5-European Programme for Public Health native or mutant sequences was measured Microbiology Training (EUPHEM) ECDC, Stockholm- Sweden in artificial membranes and amino acids involved in 3a and E proteins IC activity were identified. Then, rSARS-CoV-3aIC- and Chikungunya is endemic in some parts of rSARS-CoV-EIC- viruses were constructed. Africa, Southeast Asia and in the Indian Evaluation of rSARS-CoV-EIC- pathogenicity subcontinent. In late 2013, the first in BALB/c mice showed that E protein IC documented autochthonous transmission activity is a virulence factor. Furthermore, of chikungunya virus (CHIKV) was reported E protein IC activity is required for in the Caribbean island of Saint Martin and inflammasome activation, which triggers since then the infection has spread quickly the expression of proinflammatory in countries and territories in the cytokines leading to edema accumulation Caribbean region, North, Central and South and ARDS. In contrast, 3a protein IC activity America. With the ongoing outbreak in the was not essential for SARS-CoV virulence. Caribbean, CHIKV is increasingly becoming Therefore, the complementation between a European public health threat. proteins E and 3a does not seem to be Transmission of CHIKV to humans occurs mediated by the IC activities of these through bites of Aedes aegypti and Ae. proteins, which have different relevance in albopictus mosquitoes. In Europe virus virulence. We suggest that the PBMs Ae.albopictus is established primarily of proteins E and 3a could be responsible around the Mediterranean basin and has

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been demonstrated its competency for because the conditions for autochthonous virus transmission. This resulted in locally- transmission are met: presence of acquired infections in Italy (Emilia competent vector and a large number of Romagna, 2007), and in France (Var and travelers returning from affected areas like Montpellier, 2010 and 2014 respectively). the Caribbean and northern South In this study, we analyzed six years of America. imported chikungunya infections in Spain. During the study period (2008-2014), a 16-15-16:30h (CO 14) total of 1311 suspected chikungunya infections were studied and more than half HEMAGGLUTININ PROTEIN OF PESTE DES were in 2014 alone. From 2008 to 2013, 30 PETITS RUMINANTS VIRUS ACTIVATES THE laboratory-confirmed (PCR, IgM/IgG) INNATE IMMUNE RESPONSE VIA TOLL- chikungunya infections were imported LIKE RECEPTOR 2 SIGNALING 1 2 whereas in 2014 there were 195 confirmed E. PASCUAL , S.R. WATTEGEDERA , C. 3 1 2 cases. The presence of IgM or IgG SANTIAGO , V. MARTÍN , G. ENTRICAN 1 antibodies against chikungunya was AND N. SEVILLA performed by immunofluorescence on 830 1 Centro de Investigación en Sanidad Animal (CISA)- out 1311 suspected patients with 188 INIA, Madrid, Spain 2 positive for IgM (22.6%) with or without Moredun Research Institute, Edinburgh, Scotland 3 IgG testing. Molecular diagnosis (RT-PCR) Centro Nacional de Biotecnología-CSIC, Madrid, was performed on 452 out 1311 suspected Spain patients (34.4%) with viral genome detected in 35 (7.7 %). Majority of Toll-like receptors (TLR) are a family of chikungunya cases with known travel proteins expressed in almost all cell types history in the period 2008-2013 reported that act as sentinels of the host innate travel to Asia whereas in 2014 the main immune system. TLR family comprises both travel destination were the Americas membrane and intracellular receptors that (Dominican Republic, Haiti and Venezuela). recognize different types of pathogen Virus sequencing revealed that all samples associated molecular patterns (PAMPs) from Americas felt into Asian genotype leading to the production of pro- (Caribbean Clade), although ECSA/Indian inflammatory cytokines and also Ocean genotype was detected in cases stimulating the development of long- imported from Asia and Africa. Most of the lasting adaptive immunity. TLR2 is located positive samples in 2014 clustered around in the cell surface and, although it was May and October, the activity period for initially thought to act as a bacterial the vector present in Spain. We have sentinel, it has been shown to recognize a described a 6.5 fold increase of imported number of viral glycoproteins. In this study chikungunya infections from the whole we sought to characterize the role of TLR2 period 2008-2013 (30 cases) compared to in the activation of the immune response 2014 (195 cases), the highest number by peste des petits ruminants virus (PPRV), recorded in Spain. Chikungunya is an a morbilivirus of the Paramixoviridaefamily emerging public health threat to Spain

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that causes an acute, highly contagious 16:30-16:45h (CO 15) disease in goats and sheep. POSTNATAL PERSISTENT INFECTION WITH Using 293 cells stably expressing human CLASSICAL SWINE FEVER VIRUS IN TLR2 but lacking any other TLR we found DOMESTIC PIGS AND WILD BOARS: that inactivated virus of the vaccine strain OPENING PANDORA'S BOX Nigeria/75 of PPRV induces IL-8 production L. GANGES1*, S. MUÑOZ-GONZÁLEZ1, O. in a dose-dependent manner. That CABEZÓN1,2, N. RUGGLI3, M. PEREZ-SIMÓ1, activation is only observed in cells J. A. BOHÓRQUEZ1, R. ROSELL1,4, I. expressing TLR2 and is greatly reduced MARCO2, S. LAVÍN2, A. SUMMERFIELD3 when the receptor is blocked by the AND M. DOMINGO1,5 pretreatment with a specific antibody. We 1 Centre de Recerca en Sanitat Animal (CReSA), identified hemagglutinin (H) as the Institut de Recerca i Tecnologia Agroalimentàries responsible of TLR2 activation by (IRTA), Campus de la UAB, Bellaterra, Barcelona, Spain performing the same assays with 2 recombinant, mammalian-expressed Wildlife Health Service (SEFaS) from the Veterinary School of the Universitat Autònoma de Barcelona, purified protein. The exogenous addition Bellaterra, Barcelona, Spain of H to the cell culture induces high levels 3 Institute of Virology and immunology (IVI), of IL-8 only in TLR2-expressing cells. In Mittelhäusern, Switzerland 4 order to assess whether TLR2 signaling can Departament d'Agricultura, Ramaderia, Pesca, also be induced by PPRV in antigen Alimentació i Medi Natural, (DAAM), Generalitat de Catalunya, Spain presenting cells, we isolated ovine 5Departamento de Sanitat i d’Anatomia Animals, dendritic cells from peripheral blood Facultat de Veterinària, UAB, Bellaterra-Barcelona, monocytes and evaluated cytokine Spain production upon stimulation with either inactivated virus or purified H protein by It is well established that trans-placental RT-qPCR. In both cases, we observed a transmission of classical swine fever virus significant increase in IL-8 production, (CSFV) during mid-gestation can lead to suggesting activation by TLR2 engagement. persistently infected offspring. The aim of The involvement of these results on the this work was to evaluate the ability of host immune mechanisms in the control of CSFV to induce viral persistence upon early PPRV infection will be discussed. postnatal infection in wild boars and domestic pigs. Ten new-born domestic piglets and fifteen new-born wild boar were infected intranasally within the first 10 hours after birth with the Catalonia 01

strain (Cat01, CSFV of moderate virulence). Viral replication, innate and specific immune responses were evaluated. During six weeks after postnatal infection (duration of the experiment), most of the piglets remained clinically healthy, despite

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persistent high virus titres in the serum, the induction of persistent infection in wild organs and body secretions. The levels of boar provides new insights towards viral RNA detected were similar in both, understanding possible mechanism of domestic pigs and wild boar. The clinical maintenance of CSFV in the European signs recorded were also similar, with countries. These experiments were some temperature peaks above 40 ºC approved by the Ethics Committee for mainly during the first 14 days post Animal Experiments of the Autonomous infection. Approximately 50 percent of University of Barcelona (UAB) according to infected animals (pigs and wild boar) were existing national and European regulations. persistently infected without any clinical signs at the end of the experiment. 16:45-17:00h (CO 16) Importantly, these animals were unable to mount any detectable CSFV-specific GETTING IC-TAGGING TO WORK INTO THE humoral immune response. Four weeks ENDOPLASMIC RETICULUM 1 1 after infection, PBMCs from the N. BARREIRO PIÑEIRO , I. LOSTALÉ SEIJO , 1 persistently infected seronegative piglets J. BENAVENTE MARTÍNEZ , J. MARTÍNEZ 1 were unresponsive to both, specific CSFV COSTAS and non-specific PHA stimulation in terms 1 Laboratorio de Virología Molecular, Centro of IFN-γ-producing cells. In the case of wild Singular de Investigación en Química Biológica y boar, heterogeneous results were Materiales Moleculares, Santiago de Compostela, Spain observed in the INF-γ-producing cells after

CSFV and PHA stimulations. Although scarce, IFN-γ-producing cells were Viral factories or are structures detected upon CSFV stimulation in three where viral components are recruited to out of nine inoculated wild boar at week 4. assemble the viral progeny. A single Furthermore, high level of IFN-γ-producing nonstructural protein named muNS is cells was detected after PHA stimulation in responsible for the formation of avian four of the infected animals. However, a reovirus viroplasms. Our previous decrease or lack of IFN-γ-producing cells characterization of muNS demonstrated from week 4 to 6 against Cat01 CSFV or that a little domain (muNS-Mi) is able to PHA was observed. These results form spherical structures (microspheres) in 1 suggested the development of a state of transfected cells . We developed a immunosuppression in these postnatally molecular tagging system (IC-tagging) that persistently infected animals. Taken targets proteins to cytoplasmic muNS- together, we provided the first data derived microspheres that can be easily 2 demonstrating the feasibility of generating purified by physical methods . Thus, we a postnatal persistent CSFV infection, can produce In Vivo muNS microspheres which has not been shown for other decorated with any IC--tagged protein members of the Pestivirus genus yet. Since which have been successfully used to serological methods are routinely used in immunize mice against a viral infection 3 CSFV surveillance, persistently infected (Bluetongue virus, BTV) . The surface of animals might go unnoticed. In addition, enveloped viruses present glycoproteins

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which have acquired specific post- 3. Marín-López, A. et al. VP2, VP7, and NS1 proteins translational modifications due to their of bluetongue virus targeted in avian reovirus muNS-Mi microspheres elicit a protective immune pass through the endoplasmic reticulum response in IFNAR(-/-) mice. Antiviral Res. 110, 42– (ER), like glycosylation and disulfide bonds. 51 (2014). In an attempt to create a method to produce microspheres that might be used Parallel Session II: HIV as vaccines against enveloped viruses, we adapted the IC-tagging system to work Chairpersons: inside the ER. Our aim is to produce MANUEL LEAL AND JOSÉ ALCAMÍ microspheres inside the ER that are able to Monday June 8, 2015 capture IC-tagged viral-derived proteins WHITE ROOM that might acquire their post¬translational modifications while being incorporated in the microspheres, thus resembling the 15:00-15:15h (CO 17) surface of enveloped viruses. To do this, STUDY OF THE PROCESSIGN-BODIES (P- we introduced a signal peptide at the N- BODIES) ROLE IN THE RESTRICTION terminus of the muNS-Mi sequence, AGAINST HIV-1 IN PRIMARY HUMAN T promoting the entrance of the protein into CELLS the endoplasmic reticulum. The targeted P GIL MARTIN1, R CORREA-ROCHA2, M.A protein was able to form microspheres MUÑOZ-FERNANDEZ1 AND M PION1, 2 inside the organelle, although some 1Laboratory of Molecular ImmunoBiology, Hospital unspecific aggregation was also observed. General Universitario Gregorio Marañón; Instituto Eliminating a glycosylation site in the de Investigación Sanitaria del Gregorio Marañón. C/ Dr. Esquerdo 46, 28007 Madrid, Spain; muNS-Mi sequence increased the 2 microsphere formation efficiency while ImmunoRegulation Laboratory, Hospital General Universitario Gregorio Marañón; Instituto de drastically reducing the aggregation. Investigación Sanitaria del Gregorio Marañón. C/ Additionally, we showed that the IC-tagged Maiquez, 9, 28009 Madrid, Spain proteins can be successfully loaded onto the microspheres, raising the possibility of Naive and resting CD4+ T cells are developing biologically-generated restrictive in cases of HIV infection, while microspheres as particulate subunit activation of these cells induces a vaccines against enveloped viruses. permissive state in infection. Several 1. Brandariz-Nuñez, A., Menaya-Vargas, R., restriction factors have already been Benavente, J. & Martinez-Costas, J. Avian reovirus microNS protein forms homo-oligomeric inclusions described as acting in the early stages of in a microtubule-independent fashion, which the viral cycle. However, these restriction involves specific regions of its C-terminal domain. J. factors were generally studied in cell line Virol. 84, 4289–301 (2010). or during the replication of HIV. Only a few 2. Brandariz-Nuñez, A., Menaya-Vargas, R., works have described the very first steps of Benavente, J. & Martinez-Costas, J. A versatile HIV infection in primary human cells. It is molecular tagging method for targeting proteins to avian reovirus muNS inclusions. Use in protein interesting to note that HIV-1 Gag protein immobilization and purification. PLoS One 5, may be found associated to P-bodies e13961 (2010).

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proteins, which are cytoplasmic structures 15:15-15:30h (CO 18) in which siRNA-related silencing occurs. NEUTROPHIL MIGRATION VIA NFκB There is some debate as to whether this ACTIVATION BY MODIFIED VACCINIA union could be related to the replicative VIRUS AS A NOVEL MECHANISM TO capacity of the virus but nothing has been ENHANCE HIV-SPECIFIC T CELL RESPONSES done about whether this union could have M. DI PILATO1, E. MEJÍAS-PÉREZ1, M. an impact at the first steps of the viral ZONCA2, B. PERDIGUERO1, C. GÓMEZ1, M. cycle in primary CD4+ T cells. The aim of TRAKALA3, J. NIETO1, J. L. NÁJERA1, C. O. S. this study is to investigate whether P- SORZANO4, C. COMBADIÈRE5, bodies could constitute a new restrictive 6 2 G.PANTALEO , L. PLANELLES AND M. factor during the early stages of the viral ESTEBAN1 cycle in primary human cells. 1. Departamento de Biología Molecular y Celular, In the study, we analyzed the presence and Centro Nacional de Biotecnología, Madrid, Spain the intracellular localization of several 2. Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, Madrid, Spain proteins related to the P-bodies in naive 3 and in activated CD4+ T cells by confocal . Grupo de División Celular y Cancer, Centro Nacional de Investigaciones Oncológicas,Madrid, microscopy. Moreover, the presence of the Spain viral RNA into P-bodies was analyzed by 4. Biocomputing Unit, Centro Nacional de molecular assays after only 4 hours of Biotecnología, Madrid, Spain 5 infection. . INSERM UMR_S 945, Faculté de Médecine Pitié- Salpétrière, Laboratoire Immunité et Infection, We first demonstrated that presence and Paris, France intracellular distribution of P-bodies- 6. Division of Immunology and Allergy, Department related proteins were altered comparing of Medicine, Centre Hospitalier Universitaire non-activated or activated CD4+T cells. Vaudois, University of Lausanne, Lausanne, Furthermore, the presence of the HIV-1 Switzerland genome linked to Ago2, which is one of the P-bodies proteins associated to the RNA Neutrophils are antigen-transporting cells silencing process, was detected after just 4 that generate vaccinia virus (VACV)-specific hours of infection. T cell responses, yet how VACV modulates neutrophil recruitment and its significance The study shows for the first time that in the immune response are unknown. We during the very early stages of HIV generated an attenuate d VACV strain infection in non-activated CD4+ T cells, the (NYVAC) that expresses HIV-1 clade C virus can be found associated to P-bodies antigens but lacks three specific viral genes proteins, which may explain in part the (A52R, K7R, B15R). We found that these restriction of these cells against HIV genes act together to inhibit the NF B infection. κ signaling pathway. Triple ablation in modified virus restored NFκB function in macrophages. After virus infection of mice, NFκB pathway activation led to expression of several cytokines/chemokines that increased the migration of neutrophil

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populations (Nα and Nβ) to the infection that SAMHD1 deactivation is controlled by site. Nβ cells displayed features of antigen- CDK, which in turn, control cell activation presenting cells (APC) and activated virus- and proliferation. RNA interference of specific CD8 T cells. Enhanced neutrophil CDK2 but not CDK1, CDK4 or CDK5 trafficking to the infection site was suppressed SAMHD1 phosphorylation and responsible for increasing the T cell blocked viral replication specifically at the response to HIV vector-delivered antigens. level of proviral DNA formation. These results identify a mechanism for Importantly, we also found that CDK6 poxvirus-induced immune response and inhibition blocks SAMHD1 phosphorylation alternatives for vaccine vector design. probably through the control of CDK2, as RNA interference or pharmaceutical 15:30-15:45h (CO 19) blockade of CDK6 by palbociclib, a potent and selective CDK6 inhibitor, led to IDENTIFICATION AND CHARACTERIZATION reduced CDK2 activation, concomitant with OF THE MOLECULAR PATHWAY LEADING reduced SAMHD1 phosphorylation and TO SAMHD1-MEDIATED VIRAL blockade of HIV-1 reverse transcription RESTRICTION and virus replication. The antiviral effect of 1 1 E. BALLANA , R. BADIA , E. RIVEIRA- these inhibitors disappeared when 1 1 MUÑOZ , A. RUIZ , J. TORRES- SAMHD1 was abrogated using the HIV-2 2 1 1 TORRONTERAS , E. PAULS , B. CLOTET , R. X (Vpx), demonstrating the 2 1 MARTÍ , JA ESTÉ specificity of the mechanism of action of 1. AIDS Research Institute - IrsiCaixa, Hospital these compounds. Knockdown of the cyclin Germans Trias i Pujol, Badalona partners of CDK1, CDK2 and CDK6 showed 2 . Mitochondrial Pathology Laboratory, Institut de that cyclin D3, the catalytic partner of Recerca Hospital Universitari Vall d'Hebron, CDK6, has a major impact in SAMHD1 Universitat Autònoma de Barcelona phosphorylation, dNTP levels and HIV-1 reverse transcription and replication. Monocytes are refractory to HIV infection Finally, we investigated the effect of p21, a and only become susceptible to infection member of the Cip/Kip family of cyclin- after differentiation into macrophages, due dependent kinase inhibitors (CDKIs) to deactivation of the restriction factor specifically controlling cell cycle SAMHD1 by phosphorylation. The aim of progression through binding and activation the present work was the identification of cyclin-CDK1 or –CDK2 complexes. siRNA- and characterization of cell signaling induced downregulation of p21 strongly events leading to host SAMHD1 enhanced the phosphorylation of SAMHD1 activation/deactivation. Therefore, we followed by an increase in HIV-1 proviral have evaluated the contribution of cyclin- DNA formation and virus replication, dependent kinases (CDK) and their indicating that p21 affects SAMHD1- corresponding activating cyclins in the mediated HIV-1 restriction and further control of SAMHD1-mediated viral delineating the cellular pathway involved restriction in monocyte derived in SAMHD1-mediated viral restriction. macrophages (MDM). First, we have shown Thus, overall our results indicate a

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fundamental role of CDK2 and the CDK6- replication. However, viruses have cyclin D3 complex in SAMHD1-mediated developed mechanisms that can virus restriction in MDM during GO to G1 antagonize restriction factors. Some of the transition. The present study suggest also restriction factors known to block that agents targeting cell proliferation may replication of HIV-1 and other lentiviruses limit HIV-1 infection and hypothetically, are TRIM5alpha, APOBEC3G, BST2, might prevent the proliferation of SAMHD1, and the recently discovered persistently infected cells, offering new Mx2. However, several lines of evidence possibilities for intervention. suggest the existence of additional restriction factors that block replication of 15:45-16:00 h (CO 20) lentiviruses. INTRACELLULAR FACTORS BLOCKING To date, lentiviruses infecting New World EARLY STEPS OF THE HIV-1 REPLICATIVE monkeys have not been described. In CYCLE IN COMMON MARMOSET addition, New World monkeys are LYMPHOCYTES apparently resistant to infection by known lentiviruses. We have been studying the B. PACHECO1,2,3, L. MENÉNDEZ-ARIAS1 AND 2,3,4 susceptibility of common marmosets to J. SODROSKI HIV-1 infection and observed the presence 1 Department of Virology and Microbiology, Centro of early post-entry blocks to HIV-1 de Biología Molecular “Severo Ochoa”, CSIC-UAM, Madrid, Spain infection in peripheral blood lymphocytes 2 (PBLs) and B lymphocytic cell lines (B-LCLs). Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA The blockades present in these cells are 3Department of Microbiology and Immunobiology, dominant and phenotypically different Harvard Medical School, Boston, MA from each other. In PBLs, the blockade 4Department of Immunology and Infectious occurs at the level of reverse transcription, Diseases, Harvard School of Public Health, Boston, reducing the accumulation of early and MA late transcripts, as reported for TRIM5alpha. However, we have found that The presence of several barriers to HIV-1 marmoset TRIM5alpha doesn’t block HIV- replication in cells of many species narrows 1. In contrast, the restriction factor present the viral tropism to humans and in B-LCLs blocks HIV-1 replication at a later chimpanzees. The limited species tropism step. Additionally, we have generated a of HIV-1 is due to two types of host factors: few capsid mutants that are able to escape 1) factors that are required for HIV-1 restriction in the marmoset B-LCLs. Our replication, but that exhibit species-specific results suggest that the restriction factors changes that do not allow efficient use by responsible for the blocks present in HIV-1; and 2) dominant-acting factors that marmoset PBLs and B-LCLs are different. block replication in many hosts. The latter, We propose the existence of at least two also known as restriction factors, are part new restriction factors able to block HIV-1 of the so-called intrinsic antiviral immunity. infection in marmoset cells. The nature of Altogether, intracellular restriction factors these restriction factors is currently under can act as a powerful barrier to stop viral investigation.

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This research was supported by NIH grant characterize the mechanism of action of AI67854, the EU under the 7FP through a potential NC inhibitors (NCIs). Marie Curie Career Integration Grant A HIV-1 mutant that cannot lead to NCp9 (332623) and a Scholar Award from the and NCp7 (blocked at the NCp15 step) is Harvard University CFAR, an NIH funded not able to form a condensed NC and is program (P30 AI060354). BP is a recipient also deficient to form a conical capsid. In of a CSIC JAE-Doc contract, a program co- vitro, with purified components, NCp15 is financed by the European Social Fund. Part not able to condense a circular ssDNA of this work has been supported by template whilst NCp9 and NCp7 can. RNA EUPRIM-NetII under EU 7FP grant 262443. condensation appears much faster within a HIV-1 particle than previously thought: 16:00-16:15h (CO 21) particles at the final phase of budding are CHARACTERIZATION AND INHIBITION OF already imaged with a condensed NC at THE HIV-1 NUCLEOCAPSID MATURATION- their centre. NCp15 in vitro processing by CONDENSATION STEP HIV-1 protease occurs within minutes if 1 1 2 RNA is bound to it. This can be explained C. LORCA-ORÓ , S. LYONNAIS , S.K. SADIQ , by an original mechanism where protease C. LOPEZ-IGLESIAS3, J.M. GATELL1, A. 2,4 1 appears sequestrated by the NCp15-bound MEYERHANS , G. MIRAMBEAU RNA complex, inducing a significantly 1 .AIDS Research group, IDIBAPS, Barcelona, Spain faster turnover. Such a mechanism is 2. Infection Biology Unit, Department of Experimental and Health Sciences, Universitat consistent with a model, based on polymer Pompeu Fabra, Barcelona, Spain physics, of enhanced protease diffusion 3. Cryo-Electron Microscopy, Scientific and within the complex. Technological Centers, University of Barcelona, NCIs have been recently highlighted as Barcelona, Spain 4. Institució Catalana de Recerca i Estudis Avançats potential antiviral drugs. Members of this (ICREA), Barcelona, Spain class, designed to dock within the NCp hydrophobic pocket, efficiently inhibit in vitro NCp7, NCp9 and NCp15 coating upon The HIV-1 nucleocapsid protein (NCp) is large single-stranded nucleic acids. We highly conserved and plays multiple roles investigated their antiviral effects by two- within the HIV life cycle. The NC domain of round infectivity assays using free viruses the Gag precursor recruits full-length in TZM-bl cells or chronically HIV-infected genomic RNA to direct viral assembly. ACH-2 cells. Antiviral activity was stronger There is also an interaction with a cellular during the late steps of HIV replication and machinery for viral budding and an was within the µmolar range. Transmission interplay with the protease for viral electron microscopy showed that NCIs maturation leading within the viral core to affect maturation. HIV particles, still in condensed RNA and mature NCp (NCp7), contact with the cell membrane, display via two intermediates (NCp9 and NCp15). aberrant morphologies, for both Here, we focus on the effect of NC-RNA nucleocapsid and capsid, when cells are condensation and its inhibition on treated with NCIs. This effect suggests a maturation. The aim of the study is to defective HIV-1 NC assembly and

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maturation as the main mechanism of The HIV infection remains after decades action for NCIs. one of the most aggressive epidemics The spatiotemporal coordination for HIV around the world. Only in sub-Saharan assembly, budding and maturation Africa heterosexual transmission represent requires thousands of NC-RNA the 80% of the new infections, being the interactions. Disrupting these interactions 50% of these new infections in women. by a NCI has multimodal effects. Here we Therefore, the development of a safe, provide the first proof of concept for an effective, and low-priced topical effect on HIV maturation. microbicide to prevent the sexual transmission of HIV-1 is urgently needed. The AIDS Research group, IDIBAPS, as member of the THINPAD consortium The emerging field of nanotechnology and (thinpad.unisi.it) has received funding from its different nanosystems (i.e., dendrimers) the European Union’s Seventh Programme play an important role in addressing this for research, technological development challenge. and demonstration under grant agreement Polyanionic carbosilane dendrimer G2-S16 No 601969. has demonstrated potent and a broad- spectrum anti-HIV-1 activity in vitro.

However, its antiviral activity in humanized 16-15-16:30h (CO 22) (h)-BLT (bone marrow-liver-thymus) mice PREVENTION OF HIV-1 VAGINAL and its mode of action has not been TRANSMISSION AND MODE OF ANTIVIRAL completely elucidated. In this work, we ACTION BY TOPICAL POLYANIONIC focused on a preliminary efficacy study of CARBOSILANE DENDRIMER G2-S16 IN vaginally applied G2-S16 on h-BLT mice. HUMANIZED BLT MICE We also assessed the mechanism of D. SEPÚLVEDA-CRESPO1,2, Mª J. antiviral of action on the inhibition of HIV-1 SERRAMÍA1,2, R. GÓMEZ3, F. J. DE LA infection through a panel of different in MATA3, J. L. JIMÉNEZ2,*, Mª A. MUÑOZ- vitro antiviral assays. 1,2,* FERNÁNDEZ Topical vaginal administration of 3% G2- 1 .Laboratorio InmunoBiología Molecular, Hospital S16 prevented HIV-1JR-CSF transmission in h- General Universitario Gregorio Marañón, Madrid, BLT mice in 84% without irritation or Spain. Instituto de Investigación Sanitaria Gregorio vaginal lesions. Our results also suggest Marañón (IISGM), Madrid, Spain.Networking Research Center on Bioengineering, Biomaterials that G2-S16 exerts anti-HIV-1 activity at an and Nanomedicine (CIBER-BBN), Madrid, early stage of viral replication, as a Spain.Spanish HIV HGM BioBank, Madrid, Spain. virucidal agent and as an inhibitor of viral 2.Plataforma de Laboratorio, Hospital General entry blocking the gp120/CD4 interaction. Universitario Gregorio Marañón, Madrid, Spain. Moreover, we demonstrate the IISGM, Madrid, Spain. CIBER-BBN, Madrid, Spain 3 dendrimer’s capability to provide a barrier .Departamento de Química Inorgánica, Universidad to infection for long periods and to inhibit de Alcalá, Campus Universitario, Alcalá de Henares, Madrid, Spain. CIBER-BBN, Madrid, Spain cell-to-cell HIV-1 transmission, confirming its multifactorial and non-specific ability.

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This study represents the first Cambridge, USA.9 Advanced BioScience 10 demonstration indicating that HIV-1 Laboratories, Inc., Kensington, USA. Global Solutions for Infectious Diseases, San Francisco, vaginally infects humanized BLT mice and USA.11 The Biodesign Institute at Arizona State that transmission of the virus can be University, Tempe, USA.12University of Regensburg, efficiently blocked by vaginally applied G2- Regensburg, Germany.13 EuroVacc Foundation, S16. These results obtained in h-BLT mice Lausanne, Switzerland. 14 Division of Immunology provide a strong step forward in the and Allergy, Department of Medicine, Centre Hospitalier Universitaire Vaudois, University of development of G2-S16-based vaginal Lausanne, Lausanne, Switzerland. microbicides to prevent vaginal HIV transmission in humans. We have compared the HIV-1-specific cellular and humoral immune responses 16:30-16:45h (CO 23) elicited in rhesus monkeys immunized with HEAD-TO-HEAD COMPARISON OF two poxvirus vectors (NYVAC and ALVAC) POXVIRUS NYVAC AND ALVAC VECTORS expressing the same HIV-1 antigens from EXPRESSING IDENTICAL HIV-1 CLADE C clade C, Env gp140 as a trimeric cell IMMUNOGENS IN PRIME/BOOST released protein and Gag-Pol-Nef as Gag- COMBINATION WITH ENV PROTEIN IN induced virus-like particles (VLPs) (referred NON-HUMAN PRIMATES as NYVAC-C and ALVAC-C). The J. GARCÍA-ARRIAZA1, B. PERDIGUERO1, J. immunization protocol consisted of two HEENEY2, M. SEAMAN3, D. MONTEFIORI4, doses of the corresponding poxvirus vector C. LABRANCHE4, N. YATES4, X. SHEN4, G. plus two doses of a combination of the TOMARAS4, G. FERRARI4, K. E. FOULDS5, A. poxvirus vector and a purified HIV-1 gp120 MCDERMOTT5, S. KAO5, M. ROEDERER5, N. protein from clade C. This immunogenicity HAWKINS6, S. SELF6, J. YAO7, P. FARRELL7, profile was also compared to that elicited S. PHOGAT7, J. TARTAGLIA7, S. W. by the RV144 trial vaccine regimen BARNETT8, B. BURKE8, A. CRISTILLO9, D. consisting of two doses of the ALVAC WEISS9, C. LEE10, K. KIBLER11, B. JACOBS11, vector expressing HIV-1 antigens from B. ASBACH12, R. WAGNER12, S. DING13, G. clades B/E (ALVAC-vCP1521) plus two PANTALEO14 AND M. ESTEBAN1 doses of a combination of ALVAC-vCP1521 1Department of Molecular and Cellular Biology, and HIV-1 gp120 protein from clades C or Centro Nacional de Biotecnología, Consejo Superior B/E. The results showed that immunization de Investigaciones Científicas (CSIC), Madrid, of macaques with NYVAC-C stimulated Spain.2Department of Veterinary Medicine, more potent HIV-1-specific CD4+ and CD8+ 3 University of Cambridge, Cambridge, UK. Division T-cell responses than those induced by of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, ALVAC-C. Furthermore, NYVAC-C induced a USA.4 Duke University, Durham, USA. 5 Vaccine trend toward higher levels of binding IgG Research Center, National Institute of Allergy and antibodies against clade C HIV-1 gp140, Infectious Diseases (NIAID), National Institutes of gp120 or MuLV gp70-scaffolded V1/V2 and 6 Health (NIH), Bethesda, USA. Statistical Center for toward best cross-clade binding IgG HIV/AIDS Research and Prevention, Fred Hutchinson Cancer Research Center, Seattle, USA. 7 Sanofi responses against HIV-1 gp140 from clades Pasteur, Swiftwater, USA.8 Novartis Vaccines, A, B and group M consensus, compared to

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ALVAC-C. Most of these binding IgG is yet available. At the post-treatment time responses were directed against the V3 point, when these patients have no loop in all immunization groups. restored CD4 levels and persist with very Additionally, NYVAC-C and ALVAC-C also low CD4 T cell counts, they show critical induced similar levels of HIV-1 neutralizing immunovirological alterations. Thus, they antibodies and antibody-dependent finally show increased T-cell activation, cellular cytotoxicity (ADCC) responses. senescence and apoptosis, lower thymic Interestingly, binding IgA antibodies function, increased Treg frequency and against HIV-1 gp120 or MuLV gp70- their virus are more likely X4-tropic. scaffolded V1/V2were absent or very low However, whether these factors are cause in all immunization groups.Overall, these or consequence of the persistence of low results provide a comprehensive survey of CD4 T cell counts is unknown. the immunogenicity of NYVAC versus To date, no previous study has focused on ALVAC expressing HIV-1 antigens in non- their potential early immunovirological human primates and indicate that NYVAC alterations. This information could be may represent an alternative candidate to crucial to help to prematurely recognize ALVAC in the development of a future HIV- these patients by clinicians and for better 1 vaccine. understanding subjacent mechanism of such failure. Our more recent research 16:45-17:00h (CO 24) address pre-treatment samples from HIV- IMMUNOVIROLOGICAL TRAITS OF HIV infected subjects with late diagnosis, SUBJECTS WITH DELAYED INITIATION OF focusing on groups of subjects with later cART AND SUBSEQUENT POOR CD4 poor CD4 restoration and good CD4 RESTORATION. STUDY ON PRE- restoration, in response to cART. TREATMENT SAMPLES Importantly, at the onset of the cART 1 1 1 initiation, both groups had similar, but low, I ROSADO , M LEAL , YM PACHECO CD4 levels and viral loads (and were also 1 Laboratory of Immunovirology, Clinic Unit of matched by age and sex). With a very Infectious Diseases, Microbiology and Preventive Medicine, Institute of Biomedicine of Seville (IBiS), complete study, including Virgen del Rocío University Hospital /CSIC immunophenotyping of several cell types /University of Seville, Seville, Spain. (monocytes, T cells, dendritic cells,…), an exhaustive profile of soluble markers, and One out of four HIV-infected subjects with novel omics approaches (such as delayed initiation of combined metabolomics and transcriptomics), we are antiretroviral therapy (cART) (below 200 getting involved in a very innovative and CD4 cells/μL) does maintain low levels of interesting project which is yielding CD4 T cells (below 250 CD4 cells/μL) relevant information. This is a major despite a subsequent long-term effective collaborative project of the Spanish AIDS treatment. These HIV-infected subjects are Research Network (RIS); belonging to the at an increased risk of clinical progression WP3 “Immunological damage and and death, and no therapeutic alternative reconstitution”, inside the research programme “HIV immunopathogenesis and

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vaccines”. Samples for the study have been and subgenomic RNAs. Some of them obtained from the Spanish HIV Biobank. contain in their 3´-UTRs RNA elements able to enhance their cap-independent Parallel Session III: PLANT VIRUS translation (3´-CITEs). We have shown that cap-independent translation of Melon Chairpersons: necrotic spot virus (MNSV, family JESÚS NAVAS AND VICENTE PALLÁS Tombusviridae, genus Carmovirus) RNA is Monday June 8, 2015 controlled in cis by a 3´-CITE (Truniger et AUDIOVISUAL ROOM al., 2008.Plant J. 56:716-727). Remarkably, MNSV 3´-CITEs are diverse, including at

least Mα5TE, M264TE and CXTE (Truniger 15:00-15:15h (CO 25) et al., 2008; Miras et al., 2014, New Phytol. STRUCTURAL AND FUNCTIONAL 202:233-246). The CXTE has been acquired DIVERSITY OF PLANT VIRUS 3´-CAP- by recombination with an Asiatic isolate of INDEPENDENT TRANSLATIONAL Cucurbit aphid-borne yellows virus (CABYV; ENHANCERS family Luteoviridae, genus ), V. TRUNIGER1, M. MIRAS1, A.M. suggesting that 3´-CITEs are modular, RODRÍGUEZ-HERNÁNDEZ2, C. ROMERO- interchangeable structural elements. Here LÓPEZ3, A. BERZAL-HERRANZ3, M.A. we show that CABYV Asiatic and European ARANDA1 isolates have two different translational 1. Grupo de Patología Vegetal. Centro de Edafología enhancers. For all different 3´-CITEs that y Biología Aplicada del Segura (CEBAS-CSIC). Apdo. we have identified, we analysed their correos 164, 30100 Espinardo, Murcia, Spain secondary structure and showed that their 2. Centro de Investigación en Química Aplicada (CIQA). Consejo Nacional de Ciencia y Tecnología depends on the eukaryotic translation (CONACYT). Blvd. Enrique Reyna Hermosillo 140, 25294, Saltillo, Coahuila, México. initiation factor eIF4E, the other four are 3. Instituto de Parasitología y Biomedicina López- eIF4E-independent, conferring Neyra. Consejo Superior de Investigaciones translational competence to RNAs in the Científicas (IPBLN-CSIC). Av. Conocimiento s/n. absence of this factor. On the other hand, 18016. Armilla (Granada). Spain we showed that the translation enhancer activity of all five 3´-CITEs depends on the Viral mRNAs have evolved numerous presence of the 5´-UTR in cis. For the mechanisms to recruit the host translational machinery, allowing them to RNA circularization is achieved by long- compete with host mRNAs and avoid distance interactions between the 5´- and defence mechanisms that act at the level 3´-ends based on sequence of translation. Thus, while most plant- complementarity. This interaction involves encoded mRNAs contain a 5´-cap and a nucleotides of the 3´-CITE that have two poly(A)-tail that act synergistically to complementary nucleotide stretches at the stimulate translation, ~80% of known 5´end, one in the first 20 nucleotides of the positive-strand RNA plant viruses lack one 5´-UTR, the other at the beginning of the or both of these features in their genomic coding region of the first ORF. For the

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this ORF could result in a putative new 5´end sequence is required in vivo for its gene product P1N-PISPO. translational enhancer activity, and it is The presence of the P1N-PISPO protein has therefore essential for virus viability. been investigated in Ipomoea batatas plants infected with a Spanish isolate of 15:15-15:30h (CO 26) SPFMV. The genome sequence of this EXPRESSION AND FUNCTION OF THE isolate was assembled from NGS data, TRANS-FRAME P1N-PISPO GENE PRODUCT showing that the expected trans-framed OF THE POTYVIRUS SWEET POTATO PISPO sequence was present, preceded by FEATHERY MOTTLE VIRUS (SPFMV) a G2A6 domain. The predicted size for the 1 2 3 P1N-PISPO protein was 72.7 KDa. Analysis A. MINGOT , A. VALLI , B. RODAMILANS , of the viral gene products present in D. SAN LEÓN3, D.C. BAULCOMBE2, J.A. 3 1 infected plant tissues was performed using GARCÍA , J.J. LÓPEZ-MOYA LC-MS/MS after separation in SDSPAGE, 1 Center for Research in Agricultural Genomics focusing in products >50KDa. Detected CRAG, CSIC-IRTA-UAB-UB, Campus UAB Bellaterra, Cerdanyola del Vallès, 08193-Barcelona, Spain viral peptides corresponded to proteins 2 such as CI (72 KDa) and HCPro (52.1 KDa), Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, all of them translated from the viral United Kingdom ORF.Moreover, peptides corresponding to 3Centro Nacional de Biotecnología CNB, CSIC, the P1 protein were detected from both Darwin 3, 28049-Madrid, Spain the N-terminal portion (11 different peptides, 39% coverage), before the Potyviruses (genus Potyvirus, family frameshifting signal and therefore ) are plus strand RNA viruses common for P1 and P1N-PISPO, and in the with a genomic organization similar to C-terminal part (2 peptides exclusive for Picorna-like viruses. The genome of P1, 10% coverage). Interestingly, four potyviruses is characterized by the peptides exclusive of PISPO, in its unique presence of a large ORF yielding a ORF (21.3% coverage), were also found. polyprotein, later autoproteolitically These results indicated that both products processed into several mature gene P1 and P1N-PISPO were expressed in products (P1, HCPro, P3, 6K1, CI, 6K2, VPg- SPFMV infected plants. NIa, NIb and CP). In addition, a short ORF To determine the possible function of the named PIPO can be found embedded P1N-PISPO product during infection, within the P3 region in another frame, constructs adequate for transient starting at a conserved G1- 2A6-7 motif. This expression were prepared and tested for coding sequence is able to generate an RNA silencing suppressor (RSS) activity. essential P3N-PIPO product. Recently, While in other potyviruses the RSS function another additional ORF named PISPO was is associated to HCPro, our results showed identified in silico within the P1 region in RSS activity for the P1N-PISPO product. The the genome of Sweet potato feathery mode of action of this new RSS compared mottle virus (SPFMV) and several related to other RSS from members of the sweet potato potyviruses. Expression of Potyviridae family will be discussed.

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(Work funded by Mineco grant AGL2013- exception is the case of the CP from 42537-R. A. Mingot received FPI fellowship Papaya mosaic virus (PaMV) whose Xray BES-2011-045699). structure was reported. Nevertheless, the atomic data for PaMV CP was obtained 15:30-15:45h (CO 27) with purified protein, not from virions, and lacking around 20% of the protein mass at INHALED DELIVERY OF PEGYLATED its C-terminal domain (Yang et al., 2012, J. DENDRIMERS: PHARMACOKINETICS AND Mol. Biol. 422 p. 263). THERAPEUTIC POTENTIAL 1 2 We report here the structure of intact X. AGIRREZABALA , F.E. MÉNDEZ , G. PepMV virions by cryoEM at 4Å resolution. LASSO3, M.A. SÁNCHEZ-PINA2, M.A. 2 1 The results and the resolution achieved ARANDA AND M. VALLE allowed the modeling of the CP, the 1. Structural Biology Unit, CIC bioGUNE, Derio, Spain (+)ssRNA and their relative interactions. 2. Dpto. de Biología del Estrés y Patología Vegetal, CEBAS-CSIC, Murcia, Spain The CP was modeled starting with the CP 3.Department of Biochemistry and Molecular from PaMV, clearly showing a similar Biophysics, Columbia University, New York, USA folding of the α-helical domain for the family. The ssRNA is Flexible filamentous viruses belonging to allocated and protected in a continuous the Alphaflexiviridae family cause from groove of high electropositive potential severe to mild diseases in agricultural built up by the CP in helical arrangement. crops, often reducing yield and crop The CP polymerize through a flexible N- quality. Viral particles from the terminal arm providing the structural basis Alphaflexiviridae group are formed by a for the flexibility of the virus. Interestingly, (+)ssRNA molecule encapsidated by single the overall structure and organization of capsid protein (CP) monomers arranged in CP from PepMV is similar to the helical symmetry. Pepino mosaic virus organization of nucleoproteins from the (PepMV) is a flexible filamentous plant Bunyaviridae family, a group of enveloped virus belonging to the genus (-)ssRNA viruses. Common features included in the family Alphaflexiviridae include: the folding and arrangement of which genome consists of a ~6.4 kb the α-helical main domain; the groove for (+)ssRNA and encodes five proteins. the ssRNA; the N-terminal arm for In sharp contrast with rigid plant viruses polymerization; and the relative position such as Tobacco mosaic virus, little is between all these elements. Although known about the structure of flexible structural homology between viruses filamentous plant viruses at high revealed by their atomic structures is resolution. Their intrinsic flexibility common, in the current case, the different precludes high-resolution studies by fiber nature of capsid protein and nucleoprotein diffraction or Xray crystallography, and might have profound evolutionary reported cryoEM structures stay at implications. resolutions above 1 nm (Kendall et al., 2012, Virology 436 p.173). A clear

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15:45-16:00h (CO 28) interactors of p23, an initial yeast two- GLYCERALDEHYDE 3-PHOSPHATE hybrid (Y2H) screening of an expression DEHYDROGENASE IS CO-OPTED FOR library of Nicotiana benthamiana (in which REPLICATION OFCITRUS TRISTEZA VIRUS at least one CTV isolate replicates and VIA INTERACTION WITH THE VIRAL- incites symptoms) led to the identification ENCODED PROTEIN P23 of the cytoplasmic glyceraldehyde 3- S. RUIZ-RUIZ1, R. SPÀNO1, S. DAVINO1, L. phosphate dehydrogenase (GAPDH), NAVARRO2, P. MORENO2, L. PEÑA1, R. further confirmed in 1-by-1 Y2H tests. FLORES1 Bimolecular fluorescence complementation assays in planta 1Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones corroborated the previous results and Científicas-Universidad Politécnica de Valencia, provided new insights. Briefly, p23 Spain interacts with itself in the nucleolus, Cajal 2Instituto Valenciano de Investigaciones Agrarias, bodies and plasmodesmata, and with Moncada, Valencia, Spain GAPDH (in the cytoplasm forming aggregates) and in plasmodesmata. This Citrus tristeza virus (CTV), genus latter interaction was preserved in a p23 , family , is an deletion mutant affecting the C-terminal important pathogen that has killed more domain, but not in two other deletion than 50 million citrus trees in Spain. CTV mutants affecting the Zn-finger domain genome is a positive-sense RNA of and one internal basic motif. Most approximately 19.3 kb organized in 12 importantly, qRT-PCR and RNA gel-blot open reading frames (ORFs) potentially hybridization showed that virus-induced coding for at least 17 proteins. One of gene silencing of GAPDH mRNA resulted in them, p23, encoded by the 3’-terminal a significant decrease in CTV titer. ORF, has no homologues in other Altogether these data suggest that, closteroviruses and, therefore, is paralleling the situation observed in a distinctive. CTV-p23 has also peculiar tombusvirus (Wang and Nagy, Cell Host features: i) it is an RNA-binding protein of and Microbe 2008), CTV co-opts GAPDH 209 amino acids with a putative Zn-finger through p23 to convert the host cell into a domain and some basic motifs, ii) it viral factory. The finding that two very accumulates mainly in the nucleolus and different viruses co-opt for their replication Cajal bodies, and in plasmodesmata, and the same host protein (GAPDH) suggests iii) it mediates many functions including that this protein is endowed with an the asymmetric accumulation of CTV RNA intrinsic feature, possibly its RNA-binding strands, the intracellular suppression of ability, that facilitates the process. RNA silencing, and the induction of some CTV syndromes when expressed from the virus, and of CTV-like symptoms and enhancement of systemic infection when expressed ectopically as a transgene in several Citrus spp. To search for host

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16:00-16:15h (CO 29) natural malvaceous hosts. Replication of BIOLOGICAL CHARACTERIZATION OF NON- these satellites is also supported by other CODING DNA SATELLITES ASSOCIATED TO geminiviruses, including the monopartite NEW WORLD BEGOMOVIRUSES New World Tomato leaf E.FIALLO-OLIVÉ, R. TOVAR, J. NAVAS- deformation virus. Our results confirmed CASTILLO that these molecules are indeed satellites of New World bipartite begomoviruses and Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga constitute a novel class of such subviral - Consejo Superior de Investigaciones Científicas agents. (IHSM-UMA-CSIC), Estación Experimental "La Mayora", 29750 Algarrobo-Costa, Málaga, Spain 16-15-16:30h (CO 30)

INTERFERENCE OF SINGLE AND DUAL Begomoviruses (genus Begomovirus, family BIOTIC STRESSES ON HOST DNA ) cause serious diseases in a METHYLATION PATHWAYS number of economically important crops, 1 2 mostly in tropical and subtropical regions. E.M. TORCHETTI , M. PEGORARO , B. NAVARRO1, M. CATONI3, E. NORIS2, F. DI They are plant ssDNA viruses that are 1 transmitted by the whitefly Bemisia tabaci SERIO 1 (Hemiptera: Aleyrodidae). Begomoviruses .Istituto per la Protezione Sostenibile delle Piante, Consiglio Nazionale delle Ricerche, UOS Bari, Italy have been shown to be helper viruses for a 2 number of distinct DNA satellites, including .Istituto per la Protezione Sostenibile delle Piante, Consiglio Nazionale delle Ricerche, Torino, Italy betasatellites and alphasatellites. During a 3.Sainsbury Laboratory, University of Cambridge, survey in Cuba, we found two malvaceous Cambridge, UK species, Malvastrum coromandelianum and Sidastrum micranthum, infected with bipartite begomoviruses associated with DNA methylation (DM) pathways play ssDNA molecules of a quarter the size of major roles in preservation of genome the begomoviral genome components. integrity, transposon stability and These molecules shared some genetic regulation of gene expression. In plants, features with betasatellites and ToLCV-sat DM has also been involved in responses to such as an A-rich region, but also abiotic and biotic stresses, including contained nucleotide stretches of defense against geminiviruses (GV), a large begomoviral origin, presumably the group of viruses with a single-stranded remains of recombination events involved DNA genome that replicates in the nucleus in their origin (Fiallo-Olivé et al., 2012). In forming minichromosomes associated with this work we have developed infectious cellular histones. It is proposed that host clones of two ssDNA satellites, from M. DM machinery impairs viral accumulation coromandelianum and S. micranthum, in the infected tissues by targeting GV DNA respectively. Agroinoculation of satellites for methylation. In contrast, whether DM together with their helper begomoviruses is involved in the molecular interplay showed that satellites were replicated in between plants and nuclear replicating Nicotiana benthamiana plants and in their viroids, which are infectious non-protein-

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coding RNAs frequently inducing severe 16:30-16:45h (CO 31) diseases in plants, is still unclear. Viroid UNRAVELLING THE RNA2-ENCODED RNAs are targeted by host enzymes POLYPROTEIN CLEAVAGE SITES OF involved in DM pathways, but whether the TOMATO-INFECTING TORRADOVIRUSES genes implicated in this pathways are USING N-TERMINAL PROTEIN differentially regulated in response to SEQUENCING AND A REVERSE GENETICS viroid infection is unknown. In addition, SYSTEM whether DM pathways may differentially I. FERRIOL1, D. MARQUES DA SILVA2, M. target host and GV DNA depending on the TURINA3, E. J. ZAMORA-MACORRA4, B. W. presence or absence of a nuclear infecting 1 FALK viroid is also not known. To further explore 1 Plant Pathology Department, University of the interference of single and dual California Davis, 95616, Davis, CA, USA infections by nuclear replicating infectious 2 CAPES Foundation, Ministry of Education of Brazil, agents, we have developed an Brasilia-DF, 70040-020, Brazil experimental system based on tomato 3 Istituto per la Protezione Sostenibile delle Piante, plants infected by the geminivirus Tomato Sez. di Torino, CNR, Turin, Italy yellow leaf curl Sardinia virus (TYLCSV) 4 Colegio de Postgraduados-Campus Montecillo, and/or the nuclear-replicating Potato 56230, Texcoco, Mexico spindle tuber viroid (PSTVd). DNA methylation profiles of TYLCSV DNA and of In a time span of just two decades, a new two host genomic targets were tested as group of RNA plant viruses, the molecular sensors of host DM under stress torradoviruses, has been discovered conditions. Moreover, expression of genes affecting tomatoes and other plant involved in DM was investigated at species. The genus includes transcriptional level by quantitative RT-PCR three species: i) Tomato torrado virus assays. Our data show that both TYLCSV (ToTV), first found in Europe, and and PSTVd interfere with the regulation of afterward in Central America and Australia; most host genes involved in DM pathways ii) Tomato marchitez virus (ToMarV) (also and, interestingly, that the plant response called Tomato apex necrosis virus,ToANV), to a single stress strongly differs from that present in Mexico; and iii) Lettuce necrotic to dual stresses, with synergistic effects. leaf curl virus (LNLCV) infecting lettuce in the Netherlands. Six new tentative species have been discovered: i) tomato chocolate spot virus(ToChSV) and tomato chocolàte virus(ToChV), both found infecting tomato in Guatemala; ii) tomato necrotic dwarf virus (ToNDV), which infected tomato in California in the mid-80s; iii) cassava torrado-like virus (CsTLV) infects cassava in Colombia; iv) motherwort yellow mottle virus (MYMoV) infects motherwort in Korea; and v) carrot torradovirus 1 (CTV-1),

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which infects carrot in UK. All known 16:45-17:00h (CO 32) tomato-infecting torradoviruses are ANALYSIS OF TOLERANCE MECHANISMS transmitted by whiteflies. Torradoviruses AND TRADE-OFFS IN PLANT-VIRUS have a bipartite genome consisting of two INTERACTIONS single-stranded plus-sense RNAs. RNA1 is V. VIJAYAN& I. PAGAN ca. 7 kb and has one open reading frame Centro de Biotecnologia y Genomica de Plantas (ORF), which encodes replication- (UPM-INIA), Madrid associated proteins including the protease,

helicase and RNA-dependent RNA polymerase (RdRp). RNA2 is ca. 5 kb and Viruses are important selective forces for has two ORFs. The ORF1 in RNA2 is unique their hosts. As a consequence, hosts have for torradoviruses, but the functions of its developed a variety of mechanisms to encoded protein are still unclear. The ORF2 prevent or limit virus infection has coding regions for a putative (Resistance), and to reduce the effect of movement protein and the three capsid virus infection in the host fitness proteins. Little is known about the (Tolerance). Although resistance has been functions of torradovirus proteins, their extensively studied, comparatively less is replication and polyprotein strategies. known about which, and how specific, are Here, we developed an agroinoculation the host tolerance mechanisms. Because system for the tomato-infecting biological fitness cannot be maximized in torradovirus,ToANVthat can trigger every situation, mechanisms that increase infection in Nicotiana benthamiana, tolerance to infection by a given virus may tomato and tomatillo plants, and will allow come at the cost of reduced tolerance us to elucidate torradovirus protein upon infection with other viruses with functions. To better understand the different life-history traits, i.e., tolerance tomato-infecting torradovirus polyprotein trade-offs. However, how tolerance is processing, the cleavage sites in the RNA2 achieved and the potential trade-offs of ORF2-encoded proteins of two tomato- tolerance to different viruses have been infecting torradoviruses (ToANV and seldom analyzed. ToChSV) were determined by N-terminal We have analyzed trade-offs in tolerance sequence analysis. These results showed to infection by Cucumber mosaic virus that the amino acid at the -1 position of (CMV) and Turnip mosaic virus (TuMV) in the cleavage sites is a Gln (Q). Amino acid their natural host Arabidopsis thaliana. In sequence comparison of different isolates Arabidopsis, tolerance to CMV is achieved of ToANV confirmed that this Gln (Q) is by resource reallocation from vegetative to also conserved among different isolates of reproductive structures of the plant. Such ToANV, and among other members of the phenotypic plasticity is a characteristic of genus Torradovirus. Finally, site-directed accessions with longer life cycles and mutagenesis of the RNA dependent RNA slower growth rates. However, this polymerase and protease abolished the tolerance mechanism might not be replication and polyprotein processing of effective against more virulent viruses, as ToANV. TuMV, which may not give the plant

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enough time to reallocate resources. To Parallel Session IV: ANIMAL VACCINES address this subject, 19 accessions for Chairpersons: which tolerance to CMV has been FERNANDO RODRIGUEZ AND previously analyzed were challenged against TuMV. In each accession, virus ALEJANDRO BRUN multiplication, virulence (as effect of virus Monday June 8, 2015 infection on plant’s seed production), AUDITORIUM REAL CASA DE LA MONEDA effect of TuMV infection on plant growth, and length of vegetative and reproductive 17:30-17:45h (CO 33) periods were quantified. This data was A NOVEL STRATEGY FOR MULTISEROTYPE compared with values obtained upon CMV PROTECTION AGAINST BLUETONGE VIRUS infection. USING muNS-Mi MICROSPHERES AND Results indicated that TuMV multiplication RECOMBINANT MVA EXPRESSING VP2, VP7 was generally similar in the 19 accessions AND NS1 PROTEINS analyzed. Accessions with shorter life A. MARÍN-LÓPEZ1, I.OTERO-ROMERO2, F. cycles and faster growth rates were more DE LA POZA1, R. MENAYA-VARGAS2, E. tolerant to TuMV infection than those with CALVO-PINILLA1, J. BENAVENTE2, J. M. larger life cycles and slower growth rates. MARTÍNEZ-COSTAS2 AND J. ORTEGO1 Therefore, these results were at odds with 1. Centro de Investigación en Sanidad Animal, INIA- response of the same accessions to CMV CISA, Valdeolmos,Madrid, Spain. infection, and are compatible with trade- 2. Centro de Investigación en Química Biológica y offs of tolerance to TuMV and CMV Materiales Moleculares (CIQUS), Universidad de infection. Interestingly, infected plants of Santiago de Compostela, Spain. the accessions that were tolerant to TuMV infection showed shorter vegetative Recent worldwide outbreaks ofbluetongue periods than the corresponding control virus (BTV) reveal the necessity of individuals, suggesting that this could be controlling and preventing this an alternative method of achieving hemorrhagic disease that affects tolerance. ruminants. One of the most effective In summary, our results provide evidence measures against infection is vaccination. of tolerance trade-offs in plantvirus The inactivated BTV vaccines that are now interactions, and suggest that Arabidopsis being used in Europe are effective in plants may achieve tolerance by preventing outbreaks of BTV but they are mechanisms other than resource serotype-specific and secondary effects reallocation. have been associated with repetitive inoculation of aluminum-containing adjuvants. There are 27 known and two further putative BTV serotypes. Consequently, the need to develop, multiserotype, safer and more efficacious vaccines with differential diagnostic capability have re-ignited the interest in

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developing improved vaccination 17:45-18:00h (CO 34) strategies against BTV. We have TRIAL FOR CHECKING THE PROTECTIVE engineered a subunit BTV vaccine IMMUNITY OF A COMMERCIAL VACCINE candidate based on proteins VP2, VP7, and AGAINST THE “NEW VARIANT” OF THE NS1 of BTV-4 incorporated into avian RABBIT HAEMORRHAGIC DISEASE VIRUS reovirus (ARV) muNS-Mi microspheres with M. DURÁN FERRER, A. SÁNCHEZ SÁNCHEZ, potent intrinsic adjuvant activity (MS- J.I. VARO JIMÉNEZ, ELENA SAN MIGUEL VP2/MS-VP7/MS-NS1) and recombinant IBÁÑEZ, R. VILLALBA MARTÍNEZ, ISABEL modified vaccinia virus Ankara (rMVA) GONZALO PASCUAL, F. GARCÍA PEÑA, M. expressing VP2, VP7 and NS1 proteins from AGÜERO GARCÍA BTV-4 (rMVA -VP2/ rMVA -VP7/ rMVA- Laboratorio Central de Veterinaria. MAGRAMA. NS1). Algete, Madrid. Spain. IFNAR(-/-) mice immunized with MS- VP2/MS-VP7/MS-NS1 in a homologous Rabbit haemorrhagic disease is an acute, prime-boost vaccination generated highly contagious and fatal viral disease significant humoral and cellular immune caused by a member of the genus response. Immunized mice were fully and family that protected against a homologous challenge affects wild and domestic members of with a lethal dose of BTV-4 and partially species Oryctolagus cuniculus. Many cross-protected against a heterologous strains of RHDV with distinct challenge with a lethal dose of BTV-1. epidemiological and genetic characteristics The combination of these two antigen appear to circulate in nature. Although a delivery systems, microspheres and rMVAs, single serotype has been described until in a heterologousprime-boost vaccination now, three major subtypes have been strategy maintained the induction of reported: RHDV, antigenic variant RHDVa significant levels of neutralizing antibodies. and antigenic variant RHDVb. The RHDVb, Interestingly, this strategy elicited a stronger also called “RHDV new variant”, infects cellular immune response than the farmed adult individuals previously homologous immunization with immunized with vaccines against the other microspheres, and fully protected subtypes and singularly induces clinical immunized IFNAR(-/-) mice against disease in young rabbits. homologous and heterologous challenges The study presented here checks the with lethal doses of BTV-4 and BTV-1. These efficacy of a vaccine temporarily licensed results support the strategy based on in Spain for clinically protecting 36-39- microspheres in combination with rMVAs days-old New Zealand White rabbits expressing BTV antigens as a promising (vaccinated 7 days before), against a wild multiserotype vaccine candidate against strain of the RHDVb. The experimental BTV. design followed the European Pharmacopoeia (8.0 version) and minimized the number of individuals due to animal welfare reasons.

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Experimental groups were as follows: 18:00-18:15h (CO 35) vaccinated group (VG, n=12), control group SYLVATIC RABIES IN THE NORTH-EAST OF 1 (CG-1, n=7), control group 2 (CG-2, n=6) ITALY: MONITORING AND EVALUATION and witness group (WG, n=5). VG was OF THE EFFECTIVENESS OF PROPHYLAXIS vaccinated following the manufacturer IN WORKERS AT RISK AND TRAVELERS instructions (0.5 ml/SC route). Seven days N. INGLESE1, C. SALATA1, P. DE after, the VG and the CG-1 were BENEDICTIS2, G. CARPENÈ3, R. MEL3, M. challenged (0.2 ml/IM) with full dose of BEVILACQUA4, S. SCHMORAK4, P. ROSSI5, E. 5 2 1 virus (100 x LD50) and the CG-2 with a PAGANI , P. MULATTI , G. PALÙ , L. 2 2 6 reduced dose (1 x LD50). The inoculum BONFANTI , F. MUTINELLI , P. D’AGARO , consisted of diseased young rabbit liver D. SANTON6, T. GALLO7, S. MARANGON2, diluted homogenates that were P.TOMAO8,N. VONESCH8. characterized as RHDV new variant by a 1. University of Padua, Department of Molecular specific rt-RT-PCR previously developed Medicine, Padua, Italy (Rocha et al., 2013). The WG did not 2. Istituto Zooprofilattico Sperimentale delle Venezie received any intervention. (IZSVe), Legnaro, Italy 3. UlSS1di Belluno, Servizio di Igiene e Sanità The animals were clinically monitored Pubblica, Belluno, Italy during 7 days (CG-1, CG-2) and 13-14 (VG 4. Azienda Sanitaria dell’Alto Adige, Comprensorio and WG) after challenge, respectively, and Sanitario di Merano, Servizio Igiene e Sanità Pubblica, Merano, Bolzano, Italy the survivors euthanized and subjected to 5 necropsy. Sera and liver samples were . Azienda Sanitaria dell’Alto Adige, Comprensorio Sanitario di Bolzano, Laboratorio Aziendale di taken for antibodies (ELISA, HI test), Microbiologia e Virologia, Bolzano, Italy antigen (ELISA, HA test) and nucleic acid 6. University of Trieste, Department of Medical, detection (rt-RT-PCR), as well as for Surgical and Health Sciences; I.R.C.C.S. “Burlo histological studies. Garofolo”, Trieste, Italy 7. Azienda per servizi sanitari n.4 (ASS4), “Medio The trial resulted valid: 100% (7/7) Friuli”, Dipartimento di Prevenzione, Udine, Italy. morbidity and mortality in the CG-1; 50% 8. INAIL,Istituto Nazionale per l'Assicurazione contro (3/6) morbidity and mortality in the CG-2; gli Infortuni sul Lavoro SETTORE RICERCA - 100% (5/5) survival in the WG; and the Dipartimento di Medicina del Lavoro, Monteporzio Catone , Rome, Italy vaccine showed its efficacy for protecting

100% (12/12) of young rabbits against the Rabies is a global zoonotic disease that clinical disease. occurs in developing and developed countries, producing consistently fatal encephalitis in humans and animals. Rabies virus infects mammals through infected saliva via bites or scratches, although atypical exposures have been documented. In late 2008, wildlife rabies re-emerged in Northeastern Italy in an area bordering Slovenia, spread to Veneto region (Belluno province) and to the autonomous province

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of Trento and Bolzano. Since then, 287 18:15-18:30h (CO 36) animal cases have been detected in wild ROLE OF INFLUENZA VIRUS SMALL RNAs and domestic animals; the last one has CONTROLLING PATHOGENICITY IN VIVO been diagnosed in a red fox in February J. VASILIJEIVC1, 2, G. GÓMEZ1, A. NIETO1, 2 2011. No human cases have been reported AND A. FALCÓN1, 2 linked to the recent epidemic and Italy has 1.Centro Nacional de Biotecnología (CNB-CSIC), been declared as free from rabies in Madrid, Spain February 2013. Several oral fox vaccination 2Centro de Investigaciones Biomédicas en Red de campaigns accompanied by efficacy Enfermedades Respiratorias (Ciberes), Spain. monitoring and extensive surveillance of territories affected by the epidemic have Influenza virus particles contain small RNAs been implemented together with (svRNAs) corresponding to large internal education and preventive vaccination of deletions of viral segments generated workers at risk of viral exposure (i.e. during in vitro serial passages, which are forestry and wildlife workers, known as defective interfering particles veterinarians, shelters ‘operators and (DIs). The presence of DIs, potentiate the laboratory personnel). The aim of this work immune response both in cell cultures and was the evaluation of the rabies antibodies animal models, possible through level and persistence in workers at risk of recognition of double-stranded RNA by exposure and travelers. A total of 347 different receptors activating antiviral serum samples were collected: 169 after signaling cascades. Accordingly, a single pre-exposure prophylaxis and 178 after dose of the DIs given several days before post-exposure prophylaxis performed with inoculation, protects elderly mice and different immunization schedules. All sera reduces a severe and fatal disease to have been tested to detect rabies virus subclinical and mild infection, probably anti-glycoprotein antibodies by a through activation of antiviral response. commercial quantitative indirect ELISA Recently the presence of svRNAs in (Platelia TM Rabies II kit; Biorad) and with pH1N1 infected patients has the reference method FAVN (Fluorescent been reported, but at present nobody has Antibody Virus Neutralization), according evaluated their possible correlation with to the procedure recommended by the pathogenicity in humans. WHO. The results on the protection level, persistence of antibodies and the We have analyzed differential virulence comparison between the ELISA and FAVN among pandemic pH1N1 circulating test will be discussed. viruses, comparing the pathogenicity of a virus from a fatal case (F), with a virus that caused mild symptoms (M), both of them without any known co-morbid condition. Higher rate of replication in cell cultures and increased mortality in infected mice was found in the F virus. The high- throughput sequence of virions from these

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two strains and six more pandemic isolates 18:30-18:45h (CO 37) showed the presence of small viral RNAs BLOCKING OF TYPE I IFN PATHWAY BY (svRNAs) in all samples, but interestingly PESTE DES PETITS RUMINANTS VIRUS the fatal virus contained much lower (PPRV) amounts of these svRNAs. These svRNAs M. AVIA, G. RANGEL, E. PASCUAL, V. possess the 5’ and 3’ ends of the parental MARTÍN AND N. SEVILLA RNA segments and most have large, single, Centro de Investigación en Sanidad Animal (CISA)- central deletions. They have been found INIA, Madrid, Spain. for all segments, but the majority of them belong to PB2 and PB1 segments. Peste des petits ruminants virus (PPRV) is Activation of innate immune response has the causative agent of an economically been evaluated in M or F virus-infected significant and highly contagious disease of human lung epithelial cells. This study small ruminants, peste des petites showed that F virus induces lower amounts ruminants (PPR). PPRV belongs to the of type I interferon and interferon family , genus stimulated genes than M virus. ,which includes important Recombinant viruses with the individual pathogens as Measles virus (MeV) in changes found in the F virus were humans or Rinderpest virus (RPV) in obtained. That one having D529N mutation animals. The P gene of Morbillivirus in the PA polymerase subunit produced encodes for the P protein, and also for less amount of svRNAs in cell culture and three non-structural proteins: the V and W was even more pathogenic than the proteins, that are produced by co- original F isolate in the mice. In addition, transcriptional insertion of additional G deep sequencing of RNA from lungs of residues into a fraction of the mRNAs mutant PA D529N or control virus-infected transcribed from the P gen, and the C mice has been performed and we are now protein, that results from translation of an evaluating their differential genome alternative open reading frame. Previous expression profile. studies with MeV and RPV have shown These results suggests that a specific that these non-structural proteins play a residue in the viral polymerase, found in a role in blocking IFN pathway signaling. In virus isolated from a fatal case, is the case of PPRV only V protein seems to responsible for svRNAs synthesis and interfere with both type-I and type-II IFN establishes a correlation between amount signaling pathways. However, there is no of svRNAs and virus pathogenicity in the evidence of the inhibitory activity of C, P mice model and possibly in humans. and W proteins. To improve our understanding of the mechanisms involved in the ability of PPRV proteins to block IFN action, we have studied the inhibition of the activation of IFN stimulated response elements (ISRE), using luciferase reporter assays, by V, C, P

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and W proteins. First, we have shown that that causes high mortality in naive ICV’89 and India/94, two virulent PPRV populations of horses. The disease is strains, and the vaccine strain Nigeria/75, endemic in sub-Saharan Africa, but are highly effective in blocking the action outbreaks also occur in North Africa, Asia of type I IFN by significantly reducing the and Europe, leading to massive economic activation through the ISRE promoter. losses to the equine industry. Previous Thereafter, we have cloned and sequence studies in our group showed that active for the first time the PPRV W protein, immunisation with a recombinant modified needed for subsequent assays. In vitro vaccinia Ankara virus expressing VP2 experiments demonstrated that V appears (MVA-VP2), the major virus neutralisation to be the dominant inhibitor of IFN antigen of AHSV serotype 4, induced virus signaling. The W effect was weaker than neutralising antibodies and complete the inhibition observed with the V protein protection against lethal challenge in but still significant. Finally, P protein seems interferon alpha receptor gene knock-out to weakly block IFN activity and C shows no mice (IFNAR -/-) and horses. In addition, blocking effect on the stimulation through passive transfer of MVA-VP2 antiserum the ISRE promoter. The effect of each was found to protect mice and significantly protein in STAT1/2 phosphorylation and/or decrease levels of viral replication when nuclear translocation is currently under administered 1h before AHSV challenge. study. In summary, this study highlights We have extended these studies to further the ability of PPRV proteins to block the characterise protective role of the IFN response as a mean to control the host antibody responses induced by MVA-VP2 immune response. vaccination. Thus, recipient mice were transferred with antiserum or splenocytes 18:45-19:00h (CO 38) derived from MVA-VP2 vaccinated mouse ADMINISTRATION OF ANTISERUM FROM donors and then challenged with virulent MICE VACCINATED WITH MODIFIED AHSV-4.The protective immunity of the VACCINIA ANKARA VIRUS EXPRESSING passively immunised mice was compared AFRICAN HORSE SICKNESS VIRUS (AHSV) with that of MVA-VP2 vaccinated and VP2 PROTEIN CONFERS PROTECTION unvaccinated animals. Antiserum WHEN ADMINISTERED BEFORE OR AFTER recipients showed high protection against CHALLENGE disease, with 100% survival rates even in EVA CALVO-PINILLA 1, FRANCISCO DE LA mice that were immunised 48 h after POZA 2, PETER MERTENS1, JAVIER ORTEGO challenge and statistically significant 2, JAVIER CASTILLO-OLIVARES 1. reduction in viraemia in comparison with the control groups. On the other hand, 1. The Pirbright Institute, Surrey, United Kigndom. mice that received splenocytes from MVA- 2. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria INIA-CISA, Madrid, Spain VP2 vaccinates, showed a small reduction in viraemia and a 40% survival rate. Results

of these experiments show the potential of African horse sickness is a fatal viral administration of MVA-VP2 hyper immune disease spread by Culicoides biting midges

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serum as an emergency treatment for While 103pfu of BA71cos (BA71 passed ten AHSV. times in Cos cells) killed 100% of the pigs within a week, the same dose of BA71Δfx 19:00-19:15h (CO 39) did not provoke any clinical signs BA71ΔFX: A LIVE ATTENUATED VACCINE compatible with ASF. Interestingly, 2 out of THAT CONFERS PROTECTION AGAINST the six pigs immunized with this low dose HOMOLOGOUS AND HETEROLOGOUS of BA71ΔfX survived the lethal challenge AFRICAN SWINE FEVER VIRUSES with the homologous virulent BA71, corresponding with those showing strong P. LÓPEZ-MONTEAGUDO1, A. GALLEI2, A. 1 1 1 antibody and specific T-cell responses (by LACASTA , M.J. NAVAS , S. PINA , F. -ELISPOT). The protection afforded ACCENSI1, J.M. RODRÍGUEZ3, M.L. SALAS3, 1 by BA71ΔfX was dose-dependent since F. RODRÍGUEZ increasing 30 times the vaccine dose 1 Centre de Recerca en Sanitat Animal (CReSA) - (3x104pfu) yielded 100% of protection Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Campus UAB, 08193 Bellaterra, Barcelona, against the BA71 lethal challenge and more Spain importantly, also against the heterologous 2 2 Boheringer Ingelheim… E75 lethal challenge . In consonance with 3Centro de Biología Molecular Severo Ochoa these results, all pigs intramuscularly 6 (Consejo Superior de Investigaciones Científicas- inoculated with 10 pfu of BA71Δfx also Universidad Autónoma de Madrid), Madrid, Spain survived, in this occasion showing no viremia at any time after challenge. Aiming The absence of safe and efficient vaccines to extend these studies to the virus strain against African swine fever virus (ASF), currently circulating in Europe, a final difficult its control. Experimental experiment was set up. Thus, a group of 10 pigs were intramuscularly inoculated with immunizations with Live Attenuated 6 Viruses (LAV) have demonstrated to induce 10 pfu of BA71ΔfX and then challenged efficient protective immune responses, with a lethal dose of Georgia 2007. In albeit most of the times circumscribed to contrast with control pigs dying by day 7 homologous ASFV challenges. The main post-challenge, all BA71ΔfX-immunized objective of our work was to explore the pigs survived Georgia 2007. While 4 of protective potential of BA71ΔfX: a them suffered short viremia and fever genetically modified LAV deficient on a key peaks after challenge, the other 6 virulence factor (factor X). BA71ΔfX was remained clear of virus and clinical signs obtained by compatible with ASF throughout the from the virulent BA71 and was grown is infection. In spite of these impressive Cos-1 cells1. Groups of 8 weeks old Large x protection results, further work is needed White male pigs were intramuscularly to increase the safety of our vaccine since transmission to sentinel pigs has been inoculated once with different amounts of 6 BA71ΔfX and 28 days later were subjected occasionally recorded when using 10 pfu to intramuscular lethal challenge using of BA71ΔfX. In conclusion, BA71ΔfX has different ASFV stains. PBS-inoculated pigs demonstrated to confer very solid were always used as controls in our assays. protection against experimental challenge

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with lethal homologous and heterologous cell expression system. This molecule was ASF viruses. previously described as capable to target fused antigens to Antigen Presenting Cells 19:15-19:30h (CO 40) enhancing humoral and cellular immune responses. The immunogenicity of VP2 EXPERIMENTAL BLUETONGUE VIRUS 4 and APCH-VP2 recombinant proteins was SUBUNIT VACCINE DELIVERED TO evaluated in guinea pigs, cattle and ANTIGEN PRESENTING CELLS IFNAR(-/-) mice. Titers of specific 1 2 D. LEGISA , A. MARÍN-LOPEZ , M. PEREZ- neutralizing antibodies in guinea pigs and 1 1 AGUIRREBURUALDE , F. GONZALEZ , V. cattle immunized with VP2 or APCH-VP2 1 1 3 RUIZ , A. WIGDOROVITZ , J. ESCRIBANO , J. were high and similar to those induced by 2 1 ORTEGO . M. DUS SANTOS . a conventional BEI-inactivated vaccine. 1. Instituto de Virología, INTA-Castelar. Buenos Even more, similar titers were reached for Aires. Argentina, treatments including BEI-inactivated 2 . Centro de Investigación en Sanidad Animal, INIA- vaccine, VP2- and the APCH-VP2-based CISA, Valdeolmos, Madrid, Spain. 3 vaccines, although a four-fold lower . Departamento de Biotecnología, INIA, Madrid, antigenic mass was used in the APCH-VP2 Spain group. The immunogenicity of recombinant proteins was further studied Bluetongue virus (BTV), the causative in the IFNAR(-/-) mouse model. Purified agent of bluetongue disease (BT) in VP2 and APCH-VP2 proteins were domestic and wild ruminants, is inoculated without use of adjuvant, in worldwide distributed and it is included in order to do not mask immunogenicity. the unified OIE list of notifiable terrestrial Here, the fusion of VP2 to APCH and aquatic animal diseases. A total of 27 enhanced the cellular immune response serotypes have been described so far, and and the neutralizing activity induced by several outbreaks have been reported VP2. along with their associated economic loss. Vaccination is critical for controlling the spread of BTV. In the last years, subunit vaccines, viral vector vaccines and reverse genetic-based vaccines have emerged as new alternatives to conventional inactivated or attenuated vaccines. In this study, we developed an experimental subunit vaccine against BTV4, with the benefit of targeting the recombinant BTV protein to antigen-presenting cells. The VP2 protein from an Argentine BTV4 isolate was expressed alone or fused to the Antigen Presenting Cell Homing (APCH) molecule, in the baculovirus insect

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Parallel Session V: of IFN-alpha and/or recombinant purified CELL-VIRUS INTERACTION IFNBP. To examine the immunomodulatory Chairpersons: activity of the IFNBP, the transcriptome analysis from cells infected witha VACV- COVADONGA ALONSO AND WR deletion mutant lacking the IFNBP was ENRIQUE VILLAR included. RNA libraries were prepared and Monday June 8, 2015 paired-end sequencing performed using WHITE ROOM the Illumina HiSeq system. After removal of low quality reads, over 100M high

quality reads per sample were obtained, 17:30-17:45h (CO 41) which could be mapped either to the VACV RNA-SEQ BASED TRANSCRIPTOME or mus musculus strain C57/BL6 reference ANALYSIS OF THE INTERFERON HOST genomes. Then, analysis of differential RESPONSE UPON VACCINIA VIRUS gene expression and GO pathway INFECTION enrichment analysis were performed to G. ALONSO1, B. HERNÁEZ1,, J.M. ALONSO- reveal the molecular mechanisms of LOBO1, D. AGUIRRE DE CARCER1, A. action. 1 2 2 RASTROJO , C. FISCHER , S. SAUER , B. We could validate the experiment 1 1 AGUADO AND A. ALCAMI . identifying the expected transcriptional 1Centro de Biología Molecular Severo Ochoa changes after IFN-induced signalling in the (CBMSO), Madrid, Spain. transcriptome from IFN-treated cells. The 2 Max-Planck-Institute for Molecular Genetics, addition of recombinant IFNBP to cells Berlin, Germany. prior to IFN completely reverted these IFN- induced changes to basal levels found in Evasion of interferon (IFN)- untreated cells. The addition of mediatedantiviral immunityis critical for a recombinant IFNBP to cell cultures did not successful virus infection. So poxviruses result in significant activation of any have evolved diverse molecular strategies cellular pathway, in spite of the IFNBP to counteract IFN host response activity at attaching to the cell surface. Finally, to different levels. In the case of VACV, one of detect and analyze those changes in host these is to encode a soluble IFN-/ gene expression after viral infection of binding protein (IFNBP) which located at cultures, treated or not with IFN, analysis cell surface binds type-I IFN molecules with of differential gene expression and GO high affinity, preventing their interaction pathway enrichment analysis were with the host cell receptor. performed and will be discussed. In the present work, we have dissected by RNA-Seq the viral modulation of the IFN- based host response at the transcriptional level. RNA from VACV-WR infected murine

L929 cells was extracted at 0, 4 and 9 h post-infection in the absence or presence

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17:45-18:00h (CO 42) corresponding host responses with a A SYSTEM BIOLOGY APPROACH REVEALS systems biology approach in vivo. Acute or GENES AND PATHWAYS INVOLVED IN T- persistent infections were established by CELL EXHAUSTION AT TISSUE LEVEL inoculating mice with different LCMV-virus J ARGILAGUET1, A ESTEVE-CODINA3, M doses, and spleen-specific transcriptomes PEDRAGOSA1, C PELIGERO1, S HEATH3 AND of mice with different infection outcomes A MEYERHANS1,2 were determined. Here, we present the results obtained through a bioinformatic 1Infection Biology Group, Department of Experimental and Health Sciences, Universitat analysis that allows us to describe the Pompeu Fabra, Dr. Aiguader 88 08003, Barcelona, kinetics of the main biological processes Spain ([email protected]) induced in response to acute and 2Institució Catalana de Recerca i Estudis Avançats persistent virus infections. We have (ICREA), Barcelona, Spain identified a set of co-regulated genes 3 Statistical Genomics, Centro Nacional de Análisis linked to the virus-specific T-cell effector Genómico, Barcelona, Catalonia, Spain. response during an acute infection, and we describe how this biological process is Viral infections can be fundamentally fragmented into several ones during a categorized as acute or persistent persistent infection at the time of according to their temporal relationships exhaustion appearance. Hub genes with their hosts. In an acute infection, temporally related to these processes were virus-specific T-cells become activated, characterized, representing control points proliferate, and differentiate into effector of the main biological pathways involved in T-cells, allowing the virus elimination infection fate at tissue level. Moreover, we within a few weeks. By contrast, persistent used a free web-based software (digital infections, such as those caused by HIV and cell quantification, DCQ) that combines HCV, are not resolved and develop when T- genome-wide gene expression data with cells become exhausted, i.e. differentiate an immune cell compendium to infer into a state with poor effector function to changes in the quantities of immune cell avoid immunopathology. There is a broad types from the transcriptome profiles knowledge of T-cell-intrinsic mechanisms obtained from spleens. This allowed us to involved in the dysfunction of virus-specific predict the immune cell subpopulations effector lymphocytes during chronic likely involved in the previously infections. However, the establishment of characterized biological processes induced a persistent infection is the result of the in response to an acute and persistent interactions between multiple immune cell virus infections. populations, and the T-cell-extrinsic mechanisms involved in the induction of T- cell exhaustion are still poorly understood. We used the Lymphocytic Choriomeningitis Virus (LCMV)-infection mouse model system, which enables us to follow the dynamics of the virus infection and the

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18:00-18:15h (CO 43) that virus entry and early gene expression AFRICAN SWINE FEVER VIRUS could be directly affected by the REPLICATION IS AFFECTED BY THE proteasome inhibitor. Our data suggests INHIBITION OF THE PROTEASOME SYSTEM thatfunctional ubiquitin-proteasome L. BARRADO GIL1, I. GALINDO 1, M.A. machinery is required during ASFV CUESTA-GEIJO1, R. MUÑOZ-MORENO1,2 infection. As in other viral models, core- AND C. ALONSO 1 associated and/or viral proteins involved in DNA replication may be targets for the 1 Departament of Biotechnology, Instituto Nacional de Investigación Agraria y Alimentaria (INIA), ubiquitin-proteasome pathway that could Madrid, Spain possibly assist to the virus in either core 2 Present address: Department of Microbiology, uncoating or DNA replication. Icahn School of Medicine at Mount Sinai, New York, USA. 18:15-18:30h (CO 44) CORRELATIVE LIGHT AND ELECTRON Ubiquitination has a central role in a MICROSCOPY TO STUDY VIRAL variety of cellular processes, such as MORPHOGENESIS AND EGRESS control of cell division, signal transduction, L.SANZ-SÁNCHEZ1, C.RISCO1 transcriptional regulation, immune 1Laboratorio de Estructura Celular, Centro Nacional response, endocytosis, cellular trafficking, de Biotecnología, CSIC, Madrid, Spain. and cell survival control. Many viruses manipulate the proteasome system for Arbovirus infections represent an their advantage. Some of them encode important percentage of all emerging proteins that can modify the host’s infectious diseases detected recently. ubiquitin machinery, and other viruses These viruses are transmitted to humans even encode their own ubiquitinating or and animals by arthropod vectors, mainly deubiquitinating enzymes. mosquitoes and ticks. Arbovirus outbreaks ASFV encodes a gene with high homology are showing up in new regions due to the with the E2 or ubiquitin conjugating (UBC) introduction of the arthropod vectors in enzyme. UBCv is expressed throughout temperate habitats. The Bunyaviridae is a ASFV infection and accumulates at late large family of RNA viruses, most of them times post infection. The presence of a Arboviruses. This family includes several viral ubiquitin conjugating enzyme in the important pathogens that cause virions implies that the ubiquitin- encephalitis or haemorrhagic fevers. The proteasome pathway could play an best characterized member of the family is important role during ASFV infection. Bunyamwera virus (BUNV). In infected cells We found that the proteasome inhibitor BUNV builds a complex factory by MG132 blocked a post entry step in ASFV recruitment of mitochondria and RER replication. In the presence of MG132, ASF elements around Golgi stacks. A viral genome replication, late gene Transmission Electron Microscopy (TEM) expression and viral production were study of cultured adherent cells´ revealed severely reduced.Moreover,we excluded two previously unreported structures

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induced by BUNV in basal regions of the will provide new means for identifying cell: complex multilamellar structures viral-cell interactions and targets for novel (MLS) and extracellular filament bundles. antiviral compounds. We have used live cell microscopy followed by Correlative Light and Electron 18:30-18:45h (CO 45) Microscopy (CLEM) to study the structural CD2v INTERACTS WITH ADAPTOR changes of cells during the late phase of PROTEIN AP-1 DURING AFRICAN SWINE BUNV infection that is egress and FEVER INFECTION propagation. Serial sections and 3D 1 1 reconstructions showed that MLS D. PÉREZ-NÚÑEZ , E. GARCÍA-URDIALES , M. MARTÍNEZ-BONET2, M. L. NOGAL1, S. exclusively contacted the plasma 1 1 1 membrane; however, these virus-induced BARROSO , R. MADRID AND Y. REVILLA 1 structures were not similar to any other Virology Department, Centro Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain plasma membrane specializations, such as 2 podosomes, filipodia or invadopodia. Hospital Gregorio Marañón, Madrid, Spain. Morphology and dimensions of MLS were reminiscent of those reported for the African swine fever (ASF) is a highly lethal nanostructures on gecko fingertips, which and economically important disease of are responsible for the extraordinary domestic pigs for which there is no control attachment capacity of these lizards. As strategy other than animal quarantine and infected cells with MLS were more slaughter. Classified as a notifiable disease resistant to detachment than control cells, by the World Organization for Animal we propose an adhesive function for these Health (OIE), ASF causes major economic structures, which would compensate for losses to the pig industry in affected the loss of adherence during release of countries. Despite the high risk new virus progeny. The filament bundles represented by the recent outbreak in the visualized in the basal regions of infected Caucasus in 2007, its subsequent cells had numerous viruses attached and propagation throughout Russia and often contacted with non-infected cells. potential dissemination to neighboring These filaments contain actin as confirmed countries, to date, no specific protection or by both confocal microscopy and vaccine against ASF is available. immunoelectron microscopy. Using a African swine fever virus (ASFV) CD2v potent inhibitor of actin polymerization, protein seems to be involved in virulence cytochalasin D, the filament bundles were enhancement, viral hemadsorption, and no longer seen and viruses remained pathogenesis, although the molecular attached to the cell surface. We propose mechanisms of the function of this viral that viruses can be transported between protein are still not fully understood. CD2v cells on these actin-based railways. We are resembles the T-lymphocyte surface currently using live cell microscopy and adhesion receptor CD2, and it contains an CLEM together with two different eGFP- extracellular N-terminal (Nt) domain tagged recombinant viruses to study BUNV composed of two immunoglobulin-like morphogenesis and egress. This approach domains, while the cytosolic C-terminal

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domain of CD2v (CD2v-Ct) shares no 18:45-19:00h (CO 46) obvious amino acid sequence with the THE MAMMALIAN CELL CYCLE REGULATES cellular CD2 cytoplasmic domain. During PARVOVIRUS NUCLEARCAPSID ASSEMBLY infection, CD2v is assumed to be cleaved JON GIL-RANEDOa,, EVA HERNANDOb, into a Nt glycosylated and a Ct non-10 LAURA RIOLOBOSa,, CARLOS DOMÍNGUEZa, glycosylated form, but both coexist in the MICHAEL KANNb, AND JOSÉ M. infected cell with the full length protein. ALMENDRALa Here we describe that CD2v localized aCentro de Biología Molecular "Severo Ochoa" around viral factories during ASFV (Consejo Superior de Investigaciones Científicas- infection, suggesting a role in the Universidad Autónoma de Madrid), 28049 b generation and/or dynamics of these viral Cantoblanco, Madrid, Spain. University of structures and hence in disturbing cellular Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France. traffic. We show that CD2v targeted the regulatory trans-Golgi network (TGN) protein complex AP-1, a key element in Cellular and viral life cycles are connected cellular traffic. This interaction was through multiple though poorly disrupted by brefeldin A even though the understood mechanisms. It is unknown location of CD2v around the viral factory whether the mammalian cell cycle could remained unchanged. CD2v-AP-1 binding impact the assembly of viruses maturing in was independent of CD2v glycosylation the nucleus. We addressed this and occurred on the carboxy-terminal part fundamental question using MVM, a of CD2v, where a canonical di-Leu motif reference member of the icosahedral previously reported to mediate AP-1 ssDNA nuclear parvoviruses, which require binding in eukaryotic cells, was identified. cell proliferation to infect by mechanisms This motif was shown to be functionally partly understood. Constitutively interchangeable with the di-Leu motif expressed MVM capsid subunits (VPs) present in HIV-Nef protein in an AP-1 accumulated in the cytoplasm of mouse binding assay. However, we demonstrated and human fibroblasts synchronized at G0, that it was not involved either in CD2v G1, and G1/S transition. Upon arrest cellular distribution or in CD2v-AP-1 release, VPs translocated to the nucleus as binding. Taken together, these findings cells entered S phase, at efficiencies relying shed light on CD2v function during ASFV on cell origin and arrest method, and infection by identifying AP-1 as a cellular immediately assembled into capsids. In factor targeted by CD2v and hence synchronously infected cells, the elucidate the cellular pathways used by the consecutive virus life cycle steps (gene virus to enhance infectivity. expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs

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became increasingly retained in the 19:00-19:15h (CO 47) cytoplasm hours post-stress, forming POLYANIONIC CARBOSILANEDENDRIMERS empty, mislocalized capsids in mouse PREVENT THE VAGINAL/RECTAL HSV-2 fibroblasts, thereby impairing ENTRY IN VIVO. encapsidation of the nuclear viral DNA. 1* 1* P. GARCIA-BRONCANO , R. CEÑA-DIEZ , Synchronously infected cells subjected to 1 2 R. GÓMEZ , F. J. DE LA MATA , MA. density-arrest signals while traversing early 2 MUÑOZ-FERNANDEZ . S phase also blocked VPs transport, 1. Laboratorio InmunoBiología Molecular, Hospital resulting in a similar misplaced cytoplasmic General Universitario Gregorio Marañón, Madrid, capsid assembly in mouse fibroblasts. In Spain. Instituto de Investigación Sanitaria Gregorio contrast, the above-mentioned stressing Marañón (IISGM), Madrid, Spain.Networking signals deregulating virus assembly neither Research Center on Bioengineering, Biomaterials perturbed nuclear translocation of the NS1 and Nanomedicine (CIBER-BBN), Madrid, Spain.Spanish HIV HGM BioBank, Madrid; Spanish protein nor viral genome replication HIV HGM BioBank, Madrid, Spain. occurring under S/G2 cycle arrest. The 2. Departamento de Química Inorgánica, exquisite cell cycle-dependence of Universidad de Alcalá, Campus Universitario, Alcalá parvovirus nuclear capsid assembly de Henares, Madrid, Spain. CIBER-BBN, Madrid, conforms a novel paradigm of time and Spain functional coupling between cellular and virus life cycles. This junction may Despite a significant worldwide need for determine the characteristic parvovirus effective microbicides to reduce sexually tropism for proliferative and cancer cells, transmitted diseases (STD) and genital and its disturbance could critically 2 (HSV-2) contribute to persistence in host tissues. transmissions, these microbicides are not These findings may contribute to currently available. Topical microbicides understand cellular regulations on the are applied directly to the genital tract or assembly of other nuclear eukaryotic rectum prior to intercourse to protect viruses, and to develop cell cycle-based against STD. Vaginal and rectal avenues for antiviral therapy. microbicides can reach high local drug concentrations to prevent HSV-2 transmission without toxicity due to the many potential benefits associated with the drug delivery route. We have

previously demonstrated that polyanionic carbosilane dendrimers, G1-S4 and 2G-S16 exert their inhibitory effect impeding the viral entry by different mechanisms of action; G1-S4 could bind directly on viral proteins on the surface of HSV-2 particles and inactivate HSV-2, whereas 2G-S16 carry out its inhibitory effect by binding to cellular surface molecules of host cell.G1-

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S4 and 2G-S16, showed a good safety 19:15-19:30h (CO 48) profile and did not cause histopathological RECRUITMENT OF HOST FACTORS BY THE alterations to the vaginal epithelium at CRE ELEMENT OF THE HEPATITIS C VIRUS concentrations of 8 mM and 12 mM, P. RÍOS-MARCO, C. ROMERO-LÓPEZ Y A. respectively. Our data clearly demonstrate BERZAL-HERRANZ that topical vaginal administration of 3% Instituto de Parasitología y Biomedicina López- G1-S4 or 3% 2G-S16 formulated as 2% Neyra, IPBLN-CSIC, PTS Granada, Avda. del hydroxyethylcellulose (HEC; NIH-ARRRP) Conocimiento s/n, Granada, Spain gel prevent HSV-2 transmission in BALB/c mice in 100%. This research represents the The hepatitis C virus (HCV) has a positive first demonstration that transmission of single stranded RNA genome, with an HSV-2 can be efficiently blocked by approximate length of 9.6 Kb. This genome vaginally applied of G1-S4 or 2G-S16. In contains a single ORF, flanked by addition, promising results were also conserved 5' and 3' untraslated regions obtained in the case of rectal infection, (UTRs). The 5'UTR contains the most part although without reaching the inhibition of the IRES (Internal Ribosome Entry Site), values obtained in the vaginal challenge which allows the Cap-independent assay. Therefore, further studies should be translation of a viral polyprotein, which is performed in order to increase their rectal proteolized into ten different viral protection efficiency. Our dendrimers have proteins. The 3'UTR is involved in the viral shown a synergistic profile in in-vitro when replication and translation, and is combined with other anti herpetic drugs composed of three well-defined elements: already described, such as Tenofovir, the hypervariable region, a poly U/UC against HSV-2 as well as against HIV; hence domain and the so-called 3’X-tail domain it would be worthwhile to analyze such a that is formed by three well conserved combination therapy with those antivirals stem-loop subdomains. Another element in-vivo. of structural and functional importance in These results obtained in BALB/c mice the viral genome is the cis-acting provide a strong step forward in the replicating element (CRE), located at the 3' development of G1-S4 or 2G-S16-based end of the region coding for the HCV viral vaginal and rectal microbicides to prevent polymerase (NS5B). CRE plays a role at vaginal HSV-2 transmission in humans. replication by recruiting NS5B and also exerts negative control of the IRES- dependent translation. In fact this region, of approximately 200 nucleotides, is able to establish internal contacts to specific

domains belonging to 3'UTR and 5'UTR, modulating structural changes in the genome and regulating essential processes for viral cycle progression. However, the interrelationship of the CRE element with

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cellular factors has been little studied so Parallel Session VI: ROLE OF VIRUS IN far. This work summarizes the progress of PEDIATRIC DISEASES (SEV-SEIP) our group in the identification of host [Joint Session SEV-SEIP] proteins and translational machinery with Chairpersons: capacity to interact with the CRE element. For example, by proteomic approach, we Mª ISABEL GONZÁLEZ-TOMÉ AND report a wide group of CRE-binding MARISA NAVARRO proteins such as hnRNPs, Ras GTP- Monday June 8, 2015 activating protein-binding proteins, RNA- AUDIOVISUAL ROOM helicases or splicing factors. Moreover we provide data indicating that CRE interacts with ribosomal particles and map in detail *Invited paper the nucleotides involved in such 17:30-17:45h (CO 49) interaction. Our data indicates that the IMPLICATIONS OF CONTROL OF VIRAL CRE stem-loop 5BSL3.2 plays an important LOAD DURING PREGNANCY AND RISK OF role both in recruiting host proteins and in HIV-1 MOTHER-TO-CHILD TRANSMISSION binding of the 40S ribosomal subunit. The L. PRIETO1 knowledge of associations between 1 Paediatrics Department.Hospital Universitario de cellular factors and the virus genome will Getafe, Getafe, Spain. help to understand the interaction dynamic of RNA-RNA and RNA-protein that Approximately 3.2 million of children are allows the progression of HCV viral cycle. living with HIV infection worldwide. An estimated 240,000 children were newly infected with HIV in 2014. More than 90 percent of HIV infections in children result from mother-to-child-transmission (MTCT) when the virus passed from a HIV-infected

mother to her baby during pregnancy, childbirth, or breastfeeding. Rates of HIV-1 MTCT in untreated non- breastfeeding populations in developed countries range from 15 to 40%. Nowadays, MTCT is almost entirely preventable infection when interventions

including antenatal HIV screening, antiretroviral therapy (ART), appropriate mode of delivery, neonatal antiretroviral prophylaxis and avoidance of breastfeeding are carried out. These interventions have resulted in MTCT rates

less than 2% in many developed countries.

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However, in the context of low MTCT rates, response against RSV, and the lack of questions around optimal management development of protective immunity after remain inconclusive. Maternal HIV-1 viral the first infection. Most infants admitted load is considered the best predictor of the to hospitals during each epidemic risk of MTCT. Moreover, in the era of bronchiolitis caused by RSV, are previously highly active antiretroviral treatment healthy infants without underlying (HAART), MTCT rates are directly diseases. Currently it is impossible to associated with HIV-1 RNA levels at predict the evolution of these patients. At delivery. Non suppressive maternal viral the time of admission is impossible to load at delivery regardless of HAART is know which of these RSV-infected infants associated commonly with lack of prenatal will be discharged within 24 or 48 hours care of HIV-infected women but also with and which of them are going to get worse acute HIV infection during pregnancy. and they will need ventilatory support in Rates of MTCT in these cases remain high. the following days after admission. Strategies for the control of viral load Numerous factors have been described replication during pregnancy and further that can contribute to the severity of the considerations in the management of HIV- disease, such as age, viral load, RSV infected women and their exposed infants subtype A or B or the infant immune at high risk of MTCT situations are the response. Now believed to be a scope of this review. combination of host factors and virus that likely contribute to determine the severity of the disease. *Invited paper In addition, numerous studies in children 17:45-18:00h (CO 50) hospitalized for bronchiolitis due to RSV, RSV BRONCHIOLITIS: CHALLENGES IN 2015 have shown that approximately between R. RODRÍGUEZ-FERNÁNDEZ 40 and 50% of them develop in the first Hospital Infantil Gregorio Marañón. Sección de year of life recurrent wheezing, which in Lactantes. Madrid. Spain most cases disappear within 3-4 years of life. The high frequency with which this Bronchiolitis by respiratory syncytial virus occurs, suggests that there is a relationship (RSV) is the most frequent cause of between the two events, though still hospitalization in the first year of life in today, it is unclear what the causal developed societies. It also represents the mechanism. Most hypotheses suggest that leading cause of infant mortality after RSV is directly responsible of these malaria in the first 12 months of life. persistent or recurrent wheezing, while Despite this impact on global child health, other authors propose that the virus is still do not have vaccines or antiviral simply a marker that identifies children treatments in daily clinical use. One reason predisposed to persistent wheezing in why we have not yet effective therapeutic early childhood. or preventive interventions is our A better understanding of virus and the incomplete understanding of the immune infant immune response will allow us to

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establish predictive markers of severity We are conducting a prospective study in and evolution of infants with bronchiolitis. hospitalized young children with fever without source, meningitis, encephalitis *Invited paper and clinical sepsis in 10 hospitals in Spain (PI12-00904) with the objective to know 18:00-18:15h (CO 51) the prevalence of HPeV infections in this NEUROLOGICAL AND SYSTEMIC group of patients and their clinical PARECHOVIRUS INFECTIONS IN CHILDREN characteristics. A comparison with C CALVO1, M CABRERIZO2. (EV) is also performing. 1 Pediatrics Department.Severo Ochoa HPeV was tested in those specimens (sera, 2 Hospital.Leganés. Madrid. Spain Enterovirus Unit. cerebrospinal fluid, pharyngeal frotis) National Microbiology Center (ISCIII),Majadahonda, negative for EV at the CNM, using a real- Madrid. Spain. time RT-PCR designed in the 5-NCR of the

genome. Molecular typing of detected EV Human (HPeV) are RNA and HPeV was carried out by amplification viruses belonging to the family of of 3’-VP1 or VP3/VP1 regions, respectively, Picornaviridae. Formerly described as and sequencing. echovirus 22 and 23 in the Enterovirus Although our study is ongoing, we can say genus, HPeV were reclassified into their that HPeV circulating in our country are own genus, Parechovirus, in 90’s, and were relatively common in children <3 years. In renamed as HPeV-1 and HPeV-2, infants under 3 months HPeV accounting respectively. Additional types of HPeV have for 12% of the studied infections. HPeV-3 is been reported and a total of 16 different the most common. The clinical picture of types have been recognised to date (HPeV- our patients is characterized by fever 1 to 16). The most common genotype without source or suspected sepsis, detected worldwide is HPeV-1 followed by without leukocytosis and pleocytosis, even HPeV-3. Other types such as HPeV-2 and though the virus is detected in CSF. HPeV-4 are less common. Generally, they have a good prognosis. Infections with HPeV are prevalent in For pediatricians, we consider that is of young children and have been associated interest to suspect these viruses as with mild diseases of the respiratory and etiological agents of infections in young gastrointestinal tract, mainly associated to children with the mentioned HPeV-1 and HPeV-2, but also with symptomatology and to include them in meningitis, encephalitis and sepsis in the routine virological diagnosis in order to infants. HPeV-3 might be one of the main avoid unnecessary antibiotherapy and agents causing severe neonatal prolonged hospitalizations. neurological infections in Europe, although its real incidence is unknown since HPeV detection is not routinely performed. Epidemiological and clinical data in our country are scarce.

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*Invited paper transmission and individual factors 18:00-18:15h (CO 52) regarding cCMV infection in order to CONGENITAL CYTOMEGALOVIRUS (cCMV) improve the treatment options and INFECTION: HOW, WHEN, WHERE. outcome of these children. DANIEL BLÁZQUEZ-GAMERO Hospital Universitario 12 de Octubre. Sección de Parallel Session VII: Enfermedades Infecciosas e Inmunodeficiencias. VIRAL ENTRY MECHANISMS Madrid. Spain Chairpersons: JOSÉ A. MELERO AND

JOSÉ MARÍA ALMENDRAL Human cytomegalovirus (HCMV) is the Tuesday June 9, 2015 leading cause of congenital infections worldwide, the most frequent cause of non AUDITORIUM REAL CASA DE LA MONEDA genetic hearing loss in children, and a significant cause of permanent neurologic 15:00-15:15h (CO 53) sequelae. In many countries, especially in LIPID COMPONENTS IN AFRICAN SWINE developing regions with high prevalence of FEVER VIRUS ENTRY AND REPLICATION seropositive women, cCMV is a neglected M. A. CUESTA-GEIJO1, I. GALINDO1, R. disease, and no screening during MUÑOZ-MORENO1,2, L. BARRADO-GIL1 pregnancy nor in the newborn period is AND C. ALONSO1. routinely recommended. In most cases 1 Department of Biotechnology, Instituto Nacional (90%) newborns with cCMV show no de Investigación y Tecnología Agraria y Alimentaria symptoms at birth, but up to 15% of these (INIA), Madrid, Spain asymptomatic children will develop late 2 Present address: Department of Microbiology, onset sequelae. In symptomatic newborns, Icahn School of Medicine at Mount Sinai, New York, the rate of long term sequelae is even USA higher (40-50%). Despite the global impact of this infection, there are still important Since its introduction in Europe through gaps in the knowledge of disease the Caucasus in 2007, African swine fever mechanisms regarding virus transmission, (ASF) has been spreading westwards from role of viral genotypes, immunological Russia to EU countries threatening porcine responses and individual host factors. industry. Due to the lack of an effective Today there is no effective antiviral vaccine, ASF control relies on early treatment available during pregnancy, and diagnosis and massive stamping out of other treatments, such as anti-CMV animals. hiperimmune globulin, have shown limited We searched for targets at early stages of benefits. Antiviral treatment with infection aiming to discover how African ganciclovir or valganciclovir during swine fever virus (ASFV) surpasses host cell neonatal period in symptomatic children defenses and reorganizes cellular has shown encouraging results. It is of structures to initiate replication. The virus paramount importance to increase the enters the cell by endocytosis and within knowledge about mechanisms of few minutes after infection, viral

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decapsidation occurs at the acid pH of late world. The development of safe topical endosomes. We report here that the virus microbicides, compounds that applied exit from the endosome to start replication vaginally or rectally, to protect the user requires intact cholesterol endosomal from sexually transmitted infections, is of efflux. In fact, cholesterol is required at crucial importance; especially when HSV-2 several steps of the virus life cycle starting infection is associated with a 3-fold to 4- from virus entry. ASF virus reorganizes fold increased probability of HIV cholesterol landscape of the cell to the acquisition. Nanotechnology offers novel perinuclear replication site where the viral suitable tools to develop new microbicidal factory is built. These results add to a compounds, such as dendrimers. growing body of evidence pointing out Dendrimers are a class of nanoparticles cholesterol efflux and the endosomal that have shown their potential as membrane as crucial players for the start therapeutic agents and as carriers of of viral replication in several virus models. different molecules. The safety and antiviral activity of eight polyanionic 15:15-15:30h (CO 54) carbosilane dendrimers were evaluated to POLYANIONIC CARBOSILANE select a proper candidate for the DENDRIMERS AS PROMISING development of a topical microbicide MICROBICIDE CANDIDATES AGAINST HSV- against HSV-2. All dendrimers were non- toxic at the majority of the studied 2 INFECTION: BROAD-SPECTRUM ACTIVITY AND ACTION MECHANISM concentrations. The plaque reduction 1 1 assay on Vero cells showed that 2G-S16, R. CEÑA-DIEZ , E. VACAS CORDOBA P. G1-S4 and G3-S16 present the highest GARCÍA BRONCANO1, R. GÓMEZ2, F. J. DE 2 1 inhibitory effect on the HSV-2 infection, LA MATA ,MA. MUÑOZ-FERNANDEZ . reaching values of 100%, 77% and 68%, 1 . Laboratorio InmunoBiología Molecular, Hospital respectively. Moreover, changes in pH did General Universitario Gregorio Marañón, Madrid, Spain. Instituto de Investigación Sanitaria Gregorio not alter the inhibitory profile of these Marañón (IISGM), Madrid, Spain.Networking dendrimers. Interestingly, we Research Center on Bioengineering, Biomaterials demonstrated that our dendrimers inhibit and Nanomedicine (CIBER-BBN), Madrid, the viral infection at the first steps of HSV- Spain.Spanish HIV HGM BioBank, Madrid, Spain. 2 life-cycle, impeding the binding of HSV-2 2 . Departamento de Química Inorgánica, particles to target cell surface and thus, the Universidad de Alcalá, Campus Universitario, Alcalá viral entry. G1-S4 and G3-S16 sulfate- de Henares, Madrid, Spain. CIBER-BBN, Madrid, Spain ended dendrimers bound directly on viral proteins on the surface of HSV-2 particles, inactivating HSV-2; we hypothesize that Although enormous efforts have been these dendrimers could bind to HSV-2 made to prevent the sexual transmission of glycoprotein B (gB), which has an genital herpes simplex virus 2 (HSV-2), important role in HSV-2 entry by binding to there is still neither protective vaccine nor heparan sulfate on the cell surface. cure against one of the most common However, 2G-S16 achieves its inhibitory sexually transmitted infections in the effect by binding to cellular surface

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molecules of host cell. Molecular Modeling endocytosis,the possible roles of clathrin in of the interactions of G1-S4 and 2G-S16 other steps of the viral cycle remain with HSV-2 surface protein gB showed that unexplored. Thus, we studied whether cell G1-S4 binds better in general to selected culture-derived HCV (HCVcc) exocytosis binding sites on gB surface than 2G-S16 was altered after clathrin interference. and moreover it is better suited to reach Knockdown of clathrin or the clathrin worse accessible binding sites. Significantly adaptor AP-1 in HCVcc-infected human better binding properties of G1-S4 were hepatoma cell cultures impaired viral found in a position important for affecting secretion without altering intracellular transition of gB trimer from its pre-fusion HCVcc levels or apolipoprotein B (apoB) to final post-fusion state and in several and apoE exocytosis. Similar reduction in positions where it could interfere with HCVcc secretion was observed after gB/gH-gL interaction.Finally, we treatment with specific clathrin and demonstrated that these nanocompounds dynamin inhibitors. Furthermore, have synergistic activity with Tenofovir and detergent-free immunoprecipitation acyclovir, two anti-herpetic drugs. Our data assays, neutralization experiments and indicate that 2G-S16, G1-S4 and G3-S16 are immunofluorescence analyses suggested promising candidates to be developed as that whereas apoE associated with vaginal microbicides. infectious intracellular HCV precursors in endoplasmic reticulum (ER)-related 15:30-15:45h (CO 55) structures, AP-1 participated in HCVcc egress in a post-ER compartment. Finally, ROLE OF CLATHRIN AND CLATHRIN we observed that clathrin and AP-1 ADAPTOR PROTEIN-1 IN HEPATITIS C knockdown altered the endosomal VIRUS EGRESS distribution of HCV core, reducing and 1,2 1,2 I. BENEDICTO , V. GONDAR , F. MOLINA- increasing its co-localization with early 1 2,3 JIMÉNEZ , L. GARCÍA-BUEY , M. LÓPEZ- endosome and lysosome markers, 4 5 CABRERA , P. GASTAMINZA , P. L. respectively. Our data support a model in 1,2 MAJANO . which nascent HCV particles associate with 1. Molecular Biology Unit, Hospital Universitario de apoE in the ER and exit cells following a la Princesa, Instituto de Investigación Sanitaria clathrin-dependent transendosomal Princesa (IP), Madrid, Spain secretory route. 2. CIBERehd, Instituto de Salud Carlos III, Madrid 3. This is a significant finding, since to date it Liver Unit, Hospital Universitario de la Princesa, Instituto de Investigación Sanitaria Princesa (IP), has been proposed that HCV and very low- Madrid, Spain density lipoproteins (VLDL) follow similar 4. Centro de Biología Molecular Severo Ochoa, CSIC- exocytic routes. Given that lipid UAM, Madrid, Spain metabolism has recently emerged as a 5. Centro Nacional de Biotecnología-CSIC, Madrid potential target against HCV infection, our data could help to design new strategies to Althoughit is well established that hepatitis interfere specifically with HCV exocytosis C virus (HCV) entry into hepatocytes without perturbing cellular lipid depends on clathrin-mediated

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homeostasis, with the aim of achieving is the fine-tuned product of the interaction more efficient, selective and safe antivirals. between residues from different capsid proteins, in particular those located within 15:45-16:00h (CO 56) the N terminus of VP1 or close to the THE pH STABILITY OF FOOT-AND-MOUTH pentameric interface. DISEASE VIRUS PARTICLES IS MODULATED BY RESIDUES LOCATED AT THE 16:00-16:15h (CO 57) PENTAMERIC INTERFACE AND IN THE N ANALYSIS OF THE ORIGIN AND TERMINUS OF VP1 INFECTIVITY OF INFECTIOUS F. CARIDI, A. VÁZQUEZ-CALVO, F. SOBRINO, MICROVESICLES DERIVED FROM SEMLIKI M.A. MARTÍN-ACEBES FOREST VIRUS DEVOID OF CAPSID Departamento de Virología y Microbiología, Centro M. RUIZ-GUILLEN1,2, E. GABEV1,2, J.I. de Biología Molecular “Severo Ochoa” (CSIC-UAM), QUETGLAS1,2, E. CASALES1,2,J. POUTOU1,2, Madrid, Spain M.C. BALLESTEROS1,2, A. ARANDA1,2, J. BEZUNARTEA1,2, M. ONDIVIELA3, R. The picornavirus foot-and-mouth disease HERNANDEZ-ALCOCEBA1,2, N. ABRESCIA3,4, virus (FMDV) is the etiological agent of a C. SMERDOU1,2 highly contagious disease that affects 1Division of , CIMA, University of important livestock species. FMDV capsid Navarra, Pamplona, Spain. is highly acid labile and viral particles lose 2IdiSNA, Navarra Institute for Health Research, infectivity due to their disassembly at pH Pamplona, Spain values slightly below neutrality. This acid 3Structural Biology Unit. CIC bioGUNE, sensitivity is related to the mechanism of CIBEREHD .Bizkaia Technology Park, Derio, Spain 4 viral uncoating and genome penetration IKERBASQUE, Basque Foundation for Science, from endosomes. In this work, we have 48011 Bilbao, Spain analyzed the molecular basis of FMDV acid-induced disassembly by isolating and Alphaviruses are enveloped viruses that characterizing a panel of novel FMDV contain a positive-strand RNA genome mutants differing in acid sensitivity. Amino packaged into a nucleocapsid of acid replacements altering virion stability icosahedral symmetry. We have previously were preferentially distributed in two observed that alphaviruses derived from different regions of the capsid: the N Semliki Forest virus (SFV) and Sindbis virus terminus of VP1 and the pentameric are able to propagate in the complete interface. Even more, the acid labile absence of capsid sequences. This phenotype induced by a mutation located propagation seems to be mediated by the at the pentameric interface in VP3 could release of infectious microvesicles (iMVs) be compensated by introduction of an that contain viral RNA inside and viral amino acid substitution in the N terminus envelope proteins on their surface. In the of VP1.These results indicate that the acid case of SFV, these iMVs are pleomorphic sensitivity of FMDV can be considered as a and have a larger size and density than wt multifactorial trait and that virion stability virus. In the present work we have

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analyzed in detail the cellular origin of 16:15-16:30h (CO 58) iMVs, their mechanism of infection, and TYPE I INTERFERON RESPONSE IS DELAYED their infectivity in mice. These studies IN HUMAN ASTROVIRUS INFECTED CELLS showed that while iMVs lack exosomal and S. GUIX1,2, A. PÉREZ-BOSQUE2,3, LL. endosomal/lysosomal markers, they are MIRÓ2,3, M. MORETÓ2,3, A. BOSCH1,2, R. M. highly enriched in components of the PINTÓ1,2 plasma membrane, indicating that they are 1 Enteric Virus Group, Department of Microbiology, most likely derived from this University of Barcelona, Av. Diagonal 643, 08028 compartment. In addition, they lack most Barcelona, Spain of the viral replicase components, 2 Nutrition and Food Safety Research Institute (INSA- suggesting that they are not derived from UB), University of Barcelona, Av. Prat de la Riba viral replication complexes, which have 171, 08921 Santa Coloma de Gramanet, Spain been described to localize at the plasma 3 Digestive Physiology and Nutritional Adaptations membrane at early times post-infection. Group, Department of Physiology, University of Infectivity studies showed that iMVs enter Barcelona, Av. Joan XXIII S/N, 08028 Barcelona, Spain the cells through the endosomal pathway and can co-localize with markers of this cellular compartment at early times Type I interferon (IFN) activation and its postinfection. Furthermore, iMVs were not subsequent effects are important in the pathogenic to mice when injected response to viral infections. Human intravenously but were able to efficiently (HAstV) are recognized as infect different organs like lungs and heart. common viral pathogens causing However, in contrast to wtSFV, they were gastroenteritis in infants and young not able to cross the blood-brain barrier. children, with very few reports of disease Finally, we have evaluated the possibility in normal healthy adults, and some reports of using iMVs as gene transfer vectors. of severe disease after dissemination to These experiments showed that iMVs are extra-intestinal tissues in able to transfer and mediate propagation immunocompromised patients.They are of heterologous genes like GFP and non-enveloped positive-strand RNA viruses interleukin-12, making them interesting for containing a 6.8 kb polyadenylated gene therapy or vaccination applications. genome linked to a VPg protein on the 5’end.

Our study shows that HAstVsinduce a mild

and delayed IFN response upon infecting CaCo-2 cells. Although IFN-β mRNA is detected within infected cells and supernatant from infected cells show antiviral activity against the replication of other well-known IFN-sensitive viruses,

these responses occur at late stages of infection once genome replication has taken place. Interestingly, synthesis of type

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III IFN-λ mRNA, which plays an important vitro, regula el tamaño de placa de lisis. Los role in controlling other gastrointestinal mutantes de IBDV carentes VP5 presentan viral infections,also takes place within reducciones en el tamaño de placa HAstV-infected CaCo-2 cells. superiores al 90% con respecto al virus On the other hand, HAstV replication can parental. La proteína VP5 se asocia a la be partially reduced by the addition of cara citoplásmica de diferentes exogenous type I IFN, and inhibition of IFN compartimentos membranales, incluyendo activation by BX795 enhances viral la membrana plasmática, a través de su replication, indicating that HAstVs are IFN- interacción con fosfoinosítidos. Esta sensitive viruses. Finally, different levels of interacción está mediada por un dominio IFN response were observed in cells electropositivo localizado en la región C- infected with different HAstV mutants with terminal de la proteína. Mutaciones que changes in the hypervariable region of afectan a la funcionalidad de este dominio nsP1a/4, suggesting that nsP1a/4 genotype alteran la distribución subcelular de la may potentially have clinical implications proteína y provocan reducciones due to its correlation with the viral significativas en el tamaño de placa. replication phenotype and the antiviral Los resultados obtenidos sugieren que la responses induced within infected cells. eliminación selectiva de la proteína VP5 no afecta significativamente a la replicación 16:30-16:45 (CO 59) del genoma, a la expresión de otras proteínas virales o al ensamblaje del virus. LA PROTEÍNA VP5 JUEGA UN PAPEL Sin embargo, se observa un efecto ESENCIAL EN LA DISEMINACIÓN DEL dramático sobre la cinética de liberación VIRUS DE LA BURSITIS INFECCIOSA de partículas infectivas al medio F. MÉNDEZ, L.L. CUBAS, D. RODRÍGUEZ, J.F. extracelular. Nuestros datos sugieren que, RODRÍGUEZ al igual que lo observado recientemente en Departamento de Biología Molecular y Celular, otros virus carentes de envuelta lipídica, Centro Nacional de Biotecnología-CSIC, Madrid, IBDV podría emplear un mecanismo de Spain liberación de virus no asociado a lisis celular cuyo funcionamiento sería El virus de la bursitis infecciosa (IBDV) es el dependiente de la proteína VP5. La agente etiológico de una grave eliminación de este mecanismo podría enfermedad inmunosupresora que afecta a explicar la supresión de virulencia pollos domésticos responsable de graves documentada en mutantes de IBDV pérdidas a la industria avícola mundial. Los carentes de la proteína VP5. viriones de IBDV son icosaedros no envueltos con un genoma formado por dos segmentos de dsRNA.

Estudios recientes llevados a cabo en nuestros laboratorios demuestran que VP5, una proteína asociada a virulencia y prescindible para la replicación del virus in

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16:45-17:00h (CO 60) extracellular release of enveloped particles A MODEL FOR HEPATITIS A VIRUS was higher in Huh7-A1 than in FRhK-4 cells TRANSCYTOSIS IN HEPATOCYTES for all viruses, but particularly with mutant M. DE CASTELLARNAU, S. GUIX, F.J. PÉREZ- viruses. In polarized Huh7-A1 cells, their RODRÍGUEZ, L. D’ANDREA, A. BOSCH, R.M. preferential exit route was through the PINTÓ apical membrane and the viruses present in the canalicular side were enveloped Enteric Virus Laboratory, Department of Microbiology, University of Barcelona, Barcelona, particles. In contrast, those few viruses Spain released through the basolateral membrane were mostly free particles. A new model of HAV entry and exit from Hepatitis A is the most common infection human hepatocytes will be presented. of the liver worldwide and is fecal–orally transmitted. Its incidence tends to decrease thanks to improvements in Parallel Session VIII: SPECIFIC IMMUNITY hygienic conditions and vaccination Chairpersons: MARGARITA DEL VAL AND campaigns, but at the same time, its YOLANDA PACHECO severity increases due to a higher infection Tuesday June 9, 2015 rate in adults. WHITE ROOM Hepatitis A virus is a unique picornavirus with very special molecular features. Recently, an unexpected exit pathway 15:00-15:15h (CO 61) through its envelopment into exosomes THE STRUCTURALLY RELATED FUSION has been described. In the present work, PROTEINS OF HUMAN RESPIRATORY we have studied the exit process in two SYNCYTIAL VIRUS AND different cellular models: FRhK-4 cells, METAPNEUMOVIRUS ARE ANTIGENICALLY which represent a traditional cell culture AND IMMUNOGENICALLY DISSIMILAR system utilized to grow HAV in vitro, and L. RODRIGUEZ1, E. OLMEDILLAS1, V. MAS1, Huh7-A1 cells, which are more permissive C. PALOMO1, A. TRENTO1, M. VÁZQUEZ1, for HAV infection than the parental Huh7 O. CANO1, B.S. GRAHAM2, B. VAN DEN cells from which they derive and which HOOGEN3, J.S. MCLELLAN4 AND J.A. represent the natural hepatocyte model. MELERO1 Additionally, we took advantage of two 1Centro Nacional de Microbiología and CIBERES, HAV mutants bearing a VP2 replacement, ISCIII, Madrid, Spain; 2Vaccine Research Center, NIH, which potentially affects its binding to ALIX Bethesda USA; 3Erasmus Medical Center, 4 and thus its invagination into Rotterdam, The Netherlands, Geisel School of Medicine, Dartmouth, USA. multivesicular bodies and the subsequent generation of the “quasienveloped” particles. Huh7-A1 cells were shown to be The fusion (F) proteins of human less efficient in supporting HAV replication, respiratory syncytial virus (hRSV) and the although the mutants gave higher titers in highly related human metapneumovirus single round replication events. Overall, (hMPV) enable fusion of the viral and cell

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membranes at the initial stages of their 15:15-15:30h (CO 62) respective infectious cycles and are the VIH-1 INDUCE A DEREGULATION IN B-CELL main target of neutralizing antibodies. POPULTIONS THROUGH A PARTIAL Both hRSV_F and hMPV_F experience REGULATORY B-CELL PHENOTYPE IN remarkable structural changes during VITRO transit from the metastable prefusion S MARTIN DELGADO1, J LOPEZ-ABENTE2, conformation to a highly stable postfusion M.A MUÑOZ-FERNANDEZ1, R CORREA- form. Significant structural information has ROCHA2 AND M PION1 recently been gained about the refolding 1Laboratory of Molecular ImmunoBiology, Hospital process of hRSV_F by solving the structures General Universitario Gregorio Marañón; Instituto of soluble prefusion and postfusion forms. de Investigación Sanitaria del Gregorio Marañón. C/ Dr. Esquerdo 46, 28007 Madrid, Spain; Although much less is known about 2 hMPV_F, partial information indicates that ImmunoRegulation Laboratory, Hospital General Universitario Gregorio Marañón; Instituto de it shares structural characteristics with Investigación Sanitaria del Gregorio Marañón. C/ hRSV_F. Maiquez, 9, 28009 Madrid, Spain Despite the noted structural resemblance and partial amino acid identity, both HIV-1 patients usually show general hRSV_F and hMPV_F have limited antigenic immune system deregulation and hyper- identity reflected in few cross-neutralizing activation. At the humoral immunity level, monoclonal antibodies (MAbs). HIV infection induces hyper- Furthermore, only very limited cross- gammaglobulinemia and loss of memory B reactivity is seen in polyclonal responses cells. In our group, we have previously against various forms of prefusion or reported that HIV-1 particles caused a postfusion F proteins. More importantly, direct marked effect on the activation, while depletion of human sera with proliferation and phenotype of B cells. This postfusion hRSV_F removes only a small deregulation could explain why efforts to fraction of neutralizing antibodies, the develop an efficient vaccine against HIV same procedure depletes most of the have been unsuccessful. Therefore, it is neutralizing antibodies directed against essential to determine the B-cell hMPV_F, indicating substantially different deregulation mechanism so as to improve conformational requirements. All these the design of an anti-HIV vaccine and data provide new insights into hRSV_F and obtain an efficient response of the immune hMPV_F based on structural studies that system. should contribute to the development of In the present study, we considered the efficient vaccines against these important phenotype and functions of HIV-treated B- human pathogens. cell. B cells were extracted from buffy coat and treated with HIV-1 or different stimuli. Using quantitative PCR, we analyzed the expression of different cytokines in B-cell and, by flow cytometry; we analyzed the phenotype of these cells. Moreover, the

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function of B cells on CD4+ or CD8+ T cells 15:30-15:45h (CO 63) were determined after co-culture ENGINEERED THERMOSTABLE EMPTY experiments in vitro. CAPSIDS OF FMDV FOR IMPROVED Surprisingly, B cells exposed to HIV showed VACCINES a higher level of mRNA for IL-10, IL-6, EBI3 S. LÓPEZ-ARGÜELLO1, V. RINCÓN1, A. or IL-12(p35) in vitro, presenting a RODRÍGUEZ-HUETE1, E. MARTÍNEZ-SALAS1, potential immunosuppressive profile. In G. BELSHAM2, M.G. MATEU1 addition, HIV-treated B cells exposed to 1Centro de Biología Molecular Severo Ochoa (CSIC- lymphocytes were able to reduce the UAM), Universidad Autónoma de Madrid, Madrid, proliferative capacity of CD4+ and CD8+ T Spain, and 2Technical University Denmark. cells, confirming the immunosuppressive profile of these B cells. However, HIV- Foot-and-mouth disease virus (FMDV) is treated B cells did not show a specific the causative agent of one of the ability to reduce the production of TNFα economically most important animal from CD4+ or CD8+ T cells as results of co- diseases worldwide. Novel vaccines based culture experiments and the phenotype of on recombinant FMDV empty capsids are the B cells studied by flow cytometry were being investigated to avoid the risks of not conclusive. virus escape or deficient inactivation We have already established that HIV- during vaccine production, while still treated B cells show a deregulated preserving the full immunogenicity and phenotype and function. But looking antigenic spectrum of current, virion-based deeply at this deregulation, we showed vaccines. FMDV empty capsids can be that HIV-treated B cells displayed a partial more acid-resistant than virions, but they immunosuppressive phenotype and appear to be even less termostable. Thus, function. These results may explain the there is a clear need to increase empty general deregulation of the immune capsid thermostability for the system and the high concentration of IL-10 development of capsid-based, infection in plasma observed in HIV patients. These risk-free FMD vaccines. preliminary outcomes are highly promising We have engineered and produced mutant as a means of understanding the hyper- recombinant FMDV empty capsids carrying activation of the immunity in HIV patients different mutations that were chosen and further experiments ex vivo are based on the FMDV atomic structure and needed to confirm these results. our previous work. The empty capsids were produced in eukaryotic cells using an expression system based on recombinant vaccinia virus. The thermal and acid resistance of these mutant capsids against dissociation into pentameric subunits was compared to that of the non-mutated (natural) capsid. Four tested mutants showed substantially increased thermal

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resistance although only some of them TLR-3, STAT1, ISG15, ISG20, IFIT1, IFIT2, showed increased acid-resistance. The IFIT3, MX, and OAS. However, we found results are helping to elucidate the other genes such as GBP5, GBP6, IFI6, structural basis of the thermal sensitivity of IFI27, IFI35, IFI44, TRIM22, and TRIM34, FMDV, and the effects of compensatory which were transcriptionally induced after mutations that were found to restore the influenza virus infection, but their role viability of FMDV lethal mutants. In during the virus cycle is unknown. Using addition, the empty capsids of increased siRNAs to specifically knockdown the thermostability we have engineered could expression of the proteins, and plasmids to provide a basis for the development of overexpress these proteins, we have improved empty-capsid-based FMD identified that genes IFI6, IFI27, IFI35, and vaccines. IFI44, increase viral replication of influenza virus, and also of other viruses such as 15:45-16:00h (CO 64) lymphocytic choriomeningitis virus (LCMV). ROLE OF INTERFERON STIMULATED In addition, we have shown that these GENES IN INFLUENZA VIRUS PRODUCTION genes act as negative feedback regulators AND ANTIVIRAL SIGNALLING of cellular IFN antiviral responses induced by different viruses, by the analog of M. L. DEDIEGO, L. MARTINEZ-SOBRIDO, D. dsRNA polyinosinic polycytidylic acid, and J. TOPHAM by IFN itself, most probably accounting for Department of Microbiology and Immunology, and the differences in the virus titers observed. David H. Smith Center for Vaccine Biology and Immunology. University of Rochester, Rochester, As far as we know, the function of these NY, USA genes during influenza virus infection has been described for the first time. In addition, the role of IFI6, IFI27, and IFI44 in Influenza A (IAV) and B (IBV) viruses are regulating the cellular IFN responses is considered one of the most common novel for these proteins. causes of respiratory virus infections in humans causing annual epidemics and occasional of considerable public health and economic impact. The innate immune system, leading to the expression of IFN-stimulated genes (ISGs), is the first line of defense against virus infections. To study the IFN-stimulated genes (ISGs) induced during influenza virus infection and their roles in virus production, cellular gene expression in epithelial cells infected with influenza A and B viruses was analyzed. Many well- known ISGs were induced during influenza virus infection, such as RIG-I, MDA5, IRF-7,

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16:00-16:15h (CO 65) epidemiológica 46/2012 (11/2012) a la EPIDEMIOLOGÍA MOLECULAR DE GRIPE A 16/2013 (4/2013), en cinco hospitales de Y B EN ENFERMEDAD RESPIRATORIA referencia. Se obtuvieron frotis GRAVE Y ESTADO VACUNAL nasofaríngeos y nasales (< 14 años) o J PUIG-BARBERÀ (1, 2), KARINA faríngeos (>=14 años). Se llevó a cabo la SALVATIERRA (3), SILVIA SANCHO-TELLO (3, detección de 14 virus respiratorios (RT- 4), JAVIER DÍEZ-DOMINGO (1), F XAVIER PCR). De forma sistemática se secuenció el LÓPEZ-LABRADOR (3, 5, 6) gen completo de la hemaglutinina en asilados de gripe A y gripe B, para (1) Area de Vacunas, Centro Superior de Investigación en Salud Pública (FISABIO-Salud compararlo con el de las cepas vacunales. Pública), Conselleria de Sanitat, Valencia, España.. Resultados: De 1.034 pacientes incluidos (2) Centro de Salud Pública de Castellón, Conselleria en el estudio, en 510 se detectó como de Sanitat, Castellón, España. mínimo algún virus por RT-PCR, de los que (3) Laboratorio de Virología, Area de Genómica y 242 fueron positivos para gripe. Salud, Centro Superior de Investigación en Salud Observamos dos olas, la primera de gripe B Pública (FISABIO-Salud Pública), Conselleria de Sanitat, Valencia, España. donde casi todos los asilados pertenecían al linaje B-Yamagata (n=151; 30%; B- (4) Servicio de Microbiología, Hospital Clínico Universitario de València, España. Yamagata n=145) y la segunda de gripe A, (5) Unidades Mixtas Infección-Salud Pública y donde casi todos los aislados fueron Genómica-Salud (FISABIO-Salud Pública/Universtat A(H1N1)pmd09 (n=85; 35%) y sólo 5 de València) A(H3N2). Los aislados de gripe B Yamagata (6) CIBER-ESP (Centro de Investigación Biomédica en (54 secuenciados) se distribuyeron en dos Red en Epidemiología y Salud Pública), Instituto de clados: (i) clado 2, B/Brisbane/3/2007-like, Salud Carlos III, España. cercano a B/Massachusetts/02/2012; y clado 3, В/Wisconsin/1/10 (cepa vacunal)- Introducción: El papel de la vacuna de la like, cercano a B/England/709/2012. La gripe en la prevención de ingresos por mayoría de los virus A(H1N1)pdm09 (37 enfermedad respiratoria es objeto de secuenciados) pertenecían al clado 6 controversia. Dentro de un estudio (A/St.Petersburg/27/2011-like), y sólo prospectivo basado en la vigilancia de cuatro aislados pertenecían al clado 7 hospitalizaciones en las tres provincias de (A/St.Petersburg/100/2011-like). Un total la Comunitat Valenciana (cubriendo de 18 (34%) vs. 34 (65%) aislados de gripe 1,266,899 habitantes, 27% de la B, ó 12 (32%) vs. 25 (68%) de gripe A, población), intentamos relacionar las tasas provenían de pacientes vacunados o no de hospitalización debido a enfermedad vacunados, respectivamente. No se respiratoria con la prevalencia de virus encontró un agrupamiento filogenético respiratorios durante la temporada 2012- diferencial de los aislados según el estado 13, según el estado de vacunación para de vacunación, ni para gripe A, ni para gripe (vacuna administrada mínimo 14 días gripe B. antes del ingreso) y la cepa del virus. Conclusiones: No se pudo relacionar Métodos: El cribado se realizó en todas las ningún grupo filogenético de gripe B- admisiones por urgencias de la semana Yamagata o A(H1N1pdm09) con fallo

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vacunal en ingresos por enfermedad early antigen expression by VACV and respiratoria grave durante la temporada provide alternative strategies for the 2012-13. Otros factores, distintos al clado design of poxvirus-based vaccines. del virus, pueden contribuir a la poca efectividad de la vacuna en una porción 16:30-16:45h (CO 67) sustancial de casos. HIGHLY EFFICIENT INSECT CELL-BASED

PLATFORM FOR VIRUS-LIKE PARTICLE 16:15-16:30h (CO 66) VACCINES PRODUCTION USING AN MODIFICATION OF PROMOTER SPACER IMPROVED BACULOVIRUS VECTOR LENGTH IN VACCINIA VIRUS AS J. LÓPEZ-VIDAL1*, S. GÓMEZ-SEBASTIÁN1*, ASTRATEGY TO CONTROL THE ANTIGEN J. BÁRCENA2, M. C. NUÑEZ1, D. MARTÍNEZ- EXPRESSION ALONSO3, B. DUDOGNON1, E. GUIJARRO4 1 1 M. DI PILATO , L. SÁNCHEZ-SAMPEDRO , E. AND J. M. ESCRIBANO4 1 2 MEJÍAS-PÉREZ , C. O. S. SORZANO AND M. 1Alternative Gene Expression S.L., Pozuelo de 1 ESTEBAN Alarcón, Madrid, Spain 1 . Departamento de Biología Molecular y Celular, 2CISA-INIA, Valdeolmos, Madrid, Spain Centro Nacional de Biotecnología, Madrid,Spain 3CBMSO, Canto Blanco, Madrid, Spain 2 . Biocomputing Unit, Centro Nacional de 4Departamento Biotecnología, INIA, Madrid, Spain Biotecnología, Madrid, Spain * Contributed equally

Vaccinia viruses (VACV) with distinct early Vaccines based on virus-like particles (VLP) promoters have been developed to have proved their success in human and enhance antigen expression and improve animal health. Insect cells platform, based antigen-specific CD8 T cell responses. It has on the use of recombinant baculoviruses, is not been demonstrated how the length of one of the most used technologies to the spacer between a gene and its early generate this kind of highly immunogenic promoter motif influences antigen vaccines. Because production cost is a very expression, and whether the timing of relevant constraint to extend the use of gene expression can modify the antigen- these vaccines to human and animal specific CD4 T cell response. We generated populations, any improvement in several recombinant VACV based on the productivity may be a relevant factor to attenuated modified vaccinia Ankara reduce the vaccine costs. Here we describe (MVA) strain, which express GFP or the the use of a novel baculovirus expression Leishmania LACK antigen under the control cassette, denominated TopBac, to model of an optimized promoter, using different the production in insect cells of two well spacer lengths. Longer spacer length defined VLPs. Capsid proteins, from increased GFP and LACK early expression, porcine type 2 (Cap) and from which correlated with an enhanced LACK- the calicivirus producing the rabbit specific memory CD4 and CD8 T cell hemorrhagic disease (VP60), were response. These results show the expressed in insect cells using importance of promoter spacer length for baculoviruses genetically engineered with

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the TopBac expression cassette. RNAs produced during infection. Once Productivities were compared to that activated, the pathways leads to the obtained by their conventional counterpart induction of type I IFN and vectors expressing the proteins under the proinflammatory cytokines, leading to a control of polyhedrin promoter. Results cellular antiviral state. In a non-infected demonstrated that production yields cell, RIG-I is in an inactive form where a obtained for these vaccine proteins under helicase intermediate domain is interacting the control of the TopBac cassette were with the CARDs domains. Upon recognition increased by more than 300%. In both of the RNA by the RD, RIG-I hydrolyzes ATP cases the recombinant protein was fully and changes its conformation to an active functional, forming identical VLPs in size state. The CARD domains are then exposed and shape than those produced by the and become K63-linked polyubiquitinated conventional baculovirus, as determined by E3-ligases, such as TRIM25. by electron microscopy analysis. The use of The activation of this pathway is complex the TopBac expression cassette and well characterized, but most of the represents a simply modification of the spatio-temporal events, and the baculovirus vectors that significantly subcellular localization where the essential improves the cost efficiency of VLP-based proteins interact, are still under vaccines production, facilitating the broad interrogation. Through different application of these vaccines in human and techniques, we analyzed how these animal health. proteins form complexes that are distributed and reorganized spatially 16:45-17:00h (CO 68) within the cell in order to create an efficient antiviral state. VIRAL PROTEINS TARGET COMPLEXES IN THE RIG-I LIKE RECEPTOR RIG-I is the main sensor for recognition of many ssRNA viruses such as M.T. SANCHEZ-APARICIO1,2, J. AYLLON1,2, A. Paramyxoviruses, Flaviviruses, GARCIA-SASTRE1,2,3. 1 Rhabdoviruses and Orthomyxoviruses. Department of Microbiology, Icahn School of Many of them have developed numerous Medicine at Mount Sinai, New York, New York, United States of America. 2 Global Health and and different strategies to overcome the Emerging Pathogens Institute, Icahn School of activation of the RLR pathway. Medicine at Mount Sinai, New York, New York, 3 We will discuss and show new insights on United States of America. Department of how, where, and when, viral proteins can Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, New counteract the activation of the RLR York, United States of America. pathway. NS1 of Influenza A virus, NS34A of Hepatitis C Virus and NSs of Severe Fever with Thrombocytopenia Syndrome The innate immune response relies on a Virus, are IFN antagonistic viral proteins set of Pathogen Recognition Receptors that interact with specific complexes in (PRRs) that sensor pathogen patterns very well defined areas in the host cell in (PAMPs). RIG-I is a cytosolic PRR that detects 5’-triphosphate double-stranded

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order to inhibit the antiviral state in an Project in USA, have been created with the infected cell. aim of to define a standardized “normal gut microbiota composition”. These Parallel Session IX: platforms have reported the quantitative and qualitative differences in the MICROBIOME AND HEALTH microbiota through the different races and [Joint Session SEV-SEM] age stages, as well as the existence of a Chairpersons: ALBERT BOSCH AND universal “core” conserved in almost all ROSA DEL CAMPO humans. Tuesday June 9, 2015 A healthy gut microbiota plays many crucial digestive and metabolic functions in AUDIOVISUAL ROOM the host, whereas alterations in their composition or reductions in the microbial *Invited paper diversity are highlighted in several 15:00-15:30h (CO 69) pathologic status and diseases. Those SCIENTIFIC EVIDENCES OF GUT modifications in the intestinal microbiota MICROBIOTA IMPLICATIONS IN HUMAN composition and function have been linked DISEASES to diseases or pathological status, including functional gastrointestinal disorders, R. DEL CAMPO obesity and diabetes. Nevertheless, in all Microbiology Department, University Hospital Ramón y Cajal, Madrid, SPAIN cases it is important to define if the alterations observed in the microbiota are

the cause or the consequence of the In the recent years, numerous scientific disease. works have pointed to the human gut The most recent studies revealed the microbiota as a significant metabolic organ existence of a brain-gut axis in which the with relevant repercussions in the global metabolic molecules produced by the human health. Our gastrointestinal tract bacteria are structurally similar to harbours a considerable microbes neurotransmitter, and due to that might ecosystem, constituted by bacteria, have neuroactive functions. On the other parasites, viruses, and archaeas, which hand, the brain has a connection with the interact with the human eukaryotic cells, gut microbiota through the enteric nervous and the host immune system. system, when released in the intestinal The recent technological advances in lumen and consequently signal brain molecular tools such as metagenomics and function and behaviour. next-generation sequencing have allowed Dietary supplementation with probiotics increasing the known of the complete and prebiotics are the most widely used diversity in the gut microbiota ecosystem, dietary adjuncts to modulate the gut including the non-cultivable microbiota. In some particular diseases, microorganism, which are the majority. the faecal transplantation might be Some international alliances, as META-HIT in Europe and The Human Microbiome

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indicated for a complete gut microbiota shown that gestational age and weight at restoration. birth also exert a strong influence on this process. Numerous studies monitoring the *Invited paper bacterial communities in preterm infants indicated that the fecal microbiota of 15:30-16:00h (CO 70) premature infants is different compared INITIAL BACTERIAL COLONIZATION: with that of term infants. In fact, the gut IMPACT ON HUMAN HEALTH colonization pattern of preterm infants has E. JIMÉNEZ QUINTANA been described as delayed and aberrant. ProbiSearch, SL, Tres Cantos, Spain; Departamento Abnormal intestinal colonization during the de Nutrición, Bromatología y Tecnología de los first weeks of life may alter the barrier, Alimentos. Universidad Complutense de Madrid. nutritional and immunological functions of Spain. the host microbiota and, as a consequence, increases susceptibility to The microbial colonization of the infant disease. Necrotizing enterocolitis, gastrointestinal tract is an essential inflammatory bowel disease, obesity, process in the human lifecycle since asthma, atopy and even autism are interactions established between the diseases that have been related with the microbiota and the host have important microbial gut composition in infants. The consequences for human health and use of probiotics and/or prebiotics for disease. Traditionally, it has been potential modulation of the microbiota considered that the intestinal tract was early in life may have a long lasting impact sterile at birth, being rapidly colonized with on health and could be of great microorganisms from the mother and the importance for disease prevention. surrounding environment. However, culture dependent analyses have detected microorganisms in amniotic fluid, fetal membranes, umbilical cord and placenta, even in cases where no rupture of membranes has occurred and in elective C- sections. Other studies suggest that, actually, the meconium, the newborn’s first intestinal discharge, composed of material that has been ingested or secreted in the gut during fetal life from healthy hosts is not sterile suggesting that gut colonization may start before birth. Different factors, such as mode of delivery, antibiotherapy, diet or environment, affect infant gut colonization although their actual contribution to shape the infant microbiota remains unclear. It has been

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*Invited paper supragingival plaque (nine patients with 16:00-16:30h (CO 71) active caries and eight healthy subjects) or METAGENOMIC ANALYSIS OF VIRUSES IN oral mucosa swabs (seven patients with THE HUMAN ORAL CAVITY recurrent aphthous stomatitis and eleven healthy subjects) were sequenced to M. PARRAS MOLTÓ1, P. SUÁREZ 1 1 generate >59 million paired-end reads (35 RODRÍGUEZ , A. RODRÍGUEZ GALET , A. Gbp). In average, 78% of the reads were SIMÓN-SORO2, A. EGUIA3, JM. AGUIRRE 3 2 1 assembled in viral contigs based on Blast URIZAR , A. MIRA , A. LÓPEZ BUENO . similarities to comprehensive databases. 1 Centro de Biología Molecular Severo Ochoa Oral viromes were dominated by tailed (Consejo Superior de Investigaciones Científicas- Universidad Autónoma de Madrid), Madrid, Spain. mainly from the 2 and families. Due FISABIO Foundation, Center for Advanced Research in Public Health, Valencia, Spain. to the unprecedented sequencing depth 3 Dpto. de Estomatología II, UFI 11/25 Universidad we have assembled 150 different nearly del País Vasco/EHU, Leioa, Spain. full-length genomes with an average length of 37 Kbp and average coverage of 357x. Importantly, most of them correspond to The oral cavity is not only the main route novel phages poor related to those for bacteria and virus entry but also deposited in databases. In addition, we harbours several microbial ecosystems have detected high prevalent eukaryotic whose composition and function remained viruses including human herpevirus 4 and poorly understood. A healthy oral 7, and the complete circular genome of commensal flora protects against seven human papillomavirus (four novel pathogenic microorganisms, and some types), five Circovirus-like viruses and 14 authors propose that changes in stress anelloviriridae including two new human level, diet or hygiene habits might alter viral species (Parras-Moltó et al. 2014). their ecological equilibrium and trigger pathological processes. Although The comparison of the viromes reveals that bacteriophages are key players in the in spite of the high degree of interpersonal regulation of microbial communities, their variation, there are few abundant contribution to oral dysbiosis has not been bacteriophages widely distributed along investigated yet. The development of new dental plaque and oral mucosa samples. techniques of high-throughput sequencing Their closest relatives in databases are and their applications to the study of phages that infect hosts normally found in natural microbial communities the mouth and some of them involved in (metagenomics) is providing valuable the development of oral diseases. Finally, information about human microbiomes. although we have not detected statistically However few studies have focused on significant differences among viromes on indigenous viruses from our microbiota. the basis of oral health status (healthy versus caries or oral ulcers), some phages Here we report a metagenomic survey of are mainly associated with sickness or viruses in the mouth of 35 unrelated healthy status. human subjects. Viral DNA purified from

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This study represents the largest virus. Indeed, this is not exceptional since metagenomic study of virus in the human most commercial vaccines should be mouth and enhances our understanding considered as “imperfect” from a about this complex biological system. virological point of view, since they do not provide full protection from pathogen *Invited paper infection but protect individuals and populations from disease expression. Such 16:30-17:00h (CO 72) “imperfect” vaccines, however, may affect VACCINE DRIVEN EVOLUTION OF PORCINE and selection of viral CIRCOVIRUSES variants in vivo. Our research group is T. KEKARAINEN interested on vaccine driven evolution of Centre de Recerca en Sanitat Animal (CReSA) - animal infecting viruses. By in depth Institut de Recerca i Tecnologia Agroalimentàries molecular analysis using next generation (IRTA), Bellaterra, Spain sequencing and subsequent bioinformatics analysis, we have shown that PCV2 variant Porcine circovirus 2 (PCV2, family populations are circulating in commercial ) is one of the smallest known farms and that this variability differs viruses with single-stranded circular DNA between vaccinating and non-vaccinating genome of 1.7kb long. It is one of the farms. most important pig infecting viruses causing great economical losses to pig Parallel Session X: TEACHING AND industry. PCV2 is linked to several diseases DISSEMINATION OF THE VIROLOGY named as porcine circovirus diseases Chairpersons: ESPERANZA GÓMEZ-LUCÍA (PCVD). The most known PCVD is PCV2- AND JOSE A. LÓPEZ-GUERRERO systemic disease (PCV2-SD), also known as postweaning multisystemic waisting Tuesday June 9, 2015 syndrome (PMWS). PCV2 targets the AUDITORIUM REAL CASA DE LA MONEDA lymphoid tissues, which leads to lymphoid depletion and immunosuppression in pigs. *Invited paper The virus modulates the function of 17:33-17:41h (CO 73) immune cells, upregulate IL-10 and proinflamatory cytokines. First vaccine TEACHING VIROLOGY AT A against PCV2 was marketed 2004 and PREUNIVERSITARY LEVEL nowadays several pharmaceutical F.J. MEDINA DOMÍNGUEZ companies offer PCV2 vaccine. It is Departamento de Ciencias Naturales, IES ALPAJÉS estimated that up to 90% of Spanish pigs (Consejería de Educación, Comunidad de Madrid), are vaccinated against PCV2. All marketed Aranjuez, Madrid, Spain vaccines are based on the viral capsid protein and they provide high levels of Teaching microbiology and virology into efficacy and excellent return on secondary courses present some main investment. However, despite of difficulties concerning, primary, the vaccination, pigs still get infected with the dimensional problem: understanding what

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to be small means in terms of scaling s/inicio) , a blog intending to keep our correctly the different microscopic students in touch with the actuality on structures and living beings and to health (http://sos- understand life concepts as span life, alpajes.blogspot.com.es/ ) and a wiki ( metabolism rate… http://sosalpajes.wikispaces.com/ ) , build In addition, viruses represent the boundary by students of 3º ESO (14 years old), between molecular and cellular level, an replacing the role of the traditional book in aspect that connect directly with the very our classes. definition of a living being. Understanding reducing the formation of defective how some molecular association may products, various methods have been present complexes activities usually causes suggested and solidphase organic difficulties to our students. syntheses (SPOS) is one of the popular At a third level, viruses and bacteria are approaches. conceptually linked to our students as Several SPOS approaches have been infectious agents. Separate both groups reported for the preparation of PAMAM requires a special attention in order not to dendrons, but similar by-products are commit major errors like consider, for observed while the original protocol instance, antibiotics as a therapeutic tool developed remains used. Consequently, a against viral infections. new approach is need in the preparation of PAMAM dendrons. Herein, a new SPOS Finally, the presence of laboratory approach to the production of inverse poly activities is reduced in our high school due (amidoamine) dendrons was developed. A to the fact that these practices aren’t newly designed AB2 building block considered too important by our educative provided the focal point of the dendrons authorities, despite the evidence that a and provided a means to reduce the lack of a minimum of a laboratory formation of side products. A comparison experience may compromise the scientific with the classical approach revealed that knowledge and the setting of good bases fewer reaction steps were needed and a to follow further studies. smaller amount of the building blocks was To face these pedagogic challenges we are required to build dendrons of similar size. developed a didactic programme witch This also leads to the more efficient combine laboratory activities, aiming to approach. For example, construction of a create a trajectory in this field in ours G5 dendron can be done in 5 days and students, and micro-research projects, filtration is the only necessary purification looking for setting a solid basement, which procedure. This approach was also applied allow adding new knowledge avoiding to the preparation of other dendrons. This misconceptions. protocol was accomplished on a solid- The result of these experiences are phase synthesizer that can reduce the collected in different educational costs and time associated with preparing platforms: a magazine of science named dendrons, especially for high generation of “Argos” dendrons. (http://www.educa2.madrid.org/web/argo

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*Invited paper *Invited paper 17:41-17:49h (CO 74) 17:49-17:57h (CO 75) THE RADIO AS A VIROLOGY VIROLOGY FOR ALL IN FREE AND ONLINE INFORMATION DIFFUSOR DIVULGATION JOURNALS M. SEARA VALERO A. DOMÉNECH GÓMEZ 1, A. IRURZUN 2 Director del Programa “A hombros de gigantes”. IPIÉNS Radio Nacional de España. Prado del Rey. 28223 1 Dpto. Sanidad Animal, Facultad de Veterinaria, Madrid. Spain. Universidad Complutense, Madrid, Spain 2 Editorial Hélice, Madrid, Spain. Viral diseases news appear occasionally in www.editorialhelice.com the media; diseases like AIDS, bird flu, human papillomavirus, measles or, more Nowadays there is a general growing recently, Ebola, have had a notorius interest in virus and diseases they cause. In position in newspaper covers. Some, such this sense, scientific societies can play an as African swine fever, have caused important role as they can give that significant economic losses in the livestock information in a clear and trustable way sector. using confirmed scientific data, and Moreover, in latest years, groups against avoiding unjustified alarms. There are children vaccination have appeared. This different ways to transmit that entails a serious health threat for children information, as courses and and, eventually, the whole population. congresses/conferences or scientific The media have a fundamental role in publications (usually for their members reporting these diseases to avoid and other researchers). However, these unnecessary alarmism Putting in the right technical forms may be difficult to perspective the scope and the understand for people not specialist in the consequences is also required. Fighting topic. For that reason, publications pseudoscientific movements or inaccurate directed to general public, with different theories is essential to avoid this kind of levels of education, are more needed and threats to our society. important, and especially those free available in internet and social media (as That's at least what we have tried with the blogs, twitter or facebook) that may be "A hombros de gigantes" broadcast easily looked upon. program: With the aim of approaching virology to (http://www.rtve.es/alacarta/audios/a- society, in 2010 the Spanish Society for hombros-de-gigantes/) that I run in RNE Virology (SEV) created “Virología", a free since September 7, 2007. online divulgation journal hosted on the SEV web site (http://sevirologia.es). The journal intends to reach anyone, from expert virologists to general public, which wants to read or learn more about virus, diseases they cause, new treatments,

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diagnostic methods or vaccination In this brief time I will speak about finding developed to fight against them. Readers ways to live science in a society that does may find in the journal, among other not want any more scientists, and in a issues, reviews about present topics in scientific community marked with virology, written in an easily competitiviness and a unthoughtful understandable way, interviews with meritocratic system. Also, I will provide outstanding virologists, curiosities and insight on how this generation is stories in virology history, or discover the developing towards science, with the new presence of virus in poetry, painting, opportunities that it has spawned and the philosophy, museums or even in a football new job positions taken over by this group world championship!!. In conclusion, the of people in the fields of advertisement, main goal of “Virología” is to disseminate markenting, knowledge transfer or even the scientific knowledge of the amazing teaching at various levels. viral world in an accessible and enjoyable way. *Invited paper

18:05-18:13h (CO 77) *Invited paper DISSEMINATION OF VIROLOGY THROUGH 17:57-18:05h (CO 76) BLOGS AND OTHER SOCIAL MEDIA YOUNG VIROLOGISTS TO RECEIVE THE MIGUEL ÁNGEL JIMÉNEZ-CLAVERO BATON INIA-CISA, Valdeolmos, Spain. RAFAEL N. AÑEZ. Former student of the Master’s Degree in Virology, Science is an important part of our culture, Universidad Complutense de Madrid, Madrid, Spain; English teacher, Native Professional Teachers, and as such its dissemination must be Madrid, Spain; English teacher, Galea Consulting, considered among the top priorities in the Madrid, Spain; Secondary teacher in training, IES agenda of R&D activities in any advanced Alpajés, Aranjuez, Spain society. For that, the involvement of institutions and civil society in general is Nowadays, the new generation of badly needed, but a special effort from scientists is finding itself at a crossroads researchers is particularly required, when it comes to find a new employment. because nobody like them has a deep Under the promise of green pastures and knowledge on the scientific disciplines they plenty of work it lies the ugly truth of free- cultivate and its latest developments. New from-salary jobs and unpaid scholarships. technologies, especially the Internet and This generation finds itself between the social media (blogs, twitter, facebook and rock that the old-school scientists other social networking platforms) make represent, not giving enough consideration available to scientists new tools that to any activities beyond the universitarian facilitate the dissemination of knowledge curriculum, and the proverbial hard place from the laboratory directly to the citizens. of the shortage of investment that the These new tools should be considered as economical crisis has brought upon us. complementary to the more traditional

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popular science media (books, magazines, mergentes/) reports since 2012 on current specialized sections in the press and mass issues around emerging viruses and the media, documentaries, lectures, etc) and diseases they produce, in a context of all should occupy their own place in the global change. With 70 posts published up knowledge society, constituting an to now and around 100,000 visits received, ecosystem in which contributing active it has been awarded in the last two scientists coexist and interact with editions of the madri+d Awards for Science journalists and communication experts Communication. with specialized knowledge in the different scientific disciplines. *Invited paper In the case of virology, dissemination of 18:13-18:21h (CO 78) knowledge based on scientific evidence deserves special significance due to the GAMES AS TOOLS FOR TEACHING wide public impact of epidemics caused by VIROLOGY viruses, notably those producing high E. GÓMEZ-LUCÍA mortality, such as the recent Ebola virus Departamento de Sanidad Animal, Facultad de disease epidemic. Very often, news on Veterinaria, Universidad Complutense (UCM), biological alerts concerning virus epidemics Madrid, Spain raise fear and insecurity among the population in a most irrational way. This Traditionally, teaching virology (as well as pitfall clearly indicates that special efforts other subjects) has orbited almost are certainly needed to give reliable, exclusively around lectures. These are scientifically based information to provide usually followed by the student studying the citizens with the knowledge necessary his notes or checking books, and the to "metabolize" the flood of information professor evaluating the acquisition of the that occur from time to time associated information. This system, although it is with virus emergencies. Though, an good for reaching a large number of important problem arising with social students, fails to really motivate the media is that not all the information student, who, as a result, will not store a published through these networks is long term memory of the information reliable, so in order to make these new received. New approaches are being communication tools useful, it is continually tested, in order to promote increasingly necessary to establish quality what is called “life-long-learning”, or in systems that allow the public to identify other words, stimulate the pleasure trustworthy information through the obtained from learning. Since playing is an Internet. emotional need, and young animals use it With the aim to contribute to disseminate for their learning, currently, in many relevant topics in the virology area, schools (especially up to high school, but targeting a broad Spanish-speaking public, also at the university level), games are the blog "Emerging Viruses and Global being implemented for teaching. The game Change" hosted on the web "madri+d" is a way of using the mind, or even an (http://www.madrimasd.org/blogs/viruse attitude about how to use the mind.

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Games may seem irrelevant at first sight, Parallel Session XI: ANTIVIRAL DRUGS but they help the student to fix concepts in Chairpersons: JULIÁ BLANCO AND a subliminal way. Some of the games used RAFII MOHAMED to teach microbiology, virology or infectious diseases represent real-world Tuesday June 9, 2015 scenarios of disease outbreaks, in which WHITE ROOM the student has to carry out virtual research on the virus and the host defence 17:30-17:45h (CO 79) mechanisms. Other games test on aspects CHD1 CHROMATIN REMODELER IS A of these subjects, being informative and POSITIVE MODULATOR OF INFLUENZA with a self-assessment component. In VIRUS REPLICATION THAT PARALLELS general, the advantages of using games are RNAP II DEGRADATION IN THE INFECTED numerous: they are motivating, relaxing CELLS for the student, distancing him from his LAURA MARCOS-VILLAR 1, 2 , ALEJANDRA normal activity, and improve self-esteem, 1, 2 1, 2 they generate pleasure, mobilize the PAZO AND AMELIA NIETO 1 subject, develop creativity, curiosity and Centro Nacional de Biotecnología-CSIC, Madrid, Spain. imagination, activate the divergent 2 thinking, promote communication, Centro de Investigaciones Biomédicas en Red de Enfermedades Respiratorias (Ciberes), Spain. integration and group cohesion. The presentation will discuss two games for learning and self-assessment of Virology: Influenza A virus polymerase associates Viropolis with chromatin components of the (http://www.interbionet.com/viropolis/jue infected cell, such as the CHD6 chromatin go/) and VirTUal epidemic remodeler. Here we show that CHD1, a (epidemia.sevirologia.es). member of the same family, also interacts with the viral polymerase complex and

positively modulates viral replication. Silencing of CHD1 causes reduction on viral polymerase activity, viral RNA transcription and production of infectious particles. Similar results are obtained during infection with H1N1 and H3N2 influenza

virus subtypes, but not with Vesicular stomatitis virus or Adenovirus 5, indicating that CHD1 is an important protein for influenza virus replication and that chromatin plays a significant role on influenza virus life cycle.

Influenza virus transcription requires a

functional coupling with cellular transcription for the cap-snatching

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process. Despite that, the RNAP II is Several RNA viruses initiate translation degraded during the infection in a process using a cap-independent mechanism triggered by the viral polymerase, once mediated by the internal ribosome viral transcription is finished and on-going entry site (IRES) that is located at their cellular transcription is not required. Since 5´ untranslated genomic region. CHD1 specifically modulates influenza virus Picornavirus IRES activity is highly RNA transcription, and associates with dependent on both its structural Mediator, a transcriptional coactivator organization and its interaction with complex of RNAP II–mediated host factors. Small molecules able to transcription, its possible degradation interfere with RNA function are valuable during influenza virus infection was candidates as antiviral agents. Here we evaluated. Reassortant viruses from strains show that IRAB, a small molecule based that induce or not RNAP II degradation on a benzimidazole compound, have allowed the identification of PA and inhibited foot-and-mouth disease virus PB2 subunits as responsible for the (FMDV) IRES-dependent protein degradation process, the involvement of synthesis in RNA-transfected cells specific residues within these subunits and leading to a decrease of the virus titer. the correlation between absence of RNAP Interestingly, IRAB preferentially II degradation and attenuation of inhibited IRES-dependent translation in pathogenicity in mice. Here we show that cell free systems in a dose-dependent CHD1 associates with RNAP II and strictly manner. RNA structural analysis by parallels its degradation pattern during Selective 2´-Hydroxyl Acylation analyzed influenza virus infection, suggesting that by Primer Extension (SHAPE) degradation of both host factors is demonstrated an increased local involved in viral pathogenicity. flexibility of the IRES structure upon incubation with IRAB, which affected 17:45-18:00h (CO 80) four stem-loops (SL) located on the apical region of domain 3. Fluorescence LOCAL RNA FLEXIBILITY PERTURBATION binding assays conducted with OF THE IRES ELEMENT INDUCED BY A individual aminopurine-labeled NOVEL LIGAND INHIBITS VIRAL RNA oligoribonucleotides indicated that the TRANSLATION SL3A binds IRAB (EC50 18 µM). 1 1 2 G. LOZANO , J. RAMAJO , A. TRAPOTE , Additionally, the results derived from 2 2 J. ROBLES , E. PEDROSO AND E. fluorescence binding assays suggested 1 MARTÍNEZ-SALAS that the target site of IRAB within the 1 Centro de Biología Molecular Severo Ochoa, CSIC- FMDV IRES might be a folded RNA UAM, Cantoblanco 28049 Madrid, Spain structure that involves the entire apical 2 Departament de Química Orgànica and IBUB, region of domain 3. Our data suggest Facultat de Química, Universitat de Barcelona, that the conformational changes 08028 Barcelona, Spain induced by this compound on a specific region of the IRES structure which is essential for its activity is, at least in

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part, responsible for the reduced IRES contribute to eradicate this important efficiency observed in cell free lysates human pathogen. and, particularly, in RNA-transfected Polyanionic carbosilane dendrimers (PCDs) cells. have demonstrated potent and broad- spectrum anti-HIV-1/HSV-2 activity in vitro 18:00-18:15h (CO 81) and in vivo. However, the potential anti- ANTIVIRAL ACTIVITY OF POLYANIONIC HCV effect of PCDs and their mode of CARBOSILANE DENDRIMERS AGAINST antiviral action remain to be determined. HEPATITIS C VIRUS IN CELL CULTURE In this study, we used an unbiased, cell- 1,2† based system to screen a battery of PCDs DANIEL SEPÚLVEDA-CRESPO , PEDRO L. aiming at identifying non-toxic antiviral MAJANO3,4†, RAFAEL GÓMEZ5, FRANCISCO 5 2 compounds targeting different steps of the JAVIER DE LA MATA , JOSÉ LUIS JIMÉNEZ *, HCV lifecycle. We selected the PCDs Mª ÁNGELES MUÑOZ-FERNÁNDEZ1,2*, 6 displaying the best 5 selectivity indexes to PABLO GASTAMINZA* characterize them by determining the † These two authors contributed equally to this genotype spectrum and step targeted in work. * Corresponding authors 1 the HCV lifecycle using HCV-pseudotyped Laboratorio InmunoBiología Molecular, IISGM, retroviral particles (HCV ) and trans- BioBank VIH HGM, CIBER-BBN, Madrid, Spain pp 2 complemented, spread-defective HCV Plataforma de Laboratorio, Hospital Gregorio Marañón, IISGM, BioBank VIH HGM, CIBER-BBN, virions (HCVtcp). Madrid, Spain Our results show that PCDs inhibit 3 Molecular Biology Unit, Hospital Universitario de la infection by genotype 2a HCVtcp and Princesa, Instituto de Investigación Sanitaria HCVpp of the major genotypes (1, 2, 3, and Princesa (IP), Madrid, Spain 4 4) at nanomolar concentrations with no CIBERehd, Instituto de Salud Carlos III, Madrid associated cellular toxicity. Given the fact 5 Departamento de Química Inorgánica, Universidad that virocidal activity against other viruses de Alcalá, Campus Universitario, Alcalá de Henares, CIBER-BBN, Madrid, Spain has previously been ascribed to members 6 of this class of molecules, we investigated Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Campus their impact on HCV virion stability. While Cantoblanco, 28049 Madrid, Spain exposure to most compounds did not alter virion infectivity, one of the PCDs irreversibly destabilized infectious virions, Hepatitis C virus (HCV) infection is a major making infectivity undetectable and worldwide biomedical problem. Although strongly reducing viral RNA integrity, even new direct antiviral agents have been after PCD removal. successfully developed for the treatment of chronic HCV infection, the potential In summary, PCDs are identified as novel threat of resistance of this genetically anti-HCV agents that target either the diverse family of viruses and the difficulties virion itself or early aspects of HCV to make treatment available to all infected infection and constitute a step forward in patients worldwide should fuel the study the development of PCDs as nanotools to of novel antiviral agents that may prevent HCV transmission in humans.

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18:15-18:30h (CO 82) mRNAs is strongly inhibited in influenza APTAMERS DESIGN AS ANTIVIRAL AGENTS virus-infected cells while viral mRNAs are AGAINST INFLUENZA VIRUS actively translated. Thus, the inhibition of P. RODRÍGUEZ-RODRÍGUEZ1,2,, V.M. NS1-PABPI interaction or its destabilization GONZÁLEZ3, M.E. MARTÍN3 AND A. can be potentially used as an antiviral NIETO1,2. strategy. 1 Centro Nacional de Biotecnología-CSIC, Madrid, Recently, nucleic acid aptamers have been Spain. put forward for use as therapeutic agents 2 Centro de Investigaciones Biomédicas en Red de against many human diseases due to their Enfermedades Respiratorias (Ciberes), Spain. inhibitory ability and target specificity. In 3 IRYCIS-Hospital Ramón y Cajal, Madrid, Spain. the present study, we have selected ssDNA aptamers specific to the human PABPI, as Influenza A virus (IAV) causes respiratory possible inhibitors of IAV mRNA translation disease and continue to be one of the and as potential antivirals for influenza largest global threats to human health. The virus replication. virus possesses a negative-oriented Two aptamers (ApPABP#7 and segmented RNA genome that encodes for ApPABP#11), which bind PABPI with high its own RNA-dependent RNA polymerase, affinity have been selected and which is error-prone. In addition, its characterized. ApPABP#11 inhibits the segmented genome allows for exchange of polyA-PABPI binding and moreover, the RNA segments between genotypically translation of CAP and IRES-dependent different influenza viruses. These features cellular mRNAs in in vitro assays. Both confer a high genetic diversity and lead to aptamers inhibit cellular and viral generation of novel strains or/and translation in in vivo experiments but, subtypes and thus contribute to the under specific experimental conditions, permanent exposure of the human viral translation is specifically inhibited population to newly emerging and re- while cellular one is preserved. emerging influenza viruses. Accordingly, under these experimental Viruses have developed different strategies conditions, both aptamers reduce two to allow selective translation of their logarithms influenza viral replication in mRNAs using cellular translation factors as multistep curves, independently of the targets. Influenza virus mRNAs are formally H1N1 or H3N2 subtype and reduced viral equivalent to the cellular mRNAs and a protein accumulation in single infection sophisticated strategy has been selected curves. by the virus to enhance specifically the These results provide support for a translation of viral mRNAs. Previous work potential use of aptamers targeting viral- has demonstrated that NS1 viral protein cellular interactions as novel antivirals interacts directly and specifically with against influenza virus replication. eIF4GI translation initiation factor and with the polyA binding protein 1 (PABPI). Consequently, translation of cellular

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18:30-18:45h (CO 83) were extracted after 72 hours. Gene and SIMVASTATIN AND METFORMIN INHIBIT protein expression were analyzed by qRT- CELL PROLIFERATION AND HEPATITIS C PCR (Quantace, Bioline) and Western-blot REPLICATION IN VITRO, BY using standard protocols. DOWNREGULATING TCTP AND Results INCREASING PTEN Simvastatin (4µM) and metformin (10mM) M. GARCÍA-VALDECASAS1, A. GIL-GÓMEZ1, treatments inhibited Huh7.5 cells A. ROJAS1, J. AMPUERO1, R. GALLEGO- proliferation 58±8.6% and 38±2.2% DURÁN1, B. FOMBUENA RUBIO1, J. respectively, in a dose-dependent manner MUNTANÉ2, FJ. PADILLO2, M. ROMERO- after 72h. In cells treated with metformin, GÓMEZ1, JA. DEL CAMPO1 TCTP, PTEN1, and MAPLC3B gene 1 Digestive Disease Departament and CIBER- expression was increased whereas MTOR ehd.Valme University Hospital. Seville. and TCTP protein expression was down- 2Oncology Surgery Department, Cell Therapy and regulated (2.08±0.28 and 1.89±0.02 fold Transplant Organs Departament.IBiS. Seville respectively). Simvastatin treatment inhibited mTOR protein (2.11±0.73), and Introduction could induce PTEN and TCTP proteins Chronic hepatitis C infection (HCV) induces (1.48±0.05 and 1.96±0.03 fold, fibrosis, cirrhosis and hepatocellular respectively). carcinoma (HCC). Statins and metformin In Huh7.5 cells infected with JFH1, TCTP, have been shown to delay the PTEN1 and MAPLC3B gene expression was development and improve the prognosis of increased. JFH1 infection inhibited PTEN1 HCC. MTOR pathway is frequently protein expression (1.75±0.04) and deregulated in cancer, and represents a increased TCTP protein level (2.12±0.36). suitable therapeutic target for HCC. PTEN1, TCTP and MTOR proteins were AIM: To evaluate the effect of simvastatin down-regulated in infected cells treated and metformin on mTOR pathway, using with metformin (2.32±0.03, 2.00±0.18 and an in vitro model and primary hepatocytes. 3.04±0.61, respectively). On the other hand, simvastatin treatment increased Methods TCTP (2.76±0.38) and decreased MTOR Huh7.5 cells were grown in supplemented (3.5±0.42). DMEM at 37ºC, 5%CO2. Human A significant effect on viral replication was hepatocytes were prepared from liver observed: metformin and simvastatin biopsies obtained from patient submitted treatment could inhibit JFH1-RNA levels to a tumor resection. Hepatocytes isolation (52.4%±6.39) and Core protein expression was based on the two-step collagenase (60.0±10.0 fold). procedure. Huh7.5 cells were infected with JFH1 (1 particle/cell) and treated with When primary hepatocytes were treated metformin (2-10 mM) and simvastatin (2-4 with metformin (2mM) mTOR and TCTP µM) 3 hours after cell seeding. Cell protein levels were found reduced (1.6 and quantification was performed using a 1.5 fold, respectively). By contrary, Caspase Neubauer chamber. Total RNA and protein 3 was found induced (2.02 fold).

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CONCLUSIONS three-dimensional structure of this Simvastatin and metformin inhibited cell membranous web in whole infected cells is proliferation and HCV replication in vitro, still unknown. decreasing TCTP levels (oncogene) and In this study we have performed full-field increasing PTEN1 (tumor suppressor). cryo soft X-ray tomography (cryo-SXT) in MAPLC3B gene expression, a marker for the water window photon energy range to autophagy, is found increased after investigate in whole, unstained cells, the metformin treatment. Simvastatin and morphology of the membranous metformin could contribute to the rearrangements induced by the HCV prevention and therapy for HCV-related replicon in conditions close to the living HCC, although in vivo experiments are physiological state. We have obtained the needed to assess this role. first complete cartography of the dramatic cellular modification caused by the stable 18:45-19:00h (CO 84) subgenomic HCV replicon transfected in cell culture. Moreover, in order to identify HEPATITIS C VIRUS REPLICATION FACTORY the viral proteins allocation in the different STUDIED BY CRYO SOFT X-RAY subcellular compartments, we have TOMOGRAPHY: PLATFORM FOR correlated the three-dimensional structure PHARMACEUTICAL TRIALS OF NEW obtained with X-rays with electron ANTIVIRAL DRUGS AT CELLULAR LEVEL microscopy immunelabeling and confocal 1 2 AJ PEREZ-BERNA , MJ RODRÍGUEZ , FJ immunofluorescence. The morphology of 2 2 CHICHON , M FRIESLAND , A the membranous HCV factory web is a 1 2 SORRENTINO , JL CARRASCOSA , P cytoplasmic accumulation of large and 2 1 GASTAMINZA AND E PEREIRO small heterogeneous vesicles, 1ALBA Synchrotron Light Source, MISTRAL Beamline mitochondria and lipid drops. – Experiments Division, Cerdanyola del Vallès, Barcelona, Spain Using this system as a platform we test the 2 consequences of the treatment of infected Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Campus culture cells with different drugs against Cantoblanco, Madrid, Spain HCV. The reversion of pathological ultrastructural alterations at cellular level has never been carried out. In conclusion, Hepatitis C virus (HCV) is a major cause of cryo-SXT provides a powerful new tool for chronic liver disease, with an estimated the analysis of host-virus interactions and 170 million people infected worldwide. facilitates the trial of new antiviral drugs Low yields, poor stability and inefficient and vaccines at cellular level. infection systems have severely limited the analysis of the HCV life cycle and the development of effective antivirals and vaccines. HCV is a positive strand RNA that replicates its genome in intracellular membranes forming a complex membranous web. Nevertheless, the

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19:00-19:15h (CO 85) genotype 1a and co-infected with HIV-1. DETECTION OF A SEXUALLY TRANSMITTED Patient A was treated with Telaprevir + HEPATITIS C VIRUS PROTEASE INHIBITOR- IFN-PEG + Ribavirina, experienced a viral RESISTANCE VARIANT IN A HUMAN breakthrough, and therefore stop IMMUNODEFICIENCY VIRUS-INFECTED treatment. Patient B, his sexual partner, HOMOSEXUAL MAN was cured for treatment with S. FRANCO1,M. NEVOT1,C. TURAL2-5,J. Daclatasvir+IFN-PEG+Ribavirina, but at MOLTÓ2-5,J. K. ROCKSTROH6,B. CLOTET1- week 48 after therapy this patient 5AND MA. MARTINEZ1 experienced an AHC. We amplified three regions of the HCV (NS3, NS5B and E2) by 1. Fundació irsiCaixa, Badalona, Spain. RT-PCR. The sequences obtained were 2.Department of Internal Medicine, Hospital Universitari Germans Trias i Pujol, Badalona, Spain. analysed with sequences described in a 3 previous work of our group. The analysis of .Universitat Autònoma de Barcelona (UAB), Spain. 4 the NS3 protease quasispecies revealed a .Universitat de Vic (UVic) Barcelona, Spain. resistant variant to telaprevir (V36M) in 5.Fundació Lluita contra la SIDA, Badalona, Spain. patient A detected after stopping 6.Department of Medicine I, Bonn University Hospital, Bonn, Germany. treatment. Phylogenetic analysis of the NS3 showed that patient A, who developed

resistance, transmitted the virus to his In the last decade, an increase in the sexual partner (B). Analyses of other number of cases of acute infections (AHC) regions of the HCV (E2 and NS5B) for hepatitis C virus (HCV) in men who have confirmed that the resistant virus belonged sex with men (MSM) co-infected with virus to an epidemiological network of human immunodeficiency type 1 (HIV-1) transmission. have been detected. Previous studies have These results confirm the sexual demonstrated the existence of transmission of resistant variant to epidemiological networks of transmission telaprevir and establish that this resistant among this group of patients. Recently, virus belongs to an international network new direct acting antiviral agents (DAA) of HCV transmission among MSM. Since have been approved to increase the there is an international epidemic of HCV response to HCV treatment. In this study, infection among HIV-infected MSM with a we describe the first documented case of high rate of re-infections, attention should transmission of a HCV DAA resistant be paid to the transmission of HCV DAA- variant from a patient co-infected with HIV resistant variants, which may impair future who was treated with telaprevir to his therapeutic interventions. In addition, this sexual partner and its relation to the case history strongly underlines that transmission of HCV as described successful treatment of HCV does not previously between MSM and co-infected preclude HCV re-infection and therefore HIV-1. emphasizes the need for behavioural We analysed the baseline and post- interventions in patients at increased risk treatment plasma samples of two patients for HCV re-infection in order to preserve of our clinical unit infected with HCV

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cost-effectiveness of HCV treatment through the presence of a mutagenic strategies. nucleoside analogue (5-azacytidine or AZC). Then, these populations were 19:15-19:30h (CO 86) exposed to a new selective pressure that consisted in an increase in the replication TRANSIENT INCREASES IN THE ERROR temperature. After a number of transfers RATE CAN OPEN NEW ADAPTIVE under the new conditions (42º C), we PATHWAYS IN AN RNA VIRUS determined the degree of adaptation 1 1 1 E. LÁZARO , L. CABANILLAS , M. ARRIBAS reached and the mutations fixed in the Centro de Astrobiología (INTA-CSIC), Madrid (Spain) consensus sequences. The results obtained show that The replication error rate is one of the populations previously exposed to an main factors influencing the extension of increase in the error rate rapidly fixed genetic diversity contained in a population. several mutations that confer advantages RNA viruses together with viroids are the when replication takes place at 42º C. biological entities with the highest error These mutations were not detected in the rates found in nature, which is associated populations that always had replicated at to a wide exploration of the genotype standard error rate, suggesting that space and a great ability to adapt to new transient increases in this parameter can selective pressures, including the immune open new adaptive pathways. We are response and antiviral treatments. currently investigating whether the However, since most mutations are expansion of the mutant spectrum that deleterious, there must be an upper limit takes place as a consequence of the for the error rate, and additional increases increase in the error rate also offers above this value can compromise both advantages for adaptation to other survival and adaptability of populations. selective pressures that are different from These considerations have inspired a new temperature changes. antiviral therapy, named lethal mutagenesis that consists in the artificial increase of the error rate through the use of nucleoside mutagenic analogues. One of the potential problems of lethal mutagenesis is whether transient increases in the error rate, which can occur when a mutagenic treatment fails to extinguish virus infectivity, could improve virus adaptability to new selective pressures. To get a deeper insight into this point, we propagated an RNA virus, the bacteriophage Qβ, under different transmission regimes that included transient increases in the error rate

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Parallel Session XII: non-structural NS1 protein with the REPLICATION MECHANISMS regulatory subunit of the PI3K, p85β. The Chairpersons: AMELIA NIETO AND mechanism by which this interaction leads to PI3K activation is not fully understood. LUIS MENÉNDEZ Here we show that NS1 inhibits p85β Tuesday June 9, 2015 SUMOylation, increases p85βinteraction AUDIOVISUAL ROOM with Src tyrosine kinases, and promotes the tyrosine phosphorylation of the regulatory subunit. These findings highlight 17:30-17:45h (CO 87) the relevance of SUMO modification in the MODULATION OF P85β ACTIVITY BY regulation of cellular signalling pathways, SUMO such as the one controlled by PI3K, and 1 C.F. DE LA CRUZ-HERRERA , M. BAZ- provide an example of virus-host 2 3 2 MARTÍNEZ , V. LANG , A. EL MOTIAM , J. interaction in which influenza A takes 4 4 4 BARBAZÁN , R. COUCEIRO , M. ABAL , A. advantage of the host SUMOylation 2 1 VIDAL , M. ESTEBAN , C. MUÑOZ- machinery 5 1 3 FONTELA , A. NIETO , M. S RODRÍGUEZ , M. COLLADO4, C. RIVAS1,2. 17:45-18:00h (CO 88) 1 Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología-CSIC, Madrid, EXPRESSION OF PSEUDORABIES VIRUS Spain IE180 PROTEIN UNDER THE CONTROL OF 2 Centro de Investigación en Medicina Molecular HUMAN TUMOR-SPECIFIC PROMOTERS (CIMUS), Universidade de Santiago de (hTERT AND CEA): I.- APPLICATION TO Compostela, Instituto de Investigaciones OBTAIN CITOLYTIC VECTORS IN TUMOR Sanitarias (IDIS), Santiago de Compostela, Spain. CELLS 3 Ubiquitylation and Cancer Molecular Biology laboratory, Inbiomed, San Sebastián-Donostia, L. LERMA, B. MARTÍN, B. SAINZ JR., E. Gipuzkoa, Spain TABARÉS. 4 Instituto de Investigación Sanitaria de Santiago Department of Preventive Medicine, Public Health de Compostela (IDIS), Complexo Hospitalario and Microbiology.School of Medicine.Autónoma Universitario de Santiago de Compostela University of Madrid. Spain. (CHUS), SERGAS, Santiago de Compostela, Spain 5 Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany Pseudorabies virus (PRV), which belongs to the viral subfamily , has

been proposed for use as a vector in Virus infection activates host cellular cancer virotherapy since it shares the same signaling pathways, including the advantages described for other anti-cancer phosphatidylinositol 3-kinase (PI3K/AKT) viral vectors such as herpes simplex type 1 signaling, which regulates many cellular (HSV-1), but at the same time has processes such as protein synthesis, additional inherent advantages including metabolism, cell survival and proliferation. the absence of virulence, recombination The activation of this pathway by influenza and seroprevalence in the human A depends on the interaction of the viral population. Furthermore, PRV has a single

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immediate early gene, encoding the IE180 between 3 and 4 logs for the recombinant protein, which controls viral temporal virus relative to the parental virus replication and thus represents a more vBecker2 in control FP7 cells and only a 1 simplified system for the development of to 3 log reduction in U2OS, HeLa, HT29, anti-cancer viral vectors. 215 and 354 cells. Interestingly, This study focused on enhancing the tumor recombinant virus growth was selectivity and cytolytic efficacy of undetectable in HPDE and 185 cells. These recombinant PRV for tumor cells by data indicatethat recombinant PRV viruses controlling IE180 gene transcription using may represent potential candidates for the cellular promoters preferentially active in design of anti-cancer specific oncolytic tumor tissues: human telomerase reverse viruses. transcriptase (hTERT) and carcinoembryonic antigen (CEA). 18:00-18:15h (CO 89) PRV-TER and PRV-CEA viruses were THE EXONUCLEASE XRN1P IS SPECIFICALLY obtained by homologous recombination REQUIRED FOR THE TRANSLATION OF with PBAC90. They have a single copy of BROME MOSAIC VIRUS the IE180 gene under the control of a B. BLASCO-MORENO1, J. JUNGFLEISCH1, S. tumor-specific promoter in their genome. LEIDEL2, J. DÍEZ1 PRV-T180TER and PRV-T180CEA viruses 1. Virology Unit, CEXS, Universitat Pompeu Fabra were obtained by homologous (UPF), Barcelona, Spain recombination with PBAC80. They have 2. RNA Biology Group, Max Planck Institute for two copies of the IE180 gene, one of them Molecular Biomedicine, Münster, Germany. under the control of a tumor-specific promoter and the other under the control The exonuclease Xrn1p is a crucial factor in of a tetracycline-inducible (Ptet) promoter. controlling the degradation of most Genomes of recombinant viruses were messenger RNAs (mRNAs). Given that characterized by PCR and IE180 mRNA positive-strand RNA viruses mimic cellular expression was assessed by RT-PCR. Virus mRNAs, Xrn1p is expected to be a growth was studied in the human restriction factor for this group of viruses. osteosarcoma (U2OS), cervical cancer Such role has already been proven for (HeLa) and colon cancer (HT29) cell lines, hepatitis C virus. However, other viruses using lung fibroblasts (FP7) as a normal such as Dengue use Xrn1p to precisely cells line reference. In addition, this study degrade the genomic RNA and produce a was also carried out in primary pancreatic pathogenic subgenomic RNA. Here, we ductal adenocarcinoma cultures (185, 215 report a new function of Xrn1p in the and 354) with human pancreatic duct translation of the [(+)-RNA] brome mosaic epithelial cells (HPDE) as a non-tumor cell virus (BMV) in yeast. control. Recombinant virus growth was compared to the parental virus vBecker2 We demonstrate that translation of BMV and to N1aHTK (which expresses the HSV-1 RNA is highly and specifically inhibited TK protein) and its parental PRV-NIA3 when Xrn1p is deleted, in spite of the over- virus. Results show a titer reduction accumulation of viral RNA. By sequential

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deletion analysis, we identify the 5’UTR double-stranded RNA (dsRNA) genomes. and a stem-loop structure in the ORF as IBDV infects different bird species and the main determinants in the Xrn1p- causes an acute immunosuppressive dependence for translation. Moreover, disease, known as IBD or Gumboro disease, polysome profiling analyses indicate that that affects domestic chickens (Gallus Xrn1p is needed for efficient BMV RNA gallus), and is responsible for major translation initiation. economic losses to the poultry industry Three main conclusions arise from studying worldwide. The most obvious pathogenic mutants targeting key Xrn1p features. sign is the atrophy of the bursa resulting First, both the Xrn1p exonuclease activity from the infection and destruction of pre-B and its capability to bind 5’ uncapped lymphocytes. Although the molecular bases mRNAs are required for efficient BMV RNA for IBDV pathogenesis are still poorly translation. Second, expression of the understood, it has been suggested that an nuclear exonuclease in the cytoplasm exacerbated innate immune response that (Rat1∆NLS) does not rescue BMV RNA leads to a massive production of translation in xrn1∆. Importantly, proimflamatory cytokines is related with Rat1∆NLS can recover normal growth and IBDV-induced pathogenicity. steady-state BMV RNA levels. Third, an A crucial component of the host innate Xrn1p mutant unable to be imported to immune response is the IFN system. IFNs the nucleus is still capable of promoting have been extensively studied in the viral RNA translation. This indicates that context of host defense against viral the role of Xrn1p in translation is infection. However, type I (IFNα/β) and independent from its recently described type II (IFNγ) may have dual biological roles: function in transcription. Together, our elicit an antiviral state in uninfected cells data suggest a novel function of Xrn1p in through the transcriptional activation of the specific regulation of BMV RNA anti-viral proteins such as PKR, OAS and translation. Mx, while selectively inducing apoptosis in virus-infected cells, thus limiting viral 18:15-18:30h (CO 90) replication and spreading of the infection. IFN-α TREATMENT CAUSES A MASSIVE We focus our study on the interaction APOPTOSIS IN IBDV INFECTED CELLS between IBDV and the host innate immune response. In this respect, we have L.L. CUBAS, M. CISCAR, J.F. RODRÍGUEZ, D. observed a generalized apoptosis in RODRÍGUEZ. cultures infected with IBDV and treated Departamento de Biología Molecular y Celular, with IFN at different times post-infection. Centro Nacional de Biotecnología-CSIC. Madrid, Spain. As observed by different assays, the apoptotic effect is milder when IFN is

added at later times post-infection. The infectious bursal disease virus (IBDV) is Significantly, in cells that do not express the best characterized member of the PKR (siRNA) the IFN treatment after IBDV Birnaviridaefamily, that groups naked infection does not cause extensive icosahedral viruses with bi-segmented apoptosis. To further analyze the cellular

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response, we studied the expression of Order). The presence of DMV different ISGs and pro/-anti-apoptotic clusters, considered to be involved in the genes by qRT-PCR. In these assays, we replication process, is a common feature in determined that TNF-α is upregulated in Nidovirus infected cells. Other structures cultures infected with IBDV and treated that have also been related with the with IFN at earlier times post-infection, but replication factories, such as convoluted this effect is not observed in PKR-silenced membranes, are present only in some of cells. These results suggest that IFN the examined coronaviruses. In addition, in secreted by infected cells may contribute cells infected with the infectious bronchitis to trigger TNF-α mediated apoptosis which, virus (IBV), a member of the in turn, could explain the destruction of gammacoronavirus, new structures known the bursa of Fabriciusin IBDV-infected as spherules, which strongly resemble the chickens, which leads to replication sites of other positive- immunosuppression. strandedRNA viruses, have been recently described. Our purpose was to perform an 18:30-18:45h (CO 91) in-depth ultrastructural analysis of cells infected with BEV to characterize the BIOGENESIS AND DYNAMICS OF architecture of torovirus replication TOROVIRUS REPLICATIVE STRUCTURES factories, and to learn about their 1 2 3 G. ÁVILA , M.T. REJAS , F.J. CHICHÓN , M. biogenesis and dynamics during the 2 3 GUERRA , J.L. CARRASCOSA , J.J. infection. Previous analysis by 3 1 FERNÁNDEZ , D. RODRÍGUEZ . conventional transmission electron 1Department of Molecular and Cellular Biology and microscopy suggested that the DMVs form 3 Department of Macromolecular structures, Centro a reticulovesicular network (RVN) Nacional de Biotecnología (CNB-CSIC), Madrid. 2 resembling those described for the related Centro de Biología Molecular Severo Ochoa (CBM- severe acute respiratory syndrome (SARS) CSIC). Madrid, Spain. coronavirus and the equine arteritis virus (EAV). Here, we used serial sectioning and Plus-stranded RNA viruses replicate in the electron tomography of cells infected with cytosol of infected cells, in membrane- BEV and fixed at different post-infection bound replication complexes containing times to obtain three-dimensional images the replicase proteins, the viral RNA and of the replication factories. We confirmed host proteins. The formation of the the formation of a RVN in BEV infected replication complexes through the cells where the DMVs outer membranes rearrangement of cellular membranes is are interconnected with each other and currently being actively studied for viruses with the ER. Like in EAV, convoluted belonging to different viral families. We membranes were not observed in the previously identified double membrane RVNs. However, we observed paired or vesicles (DMVs) in the cytoplasm of cells zippered ER membranes lacking luminal infected with the equine torovirus Berne space, which in some cases are connected virus (BEV), the prototype member of the with the DMVs, and likely represent early Torovirus genus ( Family, structures that will evolve to give rise to

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DMVs. Interestingly, curled membranes key factor for its functionality. Using resembling the spherules described in IBV electron microscopy and image processing were observed at late time post-infection we have determined the structure of in BEV-infected cells. After careful native influenza RNPs derived from virions. examination of the tomograms we The basic arrangement shows a double- hypothesize that these structures probably helical conformation in which two NP/RNA represent remnants of paired membranes strands are associated each other in an unused for the formation of DMVs, which antiparallel way; both strands are accumulate at late times post-infection. connected by a loop at one end of the Hence, BEV shows important similarities, particle and associated to the polymerase but also some differences, in the at the other end. architecture of the replication factories Our group is currently conducting an other with other related viruses in the structural study of native influenza RNPs Nidovirales order. derived from virions much more detailed thanks to the use of a high-end microscope 18:45-19:00h (CO 92) equipped with a direct detector. This study STRUCTURAL BASIS OF INFLUENZA VIRUS is revealing a more complex structure in RNP ACTIVITY which we have found a broad conformational variability that could be R.COLOMA1, R. ARRANZ2, J. ORTIN1 AND J. 2 key for the movement of the polymerase MARTIN-BENITO . along the RNP during the transcription and 1 .Departamento de Biología Molecular y Celular, replication processes. Centro Nacional de Biotecnología, Madrid, España. 2.Departamento de Estructura de Macromoléculas, Centro Nacional de Biotecnología, Madrid, España. 19:00-19:15h (CO 93) THE DEAD-BOX HELICASE DHH1 The influenza A virus genome is formed by PROMOTES TRANSLATION OF HIGHLY a set of 8 ribonucleoprotein particles in STRUCTURED mRNAS which each RNA molecule is associated to J JUNGFLEISCH1, D NEDIALKOVA2, I DOTU3, the polymerase complex and many E RAINERI4, S LEIDEL2, J DÍEZ1 monomers of the nucleoprotein (NP). 1. Laboratorio Virologia Molecular, Universitat These complex molecular machines are Pompeu Fabra, Barcelona, Spain central in crucial viral processes. RNPs are 2. RNA Biology Laboratory, Max Planck Institute for transcribed and replicated inside the Molecular Biomedicine, Münster, Germany nucleus of the infected cell, from which 3. Hospital del Mar Medical Research Institute, they are exported to cytoplasm where the Barcelona, Spain 4 morphogenesis of virions takes place. As . Statistical Genomics, Centro Nacional de Analisis shown by multiple studies, most of them Genomica, Barcelona, Spain with NP mutants, the mRNA synthesis depends strongly on the correct Translation control and mRNA decay are arrangement of the whole complex, central to maintain proper gene expression showing that the structure of the RNP is a allowing to respond rapidly to

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perturbations. The group of Dhh1/DDX6 that has been hijacked by viruses to DEAD-box helicases plays a key role in control their gene expression and points these processes since its members act at out at this DEAD-box helicase as a key the interface of mRNA translation and cross-talk mediator between translation, decay They promote translation repression translational repression and decay. of cytoplasmic mRNAs that are then fed into decay or stored. Intriguingly, we have 19:15-19:30h (CO 94) previously shown that Dhh1/DDX6 activated translation of positive-strand INCREASED PATHOGENESIS OF INFLUENZA RNA viral genomes. However, the A H1N1 VIRUS LED BY A PA RESIDUE mechanism involved and whether this role DETECTED IN A FATAL CASE 1,2 1,2 is extended to cellular mRNAs is unknown. J. VASILIJEVIC , A. NIETO AND A. 1,2 By using a model system that allows the FALCON . replication of the Brome mosaic virus in 1Centro Nacional de Biotecnología-CSIC, Madrid, yeast here we show that the ATPase Spain. 2 activity of Dhh1 was required for its Centro de Investigaciones Biomédicas en Red de positive role in translation. Moreover, Enfermedades Respiratorias (Ciberes), Spain. polysome profile analyses indicated that Dhh1 promotes translation initiation. This Pandemic 2009 H1N1 (pH1N1) influenza role was linked to the concurrent presence viruses caused mild symptoms in most of the 5´ and 3´UTRs, two highly structured infected patients. However, a greater rate sequences known tocontrol translation, of severe disease was observed in healthy and of a newly determined stem-loop in young adults and children without the ORF region. Consistent with a direct comorbid conditions, suggesting that role of Dhh1 in translation, Dhh1 co- viruses with different pathogenicity could immunoprecipitated with the viral RNA cocirculate. without affecting its stability. Excitingly, Our previous data indicated that a strain of genome-wide ribosome profiling analyses pH1N1 virus isolated from a fatal case in yeast demonstrated that Dhh1 also presented enhanced pathogenicity promotes translation of a specific subset of compared to a virus isolated from a mild cellular mRNAs that are enriched in case, which circulated during the 2009 previously described Dhh1-bound mRNAs. pandemic. PB2 A221T, PA D529N, HA These mRNAs present higher base pair S127L changes appeared as particularly probablilities at their ORFs than those interesting and suggested that one or translationally-repressed or not combination of these changes could play translationally affected by Dhh1 and are an important role in increased enriched in mRNAs related to ribogenesis pathogenicity. Biological properties of processes. As a consequence modulation recombinant viruses (pH1N1 of Dhh1 activity will lead to their fast California/04/) carrying each of these coregulation, as needed for example under residues or combination of them have stress conditions. In sum, our results been analyzed both in vitro (human lung uncover a novel role of Dhh1 in the cell alveolar epithelial cells) and in vivo (murine

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model). Wild-type recombinant virus and viruses carrying PA529N, PB221T or both

changes replicated at similar rate, but HA

recombinant virus had a slightly higher replication rate at 9 and 12 hpi in cell culture. In vivo analysis showed a significantly decreased LD50 of 50 and 10 fold for PA and PA/PB2 recombinant viruses, respectively, compared to that of

the control virus. Viral titer in lungs of PA recombinant virus infected mice was higher up to 7 dpi., moreover a high proportion of mice presenting infectious virus in the heart, was found in these infected animals whose replication was detected by the presence of NEP (Nuclear

Export Protein) mRNA. Analysis of CD45+ cells in lungs of infected mice showed higher percentage of neutrophils and dendritic cells by 1 and 2 dpi, as well as rapid loss of alveolar macrophages by the 2 dpi in PA and PA/PB2 recombinant viruses infected mice compared with the control virus infected mice. These results indicate that PA529N residue leads to increased pathogenicity of influenza A H1N1 virus mediated by several biological processes.

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PO: P O S T E R S S E S S I O N

FLASH PRESENTATIONS acetylglucosamine transferases (OGT) Chairpersons: SUSANA ALVAREZ AND identified in Arabidopsis thaliana, in up to JOSÉ LUIS JIMÉNEZ seven specific threonines located towards the N-terminal region of PPV CP.O- Wednesday June 9, 2015 GlcNAcylation of CP has a positive role in AUDITORIUM REAL CASA DE LA MONEDA the infection process, probably intervening 10:20-11:30 h in virion assembly and/or stability. A “Yin-Yang” mechanism has been *Flash presentations proposed to regulate reciprocal phosphorylation and O-GlcNAcylation of (P01) different mammalian proteins. Some THE COAT PROTEIN OF THE POTYVIRUS previous evidence suggested that PPV CP PLUM POX VIRUS CAN BE could be phosphorylated. In this study, we PHOSPHORYLATED IN VIVO AND THIS made use of proteomics analyses to MODIFICATION ESTABLISHES A CROSS- demonstrate that PPV CP is TALK WITH ITS PREVIOUSLY DESCRIBED O- phosphorylated in vivo at its N-terminal GlcNAcylation region. In contrast with the classical “Yin- 1 1 S. MARTÍNEZ-TURIÑO , J. J. PÉREZ , R. Yang” mechanism, phosphorylation affects 1 1 1 NAVAJAS , S. CIORDIA , J. A. GARCÍA residues different from the O-GlcNAcylated 1. Centro Nacional de Biotecnología CNB-CSIC, ones (serines Ser25, Ser81, Ser101 and Madrid, Spain. Ser118). However, quantification by a differential proteomics strategy based on Plum pox virus (PPV), a member of the iTRAQ (Isobaric Tags for Relative and genus Potyvirus (family Potyviridae), Absolute Quantitation) of peptides of CP causes sharka, one of the most damaging from virions purified from wild type and diseases of stone fruit trees. PPV genome SEC-deficient plants led to uncover the is a positive-sense single-stranded RNA existence of some cross-talk between O- encapsidated by a single type of capsid GlcNAcylation and phosphorylation in PPV- protein (CP) in flexuous rod particles. It is CP. Accordingly we speculate that some translated into a large polyprotein, and a sort of regulation could direct reciprocal frameshift product, that are proteolytically and dynamic changes between this two processed in at least 11 final products. PTM affecting neighbouring Thr/Ser O-GlcNAcylation is a post-translational residues that are susceptible to be modification (PTM) that adds single O- modified. linked N-acetylglucosamine residues to nuclear and cytoplasmic proteins. Contrary to animals, this PTMhas been barely studied in plants. The PPV CP is the best-

characterized target of O-GlcNAcylation produced in plants. It is modified by secret agent (SEC), one of the two O-linked N-

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*Flash presentations identify compounds that restrict HAdV (P02) infection. Since different combinatorial NEW THERAPEUTIC ALTERNATIVES FOR piperazinone libraries have previously THE TREATMENT OF ADENOVIRUS been described and identified as potent INFECTIONS IN IMMUNOSUPPRESSED antiviral compounds we choose this ring as PATIENTS: DESIGN, SYNTHESIS AND the central core for the design and EVALUATION OF THE ANTI-ADENOVIRUS evaluation of a new small library. Based on ACTIVITY OF PIPERAZINE DERIVATIVES the structure of a previously reported anti- 1 HAdV piperazinone (15D8) that targeted P. MARTÍNEZ-AGUADO , M. VEGA HOLM, the HAdV replication process, we designed, A. SERNA GALLEGO1, J. I. CANDELA, J.A. 1 1 synthesized and evaluated a library of new MARRUGAL LORENZO , I. GÓMEZ-MARÍN , piperazine derivatives with potential anti- F. IGLESIAS GUERRA, J. M. VEGA PÉREZ 1 HAdV activity. We substituted the AND J. SÁNCHEZ-CÉSPEDES piperazin-2-one ring from compound 15D8 1 Institute of Biomedicine of Seville (IBiS), University by one of piperazine, moving the carbonyl Hospital Virgen del Rocío/CSIC/University of Seville, Clinical Unit of Infectious Diseases, Microbiology group at N1 position. Starting with an and Preventive Medicine, Seville, Spain initial first generation of compounds two 2 Department of Organic and Pharmaceutical more generations were designed based on Chemistry, School of Pharmacy, University of Seville, the structure-activity relationship (SAR) of Seville, Spain the active compounds obtained in each previous generation. We found five Adenoviruses (HAdV) are the cause of phenylpiperazine compounds that many different acute infections mostly in significantly inhibited HAdV infection. the respiratory and gastrointestinal tracts, These compounds showed substantial anti- as well as conjunctivitis. HAdV disease in HAdV activity at low micromolar immunocompetent individuals is mostly concentration targeting the HAdV DNA self-limiting, however, in replication process. Moreover, we found immunocompromised individuals, that the presence of a phenylpiperazine especially in pediatric units, HAdV ring and a urea group at N1 carrying infections are cause of high morbidity and electron-withdrawing groups conferred mortality. Unfortunately, despite the little or no cytotoxicity to these molecules. significant clinical impact, there are no The selected phenylpiperazines potentially antiviral agents that are approved for the represent strong hit compounds for the treatment of HAdV. Sub-optimal development of a new class of antiviral therapeutic options to treat HAdV compounds to treat HAdV infections in infections in immunosuppressed patients immunossupressed patients and could include the use of broadly acting antivirals represent a useful tool to better such as ganciclovir, acyclovir, vidarabine, understand the complex events involved in ribavirin and cidofovir, with highly variable HAdV DNA replication. results. To address this situation, we used high-throughput screening (HTS) of synthetic small molecule libraries to

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*Flash presentations interaction plays a role in the functional (P03) recruitment of the 40S subunit to the IN VITRO IDENTIFICATION OF A mRNA. STRUCTURAL MOTIF IN THE 3’ UTR REGION OF THE IFNA5 MRNA FAVOURED *Flash presentations BY miR-122 WHICH ENABLES THE (P04) RECOGNITION OF THE 40S SUBUNIT OF QUERCETIN MODIFIES LIPID DROPLET THE RIBOSOME IN THIS REGION MORPHOLOGY AND IMPAIRS HEPATITIS C 1,2 R. DÍAZ-TOLEDANO , N. CALERO- VIRAL LIFE-CYCLE STEPS FROM ASSEMBLY 1 1,2 MUÑOZ , A. ARIZA-MATEOS AND J. TO REPLICATION 1,2 GÓMEZ 1 2 1 Á.ROJAS* , S.CLEMENT , J.A. DEL CAMPO , (1) Laboratory of RNA Archeology, Instituto de M.LEMASSON3, M.GARCÍA-VALDECASAS1, Parasitología y Biomedicina 'López-Neyra', CSIC, 1 1 1 Armilla, 18100 Granada, Spain. (2) Ciberehd. L. ROJAS , A.GIL-GÓMEZ , I. RANCHAL , J.BAUTISTA4, A.R. ROSENBERG3, F.NEGRO5,

M.ROMERO-GÓMEZ1. In silicopredictions have allowed the 1. Unit for Clinical Management of Digestive detection of a microRNA binding site Diseases and CIBERehd, VALME UNIVERSITY within the non-coding 3' region of the HOSPITAL, Seville, Spain alpha interferon subtype 5 that is 2. Division of Clinical Pathology , University Hospital, specifically expressed in the liver. We have Geneva, Switzerland analysed the RNA structure in this region 3. Hepatitis C virology, University Paris Descartes, using RNases that specifically recognise Paris, France 4 single and double chain RNA. We observed . Biochemistry and Molecular Biology, Faculty of that the presence of miR-122 modifies the Pharmacy, University of Seville, Seville, Spain 5 digestion pattern of these RNases in the . Division of Clinical Pathology and Gastroenterology and Hepatology, University region predicted for their annealing. The Hospital, Geneva, Switzerland modifications affect the RNA structure in

the “stop” codon region, and suggests the possible appearance of a pseudoknot-type Background and Aims: structure. The resulting conformational Hepatitis C virus (HCV) life cycle can be change post-hybridisation with miR-122 divided into several steps: (i) entry of viral generates an RNA mimetic structure which particles, (ii) can be recognised in vitro by human RNase translation of the viral proteins, (iii) P and the ribozyme of the cyanobacterium replication of the viral genome, a step Synechocystis sp. In addition, the presence which requires the activity of HCV non of miR-122 promotes, in a mild yet specific structural proteins including the protease way, the interaction of the 40S subunit NS3, and (iv) assembly of new viral with IFNA5 mRNA in the absence of other particles, a step which requires the protein factors. We have seen that the 3' localization of HCV core and NS5A protein region of mRNA is responsible for this to lipid droplets mediated by the host binding. It is not yet known if this diacylglycerol acyltransferase type 1

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(DGAT1). noninfected cells (fold induction 2.29±0.23 Quercetin, a bioflavonoid, seems to p<0.01). However, this increase was prevent the localization of HCV core significantly inhibited by treatment with protein to lipid droplets and to inhibit HCV quercetin [63.5±2.9% (p<0.01)]. replication (Rojas et al., AASLD 2013). Here Conclusions: In the current study, we aimed at evaluating the potential of quercetin was observed to inhibit DGAT quercetin as and further activity, to decrease NS3 activity, as such, defining its mechanism(s) of action on the resulting in impairment of viral infectivity different steps of HCV life cycle. and replication. Thus, the antiviral activity Methods: To reproduce the complete HCV of this flavonoid is promising and mediated life cycle, Huh7.5 cells and primary through several viral and host mechanisms. hepatocyte were infected with JFH1and subsequently treated with doses of *Flash presentations quercetin. i) Replication of HCV genome (P05) was assessed by measuring the intracelular levels of negative-strand HCV RNA by qRT- DELAYED LIVER FIBROSIS IN HTLV-2- PCR. ii) Production of infectious virus was INFECTED PATIENTS CO-INFECTED WITH assessed by measuring the infectivity titers HIV-1 AND HCV WITH SUPPRESSIVE in filtered culture supernatants with focus- ANTIRETROVIRAL TREATMENT formation assay and by COBAS® TaqMan® ABAD-FERNÁNDEZ M, MORENO A, HCV Test v2.0. iii) NS3 protease activity in DRONDA F, DEL CAMPO S, QUEREDA vitro was measured using a comercial Kit C,CASADO JL, PÉREZ-ELÍAS MJ, MORENO S, SensoLyte® 520 HCV Protease Assay. (iv) VALLEJO A DGAT activity was analyzed using the Department of Infectious Diseases, Instituto Ramón protocol previously described by McFie y Cajal de Investigación Sanitaria (IRYCIS), Hospital and coll. (2011) in Huh7.5 cells infected by Universitario Ramón y Cajal, Madrid, Spain. JFH1. Results: Infectivity assay in Huh7.5.1 and Objectives: HIV-1 and HTLV-2 co-infection primary hepatocytes were IC50: 37.83 and is found with relatively high frequency 23.63 μM respectively. At 50μM HCV-RNA among injection drug users in North levels decreased (Huh7.5.1: 39% and PHH: America and Western Europe since 80´s. 24%). The amount of HCV-RNA (evaluated There is still no clear evidence that HTLV-2 by the quantity of viral RNA produced in causes any human disease. Nevertheless, the supernatant) was decreased as well by several studies analyzing the effects of 60%±26.7 (p<0.05) compared to the HTLV-2 on HIV-1 pathogenesis in dually supernatant from Huh7.5 infected by JFH.1 infected HTLV-2-HIV-1 individuals revealed (1MOI). In vitro NS3 activity was inhibited delayed progression of HIV-1 to AIDS. On by quercetin by 45.40%±1.15 RFU the other hand, among individuals (p<0.001) compared to the vehicle, DMSO coinfected with HIV-1 and HCV, the (no inhibition). DGAT enzyme activity in influence of HTLV-2 on HCV pathogenesis infected cells was increased relative to has been poorly studied.

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Design: Retrospective study to clarify the *Flash presentations influence of HTLV-2 in HCV pathogenesis (P06) and hepatic fibrosis among patients co- RNA-seq PROFILES FROM RAINBOW infected with HIV-1. TROUT (ONCORHYNCHUS MYKISS) HEAD Methods: This was comparative cohort KIDNEY AFTER INFECTIOUS study including 39 HTLV-2-HIV-1-HCV- HEMATOPOIETIC NECROSIS VIRUS coinfected patients and 42 HIV-1-HCV- INFECTION. coinfected patients. They were evaluated N. A. BALLESTEROS1, H. ARTAZA1, G. for transaminase levels, hepatic fibrosis PADILLA1, L. ALONSO1, S. RODRÍGUEZ stage, IL-28B genotype, Th1/Th2/Th17 SAINT-JEAN1, S. PEREZ-PRIETO1. cytokine levels, immune activation, 1. Centro de Investigaciones Biológicas, (CSIC), Dpto. inflammation, and microbial translocation. Microbiología Molecular y Biología de la infección, Results: HTLV-2-HIV-1-HCV-coinfected 28040, Madrid, Spain. patients had lower alanine aminotransferase levels (p=0.023) and Infectious hematopoietic necrosis virus hepatic fibrosis (p=0.012) compared to (IHNV) is a single-stranded, negative-sense HIV-1-HCV-coinfected patients. Moreover, RNA virus and a member of the Kaplan-Meier survival analysis showed a family. IHNV is endemic delay in hepatic fibrosis development for within both wild and cultured host up to five years (p=0.032). HTLV-2-HIV-1- populations in North-West of USA. The HCV-coinfected patients also had higher virus establishes acute, lethal infection in Th1/Th2 ratio (IFNγ/IL4 ratio, p=0.045, juvenile Pacific salmonids and results in a TNFα/IL4 ratio, p=0.011) and Th17 significant loss to hatchery programs and response (p=0.047), while lower CD8 T cell aquaculture industries every year. activation (p=0.013) and LPS level In Europe, the rainbow trout is the most (p=0.002). affected species. The aim of this work was Conclusions: Findings strongly support that to contribute to a better understanding of HTLV-2 co-infection might delay fibrosis how fish respond to the viral infection. This developmentin HCV-HIV-1 co-infected information will be essential for the patients. knowledge of immunity and to afford new perspectives in the design of oral vaccines. We have focused in the transcriptome response (physiological and pathological state) of rainbow trout at 3 and 7 days

after IHNV challenge, which correspond to asymptomatic and symptomatic (respectively) viral infection. The massive RNAseq technique was used for this study and the head kidney (main hematopoietic organ) was selected for sampling. The transcriptome of rainbow

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trout from the National Animal Genome *Flash presentations Research Program was used as reference, (P07) and the information was divided in two “VIR(TU)AL EPIDEMIC”: A GAME ABOUT different groups (Ohnologous and No VIRUSES FOR SMARTPHONES ohnologous genes) as reference. These 1 2 RNA-seq libraries were sequenced with a E. GÓMEZ-LUCÍA , L. BENÍTEZ , M.M. BLANCO1, M.T. CUTULI1, A. DOMÉNECH1, R. read length of 75 nucleotides, single-end 3 4 5 1 reads in two different lanes of an Illumina FLORES , J. QUER , J. ROMERO , R. AÑEZ 1 GAiix Format. The number of reads in each Dpto de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense Madrid group was well balanced with 16.122.108 2 reads in the infected fish at 3 days post Dpto de Microbiología III, Facultad de Ciencias Biológicas, U.Complutense Madrid infection (dpi) group, 16.378.765 reads in 3 Instituto de Biología Molecular y Celular de Plantas the infected fish group sampled at 7 dpi (Universidad Politécnica-CSIC), Valencia and 16.192.950 reads in the non-infected 4 Liver Unit. Hospital Universitari Vall d’Hebrón, fish group (control group). The results Barcelona rendered 6875 differentially expressed 5Dpto de Protección Vegetal, INIA, Madrid transcripts (DETs) at 3 dpi in the IHNV infected group and 5857 DETs at 7 dpi in A team of teachers of the Complutense comparison with the control group. In University of Madrid, virologists of the SEV addition, gene pathway analysis of the and computer science designers (Sr. differentially expressed gene set Brightside) has developed a free online highlighted several putative genes involved game (http://epidemia.sevirologia.es) for in the immune response activity. Smartphone, which also may be played The expression patterns of 7 differentially with tablets and conventional computers. expressed genes involved in immune It is aimed both for the self-evaluation of response were validated by quantitative Virology and to learn more about various real-time RT-PCR. Our results provide aspects of this science in a subliminal way, valuable information on gene functions enjoying the knowledge about viruses. At associated with IHNV infection. the moment, the game is offered in This work was supported by projects Spanish and in English. More than 200 AGL2010-18454 (Spanish Ministry of multiple choice questions inquire about Economy and Competitiveness,MINECO) what are viruses and their differences with and 2010-20E084 (CSIC). N. Ballesteros bacteria and eukaryotic organisms, the wants to thank the MINECO for their PhD diseases that they produce in man, animals student fellowship. and plants, their treatment, diagnosis and prevention, as well as about bacteriophages and the possible applications of viruses. As soon as the

question is answered, the correct answer is available, along with additional information on the treated aspects.

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The name “Vir(tu)al epidemic” alludes to *Flash presentations its design, representing a challenge to save (P08) the World from a lethal viral epidemic. At INNOVIROLOGY: THE NETWORK OF the beginning of the game, the program EUROPEAN TEACHERS/TRAINERS OF asks for the name of the player, who VIROLOGY begins with 0 points and five lives. 1 2 3 Whenever the player fails a question, E. GÓMEZ-LUCÍA , A. DOLEI , R. LAVIGNE , S. LEPODER4, C. LOGUE5, D. RADIN6, M. he/she loses one life, which can be 7 8,9 recovered when responding correctly to SZYNDEL , B. WÖLK 1 three questions. Each question has a value Dpto de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense Madrid (Spain). that is added to the score of the player. 2Department of Biomedical Sciences, Università Also, following every few correct answers, degli Studi di Sassari (Italy).3Katholieke Universiteit an encouraging message is received. The Leuven (Belgium).4Université Paris-Est. Ecole game ends when the player has responded Nationale Vétérinaire d'Alfort. UMR 1161 Virologie 5 to 20 questions, being his/her name and INRA-ENVA-ANSES (France). Public Health England, Leeds, (England). 6Faculty of agriculture, Belgrade score inscribed in a public ranking. As it is (Serbia).7Szkola Glowna Gospodarstwa Wiejskiego competitive due to this ranking, it is very (Poland).8LADR GmbH, MVZ Kramer and Colleagues, attractive and contributes to approach Geesthacht (Germany), 9Medizinische Hochschule Virology to the general public and to Hannover (Germany) students, who generally consider viruses as distant biological entities, possibly due to The European Union has funded an their small size and the many different initiative for innovation in teaching and aspects involving their study. training of Virology as well as for improving The game has been presented in High its dissemination through its programme Schools of the Community of Madrid, and Erasmus+ (Innovirology, project number in a course of the 8th edition of “Teachers 2014-1-ES01-KA203-004962) under the and Science” sponsored by the Fundació orchestration of eight institutions from Catalunya La Pedrera, Barcelona. A different European countries. The idea is to satisfaction survey has been prepared to create a network for teachers and trainers present it in the reference institutes, and of Virology to connect to each other, share to evaluate the acceptance of the project. and develop teaching materials and Funded by FECYT, SEV, UCM y Erasmus+ contribute to the general spread of knowledge in the Virology field. The specific aims include:

- To compile protocols for teaching laboratory Virology which different teachers choose to share, so that any member of the network can freely access it and select laboratory techniques best suited to the characteristics of their

group of students.

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- To share and make available to other students and general public to get more Virology teachers specific educational acquainted with the different aspects of tools developed by the network, such as Virology. (but not exclusively) information and communication technologies (computer *Flash presentations programs, games, tools for self- assessment or evaluation, etc.). (P09) - To develop online courses for life-long APPLICATION OF A RT-qPCR TECHNIQUE learning and bring Virology to those who IN THE CONFIRMATION OF CASES AND are interested in learning more about it. DEATHS BY YELLOW FEVER VIRUS 1,2 3 Online courses will be "What are M. ROSSI S. , J. MÉNDEZ R. , A 2 1 1 viruses?", "Basic and applied Virology", HERNÁNDEZ , F. LASALA , J.M. LUQUE , F. 1 2 1 "Clinical Virology", "Veterinary Virology", MOLERO , G. CÉSPEDES , A. TENORIO , 1 1 "Plant viruses", "Viral molecular M.P. SÁNCHEZ-SECO , A. NEGREDO diagnostics", "Emerging viral diseases" 1Laboratorio de Arbovirus y Enfermedades Víricas and "Food Virology". These courses will Importadas, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, España. be designed so that they can be followed 2 by anyone with a general interest in Sección de Investigaciones en Patología Ultraestructural y Biología Molecular, Instituto virology. The possibility of including, in Anatomopatológico José A. O´Daly, Facultad de addition to an online exam, a graded Medicina, Caracas, Venezuela. face-to-face test so the course grants a 3Instituto Nacional de Salud, Bogotá, Colombia. degree of official recognition, is being discussed. Also, the consortium aims to Yellow fever (YF) is an arbovirosis caused write a book on Virology, online and open by the Yellow Fever Virus (YFV), access. It is intended to have plenty characterized by the presentation of fever, illustrations, attractive to young high jaundice and haemorrhages of less than 10 school students and to the person of the days and a high lethality. In South America, street. between 1985 and 2004, more than 90% of - To create and maintain social networks cases and outbreaks of YF were described (Facebook, twitter, etc) about news on all in Peru, Bolivia, Brazil, Colombia, Ecuador fields of Virology. and Venezuela, with a total of 3,559 cases Any teacher of Virology in Europe is of sylvatic and 2,068 deaths (58% of welcome to join the network. This lethality). In Venezuela the YF-cases occurs invitation includes all Virology teachers as sporadic and self-limiting outbreaks in interested independent of the type of their the Central west region, dedicated to institution, their major field or their forestry, crop and livestock, and interest in different subspecialties of characterized by poor socio-sanitary Virology. At the end of the project, a conditions with difficulties to access to the questionnaire will be prepared to evaluate national health system. In most of the the usefulness of the different aspects of cases the aetiological of the cases must be the project, which we foresee will help done post-mortem at

138 Virología. Publicación Oficial de la Sociedad Española de Virología

immunohistochemical level due the *Flash presentations difficulties of geographic access. The aim of (PO 10) this work was to evaluate the applicability CONSERVATION OF G PROTEIN EPITOPES of a RT-qPCR technique for the IN RESPIRATORY SYNCYTIAL VIRUS confirmation of deaths caused by YFV in (GROUP A) DESPITE BROAD GENETIC human primates (HPr) from Venezuela and DIVERSITY: IS ANTIBODY SELECTION Colombia and non-human primates (NHPr) INVOLVED IN VIRUS EVOLUTION? from Venezuela, using samples of formalin- 1, 2 3 fixed and paraffin-embedded tissues A. TRENTO , L. ÁBREGO , R. RODRIGUEZ- FERNANDEZ4, M. I. GONZÁLEZ-SÁNCHEZ4, (FFPET) and freeze tissues (FT). Sections of 4 5 tissues were dewaxed according histologic F. GONZÁLEZ-MARTÍNEZ , A. DELFRARO , J. M. PASCALE3, J. ARBIZA5 AND J. A. techniques, dried at ambient temperature, 1, 2 digested with Proteinase K and aliquots of MELERO . 1 the lysate were added to vials containing .Unidad de Biología Viral, Centro Nacional de Microbiología. Instituto de Salud Carlos III, AVL previous to the extraction of the RNA. Majadahonda. Spain The amplifications of the cDNA were done 2 .CIBER de Enfermedades Respiratorias, Instituto de in a LightCycler 2.0 (Roche) and detected Salud Carlos III, Majadahonda, Spain. using TaqMan probes. Of a total of 11 HPr- 3 .Departamento de Investigación en Virología, deaths and 5 NHPr-deaths with Instituto Conmemorativo Gorgas de Estudios de la immunohistochemical diagnosis of YFV, Salud, Panamá. respectively 9 (81.82%) and 5 (100%) were 4 .Hospital General Universitario Gregorio Marañón, positive in the RT-qPCR with an average Ct Madrid, Spain. 5 of 32.95 for the FFPET of the HPr and 27.88 .Sección Virología, Facultad de Ciencias, for the NHPr samples. Of all the samples of Universidad de la República, Uruguay. FT (80%; 8/10) were positive with an average Ct of 25.97. The Ct-values of the Human respiratory syncytial virus (hRSV) is FFPET samples from NHPr suggest that its recognized as the major cause of severe viral load is higher than the viral load acute lower respiratory tract infections described for HPr-samples. The differences (ALRI) in infants and young children in the Ct-values between the samples of FT worldwide. Phylogeny of group A and FFPET should be associated with the sequences of the G glycoprotein of hRSV deleterious activity of formaldehyde on revealed diversification in major clades and nucleic acids such as fragmentation, genotypes over more than fifty years of methylol bridges formation, etc. Results recorded history. Multiple genotypes co- are discussed in the context of the circulated during prolonged periods of importance of RT-qPCR as a sensible and time but recent dominance of the GA2 specific tool in anatomic pathology as well genotype was noticed in several studies as for the detection of different lineages of and it is highlighted here with sequences YFV and its use for the confirmation of from viruses circulating recently in Spain deaths caused by this arbovirus in and Panama. Reactivity of group A viruses Venezuela. with MAbs that recognize strain-variable epitopes of the G glycoprotein failed to

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correlate genotype diversification with resulting multimeric intermediates of both antibody reactivity. Additionally, no clear polarities by cis-acting hammerhead correlation was found between changes in ribozymes. To parasitize the transcription strain-variable epitopes and predicted sites and processing machinery of its host, of positive selection, despite both traits PLMVd depends on sequence and being associated to the C-terminal third of structural motifs. In silico, in vitro andin the G glycoprotein. Hence, our data do not vivo approaches support that the most lend support to the proposed antibody- abundant PLMVd strand (arbitrarily driven selection of variants as major assigned the plus polarity) folds into a determinant of hRSV evolution. Other multibranched conformation stabilized by alternative mechanisms are considered to a kissing-loop interaction. In the course of account for the high degree of hRSV_G routine testing by real-time RT-PCR, variability. PLMVd could not be detected in a sample that reacted positively by RNA gel-blot *Flash presentations hybridization with a full-length riboprobe. These conflicting results led us to consider (PO 11) that, instead of the low variability AN ATYPIC ISOLATE OF PEACH LATENT presumed for the viroid region used to MOSAIC VIROID WITH IMPORTANT design the TaqMan probe, this region SEQUENCE CHANGES THAT PRESERVE THE might be quite different in the novel RNA CONFORMATION AND INCREASE THE isolate. Conventional RT-PCR with two BIOLOGICAL FITNESS pairs of adjacent primers of opposite P. SERRA1, E. BERTOLINI2,3, M.C. polarity derived from distinct regions of MARTÍNEZ2, M. CAMBRA2, R. FLORES1 the molecule, cloning and sequencing 1Instituto de Biología Molecular y Celular de confirmed that this was indeed the case. Plantas, Consejo Superior de Investigaciones Intriguingly, when compared with PLMVd Científicas-Universidad Politécnica de Valencia, isolates of known sequence and biological Spain 2 properties, the novel isolate presented Instituto Valenciano de Investigaciones Agrarias, extensive covariations preserving the two Moncada, Valencia, Spain stems whose capping loops form the 3Departamento de Fitossanidade, Faculdade de Agronomia, Universidade Federal do Rio Grande do kissing-loop interaction, thus upholding the Sul, Porto Alegre, Brazil functional relevance of this interaction. Sequence analysis of multiple clones of the novel isolate showed relatively low internal Viroids, despite their minimal genomes variability, and inoculation of peach (they are non-protein-coding circular RNAs seedlings with in vitro transcripts from a of about 250-400 nt), can infect and recombinant plasmid with a head-to-tail frequently incite diseases in plants. Like dimeric insert of a representative variant other members of the family revealed a non-symptomatic infection. Avsunviroidae, peach latent mosaic viroid Moreover, when in vitro transcripts of this (PLMVd) replicates in plastids by a rolling- variant and of a symptomatic variant circle mechanism involving cleavage of the (gds6) from another isolate were co-

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inoculated, the first outcompeted the with water or food contaminated with the second as revealed by real-time RT-PCR discharge of untreated or even treated (with specific TaqMan probes for each sewage. Among the most frequently variant) and by conventional RT-PCR, detected human viruses excreted in urban cloning and sequencing of the resulting sewage are the known groups of viruses progenies, which did not display such us adenoviruses, astroviruses, detectable recombination. Based on these , rotaviruses, as well as data, a new “universal” TaqMan probe was . designed for the concurrent detection of To improve the knowledge on the excreted both variant classes. Altogether these virome and the viruses that could results provide further insights into PLMVd represent a risk associated to water or variability (which preserves key structural food for the population, NGS techniques elements like the hammerhead ribozymes have been applied to study viral richness in and the kissing-loop interaction), and into urban sewage from Barcelona. The the strong interference existing between metagenomics study using Illumina coinfecting variants (possibly mediated by platform allowed the description of more RNA silencing). From a more applied than 25 different viral families. Among perspective, they also alert on diagnosis those 9 are related to human viral techniques just relying on a fragment of pathogens. The presence of the recently the RNA to be detected. described virus /klassevirus, a new genus belonging to Picornaviridae family in *Flash presentations urban sewage, has been confirmed by conventional RT-PCR. (PO 12) HUMAN VIRUS IN FECALLY The study of excreted viruses has CONTAMINATED WATER, THE VIROME OF continued by analyzing 56 clinical samples URBAN SEWAGE AND CLINICAL from patients presenting gastroenteritis GASTROENTERITIS FECAL SAMPLES without an identified etiological agent. 1 1 Using metagenomics on different pools of X. FERNANDEZ-CASSI , M. RUSIÑOL , N. samples members of the family TIMONEDA1, R.BARTOLOMÉ2, S. BOFILL- 1 3 1 caliciviridae, astroviridae, , MAS , J.F. ABRIL , R. GIRONES . picornaviridae and have been 1. Laboratory of Virus Contaminants of Water and found. These findings show that Food, Microbiology Department, Biology faculty, Barcelona University, Catalonia, Spain. conventional clinical tests designed to 2. identify known etiological agents do not Hospital Universitari Vall d’Hebron, Microbiology Service, Barcelona, Catalonia, Spain. identify many viral pathogens related to 3Computational Genomics Lab (Compgen), Genetics gastroenteritis. Metagenomics is a very Department, Biology faculty, Barcelona University, useful technique for the identification of Catalonia, Spain. etiological agents in clinical samples presenting negative results for the Many viral infectious diseases are commonly applied diagnostic tests. transmitted by consumption or contact

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*Flash presentations animals sera were used for this for study: (PO 13) vaccinated and infected animals from APPROACHES TO DIVA ASSAYS FOR WEST controlled experiments bled at different NILE VIRUS days and vaccinated and/or infected field animals. To set up the assay, the sera from B.REBOLLO1, S.LECOLLINET2, A.CAMUÑAS1, 1 1 1 the experimental infected/vaccinated E.SORIA , A.J SANZ , A.VENTEO animals were analyzed. These samples 1. INGENASA, Inmunología y Genética Aplicada, reveals a positive result in the ELISA based Madrid, Spain on E protein with the same high OD values. 2. ANSES, Animal Health Laboratory, EU on equine Nevertheless, the antibody response West Nile disease, France against NS1 in vaccinated animals showed

decreased OD values comparing with those West Nile virus infection is identified by obtained with infected animals. The ratio several diagnostic tools, being the most OD with E protein/NS1 protein was higher commonly ones focused on the than 4 in vaccinated animals whereas this identification of the agent (RT-PCR), the ratio, in infected animals, was lower than virus neutralization test or the detection of 4.Similar results were obtained using the IgM/IgG against structural proteins. Over field samples. Additionally to test the the last decade, several outbreaks caused specificity a group of 90 negative sera by West Nile virus have been detected in showed a very low signal in both assays. In different parts of Europe, which increased conclusion, We have observed a different the vaccination of horses in many antibody response to the structural E countries. The currently diagnostic protein and to the non-structural NS1 methods cannot differentiate infected protein in infected and vaccinated horses. from vaccinated animals. The main goal of Based on this difference, using an IDAS- this work is to deal with this problem when ELISA we could differentiate vaccinated both events may occur in a horse from infected horses. The design of this population at the same time. Based on the assay carried out during this study could difference of antibody response between help to the development of a DIVA assay. infected and vaccinated horses against the Further experiments are needed to adjust structural (E protein) and the the ELISA conditions and test a bigger Nonstructural (NS1 protein), a DIVA panel of infected sera in order to check the (differentiating infected from vaccinated utility of the assay in field. animals) assay has been designed. A panel of Monoclonal antibodies (Mabs) against E and NS1 proteins was obtained and the best were selected to coat ELISA plates. After that, the inactivated whole culture virus was added and each specific protein was captured by the specific Mab. Horse samples were analyzed in an indirect ELISA DAS format (IDAS). Different groups of

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*Flash presentations to be able to encode viral miRNAs, while (PO 14) the capacity of the RNA virus to encode PCV2 GENOME CAN NOT ENCODE VIRAL viral miRNAs is a matter of controversy. miRNAs IN AN EXPERIMENTAL INFECTION Herpesviruses are the best example of 1 2 virus that encodes viral miRNAs with the F. NÚÑEZ-HERNÁNDEZ , LJ. PÉREZ , G. capability to express high amounts of viral VERA3, S. CÓRDOBA3, J. SEGALÉS1,4, A. 3,5 1* miRNAs but it has also been observed in SÁNCHEZ , JI.NÚÑEZ other kind of viruses. In this study, the 1 . Centre de Recerca en Sanitat Animal (CReSA), capability of PCV2 to encode viral miRNAs UAB-IRTA, Campus de la Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès, Spain. in a subclinical infection has been tested. 2 For this purpose four pigs were intranasally . Centro Nacional de Sanidad Agropecuaria 4.8 (CENSA), La Habana, Cuba. infected with 7x10 TCID50 of PCV2 isolate 3. Departament de Genètica Animal, Centre de Sp-10-7-54-13 and two pigs received PBS Recerca en AgriGenòmica (CRAG), CSIC-IRTA-UAB- by the same route as controls. At 21 days UB, Universitat Autònoma de Barcelona, Bellaterra, p.i. pigs were euthanized. Small RNAs Barcelona, Spain. libraries were created from tonsil and 4 . Departament de Sanitat i Anatomia Animals, mediastinal lymph node and later Universitat Autònoma de Barcelona, Bellaterra, sequenced by next-generation sequencing Barcelona, Spain. techniques. For viral miRNA discovery, the 5. Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona (UAB), obtained sequences were blasted to the Bellaterra, Barcelona, Spain. viral genome considering 100% of alignment but also allowing mismatches in the extremes considering miRNAs Porcine circovirus type 2 (PCV2) is a small variability. One candidate was found with single stranded circular DNA virus of 1768- 58 copies which sequence corresponds to 9 nt long. PCV2 is the essential etiological the Vmir precursor candidate with the infectious agent of PCV2-systemic disease highest score. The posterior analysis (PCV2-SD), formerly known as postweaning revealed that the candidate was an isomir multisystemic wasting syndrome, (PMWS). of the cellular miR-29a-5p. After this study MicroRNAs (miRNAs) are 19-24 nt long we can confirm that PCV2 does not encode non- coding single stranded RNAs with viral miRNAs in a subclinical infection. On post- transcriptional regulation functions. the other hand, the study of the homology They mediate the silencing of their target of a viral sequence with a cellular miRNA mRNAs by binding to complementary sites could shed light on how miRNAs affect viral usually located in the 3’ untranslated evolution. regions (UTRs) of the mRNA. More than 28600 miRNAs have been described as shows miRBase, the miRNA database. These miRNAs are expressed by all kind of organisms, from mammals to plants and viruses. Since this time, only DNA viruses with a nuclear phase have been described

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(PO 15) chronic hepatitis delta were analyzed. 2 QUANTIFICATION OF GENOMES samples from 3 patients (1-3) followed up ENCODING LARGE AND SHORT FORMS OF for 6 months without treatment were HEPATITIS DELTA ANTIGEN TO EXPLORE included. HDV RNA was quantified the by THE REPLICATION CAPACITY OF HEPATITIS an in-house (range, 3.5-8 log copies / mL) DELTA VIRUS method. The proportions of Genomes S/L- M. HOMS(1,2), M. BLASI(1,2), A. RUIZ(2), D. Ag were determined by massive TABERNERO(1,2), P. REIMUNDO(2), J. sequencing (nt 339-891). QUER(1,3), R. CASILLAS(2), J. GREGORI(3), Results The patients showed a mean 6.3- M. RIVEIRO-BARCIELA(4), L. NIETO(2), R. HDV RNA logs copies / mL (SD, 0.76) and a ESTEBAN(1,4), M. BUTI(1,4), F. mean of 65% of genomes encoding for the RODRIGUEZ-FRIAS(1,2) short delta antigen. HDV RNA did not 1-CIBEREHD, Barcelona, Spain correlated with the percentage of genomes 2-Liver Pathology Unit, Biochemistry and S/L-Ag. microbiology Departments, Vall d’Hebron Hospital, The 3 followed patients did not present Barcelona, Spain changes in HDV-RNA, but a dynamic in the 3-Liver Diseases, Research Institute, Vall d'Hebron S/L-Ag genomes. Hospital, Barcelona, Spain Four samples (1b, 3a, 6 and 11) presented 4- Hepatology Department, Vall d’Hebron Hospital, Barcelona, Spain an enriched population of L-Ag genomes, indicating a population of defective viral

replication. Background The genome of hepatitis delta Conclusion The lack of correlation between virus (HDV) has a single open reading HDV-RNA quantification and the frame able to encode two antigens. The proportion of genomes encoding the short stop codon at position 196 is edited to and large delta antigens, and the dynamics Tryptophan and elongates the translation genomes encoding the short and large to the codon 214. The genomes with Stop delta antigens in the absence of treatment codon in 214 encodes for large delta suggest that assessment of HDV RNA alone antigen (LHDAg), which is involved in the does not reflect the replicative activity assembly and virion formation, and HDV. inhibition of HDV replication. The genomes with stop codon in196 are the only ones that can be edited, encode for the short delta antigen (SHDAg), and have replicative capacity. Aims To quantify the genomes encoding SHDAg and LHDAg (Genomes S/L-Ag) in the quasispecies of VHD and relate with viral replication. Patients and methods Sixteen baseline samples from 13 patients (1-13) with

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*Flash presentations cART. In the Spanish CoRIS cohort, we (PO 16) selected 21 male FR patients and 20 male BEFORE-TO-CART IMMUNOVIROLOGICAL control patients with successful CD4 TRAITS OF HIV SUBJECTS WITH LOW CD4 restoration (SR patients; with CD4 reaching RESTORATION more than 250 CD4 after 2 years of successful cART), who had available I. ROSADO SÁNCHEZ1, M.M. POZO- 1 1 baseline sample at the Spanish RIS BALADO , R.S. DE PABLO-BERNAL , I. Biobank. SR patients were baseline age, JARRÍN VERA2, A.I. ALVAREZ-RÍOS3, M. 1 CD4 and viral load-matched subjects. BENHNIA RAFII-EL-IDRISSI , E. RUIZ- Plasma levels of monocyte activation MATEOS1, M. LEAL NOVAL1, Y.M. PACHECO 1 (sCD14), endothelial activation (ICAM, LÓPEZ . VCAM), platelet/lymphocyte activación 1 Laboratorio de Inmunovirología del Instituto (sCD40, β2-microglobulina), IFN-γ- deBiomedicina de Sevilla (IBiS), UGC Enf. Infecciosas, Microbiología y Med. Preventiva, Inducible Protein 10 (IP-10), coagulation Hospital Universitario Virgen del Rocío, Sevilla, marker (D-Dimer), proinflammatory España markers (hsCRP, IL6) and soluble cytokines 2 Instituto de Salud Carlos III, Madrid, España (IL10, TGF-β, IFN-γ, IL4, IL7, TNF-α, IL17a) 3 Departamento de Bioquímica, Hospital were measured. Genotypic viral tropism, Universitario Virgen del Rocío, Sevilla, España cell subsets (CD3, CD4, CD25, FOXP3) and cellular markers of activation (HLA-DR, Background CD38), senescence (CD57, CD28), and cycling (ki67) were also determined. HIV-infected subjects who persistently maintain low CD4 counts despite having Results achieved undetectable viremia during Before cART initiation, FR patients combined antiretroviral therapy (cART) are compared with SR patients showed higher at an increased risk of death, and no levels of IL-6 (7.3[3.6-12.1] vs. 4.9[2.6-6.8] therapeutic alternative is available. These ρg/ml, respectively; p=0.034), a higher patients show several immunovirological proportion of patients with hsCRP>5 mg/L traits like X4 viral tropism, increased T-cell (44.4% vs. 5.6% respectively; p=0.007), a activation, senescence and apoptosis, and higher frequency of Treg (1.14[0.54-3.60] increased Treg frequency. However, vs. 0.46[0.22-1.37]; p=0.044) and a higher whether these factors are cause or frequency of CD4+Ki67+ (11.2[7.9-18.8] vs. consequence of the failed CD4 restoration 7.6[3.8-10.1] respectively; p=0.047). is unknown. This is the first study analyzing However, no differences were found in the samples from these patients before the cellular makers of activation and initiation of cART. senescence, soluble cytokines or viral Methods tropism. Patients with failed CD4 restoration (FR Conclusion patients) were patients starting cART with Increased inflammatory levels (IL6, hsCRP), less than 200 CD4/µL and achieving less Treg and cycling CD4 cells preexist in than 250 CD4/µL 2 years after suppressive patients with low CD4 restoration before

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the cART initiation, and consequently, function decline. We assayed the these factors could be involved in the activation ex-vivo and the responsiveness subjacent mechanisms causing such to TLR2 and TLR4 agonists in-vitro in the failure.However, X4 viral tropism, as well three subset of monocytes, assessing the as T-cell activation and senescence, usually intracellular production of IL1-alpha(α), associated to these patients, are more IL1-beta (β), IL-6, IL-8, TNF-α and IL-10 of probably consequence of such failed CD4 old subjects (median age 83 [67-90] years restoration. old; n=20) compared to young controls (median 35 [27-40] years old; n=20). Ex- vivo, elderly showed higher percentage of *Flash presentations classical monocytes that expressed (PO 17) intracellular IL1-α (p=0.001), IL1-β MONOCYTE PHENOTYPE AND (p=0.001), IL-6 (p=0.002) and IL-8 POLYFUNCTIONALITY ARE ASSOCIATED (p=0.007). Similar results were seen for WITH ELEVATED SOLUBLE intermediate and non-classical subsets. In- INFLAMMATORY MARKERS, vitro, higher monocyte responsiveness to CYTOMEGALOVIRUS INFECTION AND TLR2 and TLR4 agonists with aging was FUNCTIONAL AND COGNITIVE DECLINE IN observed. Multiple cytokine THE ELDERLY polyfunctionality was higher in the elderly. RS. DE PABLO1, MI GALVÁ2, R. RAMOS2, J. Single and multiple intracellular CAÑIZARES2,S. FERRANDO3, M.B. RAFII-EL- functionality ex-vivo were strongly IDRISSI1, YM. PACHECO1, M. LEAL1, E. RUIZ- associated to soluble coagulation and MATEOS1 inflammatory markers. The activation 1Biomedicine Institute of Seville, Seville, Spain. phenotype was independently associated 2Heliopolis nursing home, Seville, Spain. to anti-CMV IgG levels and functional and 3Vaccine Research Center NIAID NIH, Bethesda, MD, cognitive decline in the elderly. These data United States. demonstrate that monocytes are key cells as potential source of the high soluble Monocytes are mediators of the inflammatory levels. Our findings suggest inflammatory response. High levels of that CMV infection might be a driving soluble inflammatory biomarkers have factor of the activation of monocytes been associated to aging, CMV infection which was associated to the functional and and adverse health outcomes in the cognitive decline in the elderly. elderly. Monocytes comprise three subsets, the classical (CD14++CD16−), intermediate (CD14++CD16+), and non- classical (CD14dimCD16+). However, little it is known about the phenotypical and functional age-related changes of monocytes and the association with soluble inflammatory biomarkers, CMV infection and functional and mental

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*Flash presentations . Quadriviruses are fungal (PO 18) viruses with a multipartite genome. Each NEAR-ATOMIC RESOLUTION CRYO-EM dsRNA segment is encapsidated separately STRUCTURE OF ROSELLINIA NECATRIX in a similar particle. The ~45 nm-diameter QUADRIVIRUS 1 RnQV1 capsid is built of 60 heterodimers with two 1,356- (P2) and 1,059-residue C.P. MATA1, J. GÓMEZ-BLANCO1, D. 1,2 3 4 proteins (P4), as deduced from analytical LUQUE , N. SUZUKI , S. KANEMATSU , W. ultracentrifugation analysis. In RnQV1 HAVENS5, S. A. GHABRIAL5, B.L. TRUS6, J.L. 1 1 strain W1075, P2 and P4 are processed CARRASCOSA , J.R. CASTÓN into several peptides without altering its 1 Department of Structure of Macromolecules, structural stability, whereas in strain Centro Nacional de Biotecnología/CSIC, 28049 Madrid, Spain; 1Centro Nacional de W1118, both proteins remain nearly intact. Microbiología/ISCIII, 28220 Majadahonda, Madrid; The 3D structure of RnQV1 W1118 was 3IPSR, Okayama University, Kurashiki, 710-0046 determined by single-particle cryo-EM Japan; 4Apple Research Station, NARO, Morioka, analysis at 3.73 Å resolution. Data were 5 020-0123 Japan; University of Kentucky, Lexington, acquired in a Tecnai Titan Krios electron KY 40546-0312 USA; 6Center for Information Technology/NIH, Bethesda, MD 20892-5624, USA microscope (Laboratory of Molecular Biology, Cambridge, UK) equipped with a

direct electron detector and processed Most dsRNA viruses have an icosahedral using RELION software. Heterodimers are T=1 capsid based on 60 asymmetric coat organized following a quaternary protein dimers of a single protein (also organization similar to reovirus, referred to as a “T=2”, 120-subunit capsid). chrysovirus and . The full-atom This ubiquitous stoichiometry provides an model of the capsid showed the critical optimal framework for genome replication contacts among structural subunits that and organization. Whereas there are mediate capsid assembly, and indicated numerous structural studies of the family that P2 is processed in its C-terminal end , additional studies are needed that faces the outer surface and lacks 383 of many fungal and protozoal dsRNA residues. Despite the lack of sequence viruses. Penicillium chrysogenum virus similarity, superimposition of P2 and P4 (PcV), a of the family - , has an authentic T=1 capsid -chains matched very well. formed with 60 copies of one polypeptide; There are two preferential “hot spots” into the capsid protein is an almost perfect which structural and functional variations structural duplication of a single domain, in can be introduced by insertion of distinct which many secondary structural elements segments. Overlaying PcV and L-A (a match very well. This conserved core totivirus) capsid proteins on either of the represents a hallmark fold preserved in the P2 and P4, while maintaining the same dsRNA virus lineage. spatial arrangement in the shell, Here we describe the 3D structure of highlighted the same conserved motif and Rosellinia necatrix quadrivirus 1 (RnQV1), hot spots for insertions. The RnQV1 capsid the type species of the family

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is the first pseudo T=1 (or “P=2”) capsid the diversity was lower in MAX protease reported to date. compared with WT protease (0.0021±0.00003 vs 0.0029±0.00006, *Flash presentations p<0.0001, unpaired t-test). These differences were not observed when (PO 19) protease flanking regions were analyzed. INFLUENCE OF CODON PAIR USAGE IN THE These results suggested that WT and MAX EVOLVABILITY OF HUMAN proteases might occupy different sequence IMMUNODEFICIENCY TYPE 1 VIRUS (HIV- spaces. To explore the evolvability of the 1) codon pair re-coded protease, WT and M. NEVOT, G. MARTRUS, M. PARERA, M. MAX viruses were subjected to the CLOS, M.A. MARTÍNEZ selective pressure of PIs [atazanavir (ATV) Irsicaixa, Hospital Germans Trias i Pujol, Badalona, and darunavir (DRV)]. After the same Spain number of serial passages in MT-4 cells in the presence of PIs, WT and MAX viruses HIV-1 populations, like other RNA viruses, developed phenotypic resistance to PIs are described as a closely related mutant (IC50 14.63±5.39 nM and 21.26±8.67 nM, spectra or mutant clouds termed viral for ATV; and IC50 5.69±1.01µM and quasispecies. Mutant cloud composition 9.35±1.89 for DRV, respectively). Sequence can impact virus evolvability, fitness and clonal analysis showed the presence, in virulence. The extraordinarily large number both viruses, of previously described of possible encodings in natural genes is to resistance mutations to ATV and DRV. some extent restricted by two encoding However, a different resistance variant biases referred to as codon bias and codon repertoire appeared in the MAX virus pair bias. An unexplored aspect of the protease when compared to WT. genetic architecture of HIV-1 is how codon Specifically, the G16E substitution was only choice influences population diversity and observed in the WT protease. In addition, evolvability. Here we compared the the L10F, L33F, K45I, G48L and L89I development of HIV-1 resistance to substitutions were only detected in the re- protease inhibitors (PIs) of wild-type (WT) coded MAX protease population. The virus and a synthetic virus (MAX) carrying a differences in the mutation pattern that codon-pair re-engineered protease emerged after PIs treatment suggested sequence with 38 (13%) synonymous again that WT and MAX virus proteases mutations. A sequence analysis of 200 occupy different sequence spaces although individual clones, obtained by virus RNA both virus proteases were able to develop endpoint dilution, demonstrated that after PIs resistance. Further studies will required one passage in MT-4 cells MAX protease to elucidate whether HIV-1 sequences quasispecies diversity (p-distance) was have evolved to optimize not only the significantly higher than that of the virus protein coding sequence but also the carrying the WT protease (0.0014±0.00003 DNA/RNA sequences. vs 0.0012±0.00004, p=0.0027, unpaired t- test). However, after 15 passages (45 days)

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*Flash presentations reactions with no significantly populated (PO 20) intermediates. THE (DIS)ASSEMBLY PATHWAY OF A We have undertaken the experimental in SIMPLE VIRUS CAPSID INVOLVES A vitro study of the (dis)assembly pathway of COMPLEX SERIES OF TRANSIENT one of the simplest viral capsids known, INTERMEDIATES that of the minute virus of mice (MVM). In M. MEDRANO, M.A. FUERTES, P.J.P. a previous study we used atomic force CARRILLO, A. RODRÍGUEZ-HUETE, A. microscopy (AFM) to study the mechanical VALBUENA, M.G. MATEU. disassembly of single MVM particles. In the present study, MVM capsids were treated Centro de Biología Molecular "Severo Ochoa" (CSIC- UAM), Universidad Autónoma de Madrid, Madrid, with limited, controlled amounts of Spain. guanidinium chloride in order to stabilize transient intermediates in the (dis)assembly pathway. The disassembly Capsid assembly is an obligate step of the reaction was followed over time, and any viral cycle; the reverse process, intermediates were visualized and disassembly, is required for some viruses quantified using mainly transmission to release its genome into the host cell electron microscopy and AFM. during infection. The study of capsid assembly and disassembly is contributing The results revealed that (dis)assembly of to the development of antiviral drugs, the the MVM capsid appears to proceed production and modification of virus-like sequentially through a series of relatively particles for vaccination and other stable intermediates, whose abundance biomedical or biotechnological and structural organization were estimated applications, and the engineering of self- over time. We conclude that (dis)assembly assembling nanoparticles for technological of even a very simple virus capsid can be a uses. marginally cooperative or non-cooperative process in which a series of intermediates In principle, assembly/disassembly of a gradually appear and disappear in virus capsid can be considered a reversible succession as a consequence of a gradual, reaction, although it can later become loss (disassembly) or accretion (assembly) irreversible through a maturation process. of subunits. These results tend to confirm Thus, the study of either capsid assembly the predictions of theoretical calculations or disassembly under appropriate and simulations on assembly/disassembly conditions can provide the same of the protein homo-oligomers that form information on this reversible process. the smallest virus capsids. Theoretical studies on simplified virus models are delivering important predictions on (dis)assembly pathways. Unfortunately, the experimental study of the (dis)assembly of simple viral capsids has been severely hampered because they have been usually observed as two-state

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*Flash presentations in agreement with mammalian miRNAs (PO 21) average length. Most of the small RNA RELEVANCE OF miRNAs IN SARS-CoV- sequences differentially expressed during PATHOGENESIS SARS-CoV infection were potential unknown cellular miRNAs, whereas few L. MORALES1, J.C. OLIVEROS2, L. 1 1 small RNA sequences corresponded to ENJUANES , I. SOLA annotated miRNAs. The discrete changes, 1 Department of Molecular and Cell Biology.National around twofold, in miRNAs expression Center of Biotechnology (CNB-CSIC). Madrid. Spain. 2 induced by viral infection complicated Computational Genomics Service.National Center further validation studies. In addition to of Biotechnology (CNB-CSIC). Madrid. Spain. cell miRNAs, other small RNA sequences

specifically aligning to SARS-CoV genome Severe acute respiratory syndrome were discovered in infected tissues. Some coronavirus (SARS-CoV) causes a of these sequences might comprise viral respiratory pathology characterized by an degradation products, which are equally exacerbated inflammatory response. distributed across the viral genome. In microRNAs (miRNAs) are small noncoding contrast, a significant proportion of reads RNAs implicated in the regulation of concentrated in peaks that aligned to several cell processes, including the specific viral genome regions, representing modulation of the inflammatory response, viral-derived small RNAs (svRNAs). by silencing associated mRNA targets. Interestingly, several of these vsRNAs were Recently, a novel role of miRNAs as detected in the serum of infected mice, regulators of the host response to virus confirming their abundance in infected infection has been proposed. The tissues. The functional relevance of these relevance of miRNAs as mediators of the svRNAs in the host response to SARS-CoV host innate immune response against infection or in the viral cycle will be SARS-CoV infection is being analyzed. We validated by using inhibitors that interfere have previously shown that the mouse- with the biological effect of svRNAs. A adapted SARS-CoV lacking the envelope E deeper knowledge of miRNAs and svRNAs protein (SARS-CoV-MA15-∆E) is attenuated involved in SARS-CoV pathogenesis will and causes a milder lung inflammation and allow the development of new therapeutic pathogenicity in infected Balb/c mice as approaches to prevent mortality caused by compared to wild type SARS-CoV. miRNAs SARS-CoV. differentially expressed in lung tissues from mice infected with wt SARS-CoV and SARS-CoV-∆E have been analyzed by next generation sequencing (NGS). These miRNAs may regulate cell pathways related with the differential inflammation induced in infections by both viruses.

Bioinformatics analysis revealed that most small RNA sequences were 22 nts in length

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*Flash presentations this work, we have used the acylation (PO 22) electrophile, 2-methyl nicotinic STRUCTURAL STUDY OF FOOT-AND- acid imidazolide (NAI) to modify RNA MOUTH DISEASE VIRUS within cells and thus, to report the IRES REGULATORY ELEMENTS IN LIVING CELLS and 3’UTR RNA structure in the viral RNA context. R. DÍAZ-TOLEDANO, G. LOZANO, E. MARTÍNEZ-SALAS. Our in vivo results show differences compared with previous in vitro results. Departamento de Dinámica y Función del Genoma. Centro de Biología Molecular “Severo Ochoa” The IRES pattern observed in vivo showed (CISC/UAM), Madrid, Spain. an increased reactivity in the pyrimidine tract of domain 5 and a decreased reactivity on residues 142-147, 165-168 The genome of foot-and-mouth disease and the C-rich loop of domain 3 compared virus (FMDV) consists of a positive-sense to data observed in vitro. Furthermore, a RNA of about 8500 nts. It is organized in a marked decrease of reactivity in the apical single open reading frame flanked by loop of domain 2, the GNRA motif in the highly structured 5’ and 3’ untranslated domain 3, and the pyrimidine tract of regions (UTRs). Initiation of translation of domain 5 were apparent in the presence of the viral RNA is controlled by an internal the 3’UTR. ribosome entry site (IRES) located within the 5’UTR. In addition, the 3’UTR is RNA structure in vivo is dynamic; the essential for viral replication and directionality and velocity of transcription infectivity. Viruses are obligatory and translation, as well as trans-acting intracellular parasites. Host cell and viral factors such as proteins, small ligands or proteins cooperate with viral RNA other RNA molecules are likely to influence elements to allow virus multiplication. the RNA architecture. Knowledge of the Currently, knowledge of RNA structure in viral RNA structure in living cells would vivo is limited due to the shed light on the molecular mechanism governing virus infections. lack of suitable methodologies.

The 2’-hydroxyl group is a universal chemical feature of RNA. Selective 2´- Hydroxyl Acylation analyzed by Primer Extension (SHAPE) has become the gold standard for monitoring the secondary structure of complex RNAs. SHAPE reactivity reveals flexible regions or nucleotides constrained in a conformation where the ribose 2´-OH is susceptible to modification. Conversely, nucleotides involved in canonical base pairing or stable U:G, A:A, and A:G pairs are not reactive. In

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*Flash presentations Among the factors involved in post- (PO 23) transcriptional regulation, the poly(rC)- IN VITRO SELECTION AND binding proteins (PCBPs) are of interest CHARACTERIZATION OF RNA AND DNA due to their role in translation initiation of APTAMERS TARGETING THE RNA-BINDING some viral genomic RNAs. In particular, PROTEIN PCBP-2 translation initiation of picornavirus 1 1 genomes requires the assembly of M. MORENO , M. FERNÁNDEZ-ALGAR , J. ribonucleoprotein complexes involving FERNÁNDEZ-CHAMORRO2, J. RAMAJO2, E. 2 1,3 host cell factors, including PCBPs, on a MARTÍNEZ-SALAS , C. BRIONES . highly structured RNA functional 1 . Laboratory of Molecular Evolution. Centro de elementtermed internal ribosome entry Astrobiología (CSIC-INTA), Torrejón de Ardoz, Madrid, Spain. site (IRES), located at the 5’ untranslated 2 region (5’UTR) of the viral genome (4). . Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Madrid, Spain. We have selected and characterized in 3. Centro de Investigación Biomédica en Red de parallel ssRNA and ssDNA aptamers against Enfermedades Hepáticas y Digestivas (CIBERehd), PCBP-2. In both cases, molecular cloning of Spain. the 76 nt long evolving population after 10 rounds of in vitro selection allowed us to Aptamers are single-stranded identify the fittest aptamers according to oligonucleotides (ssRNA or ssDNA) selected quantitative amplification of the bound from combinatorial libraries by an aptamer, as revealed by the analysis of amplification-selection in vitro process their affinity constant (Kd) and maximum termed SELEX. They possess a specific binding capacity (Bmax). Additionally, gel- three-dimensional structure depending on shift analyses have confirmed that the their sequence and the physicochemical assayed aptamers bind to the target PCBP- features of the folding buffer (1). These in 2 in solution. Herein, we present sequence vitro selected nucleic acids are able to and structure comparison of the in vitro recognize and, eventually, alter the activity selected nucleic acid molecules, and of their target molecules by establishing discuss on the differential behaviour of non-covalent aptamer-target molecular RNA and DNA aptamers targeting the RNA- interactions. The current technology allows binding protein PCBP-2. generating aptamers with very high affinity and specificity for a broad range of targets 1. Ellington and Szostak (1990). Nature 346, 818. including low molecular weight 2. Cho et al. (2009). Ann. Rev. Anal. Chem. 2, 241. compounds, proteins, nucleic acids and 3. Keefe et al. (2010) Nat. Rev. Drug. Discov.9, macromolecular complexes. Therefore, 537. SELEX technology is increasingly been used 4. Martinez-Salas et al. (2015). Virus Research, in in a growing number of diagnostic (2) and press. therapeutic (3) applications over the last decade.

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POSTERS SESSION CISA, Valdeolmos (Madrid), in charge of (PO 24) the animal virology workpackage); and MEDILABSECURE: IMPLEMENTING A Italy (Istituto Superiore di Sanitá, Rome, in LABORATORY NETWORK FOR VECTOR- charge of the epidemiology workpackage). BORNE DISEASES BRINGING TOGETHER The MediLabSecure project is reinforcing ANIMAL VIROLOGY, HUMAN VIROLOGY the public health laboratory and AND ENTOMOLOGY IN THE epidemiology networks previously MEDITERRANEAN AND BLACK SEA established by the EpiSouth Plus project REGIONS THROUGH A ONE HEALTH (2010-2013) by additionally involving animal virology and medical entomology APPROACH laboratories in a fully integrated “one MIGUEL ÁNGEL JIMÉNEZ-CLAVERO1, ELISA 1 health” approach for surveillance and PÉREZ-RAMÍREZ , JEAN-CLAUDE control of emerging arboviral diseases. MANUGUERRA2, CAMILLE ESCADAFAL2, VINCENT ROBERT3, MARIE PICARD3, MARIA One laboratory per field of study (human GRAZIA DENTE4, SILVIA DECLICH4, FLAVIA virology, animal virology, medical RICCARDO4, FANNY CHERBLANC2, entomology) and per country was selected KATHLEEN VICTOIR2 in 2014. A first meeting involving the heads of laboratories was held in Paris in January 1 Center for Research in Animal Health INIA-CISA, Valdeolmos (Madrid), Spain 2015 with the aim of defining priorities and 2 Institut Pasteur, Paris, France adapting upcoming activities of the project 3 to the needs and interests of participating Institut de Recherche pour le Développement IRD, Montpellier, France countries. A “Needs assessment” 4 Istituto Superiore di Sanitá (ISS), Rome, Italy questionnaire was implemented to assess laboratory capacities and needs regarding

biosafety, diagnostic methods and As (re-)emerging viruses are threatening integration of laboratory and global health, the EU-funded epidemiological surveillance for emerging MediLabSecure project (2014-2017) aims vector-borne viruses. at enhancing the preparedness and Forty-seven laboratories were selected to response to viral threats by establishing an actively join the project. The January integrated network of animal virology, meeting allowed the project partners and human virology and entomology head of laboratories to meet and exchange laboratories in 19 non-EU countries of the on the objectives and future steps of the Mediterranean and Black Sea areas in project as well as on their experiences, partnership with 4 Institutes in three needs and expectations. Based on these countries: France (Institut Pasteur, Paris in discussions and on the responses to the charge of the coordination of the project “Needs assessment” questionnaire, the and the human virology workpackage, and first tailored training sessions will be Institut de la Recherche pour le organized in June 2015, enabling Developpement, Montpellier, in charge of laboratories to implement harmonized and the entomology workpackage); Spain up-to-date techniques to perform (1) (Center for Research in Animal Health INIA- laboratory diagnosis of relevant vector-

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borne viral diseases such as West Nile, Madeira archipelago. Both, 2010 and 2012 Chikungunya and Rift Valley Fever in outbreaks were caused by DENV-1. In humans and animals and (2) mosquito Europe, travelers can act as vectors to species determination and entomological introduce DENV, to uninfected areas or field surveys. region, as occurred previously. The present By enhancing diagnostic capacities and study is aimed to characterize DENV regional multidisciplinary cooperation, the serotypes and genotypes obtained from Medilabsecure network could represent infected travelers returning from different the cornerstone of a corporate continents with acute dengue infections. preparedness and response to vector- Samples were obtained from from 11 borne viral threats in the Mediterranean European clinics belonging from Tropnet and Black Sea regions based on a One network and participating in the Health approach. DengueTools ), from 2011 project(www.denguetools.net to 2014. Sequences of the Envelope gene (PO 25) were used for sero and genotyping of 120 GENOMIC FEATURES OF DENV IMPORTED viraemic samples. In addition, complete TO EUROPE genome sequences were obtained from A ROJAS1,2, F MOLERO1 , MD FERNÁNDEZ relevant lineages and new emerging 1,3 , L HERNÁNDEZ1, A POTENTE1, ANDREAS clades, including all DENV-1 introduced in NEUMAYR4, A WILDER SMITH5, C HATZ4, Europe in recent years. A genomic analysis ATENORIO1 ,MP SÁNCHEZ –SECO1, ANA from all studied samples will be presented VÁZQUEZ1, L FRANCO1 and discussed. 1-Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid-Spain (PO 26) 2-Master en Virología, Universidad Complutense de Madrid, Madrid-Spain NEW TOMOGRAPHIC APPROACHES TO 3-Pastuer Institute,Dakar-Senegal VISUALIZE THE STRUCTURE OF VIRUSES 4- Swiss Tropical and Public Health Institute, Basel,- AND THEIR INTERACTION WITH HOSTS Switzerland F.J. CHICHÓN1,2, G.N. CONDEZO1,2, 5-Umea University,Umea-Sweden J.J.CONESA1,2, C. SAN MARTIN1,2, E. PEREIRO3 AND J.L. CARRASCOSA1,2,4 1 Dengue is caused by 4 different related Department of Macromolecular Structures, Centro viruses, (DENV-1 ,2, 3, 4) transmitted to Nacional de Biotecnología (CNB-CSIC), 28049 Madrid, Spain humans through the bites of Aedes 2NanoBiomedicine Initiative, Centro Nacional de mosquitoes. The disease is endemic in Biotecnología (CNB-CSIC), 28049 Madrid, Spain more than 100 countries from Asia, 3ALBA Synchrotron Light Source, MISTRAL Beamline America, Africa and Oceania. In 2010, – Experiments Division, 08290 Cerdanyola del dengue re-emerged in the French Riviera Vallès, Barcelona, Spain and Croatia, with small outbreaks. Two 4Instituto Madrileñoo de Estudios Avanzados en years later, in October 2012, a sustained Nanociencia (IMDEA Nanociencia), 28049, Madrid, and explosive epidemic appeared in Spain

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In recent years, the use of electron and generate quantitative models. In this work Soft X-ray tomography are providing we show several examples of the use of virologist with very valuable data about the these techniques applied to the field of structure of viruses as well as its Virology. interaction with the host. Both electron 1. Electron tomography (ET): and X-ray tomography are based on the a. Immature Vaccinia Virus Structure. (Chichón acquisition of projections at different et al. 2009). angles. Then the projections are aligned b. Native Structure of influenza virus and combined to obtain a three- nucleoprotein. (Arranz et al. 2012) dimensional map, which must be c. Organization of the adenovirus mini- subsequently analyzed to extract biological chromosome. (Pérez-Berná et al. 2015) information in a process called 2. Soft X-ray Tomography (SXT) (Pereiro and segmentation. Both electron and soft X-ray Chichón 2014): tomography data collection can be carried a. ET vs.SXT (Carrascosa et al. 2009) out in cryo-conditions. These require a b. SXT of the infected cells (Chichon et al. 2012) complex system to maintain the sample at Arranz, R., R. Coloma, F. J. Chichón, J. J. Conesa, J. L. Carrascosa, J. M. Valpuesta, J. Ortín and J. Martín- low temperatures along the acquisition Benito (2012). "The structure of native influenza process once vitrified, but produce unique virion ribonucleoproteins."Science 338(6114): structural details in a hydrated 1634-1637. environment, avoiding the addition of Carrascosa, J. L., F. J. Chichon, E. Pereiro, M. J. contrast and fixing agents. Rodriguez, J. J. Fernandez, M. Esteban, S. Heim, P. Guttmann and G. Schneider (2009). "Cryo-X-ray Due the poor penetration of electrons into tomography of vaccinia virus membranes and inner the biological matter, limited to half a compartments."J Struct Biol168(2): 234-239. micron, the use of electron microscopy Chichon, F. J., M. J. Rodriguez, E. Pereiro, M. requires the generation of sections to Chiappi, B. Perdiguero, P. Guttmann, S. Werner, S. analyze viruses interacting with their hosts. Rehbein, G. Schneider, M. Esteban and J. L. Soft X-ray microscopy offers a new Carrascosa (2012). "Cryo X-ray nano-tomography of vaccinia virus infected cells." J Struct Biol177(2): valuable tool to study in three dimensions 202-211. viruses and cells in their hydrated state, Chichón, F. J., M. J. Rodríguez, C. Risco, A. Fraile- without sectioning or staining the sample. Ramos, J. J. Fernández, M. Esteban and J. L. Moreover, this technique presents an Carrascosa (2009). "Membrane remodelling during intermediate resolution and magnification vaccinia virus morphogenesis."Biol Cell101(7): 401- ranges between electron and confocal 414. microscopy, opening new correlative Pereiro, E. and F. J. Chichón (2014)."Cryo-Soft X-ray approaches to link functional and Tomography of the Cell." eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net. structural information in the same sample. Pérez-Berná, A. J., S. Marion, F. J. Chichón, J. J. In summary, these new techniques allow Fernandez, D. C. Winkler, J. L. Carrascosa, A. C. to obtain three-dimensional information Steven, A. Šiber and C. San Martin (2015). on the architecture of viruses or cells in a "Distribution of DNA-condensing protein complexes close-to-native state. It is therefore a great in the adenovirus core."Nucleic Acids Res"in press". advance, which allows not only to develop descriptive microscopy, but also to

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(PO 27) Nested-PCR generic for Flavivirus to FLAVIVIRUS DETECTION IN INSECT FROM amplify a fragment of 143 bp was used for THE BALEARIC ISLANDS, SPAIN, 2012. detection of the virus. Positive samples F. DEL VENTURA – VILLARROEL*1, S. were confirmed using a PCR that amplifies GUIMARÃES*1, D. ROIZ2, L. HERRERO1, E. a fragment of 1,010 bp of viral polymerase GÓMEZ-DIAZ3, A. POTENTE1, R. SORIGUER2, gene, useful to carry out studies of M.P. SÁNCHEZ-SECO1, J. FIGUEROLA2, Y A. phylogeny. Phylogenetic analysis VÁZQUEZ1. performed including sequences of different flavivirus demonstrated that the sequences 1. Arbovirus Laboratory and Imported Viral Diseases , Instituto de Salud Carlos III . Madrid, Spain . detected in this work in the mosquitoes Oc 2 .Wetland Ecology Department, Doñana Biological caspius and Oc mariae, from the Ibiza and Station, CSIC, Sevilla, Spain Cabrera islands sequences were grouped in 3. Emory University, Atlanta, USA. the insect specific flavivirus cluster, related to Ochlerotatus insect flavivirus (OcFV) previously described in Oc. caspius The Flavivirus (Flaviviridae) are viruses that mosquitoes in Spain. cause diseases in animals and humans, such as the yellow fever virus, Dengue, West Nile, Usutu and Bagaza, among (PO 28) others. They are usually transmitted by CHARACTERIZATION OF USUTU VIRUS arthropods such as mosquitoes and ticks. DETECTED IN MOSQUITOES IN SPAIN AND In this study, the molecular identification ITALY of Flavivirus was performed in a total of I. BRIOSO1, L. HERREROL1, M. GRISENTI2, J. 274 insect pools, belonging to the MORENO3, S. RUÍZ3, J. FIGUEROLA4, R. Psychodidae (Phelbotomus perniciosus, SORIGUER4, A. RIZZOLI2 M.P. SÁNCHEZ- Sergentomyia minuta) and Culicidae SECO1, A. VÁZQUEZ1. Families (Culex pipiens, Cx. modestus, Cx. 1. Laboratory of Arboviruses and Viral Imported laticintus, Cx. perexigu, Culiseta anulata, Diseases, Institute of Health ‘Carlos III’, Cs. longiareolata, Aedes (Ochlerotatus) Ctra Pozuelo-Majadahonda, Km 2, 28220 caspius, Ae. (Och) mariae and Ae (Och) Majadahonda, Madrid, Spain detritus) captured in the Balearic Islands in 2. Department of Biodiversity and Molecular 2012. The studies about arboviruses Ecology, Research and Innovation Centre, circulation in our country have been Fondazione Edmund Mach, Via E. Mach 1, 38010 carried out primarily in the Iberian San Michele all’Adige, Trento, Italy peninsula, therefore the aim of this work 3. Mosquito Control Service of Huelva (Andaluci´a), was to determine which arboviruses are Huelva, Spain. circulating in vectors from island 4. Wetland Ecology Department, Doñana Biological Station, CSIC, Sevilla, Spain territories, and then to analyze their importance and develop more direct and specific investigations. The viral RNA was Usutu virus (USUV) is a mosquito-borne extracted using the kit QIAamp Viral RNA virus that belongs to the family extraction (Qiagen, Izasa, Spain). A RT- Flaviviridae, genus Flavivirus, Japanese

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encephalitis serocomplex. The natural molecular and phylogenetic cycle involves birds as the main amplifying characterization of them. For this cause, hosts and mosquitoes (particularly of the there have been designed 17 pairs of genus Culex) as vectors. It emerged in overlapping primers, representing the Europe for the first time in Austria in 2001, complete viral genome. The obtained causing a high mortality rate in wild birds. results of the analysis of these sequences The factors that determine the severe will be discussed in this work. symptoms caused by this strain of USUV are still under discussion. Since its (PO 29) emergence in Europe, it has continued to expand, causing cases in humans as well as CONTRIBUTIONS OF TWO RT-qPCR in birds, in several European countries. In TECHNIQUES IN THE CONFIRMATION OF the summer of 2009 it was detected the DEATHS ASSOCIATED WITH THE first human case in Italy, associated with INFECTION BY DENGUE FEVER VIRUS IN neurological disease, and in 2012 in VENEZUELA (2005-2010) USING Croatia. In addition, there have been FORMALIN-FIXED AND PARAFFIN- detected antibodies in blood donors in EMBEDDED TISSUES FROM AUTOPSIES 1,2 1,3 Germany and Italy. This is the reason why M. ROSSI S. , N. MAYO , A. 2 1 until its expansion in Europe, USUV was HERNÁNDEZ , A. POTENTE VILLAFAÑE , F. 1 2 1 not considered as a potential pathogen for MOLERO , G. CÉSPEDES , A. NEGREDO , A. 1 1 human, for the reason that it has never VÁSQUEZ , M. P. SÁNCHEZ-SECO , A. 1 1 been associated to severe illness. TENORIO , L. FRANCO 1 In Spain it was firstly detected in 2006 and Laboratorio de Arbovirus y Enfermedades Víricas Importadas, Centro Nacional de Microbiología, 2009 in Culex mosquitoes from Catalonia Instituto de Salud Carlos III, Madrid, España. and Andalusia, not having been reported 2 Sección de Investigaciones en Patología human cases. As well, during 2012 virus Ultraestructural y Biología Molecular, Instituto genome has been detected on samples of Anatomopatológico José A. O´Daly, Facultad de death birds in Andalusia. The phylogenetic Medicina, Caracas, Venezuela. analysis of a partial coding section of the 3Maestría en Virología, Universidad Complutense de NS5 protein gene region indicated that Madrid, Madrid, España. USUV strains circulating in Europe come from three different clusters, African Dengue fever is the arbovirosis more strains, Spanish strains (from mosquitoes) widespread globally and responsible for and central Europe ones (including the the outbreaks and epidemics that may strain isolated from Spanish birds). affect more than 2.500 million people in Therefore, different strains are circulating urban and rural communities from tropical in Europe, with differences on its and subtropical countries. The disease is pathogenicity. caused by dengue viruses (DENV-1, 2, 3 y The objective that we set here has been to 4). In America the Infection has been amplify the complete genome of two virus estimated in 400 million of cases each strains detected on Spanish and Central year. Between 2001 and 2012, the Europe mosquitoes as well as performing a Venezuelan government agencies 659.728

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cases of DENV infection, 47.718 of which and specific tools in anatomic pathology, were haemorrhagic with a total of 32 as well as in relation with the casuistry deaths from the year 2001 to 2005. Most officially communicated and the of the fatal cases occurred in communities epidemiologic features of dengue from rural, marginal or border areas outbreaks. characterized by poor socio-sanitary conditions and with difficulties to access to (PO 30) the national health system. These conditions not only hinder the early ECOLOGICAL CONNECTIVITY SHAPES diagnosis and treatment but also QUASISPECIES STRUCTURE OF RNA conditioned the confirmation of deaths at VIRUSES IN AN ANTARCTIC LAKE anatomic pathology level. The aim of this A.LÓPEZ-BUENO, A. RASTROJO, R. PEIRÓ,

work was to evaluate the applicability of M. ARENAS & A. ALCAMÍ two RT-qPCR techniques for the Centro de Biología Molecular “Severo Ochoa” confirmation of deaths caused by DENV, as (Consejo Superior de Investigaciones Científicas- well as its serotyping, using for these Universidad Autónoma de Madrid), Madrid, Spain purposes samples of formalin-fixed and paraffin-embedded tissues. Briefly, tissue Metagenomics has revealed the enormous sections (liver, kidney, spleen and lung) diversity of viruses in nature and facilitates were recovered in vials, dewaxed the detection of minority variants. according histologic techniques, dried at Although RNA and DNA viruses appear to ambient temperature and digested with be equally abundant in aquatic Proteinase K previous to the extraction of environments, our knowledge of viral RNA the RNA´s using silica columns. The communities is scarce. RNA viruses exist as amplifications of the cDNA´s were done in complex mixtures of genotypes, known as 2 different real time platforms using quasispecies, where the evolution TaqMan probes. Of a total of 87 deaths potential resides in the whole community with presumptive pathological diagnosis of of related genotypes. Quasispecies dengue, 27 (31%) were positive for DENV structure and dynamics have been studied with an average Ct of 36.36 (CI95%: 35.85, in detail for virus infecting animals and 36.88). Positive samples showed a minimal plants but remain unexplored for those viral load between 20-200 copies/μl. Eighty infecting microorganisms in environmental one percent (22/27) of the positive samples. samples were from liver and 18.52% (5/27) Here we report a metagenomic study of from kidney. Serotyping was possible in RNA viruses in an Antarctic lake (Lake the 96.29% of positive cases, The results Limnopolar, Livingston Island). Similar to demonstrate at molecular level that the low latitude aquatic environments, this majority of deaths were due to DENV-2 lake harbours an RNA virome dominated (88.89%), one case due to DENV-1 and one by positive single strand RNA viruses from mixed infection with DENV-2 and DENV-3. the order likely infecting Results are discussed in the context of the microorganisms. Antarctic Picorna-like importance of both RT-qPCR as sensible virus 1 (APLV1), an abundant virus in the

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lake from 2006 to 2010, does not fix any and meningoencephalitis, for which no change in the consensus sequence and vaccines or antivirals for human use are shows stable quasispecies with low available. The replication of WNV takes complexity indexes. By contrast, APLV2-3 place on virus-modified membranes from are detected in the lake water exclusively the endoplasmic reticulum of the host cell in summer samples, and are major and virions acquire their envelope by constituents of surrounding cyanobacterial budding into this organelle. Consistent mats. Their quasispecies exhibit low with this view, the cellular biology of this complexity in cyanobacterial mat, but their pathogen is intimately ligated to runoff-mediated transfer to the lake modifications of the intracellular results in a remarkable increase of membranes, and the requirement of complexity that may reflect the specific lipids, as cholesterol and fatty convergence of different viral quasispecies acids, has been documented. In this study, from the catchment area or replication in a we evaluated the impact of WNV infection more diverse host community. This is the on two important components of cellular first example of viral quasispecies from membranes, glycerophospholipids and natural aquatic ecosystems and points to sphingolipids, by mass spectrometry of ecological connectivity as a modulating infected cells. A significant increase in the factor of quasispecies complexity. content of several glycerophospholipids (phosphatidylcholine, plasmalogens and lysophospholipids) and sphingolipids (PO 31) (ceramide, dihidroceramide and IMPLICATIONS OF THE CELULAR LIPIDIC sphingomyelin) was noticed in WNV- METABOLISM IN WEST NILE VIRUS infected cells, suggesting functional roles INFECTION AND CHARACTERIZATION OF of these lipids during WNV infection. THE LIPIDIC Furthermore, the analysis of the lipid COMPOSITION envelope of WNV virions and recombinant 1 1 T. MERINO-RAMOS , A.B. BLÁZQUEZ , J. virus-like particles revealed a unique 2 1 CASAS , E. ESCRIBANO-ROMERO , F. composition of their envelopes that were 3 1 SOBRINO , J.C. SAIZ , M.A. MARTÍN- enriched in sphingolipids (sphingomyelin) 1,3 ACEBES and showed reduced levels of 1Departamento de Biotecnología. INIA, Madrid. phosphatidylcholine, in a manner similar to 2Departamento de Química Biomedicinal, Instituto that of sphingolipid enriched lipid de Química Avanzada de Cataluña (IQAC-CSIC), microdomains. Inhibition of neutral Barcelona, Spain 3 sphingomyelinase (which catalyzes the Departamento de Virología y Microbiología. Centro hydrolysis of sphingomyelin into de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain. ceramide), either by pharmacological approaches or siRNA mediated silencing,

reduced the release of flavivirus virions as West Nile virus (WNV) is an emerging well as virus-like particles, suggesting a zoonotic mosquito-borne flavivirus role of sphingomyelin to ceramide responsible for outbreaks of febrile illness conversion in flavivirus budding and

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confirming the importance of sphingolipids economicconsequences. TYLCD is in the biogenesis of WNV. These results frequently controlled in commercial has allowed the identification of the tomato productionusing the Ty-1 sphingolipids metabolism as a new resistance gene. Over a 45 day period we therapeutic target for the development of studied theevolutionof infectious clones antivirals against WNV and other related from three TYLCD-associated flaviviruses. begomoviruses: Tomatoyellow leaf curl Sardinia virus, Tomato yellow leaf curl virus (PO 32) and therecombinant Tomato yellow leaf curl Malaga virus. The evolution of BEGOMOVIRUS QUASISPECIES ADAPT TO virusquasispecies was examined in HOSTS BYEXPLORING DIFFERENT susceptible tomato (ty1/ty1), resistant SEQUENCE SPACE WITHOUTCHANGING tomato(Ty1/ty1), common bean, and the THEIR CONSENSUS SEQUENCE wild reservoir Solanum nigrum. We found 1,2 S. SÁNCHEZ-CAMPOS , G. DOMÍNGUEZ- thatin addition to affecting viral 1,2 1 HUERTA , D.M. TOMÁS , J. NAVAS- accumulation kinetics, the host influenced 1 1 CASTILLO , E. MORIONES , A GRANDE- thesequence space explored by the 2 PÉREZ begomovirus quasispecies. In tomato, 1. Instituto de Hortofruticultura Subtropical y viraldynamics were not influenced by the Mediterránea "La Mayora", Universidad de Málaga- presence of the Ty-1 gene. Consejo Superior de Investigaciones Científicas Interestingly,positive adaptation of the (IHSM, UMA-CSIC), Estación Experimental "La Mayora", 29750 Algarrobo-Costa, Málaga, Spain. coat protein gene observed in the common 2. Instituto de Hortofruticultura Subtropical y bean andS. nigrum correlates with these Mediterránea “La Mayora”, Universidad de plants having viral quasispecies with the Málaga-Consejo Superior de Investigaciones highestdegree of complexity and Científicas (IHSM, UMA-CSIC), Área de Genética, heterogeneity. Our results underline the Campus de Teatinos, 29071 Málaga, Spain. importance ofthe mutant spectra of begomovirus infections, especially in wild Geminiviruses possess single-stranded reservoirs,which have the potential to give circular DNA genomes that depend rise to large numbers of emergent variants oncellular polymerases for replication in inspite of the invariance of their consensus host nuclei. In plant hosts, sequences. geminiviruspopulations behave as ensembles of mutant and recombinant genomes. Thisfavours the emergence of new geminiviruses able to cause new diseases orovercome the genetic resistance of cultivars. In warm and temperate areasseveral whitefly- transmitted geminiviruses of the genus

Begomovirus cause thetomato yellow leaf curl disease (TYLCD) with important

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(PO 33) challenge with either homologous (lineage WEST NILE VIRUS RECOMBINANT 1, NY-99) or heterologous (lineage 2, SRB- SUBVIRAL PARTICLES INDUCED Novi Sad/12) WNV strains. The cross- PROTECTION AGAINST HOMOLOGOUS reactivity of the response elicited by these AND HETEROLOGOUS CHALLENGE AND RSPs against Usutu virus (USUV) –the only CROSS-REACTIVE ANTIBODIES AGAINST other mosquito-borne flavivirus circulating OTHER FLAVIVRUSES in Europe, which shares multiple ecological E. ESCRIBANO-ROMERO1, T. MERINO- and antigenic features with WNV and that RAMOS1, A.B. BLÁZQUEZ1, R. CAÑAS- has recently caused a considerable avian 2 2 1 mortality and a few neurological human ARRANZ , F. SOBRINO , J.C. SAIZ , M.A. MARTÍN-ACEBES1,2 cases in Europe- was analyzed. Mice immunization with WNV-RSPs increased 1Departamento de Biotecnología. INIA, Madrid, Spain. specific antibody titers found upon 2 Centro de Biología Molecular Severo Ochoa (CSIC- subsequent USUV infection, proving that UAM), Cantoblanco, Madrid, Spain. RSPs prime a humoral response to this related virus. These results expand the ability of RSP-based vaccines to control the West Nile virus (WNV) is a neurovirulent two main WNV lineages and to induce mosquito-borne flavivirus that infects cross-reactive humoral responses against multiple vertebrate hosts including birds, an antigenically related flavivirus and, thus, horses and humans. Phylogenetic analyses show their potential to the control of have identified 8 different lineages, being neglected flaviviruses, as USUV, which co- lineage 1 strains considered as the most circulates with WNV. neurovirulent; however, recent outbreaks have unveiled circulation of highly neurovirulent lineage 2 strains. Since co- (PO 34) expression of flavivirus prM and E GENETIC VARIABILITY OF HUMAN glycoproteins drives the assembly of RESPIRATORY SYNCYTIAL VIRUS A IN recombinant subviral particles (RSPs) that SPANISH HOSPITALISED CHILDREN: share antigenic features with virions, a EMERGENCE OF ON1 GENOTYPE mammalian cell line stably transfected A. CALDERÓN REÑÓN 1, F. POZO with a plasmid encoding the WNV prM and SÁNCHEZ1, C. CALVO REY 2, ML. GARCÍA E glycoproteins of a neurovirulent lineage 1 GARCÍA2, M. GONZÁLEZ ESGUEVILLAS1, M. strain (NY-99) was generated. WNV-RSPs MOLINERO CALAMITA1, U. PÉREZ SAUTU1, secreted to the culture medium were I. CASAS FLECHA1 characterized and their immunogenicity 1Respiratory Virus and Influenza Unit,National was evaluated. Injection of RSPs induced a Center for Microbiology, Instituto de Salud Carlos III potent humoral response against WNV in Majadahonda, 28220 Madrid, Spain. 2Pediatrics mice with production of neutralizing Department, Severo-Ochoa Hospital, 28911 Madrid, antibodies. A single inoculation of RSPs Spain. formulated with Al(OH)3 as adjuvant protected animals against a lethal

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Human respiratory syncytial virus (HRSV) is wheezing/asthma exacerbations were the most frequently identified virus in found in 9 (24.3%). Improving virological infants hospitalized with acute lower and clinical surveillance is required to respiratory infections, and it is a significant clarify genetic diversity and transmissibility pathogen among vulnerable adults. Groups of the new ON1 genotype. A and B are serologically and genetically distinguishable among isolates. (PO 35) Phylogenetic G glycoprotein studies have MOLECULAR EPIDEMIOLOGY OF MUMPS identified numerous genotypes in both A IN SPAIN 2000-2015. GENOTYPE and B antigenic groups and demonstrated CIRCULATION AND STRAIN a complex circulation pattern during the DISCRIMINATION BASED CON same epidemic annual season. The SEQUENCING HIPERVARIABLE REGIONS. emergence and dissemination worldwide 1 1,2 1,2 of human respiratory syncytial virus group A.RUEDA , J.E. ECHEVARRÍA , F. DE ORY , A. CASTELLANOS1,2, A. FERNÁNDEZ- A, named ON1, with a72-nt insertion in the 1,2 second hypervariable region of the G gene, GARCÍA allowed us to use it as a natural tag to 1. Centro Nacional de Microbiología. Instituto de Salud Carlos III. Majadahonda. Madrid. Spain. examine the evolution of HRSV-A. The circulation pattern and the genetic 2. CIBER de Epidemiología y Salud Pública (CIBERESP).Madrid. Spain. variation in the complete G protein gene of

HRSV-A virus were analyzed during four consecutive winter seasons, from 2010 to Introduction: Mumps Virus (MuV) is 2014. Out of 2546 respiratory specimens responsible of mumps, a highly contagious taken from children 542 (21%) were disease. In Spain the vaccine was positive to HRSV, and 288 (54%) grouped introduced in 1981 with the MMR as HRSV-A, whichwas predominant during (measles, mumps and rubella) vaccine. two consecutive epidemics 2011-2012 Despite the high rates of vaccination, (74%) and 2012-2013 (95%). Complete G epidemic waves and outbreaks affecting gene sequences were obtained and the vaccinated population are still observed. phylogenetic analysis was carried out on The knowledge of the circulation patterns 122 HRSV-A virus collected among our of MuV genotypes is important for pediatric population during the study epidemiological surveillance. From 2005 to period. Two different HRSV-A genotypes now the genotype G is being the most were identified, NA1 and the recently prevalent worldwide. Genetic variation discovered genotype ON1. In 2011-2012, within the fragment of the SH gene ON1 viruses emerged in Madrid recommended for genotyping by the WHO sporadically with 2 positives and become is low and there are identical sequences predominant in the 2012-2013, with a total spread all over the world along time, so of 38 positives (95%). Clinical outcome of that it is not easy to establish circulation children positives for ON1 virus was patterns and transmission chains. The established and bronchiolitis was purpose of this work is to describe the diagnosed in 25 (67.6%) and recurrent molecular epidemiology of MuV in Spain,

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including the development of new transmission chains inside each haplotype, methods to improve strain discrimination. to improve the surveillance. Methods: A total of 1679 SH sequences of the MuV were analysed, including 397 (PO 36) from Spain and 1282 from other locations EVALUATION OF ELISA FOR THE taken from GenBank database. They were DETERMINATION OF CHIKUNGUNYA aligned (Bioedit v.7.0.5) and their VIRUS-IgG AND -IgM haplotypes determined (DNAsp v.5). F. DE ORY1,2, T. MINGUITO1, P. BALFAGÓN1, Phylogenetic analysis was carried out 1,2,3 1,2,3 through maximum likelihood (RaxML v.7 L. FRANCO , M. P. SÁNCHEZ-SECO . 1 and PhyML v.3). For the RT-PCR we use Centro Nacional de Microbiología, Instituto de Salud Carlos III, 2Virored, 3Red Colaborativa en SimPlot v.3.5.1 to look for variable regions Investigación de Centros de Enfermedades in the MuV genome and PerlPrimer v1.1.21 Tropicales. to assist the primer design. Results: We found 51 different haplotypes, In December 2013 Chikungunya virus from genotypes A (4), D (9), G (30), H (5), J, (CHIKV) was introduced in the Americas, K and N. A change on the dominant first on the island of Saint Martin, with genotype from H to G was observed subsequent spreading to other Antillean between 2003 and 2005 coincident with islands and continental countries, some of the lowest incidence ever reported in our which are important touristic destinations country. All genotype G SH sequences for Spanish travelers. This epidemic implies belong to the same phylogenetic cluster. a real challenge for Spain, since a We located three variable intergenic competent vector (Aedes albopictus) is regions (VR) which are candidates for present in the Mediterranean basin. The strain discrimination: VR1 1499-2580 nt, bite of a viremic patient would allow the NVP (genes N and P/V); VR2 2727-3669 nt, indigenous circulation of the virus, as PM (genes P and M); VR3 4180-4760 nt, recently happened in neighboring MF (genes M and F). By the moment we countries (Italy, 2007, France, 2010 and have only tested the VR1 region. 2014). Bearing in mind the symptoms and Discussion: After 2005, genotype G is the epidemiology of CHIKV and dengue virus main circulating genotype all over Europe. (DENV) differential diagnosis of both It agrees with the circulating genotype in viruses is an important issue. CHIKV Spain. Our study also confirms the viremia is intense, but very short, thus existence of importations from other serological methods for diagnosis are countries like Japan or the Unites states of required. Indirect immunofluorescence America. The main haplotype of the G (IIF) techniques are hampered by the genotype has been circulating from 2005 difficulties due to the interpretation of to 2015 all over Spain causing numerous results, since nonspecific reactivities outbreaks. The RT-PCR we are optimizing caused by autoantibodies are frequent, will allow increase the phylogenetic being required a highly experienced information to distinguish variants or personnel. ELISA techniques avoid these

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problems. The aim of the present study (PO 37) was the evaluation of indirect ELISAs using MOLECULAR EPIDEMIOLOGY AND recombinant structural protein as antigen CLINICAL ASSOCIATION OF ENTEROVIRUS to determine IgM and IgG against CHIKV D68 INFECTIONS IN SPAIN (EuroImmun, Germany). 1 2 I. TARAVILLO , M. ARANZAMENDI , M.P. A total of 158 samples (from 148 cases), ROMERO3, A. MORENO-DOCÓN4, C. received in our lab in January-September, MUÑOZ-ALMAGRO5, N. RABELLA6, A. 2014, for diagnosis of CHIKV infection, OTERO1, G. TRALLERO1, M. CABRERIZO1 were included in the study. Ninety nine 1 Enterovirus Unit, CNM, Institut of Health Carlos III, cases (108 samples) were classified as Madrid, Spain.2Hospital Cruces, Bilbao, Vizcaya, caused by CHIKV, since they showed a Spain. 3Hospital La Paz, Madrid, Spain. 4Hospital Virgen de la Arrixaca, Murcia, Spain. 5Hospital San positive result by PCR (23 cases) and/or 6 CHIKV positive IgM (88 cases, 95 samples). Joan de Deu, Barcelona, Spain. Hospital Santa Creu i Sant Pau, Barcelona, Spain. Thirty cases (31 samples) were classified as DENV infections, and 19 cases (19 samples) as negative to both viruses. The cases were Introduction. Enterovirus 68 (EV-D68) is classified by the determination of CHIKV member of Enterovirus genus (species D) IgM and IgG using IIF (Euroimmun) and/or within the Picornaviridae family. EV-D68 nsP4 and E1 genes amplification. DENV was firstly isolated in 1962 from a patient IgM was determined by a capture ELISA with respiratory illness. It shares features and VD IgG by an indirect one (both from with (RV) and had been Panbio, Korea). detected only sporadically associated with As compared with IIF, ELISA IgM CHIKV mild respiratory infections. In last years, assay showed overall agreement, however, the circulation of this serotype sensitivity and specificity of 94.3%, 95.8% has increased in different parts of the and 92.1%. These figures were improved to world. Specifically, during 2014, several EV- 97.2%, 97.8% and 94.1% when only CHIKV D68 outbreaks were described in the USA infection cases were considered. For IgG and Canada, causing substantial assay, the corresponding figures were hospitalization of children with severe 89.9%, 82.6% and 98.6%. The low respiratory diseases. Fatal cases were also sensitivity of this assay may be caused by reported. In Spain there are no studies the difficulties for detecting this isotype in about the incidence and characteristics of recent infection cases, in which the EV-D68 infections. sensitivity was 83.1%. This was probably Objectives. To characterize the serotype of due to the use of structural antigen with EV detected in children with respiratory more specific reactivity than those illnesses and to study the epidemiology detected in the IIF. and clinical association of EV-D68 ELISA methods are adequate approaches infections in Spain. for the serological diagnosis of CHIKV Patients and methods. Clinical samples infections. collected between October 2014 and February 2015 from 53 hospitalized

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children with respiratory symptoms were epidemiology and pathologies associated included in the study. The mean age of the with EV-D68 infections. patients (57% male) was 1.6 years. The clinical diagnosis was pneumonia (6 cases), (PO 38) bronchiolitis (5 cases), bronchospasm/wheezing/respiratory PESTE DES PETITS RUMINANTS VIRUS: distress (11 cases) and upper tract EXPERIMENTAL INFECTION WITH A respiratory infection (31 cases). All samples VIRULENT STRAIN AND VACCINE were throat swabs and were positive for PROTECTION STUDY IN A SPANISH SHEEP EV/RV by RT-PCR previously. Samples were BREED 1 1 sent to CNM for genotyping by specific RT- C. CANO-GÓMEZ ; F. LLORENTE ; P. 1 1 PCRs of species EV-A, B, C and D which FERNÁNDEZ-PACHECO ; A. ROBLES ; A. 1 1 2 amplify 3'-VP1 region of the viral genome, VILLALBA ; M. C. BARBERO ; G. LIBEAU ; 1 sequencing and phylogenetic analysis. M. A.JIMÉNEZ-CLAVERO ; J. FERNÁNDEZ- 1 Results. EV were confirmed in 29 (55%) of PINERO 1 2 the total of 53 specimens analyzed and RV INIA-CISA, Valdeolmos, Spain; CIRAD, Montpellier, France. in 24 (45%). Of the 29 EV, 22 (78%) were genotyped being EV-D68 the most frequently detected serotype, accounting Peste des petits ruminants (PPR) is an for 18% (4/22). Other serotypes identified acute animal disease affecting small were echovirus (E) -30, coxsackievirus (CV) ruminants, included in the OIE list of -B2 and E-6 (3/21, 14%), CV-B4 and CV-A8 notifiable diseases due to its high (2/21, 9%), and E-13, E-16, E-20, CV-B5, CV- economic impact. PPR is a highly CV-A10 and A5 (1/21, 5%). The mean age contagious disease which spreads rapidly of EV-D68-infected children was 2.4 years by direct contact through (range, 2.5 months-4.8 years). Three of excretions/secretions from sick animals, them (75%) were male. Clinically, they caused by a Morbillivirus of the were diagnosed with pneumonia (1 case), Paramyxoviridae family, PPRV. The severity bronchospasm/wheezing (2 cases) and of the disease depends upon the virulence respiratory symptoms with acute liver of the strain and the susceptibility of the failure (1 case). This last child and other species/breed affected. The acute form with bronchospasm required admission to may kill up to 90% in 5-10 days upon onset PICU, but none had further complications. of clinical signs in naïve populations. A Phylogenetic analysis showed Spanish subacute, milder form is known, in which sequences belonged to the same clusters the animals usually recover within a week formed by the American and European of the onset of symptoms. strains. PPR is endemic in most of Africa, the Conclusions. This study confirms the Middle East, South Asia and China. PPR is circulation of EV-D68 in Spain associated controlled by vaccination, restriction of with mild/severe respiratory illnesses. animal movements and efficient and rapid Further surveillance studies are needed to diagnosis. Attenuated vaccines, mainly improve our knowledge about the Nigeria 75/1 strain, have been commonly

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used, inducing a reported life-long latter remained aviremic and protected protective immunity in sheep and goats from the disease. against all known PPRV lineages. Recently, ACKNOWLEDGEMENTS: Work funded by lineage IV PPRV expanded geographically, INIA project RTA2011-00072-00-00 and reaching Northern Africa, close to INIA-MAGRAMA agreement EG13-020. Southern European countries, concerned by the risk of PPR emergence in their territories. In this framework, we aimed to (PO 39) study the disease pattern produced in a PEPTIDES INTERFERING 3A PROTEIN Spanish native sheep breed DIMERIZATION DECREASE FMDV (“Colmenareña”) by a pathogenic strain of MULTIPLICATION PPRV(Mor/08, lineage IV), and to evaluate MÓNICA GONZÁLEZ-MAGALDI1, ÁNGELA the protection conferred by the VÁZQUEZ-CALVO1, BEATRIZ G. DE LA immunization of this Spanish sheep breed TORRE2, JAVIER VALLE2, KATHERINE I. with the live attenuated PPRV vaccine CALDERÓN1, DAVID ANDREU2 AND Nigeria75/1 (lineage II) against a challenge FRANCISCO SOBRINO1 with the same pathogenic Mor/08 PPRV 1. Centro de Biología Molecular Severo Ochoa (CSIC- strain. For that, a group (n=4) of sheep was UAM), Cantoblanco, 28049 Madrid, Spain vaccinated (s.c.) and challenged (i.v.) 21 2. Departament de Ciències Experimentals i de la days post-vaccination, while other (n=4) Salut, Universitat Pompeu Fabra, 13 08003 was only challenged. Naïve or vaccinated Barcelona, Spain sheep (n=2 each) were kept in contact with vaccinated/challenged animals or with just Nonstructural protein 3A is involved in challenged sheep to evaluate contact relevant functions in foot-and-mouth transmission in the presence or absence of disease virus (FMDV) replication. FMDV 3A vaccination. Clinical follow-up and can form homodimers and preservation of laboratory studies (viral load by qRT-PCR in the two hydrophobic α-helices (α1 and α2) blood, swabs, faeces, necropsy specimens, that stabilize the dimer interface is and serum antibodies by ELISA) were essential for virus replication. In this work, carried out. DIVA qRT-PCR was applied to small peptides mimicking residues involved differentiate vaccine from field PPRV in the dimer interface were used to strains. interfere with dimerization and thus gain As a result, the Spanish sheep breed insight on its biological function. The dimer “Colmenareña” inoculated with PPRV interface peptides α1, α2 and that Mor/08 pathogenic strain showed spanning the two hydrophobic α-helices, generally mild clinical signs. Vaccination α12, impaired in a dose dependent manner (Nigeria75/1) protected sheep against i.v. in vitro dimer formation of a peptide challenge with Mor/08 strain. This strain, containing the two α-helices, this effect however, was able to spread by direct being higher with peptide α12. To assess contact from excretion/secretion sites to the effect of dimer inhibition in cultured naïve and vaccinated contact sheep, the cells, the interfering peptides were N- terminally fused to a heptaarginine (R7)

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sequence to favor their intracellular which each of the protein termini was translocation. Thus, when fused to R7, fused to a glycosylation acceptor tag, as interference peptides (100 μM) were able well as by their accessibility to degradation to inhibit dimerization of transiently by proteases. According to this model 3A expressed 3A, the higher inhibitions being would interact with membranes through found with peptides α1 and α12. The 3A its central hydrophobic region exposing its dimerization impairment exerted by the N- and C- termini to the cytosol, where peptides correlated with significant, interactions between viral and cellular specific reductions in the viral yield proteins required for virus replication are recovered from peptide-treated FMDV expected to occur. This 3A topology is infected cells. In this case, α2 was the only novel among picornaviruses, highlighting peptide producing significant reductions at that in FMDV this non-structural protein concentrations lower than 100 μM. Thus, shows characteristics and functions that dimer interface peptides constitute a tool differ from those of other virus family to understand the structure-function members. relationship of this viral protein and point to 3A dimerization as a potential antiviral (PO 41) target. A similar approach is being followed to study the effect of coxsackievirus MONOCYTE PORCINE CELL LINES FOR PRODUCTIVE AFRICAN SWINE FEVER

VIRUS INFECTION (PO 40) EG. SÁNCHEZ, P. FERNÁNDEZ, ML.NOGAL, MEMBRANE TOPOLOGY OF FOOT- AND- Y.REVILLA. MOUTH DISEASE VIRUS 3A PROTEIN Virology Department, Centro de Biología Molecular MÓNICA GONZÁLEZ-MAGALDI1, MIGUEL A. “Severo Ochoa”, Madrid, Spain MARTÍN-ACEBES1, LEONOR KREMER2 AND 1 FRANCISCO SOBRINO ASFV is highly pathogenic double-stranded Centro de Biología Molecular Severo Ochoa (CSIC- 1 DNA virus with a marked tropism for cells UAM), Cantoblanco, 28049 Madrid, Spain . Centro of the monocyte-macrophage lineage. Nacional de Biotecnología (CNB-CSIC), Cantoblanco, 28049 Madrid, Spain2. Although monkey cell lines such as Vero or COS allow the adaptation of ASFV strains

after several passages, a suitable porcine Foot-and-mouth disease virus non- cell line able to efficiently support ASFV structural protein 3A plays important roles infection is necessary to develop models in virus replication, virulence and host- for cell-host interaction and vaccine range; nevertheless little is known on the studies. For this purpose, four different interactions that this protein can establish porcine cell lines from monocyte- with different cell components. Here, we macrophage origin (IPAM WT, IPAM- describe a nonintegral membrane protein CD163, CΔ2+, WSL) have been tested in topology of transiently expressed FMDV order to set up the most similar conditions 3A. This topology was supported by the to the infection in primary alveolar lack of glycosylation of versions of 3A in macrophages (PAM) in terms of

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phenotype, ASFV infection susceptibility alters the cellular secretory pathway. BFA and viral production. To achieve this, we has been shown to inhibit the RNA analyzed on these lines the presence of replication of different enteroviruses CD163 and CD169 cellular surface including SVDV. In this study we have receptors since they are linked to analyzed the effect of several GBF1 differentiation and maturation of the inhibitors on SVDV production and isolated macrophages and seem to beclosely a SVDV mutant with increased resistance related to ASFV infection. ASFV to BFA. A single amino acid substitution, susceptibility was analyzed in the infected Q65H, in the 2C protein was found to be cells by the expression of the viral late responsible for the increased BFA protein p72 and viral production by resistance. titration on plaque assays. Results showed that although all porcine cell lines analyzed (PO 43) were susceptible to ASFV infection, none of them was as efficient as PAM in terms of IDENTIFICATION OF CD8 T CELL EPITOPES virus production. Future experiments will IN VP2 AND NS1 PROTEINS OF be focus on describing which cellular BLUETONGUE AND AFRICAN HORSE factors are related with the ability of SICKNESS IN IFNAR(-/-) porcine cell lines to support an ASFV 129/SV MICE 1 productive infection in order to establish a B. FERNÁNDEZ-RETUERTO , A. MARÍN- 1 1 2 suitable model of study. LÓPEZ , F. DE LA POZA , E. CALVO-PINILLA , 1 J. ORTEGO . 1. Centro de Investigación en Sanidad Animal, INIA- (PO 42) CISA, Valdeolmos, Madrid, Spain. THE AMINO ACID SUBSTITUTION Q65H IN 2. The Pirbright Institute, Pirbright, Surrey, United 2C PROTEIN OF SVDV INCREASES Kingdom. RESISTANCE TO BREFELDIN A. A. VÁZQUEZ CALVO, F. CARIDI, F. SOBRINO, Bluetongue virus (BTV) and African horse M.A. MARTÍN ACEBES. sickness virus (AHSV) are of the Centro de Biología Molecular Severo Ochoa (UAM- family Reoviridae that cause severe disease CSIC), Madrid, Spain. in ruminants and equids, respectively. Previous work in our laboratory has shown Swine vesicular disease virus (SVDV) is a the presence of CD8+ T cells specific of BTV porcine pathogen and a member of and AHSV antigens in mice immunized with the PICORNAVIRIDAE family. It is included recombinant Modified Vaccinia Ankara into the ENTEROVIRUS genus and is closely (rMVA) expressing VP2 and NS1 proteins of related to the human pathogen both orbivirus. We also observed that the coxsackievirus B5 (CVB5). Brefeldin A induction of a strong CD8+ T cell response (BFA), an inhibitor of the cellular protein is critical to induce multiserotype GBF1 (a guanine nucleotide exchange protection. We have now selected factor for small cellular GTPases Arf), potential CD8 T cell epitopes (MHC-class I induces Golgi complex disassemblly and binding peptides) for the 129 mouse strain

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corresponding to the VP2 and NS1 proteins Gastrointestinal disease is frequent in pigs, of BTV-4 and AHSV-4, using a combination and among the different etiological agents of four epitope prediction algorithms involved viruses are considered the leading (SYFPEITHI, BYMAS, NetMHC I and cause of diarrhea in this animal species. NetMHCpan). ELISPOT and Intracellular Furthermore about half of newly identified Cytokine Staining (ICS) analysis showed swine pathogens are viruses, most of that peptides NS1 (152) (GQIVNPTFI) of which may be transmitted to humans by BTV-4 as well as peptides VP2 (1052) direct contact or by indirect transmission (YTFGNKFLL) and NS1 (92) (CVIKNADYV) of pathways. In this study, the prevalence of AHSV-4 elicited IFN-γ production in astrovirus (AstV), group A rotavirus (RVA), splenocytes of MVA-VP2 and MVA-NS1 (NoV) and hepatitis E virus (HEV) immunized mice and were identified as infections in pigs were investigated. During CD8 T cell epitopes. In addition, these 2012-2014 a total of 242 fecal samples three MHC-class I-binding peptides were collected from pigs at different induced the surface expression of CD107a production stages (from 0 to 180 day-old) in CD8+ T cells, an indirect marker of in eight swine farms located in northern, cytotoxic activity. Importantly, NS1 (152) central and southern Italy. epitope of BTV-4, and VP2 (1052) and NS1 Seven out of 8 farms analyzed were (92) epitopes of AHSV-4 are highly positives for AstV, which was detected in conserved among the 27 BTV and 9 AHSV 163/242 (67.4%) samples and represented serotypes, respectively. The the most prevalent virus; 61 animals characterization of BTV and AHSV specific (37.4%) showed diarrhea. HEV, was CD8 T-cell epitopes provides useful detected in 6 farms and in 45/242 (18.6%) information for the design of novel of the samples analyzed. Twenty-three multiserotype vaccines against these two HEV infected pigs had diarrhea (45%). A orbivirus. lower prevalence was observed for RVA, only three farms resulted positives, it was (PO 44) found in 10/242 samples (4.13%), 6 out of DETECTION AND MOLECULAR these showed diarrhea (60%). On the CHARACTERIZATION OF ZOONOTIC contrary, no swine samples were found to VIRUSES IN SWINE ISOLATED IN ITALIAN be positive for NoV. This study compares PIG HERDS for the first time in Italy the occurrence of 1 1 1 astroviruses, rotaviruses and hepatitis E in M. MARINA , I. DI BARTOLO , G. IANIRO , a same population of pigs, and reports the G. ANGELONI1, C. MAGISTRALI2, F. 3 1 molecular characterization of viral strains OSTANELLO , F. M. RUGGERI . detected. 1 Department of Veterinary Public Health & Food Safety, Viral Zoonoses Unit, Istituto Superiore di The presence of enteric viruses also in Sanità, Rome, Italy. asymptomatic swine identifies a possibly 2 Istituto Zooprofilattico Sperimentale dell’Umbria e underscored risk of virus spreading among delle Marche, Perugia, Italy animals and addresses a potential source 3 University of Bologna, Dep. of Veterinary Medical of infection for humans. Further studies Sciences, Ozzano dell’Emilia (BO), Italy are required in order to understand the

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role of these viruses in gastrointestinal out of 45 slices, 250 mg each, 22.2%) and diseases of pigs. Sequence analyses may dry (1 of 23 slices, 4.3%) liver sausages, but permit to assess the zoonotic potential of viability of the virus was not the viruses detected in animal clinical demonstrated. A phylogenetic tree was samples. drawn using both RdRp and MTase fragments. Results confirmed presence of (PO 45) genotype 3 HEV strains and a correlation between the HEV genomes detected in HEPATITIS E VIRUS IN PORK LIVER liver sausages in this study with swine and SAUSAGES SOLD IN ITALY human HEV strains reported in Europe, 1 1 I. DI BARTOLO , G. ANGELONI , E. including Italy. This pilot study fosters 1 2 PONTERIO , F. OSTANELLO AND F. M. more investigations on HEV presence in 1 RUGGERI . pork-derived food, to assess the possible 1.Department of Veterinary Public Health and Food risk for the consumers. Safety, Istituto Superiore di Sanità, Rome, Italy

2. Department of Veterinary Medical Sciences, University of Bologna, Ozzano Emilia (BO), Italy (PO 46) DEVELOPMENT OF A NOVEL LATERAL Hepatitis E is an acute human disease FLOW ASSAY FOR DETECTION OF ASFV IN caused by the hepatitis E virus (HEV). In BLOOD 1 low-income countries, the virus has been P. SASTRE ANTORANZ , T. PÉREZ 1,2 3 involved in waterborne outbreaks. ESCODA , C. GALLARDO FRONTAURA , M. 3 1 Autochthonous hepatitis E cases are ARIAS NEIRA , A. MONEDERO MARCOS , T. 1 1 increasingly reported in developed RUIZ GONZÁLEZ , P. RUEDA PÉREZ . countries, where sporadic cases and small 1. Inmunología y Genética Aplicada S. A. outbreak have been reported. The disease (INGENASA), Madrid, Spain is normally self-limiting (mortality rate 1%), 2. Current address: Urano vet, Barcelona, Spain but chronic infections have recently been 3. Centro de Investigación en Sanidad Animal, INIA, observed in transplanted patients. The Madrid, Spain etiological agent HEV is a small RNA virus infecting both humans and animals. Pigs African swine fever (ASF) is an infectious and possibly other animal species are disease of domestic and wild pigs of all reservoir for HEV, and the consumption of breeds and ages, causing a wide range of raw contaminated animal meat and meat syndromes from mild disease to lethal products has been linked to sporadic cases hemorrhagic fever. The causative agent of and small outbreaks of hepatitis E in the infection, ASF virus (ASFV), is a large, humans. In the present study, we enveloped, icosahedral double-stranded investigated the presence of HEV and fecal DNA virus that belongs to the Asfarviridae cross-contamination in both fresh and dry family. The disease is endemic in Sub- pork liver sausages in Italy bought at a Saharan Africa and Sardinia. Since 2007, grocery store in Italy. The genome of HEV several cases have been declared in was detected by qRT-PCR in both raw (10 Georgia, Armenia, Azerbaijan, and in the

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Russian Federation, where the continued the early diagnosis of ASF. It does not spread presents a serious threat to the require any kind of equipment, nor skilled swine industry worldwide. Rapid detection users to perform the test. Furthermore, of infected animals is of paramount the test has been designed to be used with importance for early detection of blood, thus making the sample processing outbreaks, reducing the transmission of quite easy and feasible even at field level. the viruses to uninfected animals and All these features make these devices very subsequently spreading of the disease. suitable for small field labs or task forces, Current diagnosis of ASF is based on direct supporting in many cases local decisions, identification of the virus by polymerase especially in countries where laboratory chain reaction (PCR) or virus isolation, and infrastructure is under development or detection of antibodies by either ELISA, even missing. immunoblotting or immunofluorescence Acknowledgements assay. However, these methods are still The research was funded by the EU, rather time consuming and require well Seventh Research Framework Program equipped laboratories and personnel, FP7-KBBE-2207-2013 under grant number delaying the disease diagnosis in remote nº 311931 (ASFORCE). areas.

Ingenasa has developed a Lateral Flow Assay (LFA) for antigen detection based on (PO 47) the use of MAbs against VP72 protein of GENERATION OF PRRS VLPS BY MULTIPLE ASFV, the major viral capsid protein and PROTEIN CO-EXPRESSION IN THE considered the most immunogenic protein BACULOVIRUS SYSTEM of the virus. First experiments using VP72 M. GARCÍA DURÁN, S. COSTA, J. recombinant protein or inactivated virus SARRASECA, N. ROJA, J. GARCIA MIGUET, from tissue culture showed promising ISABEL GARCÍA, M.J. RODRIGUEZ results with a sensitivity similar to that a Inmunologia y Genetica Aplicada, S.A., Madrid, commercially available DAS-ELISA Spain. (11.PPA.K.2, Ingenasa). Moreover, these strips were tested with blood from Porcine reproductive and respiratory experimentally infected pigs at CISA-INIA syndrome (PRRS) is one of the most Level 3 Laboratory. The animals were important diseases in pig industry, causing inoculated with different viral isolates and high economic losses worldwide. Live- blood was collected at different days post attenuated viruses are the most commonly infection (pi). The sensitivity of the test 4 used PRRSV vaccines, but they are not fully allowed detecting viral loads from 10 HAU effective. Protective immune response in corresponding with day 4-7 pi. Further PRRS is based on neutralizing antibodies validation is currently ongoing in different and cellular response but, until now, there countries in Europe, Asia and Africa. is not a clear epitope or protein considered This novel pen-side test offers a rapid, the only responsible of the protective economic and simple-to-use diagnostic mechanisms. Moreover, the results seem tool suitable or field application, allowing

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to point out that several structural proteins The system presented here has the are contributing to the global immune flexibility to easily add or remove complete response. This work aimed at the structural proteins to the basic Gp5/M construction of PRRS Virus Like Particles VLPs, providing a useful approach to study (VLPs) that incorporate Gp5, M, Gp2, Gp3, the implication of particular proteins. Gp4 and E proteins. Besides, the possibility to exchange The cDNA sequences corresponding to the individual proteins to generate PRRS VLPs six structural proteins were amplified from from different strains would be a helpful Olot virus strain and cloned into the tool for the development of specific multiple expression baculovirus vector vaccines. Nevertheless, further work is pBAC4x-1 in two separate plasmids: Gp5 needed to clarify important aspects of the and M in one plasmid (pBAC4x-1A) and VLPs generated as their immunological Gp2, Gp3, Gp4 and E in another one abilities. (pBAC4X-1B). The expression of the Acknowledgements different proteins was assessed by The research leading to these results has received Western blot. The culture supernatants funding from the European Union Seventh after single infection with pBAC4x-1A or Framework Programme (FP7/ 2007-2013) under grant agreement nº 245141 after co-infection with pBAC4x-1A plus pBAC4x-1B were semi purified with a sucrose cushion. Subsequently, were (PO 48) loaded on the top of a sucrose layer LIMITED SUSCEPTIBILITY OF MICE TO gradient. The formation of VLPs was USUTU VIRUS (USUV) INFECTION AND confirmed, in single infections and in co- INDUCTION OF FLAVIVIRUS CROSS- infections, by electron micrograph of the PROTECTIVE IMMUNITY gradient fractions corresponding to PRRSV A.B. BLÁZQUEZ1, E. ESCRIBANO-ROMERO1, density (1,15-1,16 g/cc). M.A. MARTÍN-ACEBES1,2, T. PETROVIC3, J.C. Gp5 and M proteins were easily detected SAIZ1 in the two types of VLPs with the specific 1Departamento de Biotecnología. INIA, Madrid, mAbs, showing an apparent molecular Spain. weight similar to the one observed in the 2Centro de Biología Molecular Severo Ochoa (CSIC- virus. The minor structural proteins are, in UAM), Cantoblanco, Madrid, Spain. 3 general, more difficult to detect both in Scientific Veterinary Institute “Novi Sad“, Novi Sad, the VLPs and in the virus. This fact could be Serbia reflecting the stoichiometry of the virus, whose envelope is mainly composed of Flaviviruses (Flaviviridae family) are RNA Gp5/M complexes whereas the Gp2-Gp3- viruses that constitute a worrisome threat Gp4 complexes and E protein are nestled to global human and animal health. Until (Dokland 2010). In this work, Gp3 protein recently, West Nile virus (WNV) was the had an apparent size smaller than the one only mosquito-borne flavivirus circulating in the wild type virus, which can be in Europe, being responsible of numerous indicating a lower glycosylation level. outbreaks that have dramatically increased in number and severity in recent years,

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with dozens of human and horse deaths (PO 49) and a high avian mortality across the DETECTION OF PAN/FOOT AND MOUTH continent. In 2001, another flavivirus, the DISEASE VIRUS BY A MULTI-CHECK rRT- Usutu virus (USUV), was detected for the PCR STRATEGY first time in Austria and, since then, the L RIOS1, CL. PERERA1, L CORONADO1, D virus has quickly spread across Europe, RELOVA1, AM ÁLVAREZ2, L GANGES3, H causing a considerable number of bird DÍAZ DE ARCE4, LJ. PÉREZ1, JI. NÚÑEZ3. deaths and neurological disorders in a few 1. Centro Nacional de Sanidad Agropecuaria patients. Even though USUV infects (CENSA), La Habana, Cuba. multiple avian species, there is little 2 Instituto Nacional de Investigaciones Agricolas information about USUV susceptibility, (INIA), Maracay, Venezuela. pathogenicity and cross-reactive immunity. 3. Centre de Recerca en Sanitat Animal (CReSA), In the present report, the susceptibility of UAB-IRTA, Campus de la Universitat Autònoma de suckling and adult mice to USUV infection Barcelona, Bellaterra, Cerdanyola del Vallès, Spain. and the induction of cross-protective 4 Hospital italiano de Buenos Aires, Buenos Aires, immunity against WNV challenge was Argentina addressed. All adult mice infected with 2 4 either 10 or 10 pfu/mice of USUV The objective of this study was to develop survived to the infection, while only 16.6% and validate a multi-check strategy using and 8.3%, respectively, of those infected rRT-PCR (multi-rRT-PCR) based on SYBR- with similar doses of WNV did it. On the Green I for pan/foot and mouth disease other hand, in suckling mice survival rates virus (pan/FMDV) diagnosis. Based on the against USUV infection were dose in silico analyses, different primer pairs dependent (84.2% and 40%, respectively), were selected and addressed in order to but also higher than that recorded (18%) reduce the probability of viral escape and 4 after WNV (10 pfu/mice) infection. Except possible failures in the pan/FMDV 6 adult mice infected with the lower USUV detection due to the high variability of the 2 dose (10 ), all the remaining surviving virus. The analytical parameters were animals either adults or suckling resulted assessed on a large representative number protected against challenge with a high of viral strains. The repeatability of the test dose of WNV. No USUV-RNA could be and its performance on field samples were detected in any of the adult mice analysed also evaluated. The multi-rRT-PCR was able between 4 and 35 days post-infection to detect novel emergent strains of FMDV (d.p.i.). In contrast, USUV-RNA was which had circulated in South America amplified form suckling mice 7 d.p.i., but during the period 2006-2010 and on which not early (4 d.p.i.) or later (15 d.p.i.). These the individual assays failed when they were findings demonstrate that mice applied independently. We demonstrate susceptibility to USUV infection is age that the system proposed is a reliable and dependent and that the elicited antibodies rapid diagnosis method for sensitive and are cross-reactive and protective against specific detection of FMDV. Therefore, a other flaviviruses infection, such as that of validated multi-rRT-PCR assay based on WNV. SYBR Green I detection coupled to melting

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curves analysis for pan/FMDV diagnosis on antigenic properties. Finally, screening of clinical samples is proposed. This work also sera obtained from a wild rabbit highlights the need to incorporate the population suggests an elevated multi-target detection principle in the prevalence of the novel RHDV2, circulating diagnosis of viral pathogens with highly among apparently healthy individuals. variable genomes as FMDV. The VLPs from RHDV2 provide important tools to monitor circulation of the novel (PO 50) virus variant and to discriminate between RHDV and RHDV2 infection. GENERATION OF VIRUS-LIKE PARTICLES (VLPs) FROM THE NEW VARIANT RABBIT HAEMORRHAGIC DISEASE VIRUS (RHDV2) (PO 51) AND ITS APPLICATION FOR SEROLOGICAL COMPLETE GENOME SEQUENCE OF A STUDIES GENOTYPE 3 HEPATITIS E STRAIN J. BARCENA1, Y. GONZÁLEZ, B., GUERRA1, IDENTIFIED IN A SWINE FARM IN ITALY 2 1 1 J.R. CASTON , E. BLANCO AND A. ALEJO L. DE SABATO1, I. DI BARTOLO2, G. 1. Centro de Investigación en Sanidad Animal (CISA- ANGELONI2, E. PONTERIO2, M. MONINI2, INIA), Valdeolmos, 28130 Madrid, Spain. F.M. RUGGERI2, F. OSTANELLO1 2 . Dpto. Estructura de Macromoléculas, (CNB-CSIC), 1. Department of Veterinary Medical Sciences, Cantoblanco, 28049 Madrid, Spain. University of Bologna, Ozzano Emilia (BO), Italy 2.Department of Veterinary Public Health and Food The new variant of rabbit haemorrhagic Safety, Istituto Superiore di Sanità, Rome, Italy disease virus (RHDV2) has distinct biological properties and may be replacing Hepatitis E is an acute disease of humans the previously more prevalent RHDV virus caused by a small RNA virus, the hepatitis E in several European countries. Importantly, virus (HEV). Four mammalian HEV the RHDV vaccine does not confer genotypes are recognized. Genotype 1 and complete protection against RHDV2 2 are restricted to humans and consist of induced disease, which has prompted the epidemic strains circulating in developing development and adoption of a novel countries, mainly associated to waterborne inactivated virus vaccine. ELISA methods outbreaks. Genotype 3 and 4 infect both for the diagnosis of classical RHDV humans and many animal species (pig, infection are well characterized, but deer, wild boar, and rabbit) and circulate in currently, there are no available specific developed countries. These two latter serological tests for RHDV2. genotypes are considered zoonotic, for Here we describe the expression of the which pigs and less frequently other animal complete VP60 major coat protein of species (wild boar, deer) are reservoirs. RHDV2, which self assembles into virus like Based on sequence analysis and intra- particles (VLPs). These VLPs have been genotype variability, genotypes are divided used to study the specific antibody into sub-genotypes. Thegenotype 3 can be response in RHDV and RHDV2 vaccinated divided into 10 sub-genotypes (3a–3j). In animals, confirming their differential this study, hepatitis E infection was

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investigated in piglets affected with (PO 52) diarrhea in two small farms in Italy. The THERAPEUTIC MVA-B VACCINE IMPROVES virus was detected in 11 out of 14 animals THE MAGNITUDE AND QUALITY OF THE T tested. Based on sequence analysis the 6 CELL IMMUNE RESPONSES IN HIV-1 Italian strains examined belonged to two INFECTED SUBJECTS ON HAART clusters that contain both swine and C. E. GÓMEZ1, B. PERDIGUERO1, J. GARCÍA- human strains from Europe and Japan, ARRIAZA1, V. CEPEDA1, C. O. SÁNCHEZ- belonging to genotype 3 sub-genotypes e SORZANO1, B. MOTHE2, J. L. JIMÉNEZ3, M. and f. The two Italian clusters shared a A. MUÑOZ-FERNÁNDEZ3, J. M. GATELL4, J. nucleotide identity of 81.8% in the 400bp 3 C. LÓPEZ BERNALDO DE QUIRÓS , C. ORF2 (capsid protein) fragment and 87.5% BRANDER2, F. GARCÍA4 AND M. ESTEBAN1 in the 400bp ORF1 (RdRp) fragment, 1 Department of Molecular and Cellular Biology, confirming that genotypes 3 circulating in Centro Nacional de Biotecnología, Consejo Superior pigs in Italy are heterogeneous. The de Investigaciones Científicas (CSIC), Madrid, Spain. complete genome of a g3e strain and the 2 IrsiCaixa-HIVACAT, Hospital Universitari Germans Trias i Pujol, Badalona, Spain.3 Hospital Gregorio complete coding regions (partial ORF1, 4 complete ORF2 and ORF3) of a Marañón, Madrid, Spain. Hospital Clinic-HIVACAT, IDIBAPS, Barcelona, Spain. representative g3f strain were obtained and compared to other HEV full length or partial sequences available on line. Results Previous studies suggested that poxvirus- obtained revealed that porcine strains based vaccines might be instrumental in clustered together with human and swine the therapeutic HIV field. A phase I clinical strains detected in Europe. The analyses trial was conducted in 30 HIV-1-infected conducted showed that most changes in patients on highly active antiretroviral the coding regions correspond to therapy (HAART) with CD4 T cell counts 3 synonymous mutations, whereas only a above 450 cells/mm and undetectable small ORF1 region and the ORF3 showed viremia which were randomized to receive sites subjected to positive selection. 3 intramuscular injections of MVA-B 8 Further analyses are needed to understand vaccine (10 PFU/ dose) (coding for clade B the possible different clinical significance HIV-1 Env, Gag, Pol and Nef antigens) or of HEV genotypes and sub-genotypes. placebo, followed by interruption of HAART. Here, the magnitude, breadth, quality and phenotype of the HIV-1-specific

T cell responses were assayed by intracellular cytokine staining (ICS) in 22 out of 30 volunteers pre- and post- vaccination. Furthermore, a sub-study of HIV-1 viral rebound dynamics was performed in 9 out of 22 subjects during

the first 12 weeks after HAART interruption. MVA-B vaccine significantly induced the expansion and also the

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appearance of new HIV-1-specific CD4 T day. Nowadays, only in sub-Saharan Africa cell responses (mostly against Gag and heterosexual transmission represent the GPN antigens) that were high in 80% of the new infections, mainly in magnitude, broad, with an enhanced women. The development of new polyfunctionality and of T effector memory preventive treatments in the last years has (TEM) phenotype, while maintained the been carried out. Among them, topical magnitude and quality of the preexisting microbicides and nanotechnology play an HIV-1-specific CD8 T cell responses. In important role. We introduce the main addition, the MVA-B-induced immune steps of new potential microbicides responses were associated with a delayed screening in vitro and in vivo, as well as the HIV-1 plasma viral rebound in 50% of the methodology used to achieve effective vaccinees analyzed. Thus, MVA-B polyanionic carbosilane dendrons vaccination represents a feasible strategy microbicides. We research the potential to improve T cell responses in individuals topical microbicide activity against HIV-1 with preexisting HIV-1-specific immunity. and HSV-2 infection of six new different polyanionic carbosilane dendrons named (PO 53) BDCG044, BDCG046, BDCG048, BDCG050, BDCG052 and BDCG054. ANALYSIS OF DUAL ANTIVIRAL ACTIVITY The dendrons were synthesized by the AGAINST HIV-1/HSV-2 OF NEW group of Inorganic chemistry of Alcalá de POLYANIONIC CARBOSILANE DENDRONS Henares. We focused on carbosilane WITH FATTY ACIDS AT THE CORE AS A branches dendrons from first to third TOPICAL MICROBICIDE generation, with palmitic or hexanoic fatty C. GUERRERO-BELTRÁN1, R. CEÑA-DIEZ1, D. 1,2 2 acids as core and capped with sulfonate SEPÚLVEDA-CRESPO , J.L. JIMÉNEZ , R. groups. We evaluated cytotoxicity in GÓMEZ3, F. J. DE LA MATA3, Mª A. MUÑOZ- 1,2,* different cell lines in vitro (TZM.bl, PBMCs FERNÁNDEZ and VERO), inhibition of HIV-1 (X4-HIV- 1. Laboratorio InmunoBiología Molecular, Hospital 1NL4.3 or R5-HIV-1NLAD8) replication and General Universitario Gregorio Marañón, Madrid, Spain. Instituto de Investigación Sanitaria Gregorio HSV-2 333, time-of-addition experiments, Marañón (IISGM), Madrid, Spain.Networking establishment of IC50, cell fusion, HIV-1 Research Center on Bioengineering, Biomaterials internalization and binding assays, vaginal and Nanomedicine (CIBER-BBN), Madrid, irritation test and subsequent histological Spain.Spanish HIV HGM BioBank, Madrid, Spain. analysis. Different data analyses were 2 . Plataforma de Laboratorio, Hospital General performed using Calcusyn software. Universitario Gregorio Marañón, Madrid, Spain. IISGM, Madrid, Spain. CIBER-BBN, Madrid, Spain BDCG048 and BDCG054 showed high 3. Departamento de Química Inorgánica, biosafety in primary cells and different cell Universidad de Alcalá, Campus Universitario, Alcalá lines derived from vagina and uterus de Henares, Madrid, Spain. CIBER-BBN, Madrid. (maximum safe concentration at a range of 10 μM in TZM.bl cells). Moreover, these The HIV pandemic continues its spread at a dendrons showed a great broad-spectrum rate of over 15,000 new infections every antiviral activity achieving inhibitions of 99% using X4-HIV-1NL4.3 in the

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presence/absence semen, high inhibition 4 Servicio de Enfermedades Infecciosas, Hospital against HSV-2, blocked the entry of Universitario Ramón y Cajal and IRYCIS, Madrid. different HIV-1 strains, and protected the epithelial monolayer cells from cell Effective combination antiretroviral disruption. IC50-values were at the order of therapy (cART) has improved the quality nanomolar concentration. Additionally, no and life expectancy of HIV-infected irritation was detected in female mice after patients; nevertheless achieving the cure dendron vaginal administration. of HIV is still an unattainable challenge for We conclude that BDCG048 and BDCG054 the scientific community. Although cART both third generation dendrons with achieves undetectable plasma viral RNA hexanoic or palmitic fatty acids as core, and the normalization of CD4 T cell levels respectively could be effective to inhibit in almost all patients, several studies have HIV-1 and HSV-2 infection and shown that HIV remains incurable owing to transmission within genital mucosa. We the persistence of latently infected cells. can provide promising outcomes to The persistence of HIV-1 involves encourage BDCG048 and BDCG054 as a numerous overlapping cellular pathways, hopeful microbicides. Although this which are interesting from the promising results, further assays are pharmacological point of view. Thus, needed to be performed in order to lead targeting multiple steps within the virus these results to clinical trials. latency mechanisms is important to optimize the reactivation effect. Thus, recent therapeutic interventions to (PO 54) eradicate HIV are focused on the activation SYNERGISTIC ACTIVATION OF LATENT HIV- of viral production from latently infected 1 EXPRESSION BY NOVEL HISTONE cells. We evaluated the effect of different DEACETYLASE INHIBITORS AND combinations of bryostatin-1 (BRY) and BRYOSTATIN-1 novel histone deacetylase inhibitors M. MARTÍNEZ-BONET1, M.I. CLEMENTE1,2, (HDACIs) in HIV reactivation and compared M.J. SERRAMÍA1, E. MUÑOZ3, S. MORENO4, the toxicity and phenotype modifications M.A. MUÑOZ-FERNÁNDEZ1,* induced by single or combined treatment. 1 Laboratorio de InmunoBiología Molecular, The lymphocyte or monocyte/macrophage Hospital General Universitario Gregorio Marañón; latently infected cell lines J89GFP and Instituto de Investigación Sanitaria Gregorio THP89GFP, respectively, were treated with Marañón (IISGM), and Networking Research Center on Bioengineering, Biomaterials and Nanomedicine BRY, panobinostat (PNB) and romidepsin (CIBER-BBN); C/ Dr. Esquerdo 46, 28007 Madrid, (RMD) alone or in combination and the Spain viral reactivation effect was assessed as 2 Plataforma de cultivos celulares, Instituto de EGFP expression. We calculated the Investigación Sanitaria Gregorio Marañón (IISGM), combination index (CI) for each drug C/ Dr. Esquerdo 46, 28007 Madrid, Spain combination to determine synergy. 3 Departamento de Inmunología, Facultad de Primary CD4 T cell viability, activation and Medicina, Universidad de Córdoba, Avda. Menéndez proliferation profile was analyzed after Pidal, s/n. 14004, Córdoba, Spain single or combined drug treatment. In

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terms of latent HIV reactivation, we de Henares, Madrid, Spain. CIBER-BBN, Madrid, demonstrated a synergistic activity in the Spain BRY/HDACIs combinations tested, whereas nonsynergistic or additive effects were Due to the increased number of new HIV observed when PNB was mixed with RMD. infections, alternative prevention The EC75 of BRY, PNB and RMD were strategies based on the use of topical reduced in these combinations, showing a vaginally products to inhibit HIV-1 infection decrease from 20 to 4-fold respectively. in women has been established. Topical Primary CD4 T cells treated with drug microbicides are used vaginally and/or combinations presented better activation rectally, and they act at an earlier stage of and proliferation profiles in comparison HIV-1 infection.1 In the literature, with single drug at their EC75 value treated polyanionic dendrimers and copper cells. In summary, the combination complexes have shown interesting between BRY, PNB and or RMD presented biological properties as antiviral agents.2,3 a synergistic profile inducing virus Therefore, new bifunctionalized expression in both lymphocyte and carbosilane dendritic systems have been monocyte/macrophage HIV latently designed. The topology of these systems infected cells. Additionally, the allows them to have anionic peripheral combinatorial strategy presented herein groups for a therapeutic action, but in could lead to a reduction in the addiction, an excellent chelating agent at concentrations of LRAs used in vivo, the focal point, which forms extremely resulting in a diminution of adverse effects, stable complexes with a large number of limiting the local injuries, the toxicity, and metal ions.4 the inflammation, making this Firstly, copper complexes were combinations an attractive novel option for synthesized, and in vitro studies were future clinical trials. performed to evaluate the safety, biocompatibility, anti-HIV ability and (PO 55) mechanism of these bifunctionalized BIFUNCTIONALIZED CARBOSILANE carbosilane dendrons. All compounds have DENDRONS FOR THE PREVENTION OF HIV- been not toxic at the studied 1 INFECTION concentrations up to 20 μM in PBMC and S.MORENO1,2, F. J. DE LA MATA1, R. TZM.bl cells and have demonstrated GÓMEZ1, Mª A. MUÑOZ-FERNÁNDEZ1,2,* potent and a broad-spectrum anti-HIV-1 1 activity in vitro. Several experiments were . Laboratorio InmunoBiología Molecular, Hospital General Universitario Gregorio Marañón, Madrid, carried out to study the mechanism of Spain. Instituto de Investigación Sanitaria Gregorio action, finding that these systems act entry Marañón (IISGM), Madrid, Spain.Networking level, joining the virus, meaning a virucidal Research Center on Bioengineering, Biomaterials activity. and Nanomedicine (CIBER-BBN), Madrid, Spain.Spanish HIV HGM Biobank, Madrid, Spain. The next step in a near future will be 2. Departamento de Química Inorgánica, introduce other metals as gadollinium or Universidad de Alcalá, Campus Universitario, Alcalá gallium and these systems be used in

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image for “in vivo” applications, obtaining The HIV-2 ROD RT was expressed and diagnosis systems. purified using a plasmid that encoded the 1 F. Hladik et al, Elife., 2015, doi: sequences of the RT p68 subunit and the 10.7554/eLife.04525. HIV-2 protease. Using this construction, 2 D. Sepúlveda-Crespo et al, Nanomedicine: p68/p55 heterodimers with His6 tags in Nanotechnology, Biology, and Medicine, 2014, 10, their C-termini were purified. The fidelity 609–618. 3 was determined with a forward mutation M.Galán et al, Current Medicinal Chemistry, 2012, assay, in which the M13mp2 phage 19, 4984-4994. genome lacking one strand of the lacZα 4 G. J. Stasiuk et al, Chem. Commun., 2013, 49, 564- 566. gene (“gapped DNA”) was used as substrate of a gap-filling reaction.

Escherichia coli MC1061 were (PO 56) electroporated with the product of the HUMAN IMMUNODEFICIENCY VIRUS TYPE reaction and grown in M9 plates 2 REVERSERANSCRIPTASE FIDELITY OF containing 5-bromo-4-chloro-3-indolyl-β-D- DNA-DEPENDENT DNA SYNTHESIS galactopyranoside and isopropyl-1-thio-β- A. SEBASTIÁN-MARTÍN1, M. ÁLVAREZ1, L. D-galactopyranoside, with E. coli CSH50 MENÉNDEZ-ARIAS1 lawn cells. Mutants containing errors made 1. Centro de Biología Molecular Severo Ochoa by the RT while copying the lacZα region of (CSIC/UAM), Nicolás Cabrera 1, Campus de the M13mp2 DNA were identified as pale Cantoblanco, 28049, Madrid, Spain or colorless plaques and analyzed by nucleotide sequencing. For comparative In , the reverse transcriptase purposes, mutant frequencies and error (RT) is the enzyme responsible for the rates were determined in parallel for the replication of the viral genome. RTs HIV-1 BH10 RT. synthesize double-stranded DNA using The M13mp2-based assays revealed RNA and DNA as templates. Their error similar mutant frequencies for the HIV-2 rates have been estimated around 10-4- ROD RT (1.26 x 10-2) and the HIV-1 BH10 10-5 nucleotide substitutions per RT (2.02 x 10-2). The slightly increased replication cycle, and this could explain in accuracy of the ROD RT resulted from a part the large genetic variability of the lower base substitution error rate, despite human immunodeficiency virus (HIV). The its higher tendency to introduce fidelity of the HIV type 2 (HIV-2) RT has frameshifts. Mutational spectra of ROD been much less studied, compared with and BH10 RTs showed common hot spots, the HIV-1 RT. Available data are limited to such as the one located at position +88, nucleotide incorporation assays carried out and others found only in the mutational with a limited number of spectra of one of the enzymes. Hot spots in template/primers. The aim of this work is the HIV-2 ROD RT spectrum appeared in to purify a prototypic HIV-2 RT (derived clusters (e.g. from positions +75 to +90, from the ROD strain), and analyze its and +144 to +151) or at specific sites (e.g. fidelity of DNA-dependent DNA synthesis positions +115 or +130). in M13mp2 lacZ-based assays.

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Our results showed a similar accuracy for patients who received pegylated interferon HIV-2 and HIV-1 RTs in DNA-dependent alpha (IFN-α) 2a or 2b combined with DNA synthesis reactions. Future studies weight-based ribavirin, 10 of them with will be focused on the analysis of fidelity sustained virologic response (SVR), two while copying RNA templates. patients who relapsed, and three patients who did not respond (IFN patients); and iii) 9 patients who had spontaneous HCV RNA (PO 57) clearance (SHC patients). Total cell- REDUCED CELL-ASSOCIATED HTLV-2 DNA associatedHTLV-2 DNA was quantified by IN ANTIRETROVIRAL TREATED HIV-1-HCV- in-house real-time PCR. We also analyzed COINFECTED PATIENTS WHO EITHER other immune factors, including T cell RECEIVED INTERFERON-Α/RIBAVIRIN- immune activation and plasma IL-6 levels. BASED HEPATITIS C THERAPY OR HAD Results: Either IFN patients or SHC patients SPONTANEOUS HCV RNA CLEARANCE had lower level of cell-associatedHTLV-2 ABAD-FERNÁNDEZ M, DRONDA F, DNA compared to HCV patients (p=0.022 MORENO A, CASADO JL, PÉREZ-ELÍAS MJ, and p=0.040, respectively). CD8 QUEREDA C, MORENO S, VALLEJO A. percentage and had received IFN-based Department of Infectious Diseases, Instituto Ramón treatment or had HCV clearance were y Cajal de Investigación Sanitaria (IRYCIS), Hospital Universitario Ramón y Cajal, Madrid, Spain. independently associated to cell- associated HTLV-2 DNA. Immune activation

and IL-6 level were higher in HCV patients. Until recently, the standard treatment for Discussion: Our data indicate that patients hepatitis C virus (HCV) infection consisted treated with IFN-α have lower total cell- mainly of a combination of interferon α associated HTLV-2 DNA. On one hand, this (IFN-α) and ribavirin (RBV). It has been observed effect reflects changes after HCV reported that IFN-α is effective in reducing treatment and do not reflects a general HIV-1 RNA loads in naïve patients and in decline of HTLV-2 DNA since HTLV proviral patients receiving antiretroviral treatment load is very stable over time. On the other (ART). Also, a reduction of integrated HIV-1 hand, this work pointed out the DNA in CD4 T cells has been reported in importance of HCV infection upon the level two different works. One analyzed HIV-1- of cell-associated HTLV-2 DNA, since infected patients (not infected with HCV) patients with no HCV infection, due to who interrupted suppressive ART to spontaneous HCV RNA clearance had also receive treatment with IFN-α, and the lower level of cell-associated HTLV-2 DNA. other one analyzed HIV-1/HCV-coinfected patients under ART who received IFN-α and ribavirin as HCV treatment. Patients and methods: We analyzed the level of cell-associated HTLV-2 DNA in i) 37 patients with HCV infection who had never received pegylated interferon alpha (IFNα)-

based HCV treatment (HCV patients); ii) 15

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(PO 58) the different groups. This fact could be DEVELOPMENT OF A NOVEL METHOD FOR very useful for clustering and organizing TAXONOMIC CLUSTERING AND large databases of viral sequences, as well VISUALIZATION OF VIRAL GENOMIC as for quickly and accurately classifying SEQUENCES newly obtained data. S. DELGADO1, F. MORÁN2, A. MORA3,4, J.J. To process sequence data by SOM-based MERELO3,4, C. BRIONES5,6 algorithms, DNA or RNA sequences must 1.Departamento de Sistemas Informáticos, be previously transformed to fixed-size Universidad Politécnica (UPM), Madrid, Spain. numeric vectors. Therefore, novel 2. Departamento de Bioquímica y Biología representation methods are required for Molecular I, Universidad Complutense (UCM), automatically and bijectively transform Madrid, Spain. aligned nucleotide sequences into numeric 3.Departamento de Arquitectura y Tecnología de vectors, dealing with both nucleotide Computadores, Universidad de Granada (UGR), ambiguity and the presence of gaps Granada, Spain. derived from sequence alignment. We 4.CITIC, Campanillas, Málaga, Spain. 5 have developed a new DNA and RNA .Departamento de Evolución Molecular, Centro de codification method based on Euclidean Astrobiología (CSIC-INTA), Torrejón de Ardoz, Madrid, Spain. space, which has been tested using two 6.Centro de Investigación Biomédica en Red de SOM models: the classical Kohonen’s SOM enfermedades hepáticas y digestivas (CIBERehd), and Growing Cell Structures (GCS). The Spain. former is known for its usefulness in graphical exploratory data analysis (1) and, Over the last decade, the advent of next in turn, the latter produces better generation sequencing techniques has clustering results due to its flexible dramatically increased the amount of architecture (2). sequence data stored in databases. As a A dataset composed of 44 complete consequence, fast algorithms and sequences of the RT region of the HIV-1 pol classification methods are required to gene belonging to the three phylogenetic convert the growing number of nucleotide groups of this virus (M, O and N), and to all sequences into useful information (e.g., the subtypes within the group M (A, B, C, identification of data classes and clusters, D, F, G, H, J, K), has been used to test the characterization of relevant features or developed codification method. The hidden relationships among data, etc.). algorithm has revealed that the most Self-Organizing Map (SOM) is an important factor affecting the accuracy of unsupervised method used for clustering the sequence clustering is the assignment high-dimensional data without a previous of an extra weight to the presence of knowledge of the class to which they alignment-derived gaps. Our results show belong (1). Also, SOM spatially orders the that clustering of HIV-1 sequences is quick data on a two-dimensional map, what and straightforward, and the retrieved provides not only the clustering but a classification is in agreement with quantitative value of the similarity among traditional sequence-based phylogenetic reconstructions (3). This suggests a broad

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applicability of such a novel codification different samples during follow-up. method in different fields of virology. Fluctuations of different viral populations 1. Kohonen (2001). Self-Organizing Maps.3th were also analyzed. HIV-1 was isolated by edition. Springer co-culture in different samples and its 2. Fritzke (1994). Neural Networks, 7, 1441. replicative capacity was measured in 3. Delgado et al. (2015). Bioinformatics 31, 736. TZMbl cells. The relation between the replicative capacity and viral evolution, (PO 59) calculated as distance to the most recent common ancestor (MRCA), was estimated. VIRAL EVOLUTION IN A HIV-1 DUAL LTNP INFECTED PATIENT Result. Viral diversity in the quasispecies fromthe samples taken during the follow A. MOYANO1, N. PEDREÑO1, T. ÁLVARO1, C. up was related to clinical parameters. We CASADO1, I. OLIVARES, R. FUENTES 1, C. observed a statistical significance (p<0,5 ) RODRIGUEZ2, J. ROMERO2, C. LÓPEZ in the relation between the increase in GALÍNDEZ1 AND M. PERNAS1. viral diversity and years after diagnosis and 1. Servicio de Virología Molecular, CNM, Instituto de a decrease in the % of CD4+ T cells in Salud Carlos III, Majadahonda, Madrid, Spain. relation with diversity (p<0,5). No relation 2. Centro Sanitario Sandoval (CSS). Instituto de Investigación Sanitaria del Hospital Clínico San between viral diversity and viral load and % Carlos (IdISSC), Madrid 28010, Spain. CD8+T cells were observed. The replicative capacity of recombinant viruses with envelopes from the patient virus was Background.Long term controller patients related with the distance to the common (LTNP) constitute a group of HIV-1 infected ancestor but no relation was observed. patients without clinical symptoms for more than 10 years without antiviral We studied the fluctuation of the different therapy. Dual infection (DI), infection by viruses and viral populations with time. two or more different viral strains in LTNP There was a fluctuation in the presence of has been described only in a few sporadic the populations of the two viruses in the cases and there are no studies concerning patient. We detected that the viral strains viral evolution in this group of patients. In “a” decrease along the study, while the this work, we analyse the viral envelope second strain “b” became predominant. gene evolution in a DI LTNP and its relation Conclusions.These results show the with clinical and immunological presence of the different patterns of parameters. evolution of the two strains in a DI infected Methods. Samples from a LTNP dual LTNP patient, and an increase in viral infected patient were taken between 17- diversity which could be correlated with 28 years after the first HIV-1 positive. In clinical markers. order to study viral evolution, proviral DNA was amplified by limiting dilution nested PCR in the c2-v5 region of env gene. Clinical parameters were compared with the diversity of the quasispecies in the

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(PO 60) determine the mechanism by which SorA SORAPHEN A: A NATURAL PRODUCT inhibits HIV. FROM MYXOBACTERIA THAT INHIBITS HIV Methods: The anti-HIV effective THROUGH BLOCKING HOST FATTY ACID concentration 50 (EC50) and the cellular SYNTHESIS cytotoxicity 50 (CC50) of SorA were E. FLETA-SORIANO1, C. LORCA-ORÓ2, G. determined by titration using a TZM-bl cell MIRAMBEAU2, C. LOPEZ-IGLESIAS, G. infection assay. HIV production was KOUTSOUDAKIS4, J. DIEZ4, M. analyzed by measuring p24 in the BRÖNSTRUP5, J. P. MARTINEZ1 & A. supernatant or by MEYERHANS1,6 immunofluorescence. EGFP-Gag assembly 1Infection Biology Group, DCEXS, Universitat and HIV maturation were detected with Pompeu Fabra, Barcelona, Spain confocal and transmission electron 2AIDS Research group, IDIBAPS, Barcelona, Spain microscopy, respectively. Viral 3Cryo-Electron Microscopy Unit, CCiTUB, Universitat supernatants from latently infected ACH2 de Barcelona, Barcelona, Spain cells with or without SorA were analyzed 4Molecular Virology Group, DCEXS, Universitat for infectivity, p24 and gp120 content, and Pompeu Fabra, Barcelona, Spain viral-RNA. HIV binding to CD4 and HIV cell 5Department of Chemical Biology, Helmholtz Centre fusion was tested to analyze the entry for Infection Research, Braunschweig, Germany capacity of viruses produced from SorA- 6 Institució Catalana de Recerca i Estudis Avançats treated cells. (ICREA), Barcelona, Spain. Results: SorA inhibits mainly late steps of

HIV-1 in vitro with an EC50 between 0.14 Introduction: Human Immunodeficiency and 1.8 µM. Neither Gag assembly nor HIV Virus (HIV) infections continue to threaten maturation were inhibited by SorA as global human health. They require life- shown by confocal detection of gag long treatment with a combination of assembly-spots and virus particle antiviral drugs. Co- infections with the inspection with transmission electron hepatitis C virus (HCV) add a further level microscopy. Rather the amount of HIV of complexity as HCV-specific drugs have to envelope proteins per particle were be given in addition leading to intricate reduced. This correlated with a reduction drug-drug interactions. Targeting shared of virus host- cell fusion. host factors involved in the replicative Conclusions: SorA inhibits HIV by altering processes of both viruses could simplify co- the composition of virus produced in cells infection treatments. Soraphen A (SorA) is treated with the drug. Together with its a myxobacterial metabolite that inhibits anti-HCV activity, SorA is an interesting the host acetyl-CoA carboxylase, a key candidate for HIV-HCV co-infection enzyme in fatty acid biosynthesis. The anti- treatments. HIV activity of SorA was first identified in a high-throughput screen of myxobacterial metabolites. Recently, we have also described the SorA-mediated inhibition of HCV. The aim of this study was to

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(PO 61) immunodeficiency virus (HIV) represent a SORAPHEN A: A BROAD-SPECTRUM significant challenge for treatment due to ANTIVIRAL NATURAL PRODUCT WITH the necessity of combining diverse antiviral POTENT ANTI-HEPATITIS C VIRUS compounds that often leads to complex ACTIVITY drug-drug interactions. Soraphen A (SorA) G. KOUTSOUDAKIS1, I. ROMERO-BREY2*, C. is a myxobacterial metabolite that inhibits BERGER2*, G. PÉREZ-VILARÓ1, P. the acetyl-CoA carboxylase, a key enzyme MONTEIRO PERIN3,4, F. W. RUDOLF in lipid biosynthesis. We have previously VONDRAN4,5, M. KALESSE6, K. identified SorA to efficiently inhibit HIV. 7 7 8 The aim of the present study was to HARMROLFS , R. MÜLLER , J.P. MARTINEZ , T. PIETSCHMANN3, R. BARTENSCHLAGER2,9, evaluate the capacity of SorA and M. BRÖNSTRUP6, A. MEYERHANS8,10, J. analogues to inhibit HCV infection. DÍEZ1 Methods: SorA inhibition capacity was 1Molecular Virology Group, Department of evaluated in vitro using cell-culture derived Experimental and Health Sciences, Universitat HCV, HCV pseudoparticles and subgenomic Pompeu Fabra, Barcelona, Spain. replicons. Infection studies were 2Department of Infectious Diseases, Molecular performed in the hepatoma cell line Virology, Heidelberg University, Heidelberg, Huh7/Scr and in primary human Germany. hepatocytes. The effects of SorA on 3 TWINCORE - Institute of Experimental Virology, membranous web formation were Centre for Experimental and Clinical Infection Research, Hannover, Germany. analyzed by electron microscopy. 4German Centre for Infection Research (DZIF), Results: SorA potently inhibits HCV partner site Hannover-Braunschweig, Germany. infection at nanomolar concentrations. 5 ReMediES, Department of General, Visceral and Obtained EC50 values were 0.70 nM with a Transplantation Surgery, German Centre for HCV reporter genome, 2.30 nM with wild- Infection Research Hannover Medical School, type HCV and 2.52 nM with subgenomic Hannover, Germany. 6 HCV replicons. SorA neither inhibited HCV Department of Chemical Biology, Helmholtz Centre RNA translation nor HCV entry, as for Infection Research, Braunschweig, Germany. demonstrated with subgenomic HCV 7Helmholtz Institut für Pharmazeutische Forschung Saarland, Saarbrücken, Germany. replicons and HCV pseudoparticles, 8Infection Biology Group, Department of suggesting an effect on HCV replication. Experimental and Health Sciences, Universitat Consistent with this, evidence was Pompeu Fabra, Barcelona, Spain. obtained that SorA interferes with 9German Centre for Infection Research (DZIF), formation of the membranous web, the partner site Heidelberg, Germany. site of HCV replication. Finally, a series of 10Institució Catalana de Recerca i Estudis Avançats natural and synthetic SorA analogues (ICREA), Barcelona, Spain. helped to establish a first structure-activity *equally contributed to this work relationship. Conclusions: SorA has a very potent anti- Background & Aims: Co-infections by the HCV activity. Since it also inhibits HIV, SorA hepatitis C virus (HCV) and human is a promising candidate for the

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development of simplified treatments of loop shape according to isostericity HCV/HIV co-infection. matrices predicting recurrent three- dimensional motifs more conserved in (PO 62) structure than in sequence. In the present work we have applied these approaches to MAPPING THE TRANSCRIPTION eggplant latent viroid (ELVd), of the family INITIATION SITES IN THE IN VIVO RNA Avsunviroidae encompassing viroids with CONFORMATIONS OF BOTH POLARITY hammerhead ribozymes that replicate in STRANDS OF EGGPLANT LATENT VIROID plastids (mostly chloroplasts). Data from 1 2 A. LÓPEZ-CARRASCO , S. GAGO , R. the three approaches are consistent and 1 1 FLORES , S. DELGADO indicate that (+) and (-) genomic RNAs fold 1Instituto de Biología Molecular y Celular de Plantas into rod-like conformations with a (IBMCP), Consejo Superior de Investigaciones bifurcation at both termini. These two Científicas-Universidad Politécnica de Valencia, conformations, even if similar, are not Valencia, Spain 2 identical as revealed by their different Leibniz Institut für Pflanzenbiochemie, Halle (Saale), Germany electrophoretic mobility in non-denaturing polyacrylamide gels. One functional aspect

strongly depending on the structure of Having a genome just composed by a small viroid RNAs is transcription, which in the (250-400 nt) circular RNA without protein- family Avsunviroidae is catalyzed by a coding ability, viroids depend on nuclear encoded polymerase (NEP) sequence/structure motifs that are translocated into chloroplasts. Moreover, recognized by the host proteins mediating since RNA folds cotranscriptionally, the their infectious cycle (replication, initiation sites of the nascent RNA strands movement and suppression of defensive may influence the adoption of metastable, responses). These motifs are integrated in although functionally relevant structures, the compact secondary structures that like the hammerhead ribozymes that viroid RNAs adopt as a consequence of mediate self-cleaving of the oligomeric their extensive self-complementarity, in RNA intermediates generated in replication which double-stranded segments are through a symmetric rolling circle flanked by loops usually stabilized by mechanism. Applying RLM-RACE (“RNA arrays of non-canonic interactions. Viroid ligase mediated-rapid amplification of RNA structure can be essentially tackled: i) cDNA ends”) and primer extension in silico, with algorithms searching the methodologies to ELVd RNAs isolated from most stable conformations, ii) in vitro, by infected tissue, we have also determined in RNase and bisulphite probing in aqueous the present work the in vivo initiation sites solutions and, more recently, by SHAPE for both ELVd strands. Within the (“selective 2′-hydroxyl acylation analyzed conformations that these genomic RNAs by primer extension”), and iii) in vivo, by adopt in vivo, the initiation sites map at identifying either natural covariations that positions close to one of the two preserve the double-stranded segments or bifurcations, which might serve to recruit substitutions that leave unaffected the

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the NEP or some associated transcription at the log-level. However, we also found factor. that some foreign peptides severely impaired the ability of the recombinant (PO 63) virus to infect host plants. To overcome this problem, we have approached the LOG-INCREASING THE ABILITY TO SENSE production of recombinant VNPs of the HUMAN THROMBIN RECEPTOR VLP type, trough transient high-level ANTIBODIES USING RECOMBINANT expression of the recombinant CP in ELONGATED FLEXUOUS PLANT-MADE plants. VIRUS-LIKE PARTICLES Since no previous production of TuMV I. GONZÁLEZ GAMBOA, P. MANRIQUE, M. VLPs has been reported in biofactory SÁEZ, F. SÁNCHEZ, F. PONZ. plants, we undertook the expression of Centro de Biotecnología y Genómica de Plantas non-modified viral CP and of a (CBGP; UPM-INIA). Pozuelo de Alarcón (Madrid). Spain recombinant CP fused to a peptide strongly interfering with virus infection. This was

achieved in Nicotiana benthamiana plants Plant Viral Nanoparticles (VNPs) have been agroinfiltrated with Agrobacterium recently exploited in nanobiotechnology tumefaciens bearing high-expression for multiple applications, and their physical vectors of the pEAQ family. As a proof-of- and biological characterization is also being concept peptide we selected one derived actively carried out. In many cases, plant from the human thrombin receptor (TR), VNPs present characteristics quite whose ability to impair virus infection was advantageous in comparison with animal assessed. High level expressions of both CP or bacterium viruses. Most advances in the forms were achieved in plant leaves. The deployment of plant VNPs have been CPs assembled into VLPs detected under performed on viruses with icosahedral the electron microscope. The ability of the virions (CPMV, CCMV), or elongated rigid recombinant TR-VLP to log-increase the rods (TMV). Flexuous elongated VNPs sensitivity to sense antibodies specific to (family Flexiviridae or Potyviridae, for the TR-derived peptide was confirmed. instance) offer some specific traits worth These results allow overcoming the exploring, but have hitherto received less biological restriction imposed by peptides attention comparatively. interfering with virus infectivity, opening We are developing the potyvirus Turnip the door to the exploitation of plant-made mosaic virus (TuMV) as a source of VNPs flexuous elongated VNPs devoid of with different purposes. Displaying foreign infectious nucleic acids. peptides genetically fused to the N- terminus of the structural viral coat protein

(CP) in infectious virus constructs, allowed to show log-increases in peptide immunogenicity and the ability of the recombinant virus to increase its ability to be used as a peptide antibody sensor, also

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(PO 64) suggest a close relationship between both ECO-EVOLUTIONARY DYNAMICS OF PepMV types that face a strong PEPINO MOSAIC VIRUS IN TOMATO interference competition. Furthermore, PLANTS our microscopical preliminary resultsbased P. GÓMEZ1*, M. JUÁREZ2, M.A. SÁNCHEZ- on RNA in situ hybridization indicate that PINA1, J.M. GARCÍA-VILLALBA1, C. ALCAIDE1 these viruses are able to infect the same AND M.A. ARANDA1 cell types, and combining ultra-structural microscopy with biological analyses in 1 Centro de Edafología y Biología Aplicada del Segura (CEBAS)- CSIC, Departamento de Biología del Estrés y planta we will identify whether this Patología Vegetal, PO Box 164, 30100 Espinardo, antagonistic interaction comprises a direct Murcia, Spain competition within the same infected cell 2 Escuela Politécnica Superior de Orihuela, for common plant and/or viral resources. Universidad Miguel Hernández de Elche, Ctra. de Our results suggest that beyond the Beniel km 3.2, 03312 Orihuela, Alicante, Spain epidemiological circumstances that initiate * Correspondence: [email protected] epidemics, viral interactions by mixed infections in plants could profoundly While individual plants are often infected impact the outcome of viral diseases. in nature with more than one related or unrelated virus, the extent to which mixed (PO 65) infections can modulate the evolutionary THE POTYVIRIDAE P1a LEADER PROTEASE dynamics of these viruses is unclear. CONTRIBUTES TO HOST RANGE Pepino mosaic virus (PepMV) is an SPECIFICITY emerging RNA virus known to be one of 1 1 2 the most important tomato pathogens H. SHAN , F. PASIN , A. VALLI , C. 3 1 4 worldwide. Phylogenetic analyses showed CASTILLO , C. RAJULU , A. CARBONELL , C. 1 1 that PepMV populations in Spain were SIMÓN-MATEO , J A. GARCÍA , B. 1 composed of isolates of two types RODAMILANS . 1 (PepMV-CH2 and PepMV-EU), and here, .Departamento de Genética Molecular de Plantas, we show that they appear to be still co- Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3,Madrid, Spain circulating after 10 years from their first 2. Department of Plant Sciences, University of detection with high prevalence and genetic Cambridge, Cambridge CB2 3EA, UK variability. We then examined how viral 3.Laboratorio de Biotecnología y Desarrollo, interaction among both PepMV types and Instituto Nacional de Higiene "Rafael Rangel", also between PepMV and other important Caracas, Venezuela RNA viral pathogen of tomato (i.e., 4. Donald Danforth Plant Science Center, St. Louis, Cucumber mosaic virus, CMV), could affect MO 63132, USA their evolutionary dynamics. We found that an antagonistic interaction among The P1a protein of the both PepMV types may explain mixed- Cucumber vein yellowing virus is a serine infections prevalence, and this interaction protease phylogenetically related to was neither host-nonspecific nor affected potyviral P1. It is located at the N-terminal by the presence of CMV. These results end of the polyprotein and requires an

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unknown host factor for its proteolityc vitro incubation than when it is expressed activity; this might be related to host from a polycistronic RNA encoding other specificity. To help elucidate the role of viral proteins, and it can not be stabilize P1a cleavage in host range definition, a efficiently by PPV HCPro. These results series of constructs and chimeric viruses indicate that the effect of HCPro in the were built. In this work we demonstrate stabilization of CP is more pronounced that separation of P1a from the with the cooperation of other viral proteins polyprotein is essential for viral RNA and/or the corresponding RNA. In this silencing suppression and infection. We work, we have analyzed different deleted also show that this separation is host forms of the PPV RNA in search for a viral dependent. These findings support the role factor that contributes to CP stabilization. of viral proteases as important factors of As result, we have identified the factor that host adaptation. cooperates with HCPro in the region encoding the proteins P3, P3N-PIPO and (PO 66) 6K1.

A FACTOR COOPERATING WITH HCpro IN THE STABILIZATION OF ITS COGNATE (PO 67) CAPSID PROTEIN IS PLACED IN THE P3-6K1 DISSECTING THE MULTIPLE ROLES OF CODING REGION PEPINO MOSAIC VIRUS CAPSID PROTEIN A. GALLO GALLARDO1, J.J. PÉREZ1, A. VALLI2 F.E. MÉNDEZ1, L. RODRÍGUEZ-MORENO1, AND J.A. GARCÍA1. R.N. SEMPERE1, M.A. SÁNCHEZ-PINA1, M. 2 1 1. Centro Nacional de Biotecnología, Campus de la VALLE AND M.A. ARANDA Universidad Autónoma de Madrid, Madrid, Spain. 1. Dpto. de Biología del Estrés y Patología Vegetal, 2. Department of Plant Sciences, University of CEBAS-CSIC, Murcia, Spain Cambridge, Cambridge CB2 3EA, United Kingdom. 2. Structural Biology Unit, CIC bioGUNE, Derio, Spain

Plum pox virus (PPV) is a member of the Pepino mosaic virus (PepMV; genus genus Potyvirus (Potyviridae family). The Potexvirus, family Alphaflexiviridae) is a helper component proteinase (HCPro) of widespread plant virus that causes a major potyviruses is a multifunctional protein disease in tomato crops worldwide. The that is involved in diverse steps of the viral PepMV genome consists of a single infection, such as, polyprotein processing, stranded RNA of approximately 6.4 kb aphid transmission, and suppression of containing five open reading frames, host antiviral RNA silencing. Recently, it including a replicase gene, a triple gene has been described a new function of block (TGB) encoding TGBp1, TGBp2 and HCPro by which this viral factor enhances TGBp3, and a coat protein (CP) gene. Apart the stability of its cognate capsid protein for its structural role, the PepMV CP is (CP) and the yield of virus particles. known to be the elicitor of the Rx However, when PPV CP is expressed in resistance, to modulate the nature and plants from an mRNA lacking other viral severity of PepMV-induced symptoms, to coding sequences, it is less stable upon in be required for virus cell-to-cell movement

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and to be an RNA silencing suppressor. To (PO 68) have a better understanding of the THE ROLE OF TRANSLESION mechanisms underlying PepMV CP POLYMERASES IN THE GENERATION OF multiple roles, we are following a double GENETIC VARIABILITY OF AN EMERGENT approach. On the one hand, we are ssDNA PLANT VIRUS characterising CP single mutants in relation 1,2 to their ability to sustain PepMV cell-to-cell DÍAZ MARTÍNEZ, LUIS ; VIGUERA 1 movement and viral particle formation MÍNGUEZ , ENRIQUE; GRANDE PÉREZ, and/or stability. On the other hand, we 1,2 ANA have carried out genetic (Y2H, yeast-two- 1 hybrid) and biochemical (affinity Área de Genética, Universidad de Málaga, Campus chromatography) screenings to identify de Teatinos, Málaga, Spain. 2 tomato proteins that interact with PepMV Instituto de Hortofruticultura Subtropical y CP. For the Y2H, a PepMV-infected tomato Mediterránea “La Mayora” (IHSM-UMA-CSIC), cDNA normalized library was built and Málaga, Spain screened against the CP, providing three different interacting proteins in addition to Single-stranded DNA (ssDNA) viruses such the CP itself: a Glutathione-S-transferase as animal circoviruses or plant (GST), a Receptor-like serine/threonine geminiviruses are important emergent kinase (STK) and a Ribosomal protein L13 viruses. SsDNA viruses are as variable as (L13). Using the commercial One-Strep- their RNA equivalents and evolve quickly, tag fused to the CP of a PepMV with high mutation rates and mutation agroinfectious clone, following affinity frequencies around 10-3-10-5 chromatography with tomato and N. mutations/nt. Several factors are benthamiana extracts, an additional responsible for the elevated substitution interacting protein, the Heat shock cognate rates of ssDNA viruses including 70.3 (Hsc70.3), was identified. In vivo polymerase replication fidelity, mismatch interactions were validated in N. repair, exogenous and endogenous DNA benthamiana plants by bimolecular damage, nucleotide imbalances and the fluorescence complementation assay action of other cellular DNA modifying (BiFC) except for the STK/CP pair that did enzymes. Indels can also be introduced not produce yellow fluorescence emission. during replication or as a consequence of Finally, in order to identify a hypothetical recombination. Unlike RNA viruses, which role for each interactor in the viral owe their genetic variability in part to their infective cycle, all candidates were error prone RNA-dependent RNA assessed in virus-induced gene silencing polymerases, ssDNA viruses do not encode (VIGS) assays in tomato plants. Diverse DNA polymerases. They are replicated by phenotypes were observed for the unknown cellular DNA polymerases in the different interactors, including repression host nucleus via a rolling circle mechanism. of systemic PepMV accumulation. Mutation bias compatible with the deamination and oxidation of single- tranded DNA has been observed in

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geminiviruses. This kind of DNA damage is a substrate for Translesion Polymerases Ecosystem biodiversity provides (TLS), involved in lesion bypass. TLS pols of fundamental services for human welfare. It the Y family lack proofreading activity and has been proposed that one of such have low nucleotide selectivity, and thus services is the ability to reduce virus exhibit high error rates for base infection risk. According to this theory, the substitutions and indels, even in the reduction in the number of species present absence of damage. Here, we have in an ecosystem would increase the addressed the involvement of TLS density of those that are virus hosts, polymerases in the replication of the resulting in higher virus prevalence. Tomato Yellow Leaf Curl Virus (TYLCV) Experimental analyses have supported this geminivirus in Arabidopsis thaliana. To this prediction as often as not. This has led to end, wt A. thaliana, homozygous AtRev1 hypothesize that other factors, such as the and AtPolΚ, and heterozygous AtPolΗTLS ecosystem composition (identity and mutants were infected with TYLCV. TLS relative abundance of species), may also expression and viral loads were analysed at determine virus infection risk. However, 7, 14, 21 and 28 days post infection (dpi). this hypothesis has been seldom analyzed, We observed an absence of AtRev1 mainly due to the lack of well- transcripts, a significant reduction in characterized wild ecosystems. AtPolK expression, while AtPolH was To address this subject, we characterized expressed at wt levels in the corresponding the number, identity, and relative mutants. In each mutant, expression of the abundance of plant species in five locations other TLS polymerases was unaffected. In of evergreen oak forests and five of addition, viral loads in AtRev1, AtPolΗand riparian forests of the Iberian Peninsula; AtPolΚwere comparable to those of wt A. two ecosystems that account for 75% of thaliana TYLCV infections. Preliminary the wild landscape in Spain. At each results of the effect of altered TLS levels on location, we analyzed the prevalence and TYLCV variability measured by next host range of plant virus species of the eneration sequencing will be shown. genus Potyvirus in spring, summer and autumn of 2013-2014. (PO 69) Our results indicated that in all seasons ECOSYSTEM BIODIVERSITY AS A plant species richness was higher in DETERMINANT OF PLANT VIRUS riparian than in evergreen oak forests, with INFECTION RISK: PLANT SPECIES RICHNESS Potyvirus prevalence being also higher in VERSUS COMMUNITY COMPOSITION the former than in the later ecosystem. C. RODRÍGUEZ-NEVADO1, M. GARCÍA- This result is compatible with an effect of LOMANA1, R. GAVILÁN2, I. PAGÁN1 the number of host species in virus 1. Centro de Biotecnología y Genómica de Plantas infection risk. Interestingly, in both (UPM-INIA), Madrid, Spain. ecosystems most host species were 2. Departamento de Biología Vegetal II, Facultad de perennial, although community Farmacia, Universidad Complutense de Madrid, composition was evenly distributed Spain.

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between perennial and annual plants. This exacerbation of symptoms appear in the suggests that host identity is also multiple-infected hosts. important in determining virus infection A similar combination of two relevant risk. Finally, within each ecosystem, virus viruses affecting cucurbits resembles the prevalence across seasons correlated with mentioned SPVD: the potyvirus the relative abundance of host species, Watermelon mosaic virus (WMV) and the even when the number of host species did Cucurbit yellow stunting disorder not significantly change. virus (CYSDV) can coinfect the same host Hence, the present study, which is one of plants. The combined presence of the two the first of its kind, provides evidence that viruses has been shown in field surveys, not only plant species richness but also the but a detailed evaluation of their identity and relative abundance of host interaction is missing. In particular, the species may be important determinants of putative effect of this mixed infection virus infection risk. might be relevant in two essential processes of the pathogen cycles, like their (PO 70) natural dissemination by insect vectors (aphids and whiteflies for potyviruses and MIXED INFECTIONS OF POTYVIRUSES AND criniviruses, respectively), and their CRINIVIRUSES IN TWO PATHOSYSTEMS: capacity to deal with the plant defence SWEET POTATO AND CUCURBITS mechanisms, including RNA silencing A.B. MORENO, A. MINGOT, J.J. LÓPEZ- responses. MOYA The experimental approaches adopted to Center for Research in Agricultural Genomics CRAG, study mixed infections of WMV and CYSDV CSIC-IRTA-UAB-UB, Campus UAB Bellaterra, Cerdanyola del Vallès, 08193-Barcelona, Spain will be presented, including standardized inoculation methods with the natural

insect vectors, and efficient molecular Plant-pathogenic viruses are responsible of tools for viral load quantification in real relevant economic losses in agriculture time RT-PCR assays. For the controlled worldwide. In addition to numerous inoculation procedures, the use of double individual diseases caused by a single viral clip-on cage devices that allows easy infection, certain combinations of viruses transfer of insects from infected viral are known to result in severe synergistic sources to test plants will be described. effects. A good example is the Sweet Finally, the comparison with the best- Potato Viral Disease (SPVD), caused by the known SPVD case will include the simultaneous infection of host plants by divergences between the two the potyvirus Sweet potato feathery mottle pathosystems, like the presence of specific virus (SPFMV) and the crinivirus Sweet RNA silencing suppressors in the sweet potato chlorotic stunt virus (SPCSV). potato infecting viruses, not found in the Metagenomics studies are revealing the cucurbit-infecting counterparts. The frequent occurrence of mixed infections in purpose of the work is to gain a better plants that in many cases have remained knowledge on the combination of WMV unnoticed because no observable and CYSDV, which could eventually result

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in new management strategies and location and that a fraction of this TF recommendations for reducing the relocated from the nucleus to the damages caused by these viruses. nucleolus. This interaction also modulated (Work funded by Mineco grant AGL2013- its transcription activity since the 42537-R). expression of a member of the plant NEET proteins, which has been proposed to be regulated by ILR3, is reduced in infected (PO 71) plants. NEET down-regulation was INTERACTION BETWEEN A VIRAL COAT associated with changes in reactive oxygen PROTEIN AND THE BHLH TRANSCRIPTION species production which, in turn, affected FACTOR ILR3 PROMOTES PLANT DEFENCE salicylic acid and ABA signalling pathways. AND DROUGHT STRESS TOLERANCE IN Remarkably, infected plants showed NICOTIANA SPP. increased ABA biosynthesis and higher F. APARICIO AND V. PALLÁS water content under drought stress Department of Molecular and Evolutionary Plant conditions. Our results establish a Virology.Instituto de Biología Molecular y Celular de molecular link between the viral infection Plantas (IBMCP) (UPV-CSIC). Ingeniero Fausto Elio mechanism and host adaptation to specific s/n, 46022 Valencia, Spain. extreme environmental conditions, and Address correspondence to [email protected]; thus shed light on the mechanisms driving [email protected] these beneficial interactions.

During virus infection, the interaction of (PO 72) specific viral components with host factors elicits the transcriptional reprogramming INSIGHTS INTO VIROID RNA STRUCTURE of diverse cellular pathways. These AS REVEALED BY ATOMIC FORCE alterations can lead to the development of MICROSCOPY disease symptoms, although recent M. MORENO1, L. VÁZQUEZ2, M.A. LÓPEZ- evidence has revealed that some CARRASCO3, J.A. MARTÍN-GAGO1,2, R. pathogenic viruses might benefit the plant FLORES3, C. BRIONES1,4 host under specific extreme environmental 1. Department of Molecular Evolution, Centro de conditions. The molecular bases of this Astrobiología (CSIC-INTA), Torrejón de Ardoz, phenomenon are, however, poorly Madrid, Spain 2 understood. In this work we show that the . Instituto de Ciencia de Materiales de Madrid (CSIC), Cantoblanco, Madrid, Spain coat protein of Alfalfa mosaic virus directly 3 interacted with ILR3, a transcription factor . Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), Valencia, Spain (TF) belonging to the basic helix–loop–helix 4. Centro de Investigación Biomédica en Red de (bHLH) family which, in combination with Enfermedades Hepáticas y Digestivas. (CIBERehd), other members of the family, has been Spain proposed to participate in diverse metabolic pathways. Our findings indicate Viroids are small (250-400 nt), non-protein- that in Nicotiana ssp this interaction coding, circular RNAs that depend on interfered with the ILR3 subcellular sequence/structure motifs for recruiting

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the host plant proteins they need for 3D surface profile of the imaged sample replication, movement, and circumvention without requiring any staining or coating, of defensive barriers (1, 2). Data derived thus minimizing the structural disruption of from in silico and in vitro approaches, the biological entity under study. This together with in vivo evidence in specific feature, together with its nanometer cases, support that viroid RNA genomes resolution, makes AFM an increasingly are largely self-complementary, folding up used technique in different fields of on themselves into collapsed secondary virology (4). Based on our previous structures in which stretches of optimization of AFM-based protocols for nucleotides forming Watson-Crick pairs are analyzing structured RNA molecules (5), we flanked by loops without apparent have been able to conduct a comparative structure. However, sound evidence shows structural analysis of three different viroid that complex arrays of non-canonical pairs RNAs belonging to the families stabilize such loops, in particular those Pospiviroidae (PSTVd) and Avsunvioidae appearing in the rod-like secondary (ELVd and PLMVd) in different ionic structure characteristic of potato spindle conditions. The main results obtained will tuber viroid (PSTVd) and most other be presented. members of the family Pospiviroidae, 1. Diener (2003). Nat. Rev.Microbiol. 1, 75. which are critical for replication in the 2. Flores et al. (2012). Front. Microbiol. 3, 217 nucleus and systemic trafficking. In 3. Hansma et al. (2004). Curr.Opin.Struct.Biol. 14, contrast, members of the family 380. Avsunvioidae like eggplant latent viroid 4. Kuznetsov et al. (2010). Nucleic Acids Res. 38, (ELVd) and peach latent mosaic viroid 8284. (PLMVd), which replicate in plastids, adopt 5. García-Sacristán et al. (2015). Nucleic Acids Res. bifurcated or clearly multibranched 43, 565. conformations occasionally stabilized by kissing loop interactions required for viroid (PO 74) viability in vivo. However, data on the EVOLUTION OF GII.4 NOROVIRUS three-dimensional (3D) structure of viroid VARIANTS IS DRIVEN BY IMMUNE RNA genomes are still required. PRESSURE AS DETERMINED BY To get a deeper insight into viroid MONOCLONAL ANTIBODY REACTIVITY structure, we have used atomic force PATTERNS microscopy (AFM) to analyze genomic N. CARMONA VICENTE, S. VILA VICENT, J. viroid RNAs in native conditions. AFM is a RODRÍGUEZ DÍAZ, J. BUESA nanotechnology-based tool particularly Departamento de Microbiología, Facultad de well suited for the structural Medicina, Universidad de Valencia, Avda. Blasco characterization of a wide range of Ibáñez 17, 46010 Valencia, España biological entities, including RNA molecules of different lengths, RNA-RNA and RNA- Human noroviruses are responsible for protein complexes (3). One of the main most epidemic outbreaks and many advantages of AFM over electron sporadic cases of acute gastroenteritis microscopy techniques is that it provides a

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worldwide. Noroviruses are also the main showed a great blocking activity of viral water- and foodborne viruses. In the last binding to their receptors in saliva and in years, norovirus GII.4 has emerged as the Caco-2 cells, suggesting that the epitope most prevalent genotype. This high recognized by this antibody is associated to prevalence might be explained by several the receptor binding region of the virus. causes, including viral fitness and These results demonstrate that the evolutionary mechanisms that promote immune system drives the evolution of the escape from the immune system. GII.4 noroviruses, selecting new epidemic The aim of the present work was to variants that are able to escape from determine the ability of GII.4 noroviruses blocking antibodies. More interestingly, we to escape the immune response during its show that both older and newer variants evolution process. Due to the lack of a are not blocked by our monoclonal robust human norovirus replication ‘in antibody, giving the opportunity to the re- vitro’ system we cloned and expressed emergence of older variants. This fast several epidemic variants of norovirus GII.4 evolving profile of noroviruses to escape P particles in E. coli, including variants from the immune system might also explain the 1996 to 2012. We also produced VLPs from poor protection observed after natural the GII.4-2006b variant in the baculovirus infections and the high rate of reinfections expression system. Mice were immunized caused by noroviruses. with VLPs from the 2006b variant. After fusion of splenocytes and clonal selection (PO 75) of hybridomas, the 3C3G3 monoclonal antibody was obtained. A rabbit was also ROLE OF VIMENTIN FILAMENTS IN immunized with the P particles of the VA VACCINIA VIRUS ASSEMBLY AND 387 GII4 variant (1996) to obtain a rabbit MATURATION a a polyclonal antiserum. The antibodies were M.J. RODRÍGUEZ , J.CHICHÓN , S. c a characterized by ELISA and Western blot GUTIÉRREZ-ERLANDSSON , J.J.CONESA , a a,b against the repertoire of P particles. The M.ESTEBAN , JOSÉ L. CARRASCOSA ability of these antibodies to block the a Department of Structure of Macromolecules, binding of norovirus VLPs to their Centro Nacional de Biotecnología-CSIC (CNB-CSIC), receptors was also assayed by ELISA. Madrid, Spain bInstituto Madrileño de Estudios Avanzados en Although the polyclonal antibody showed a Nanociencia (IMDEA Nanociencia). Madrid, Spain. wide recognition capability, differences in c Servicio de Confocal, Centro Nacional de its reactivity towards the non-homologous Biotecnología-CSIC (CNB-CSIC), Madrid, Spain. P particles and the homologous P particles were found. More interestingly, the Vaccinia virus (VV) is one of the most monoclonal antibody 3C3G3 was very studied members of the poxvirus family. effective recognizing its homologous P The interaction of vaccinia with particles (GII.4-2006b) and the related microtubules and actin cytoskeleton has GII.4-2008 P domain, but reacted very been extensively studied. The involvement poorly against the earlier (1996) and newer of vimentin intermediate filaments with variants (2012). Furthermore, this antibody

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vaccinia virus as a support of the (PO 76) foci of VV factories has also been EFFECT OF THE INTRODUCTION OF previously suggested. CHARGED RESIDUES AT THE In our studies of cells infected with VV, the DIMERIZATION INTERFACE OF FOOT AND use of the steroidal lactone withaferin A, a MOUTH DISEASE VIRUS 3A PROTEIN potent vimentin inhibitor, has allowed us Y. A. VIEIRA, 1, F. SOBRINO, 1,2; M. F. to demonstrate the crucial importance of ROSAS, 1 this cytoskeletal intermediate filament in 1Centro de Biología Molecular Severo Ochoa, the establishment of the viral infection. We Madrid, Spain. have combined confocal microscopy and 2Centro de Investigación en Sanidad Animal, INIA, the imaging of thin sections of cultured Madrid, Spain. cells by transmission electron microscopy (TEM). These techniques allowed us to The FMDV non-structural 3A protein evaluate the role of vimentin in VV interacts with other viral and cellular assembly and maturation both in the proteins and can form intermolecular, context of the whole cell and at an antiparallel 3A dimers whose biological ultrastructural level. The confocal images function is unknown. By homology with show that, during the infection, a other picornavirus 3A proteins, the rearrangement of cytoskeletal vimentin presence of two α-helices, located at intermediate filaments takes place around residues 25-33 and 37-44, which form a viral factories. The presence of vimentin dimerization interface is predicted. bundles around the factories was According to this model, the hydrophobic confirmed by TEM using a specific interactions established between the embedding method of cell monolayers and residues M29-L41, M29-I42 and L38-L38 serial sectioning, and it was further contribute significantly to the stability of demonstrated by using vimentin-specific the dimer. Here we describe that antibodies. In addition, the use of X-Ray replacements M29D, M29R, I42D and I42R, tomography has solved the three which result in acquisition of charged dimensional cage of vimentin around the residues, did not significantly affect in vitro viral foci, in a near-to-native state of the viral RNA translation and polyprotein infected cells. The inhibition of this processing or the cellular distribution and vimentin network with withaferin A the ability to form dimers of transiently induced a significant reduction in the expressed 3A protein. However, preserving production of viral factories. the hydrophobic character of residues 29 Our work demonstrates that the and 42 was shown essential for virus establishment of the viral factory requires multiplication in cultured cells, since an active role of the vimentin cytoskeleton, transfection with RNA of these mutants which must provide an scaffold to localize only allowed recovery of infectious viruses and concentrate viral components at the that selected amino acid replacements in perinuclear site. the dimerization interface. Thus, following transfection of RNA with replacement

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I42R, the substitution selected in the virus in previous entry studies, which were recovered, R42L, restored the attributed to be of macrophage origin, hydrophobicity of the residue. Upon lacked the characteristic expression transfection with RNA containing profile.Then, we conducted our study after replacement M29R the virus recovered characterization of porcine macrophages selected first a mutation, I42L, in a residue obtained by alveolar lavage based on a set predicted to interact with M29, being an of specific surface markers including CD163 additional substitution at the C-ter of the and CD169. protein, S140F, observed after further For efficient cell entry, animal viruses growth of the virus. employ several strategies. Virus entry is a complex process in which virus particles (PO 77) should cross the cell membrane and to AFRICAN SWINE FEVER VIRUS INFECTS release their genome at the right location MACROPHAGES, THE NATURAL HOST to complete efficient transcription and CELLS, VIA CLATHRIN- AND CHOLESTEROL- replication. In this study, we used chemical DEPENDENT ENDOCYTOSIS agents and molecular methods to investigate the cellular mechanism I. GALINDO1, M.A. CUESTA-GEIJO1, K. 3 2 exploited for ASFV entry into the natural HLAVOVA , R. MUÑOZ-MORENO , L. target cell and compared ASFV (including BARRADO-GIL1, J. DOMÍNGUEZ1 AND C. 1 the ASFV adapted isolate Ba71V and a ALONSO virulent field isolate (608 VR13) with low 1 Department of Biotechnology, Instituto Nacional passage number in culture), with vaccinia de Investigación y Tecnología Agraria y Alimentaria (INIA), Madrid, Spain virus (VV), which apparently involves 2 different entry pathways. Our results show Present address: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, that ASFV uses endocytosis to infect host USA cells and takes advantage of several 3 Present address: Department of Immunology, endocytic pathways to initiate infection. Veterinary Research Institute, Brno, Czech Republic ASFV enters porcine macrophages by a dynamin-dependent endocytic pathway The main cellular target for African swine involving clathrin. The first steps of fever virus (ASFV) is the porcine infection are also strongly pH-dependent. macrophage. Since early studies, it was The presence of cholesterol in cellular known that ASFV entry in macrophages is membranes was found to be essential for a mediated by saturable binding sites on the productive ASFV infection while actin- plasma membrane. However, the dependent endocytosis and the receptor/s for the virus is not yet participation of phosphoinositide-3-kinase characterized. This virus naturally (PI3K) activity were other cellular factors replicates in porcine macrophages and required in the process of viral entry. monocytes and successful infection is These findings improved our linked to the expression of the CD163 understanding of the ASFV interactions scavenger receptor. In fact, cell lines used with macrophages that allow for successful viral replication.

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(PO 78) between the months of January (18%), GENERAL CHARACTERISTICS OF 100 February (37%) and March (14%), although PEDIATRIC PATIENTS WITH ACUTE cases were detected in every month of the RESPIRATORY INFECTION CAUSED BY year except June. The main symptoms BOCAVIRUS were fever> 38 ° C (79%), cough (38%), J.REINA1, A.IÑIGO1, F.FERRÉS2, V.LÓPEZ- cold symptoms (21%) and influenza-like COROMINAS2 symptoms (24%). The defintive clinical diagnoses were: 18% bronchiolitis, 15% 1Virology Unit, Microbiology Service, 2Pediatric Service. University Hospital Son Espases, Palma de pneumonia, and 11% bronchitis. 8% had Mallorca, Spain. some underlying disease, 3% were cancer, 2% premature and 2% asthmatics. 36 patients (58.4% female) required Introduction: Acute respiratory infections hospitalization with a mean age of 15.7 (ARI) of viral etiology are very frequent in months. In these patients bocavirus children (<15 years) during the winter. detected exclusively in 55.5% of cases and Most are caused by RSV, adenovirus and as a mixed infection in the rest; the influenza viruses. The Bocavirus have been predominant virus in these cases was also implicated in this disease with varying rhinovirus. 44% of patients received frequency depending on the country. antibiotic treatment (60% Material and Methods: A prospective study amoxiclin/clavulanic acid). No patient died (February 2013-February 2015) on this directly or associated with the bocavirus type of respiratory infections is presented. infection. All patients with suspected IRA were taken Conclusions: The acute respiratory a throat swab. The detection of bocavirus infections caused by bocavirus are not was performed by a commercial RT-PCR frequent (4.4%) which mainly affects the real time (Anyplex RV16; Seegen, South community infant population (<18 Korea). months). The predominant symptom fever Results: During the study period were and symptoms of upper respiratory tract. analyzed 4,587 respiratory specimens, of which 2,243 (48.8%) were considered positive for respiratory viruses. We detected 100 cases of ARI caused by Bocavirus (2.1% of all processed samples and 4.4% of the positive samples). In 49 cases the bocavirus was detected as a single virus and 51 cases a mixed viral infection was detected, the main co- infecting virus were rhinovirus, RSV, adenovirus and influenza A. The 51% of cases were girls and 49% boys; the mean age of patients was 16.8 months (range 3 days-14 years). 69% of all cases occurred

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(PO 79) screened using serological and molecular INFECTIOUS AETIOLOGY OF COMMUNITY approaches in plasma or in respiratory ACQUIRED PNEUMONIA IN PAEDIATRIC samples. Blood or pleural fluid cultures HOSPITALIZED PATIENTS (when thoracocentesis was indicated) were M RODRÍGUEZ-DOMÍNGUEZ 1,2, C also performed. Disease severity was VÁZQUEZ 3, L MARTÍNEZ 1; A COCA 3, D evaluated according to clinical, analytical MARTÍN 4, A TAGARRO 5, R CANTÓN 1; E and outcome data. OTHEO 3, JC GALÁN 1,2 RESULTS: An infectious agent could be 1Servicio de Microbiología. Hospital Universitario associated to CAP in 52/66 (78%) patients Ramón y Cajal. Instituto Ramón y Cajal de Virus were detected in 49/66 patients (74 Investigación Sanitaria (IRYCIS), Madrid.2Centro de %), being more than one virus detected in Investigación Biomédica en Red en Salud Pública 3 nearly 40% (19/49). Rhinovirus was the (CIBERESP). Servicio de Pediatría. Hospital most common virus (20/49, 40%), followed Universitario Ramón y Cajal. Universidad de Alcalá, Madrid. 4Laboratorio de Microbiología. Laboratorio by adenovirus (8/49, 16%), RSV and Central BR Salud. Hospital Universitario Infanta metapneumovirus (MPV) (7/49, 14%, Sofía. San Sebastián de los Reyes, Madrid. 5Servicio each). Isolated bacterial pathogens were de Pediatría. Hospital Universitario Infanta Sofía. only found in 5/66 (7.5%) cases. The San Sebastián de los Reyes, Madrid.Spain. microorganisms detected in these bacterial infections were 2 Sp, 2 Cp and 1 Mp. In BACKGROUND/OBJECTIVE; Community 6/66 samples (9%) viral and bacterial acquired pneumonia (CAP) is a major cause agents were detected simultaneously. of morbidity in paediatric population in Interestingly, 4/5 infections caused by Mp developed countries and an important were also associated with viral infection. cause of infant death in developing ones. Serological test used for Cp yielded IgM Our aim was to define current bacterial positive in 9 cases but seroconversion was and/or viral causes of CAP in paediatric not observed and only 2/9 were PCR patients admitted to two University positive for this pathogen. Regarding Hospitals in Madrid. disease severity, mixed infections of virus- METHODS: For two years (April 2012-April bacteria were significantly associated to 2014), 66 patients (2months-17 years old), complicated pneumonia (p=0,047). Among with clinical of lower respiratory tract virus-virus coinfection, the presence of infection and a radiographic pulmonary MPV was associated to a worse outcome infiltrate or condensation, were studied (complicated pneumonia and need for with a large microbiological diagnostic oxygen-therapy > 4 days) (p=0,043) yield. Streptococcus pneumoniae (Sp) PCR CONCLUSIONS: in blood and urinary antigen detection was - Viruses should be considered as a major performed at admission. Sixteen cause of CAP in paediatric population. respiratory viruses were investigated by a - Mixed infections are commonly found. multiplex PCR (Luminex xTAG RVP). Viral-bacterial and the presence of certain Microbial agents associated to atypical viruses could be related with a worse pneumonia, Mycoplasma pneumoniae (Mp) and Chlamydia pneumoniae(Cp) were

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outcome, although these items are not availability of combination antiretroviral clear nowadays. therapy (cART). Initially, cART was strongly - Attending to bacterial agents were only recommended only in infants with HIV- identified in 16% patients, a fast diagnosis related symptoms. However, during recent based on multiplexed PCR may reduce years, multiple studies have suggested the antibiotic overuse. benefit of starting early cART in all HIV-1- infected infants. Therefore, international

guidelines are now recommending (PO 80) initiation of cART in all HIV-1-infected ESTABLISHMENT AND REPLENISHMENT infants aged less than one year regardless OF THE VIRAL RESERVOIR IN PERINATALLY of clinical and immunological conditions. HIV-1-INFECTED CHILDREN INITIATING Despite cART generally suppresses the VERY EARLY ANTIRETROVIRAL THERAPY replication of HIV-1, it does not cure the M. MARTÍNEZ-BONET1, M.C. PUERTAS2, C. infection, because proviruses persist in FORTUNY3, D. OUCHI2, J. MELLADO4, P. stable latent reservoirs. In addition, it has ROJO5, A. NOGUERA- been proposed that low-level proviral JULIÁN3, M.A. MUÑOZ-FERNÁNDEZ1,*, J reservoirs might predict longer virologic MARTINEZ-PICADO 2,6,7,* control after discontinuation of treatment. 1 Hospital General Universitario Gregorio Marañón, Our objective was to evaluate the impact Madrid, Spain. Instituto de InvestigaciónSanitaria of very early initiation of cART and Gregorio Marañón, Madrid. Networking Research temporary treatment interruption on the Center on Bioengineering,Biomaterials and size of the latent HIV-1 reservoir in Nanomedicine (CIBER-BBN). Spanish HIV HGM vertically infected children. This BioBank, Madrid, Spain retrospective study included 23 perinatally 2 AIDS Research Institute IrsiCaixa, Institut d’Investigació en Cièncias de la Salut GermansTrias i HIV-1-infected children who initiated very Pujol, Universitat Autònoma de Barcelona, early treatment within 12 weeks after birth Badalona, Spain (n=14), or early treatment between week 3 Unidad de Enfermedades Infecciosas, Servicio de 12 and 1 year (n=9). We measured the Pediatría, Hospital Sant Joan de Déu,Universitat de proviral reservoir (CD4+ T-cell–associated Barcelona, Esplugues del Llobregat, Spain HIV-1 DNA) in blood samples collected 4 Servicio de Pediatría Hospitalaria y E. Infecciosas y beyond the first year of sustained virologic Tropicales Pediátricas. Hospital suppression. We found a strong positive Universitario Infantil LA PAZ- H. Carlos III, Madrid. correlation between the time to initiation 5 Servicio de Pediatría. Hospital 12 de Octubre, of cART and the size of the proviral Madrid, Spain reservoir. Children who initiated cART 6 Universitat de Vic – Universitat Central de Catalunya (UVic-UCC), Vic, Spain within the first 12 weeks of life showed a 7 proviral reservoir sixfold smaller than Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain children initiating cART beyond this time (p<0.01). Rapid virologic control after initiaton of cART also limits the size of the AIDS-related mortality in children has viral reservoir. However, patients who decreased significantly with the wide underwent transient treatment

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interruptions showed a dramatic increase The samples were subjected to the in the size of the viral reservoir after detection of different respiratory viruses discontinuation. In summary, our study by a commercial molecular amplification highlights the importance of very early technique RT-PCR real time (Anyplex RV16, initiation of cART, if possible within the Seegen, South Korea). first 12 weeks of life, and the benefit of Results: Over the three epidemic seasons optimal virologic control during the first we have detected 601 cases of acute years of life in order to limit the size of the respiratory infection caused by RSV. viral reservoir. Moreover, this study Although overall 83.2% of all cases suggests that treatment interruptions occurred in children under two years, should be undertaken with caution, as they there have been differences in seasons might lead to fast and irreversible with a possible trend towards older than replenishment of the viral reservoir. this age. Last season analyzed 78.6% of all cases (PO 81) occurred in children under 2 years AGE DISTRIBUTION OF PEDIATRIC ACUTE compared to 83.3% and 87.3% of the RESPIRATORY INFECTIONS CAUSED BY previous two seasons. The 21.3% of RESPIRATORY SYNCYTIAL VIRUS patients with ARI caused by RSV in the J.REINA1, A.IÑIGO1, F.FERRÉS2, V.LÓPEZ- 2014/2015 season had over 2 years of age. COROMINAS2 Also since last season has been observed, although not significant, increase in cases 1Virology Unit, Microbiology Service, 2Pediatric Service. University Hospital Son Espases, Palma de detected in patients over four years. The Mallorca, Spain. percentage of cases that occur in the first month of life, with 11.4% overall seems to have stabilized. This group together, that Introduction: Acute respiratory infections of children under 6 months (39.7%), (ARI) caused by RSV is the leading cause of represent 51.4% of all cases. this type of infection during the winter months, especially in the lower age at 2 Conclusions: Studies of age distribution of years. Classically they preferably presented RSV infections are very important to in children under 6 months and its prevent the possibility of infection by spectrum is extended to 2 years old. In vaccination of pregnant mothers. In our recent years we have detected a slight slip study, this type of vaccination would of the affected population to higher ages. prevent hypothetically and with a vaccine efficacy of 100%, 51.4% of cases detected Material and Methods: From this in the first six months of life, reducing by observation a prospective study on the age half the workload of this respiratory distribution of ARI cases caused by RSV disease. was conducted along epidemic seasons 2012/13, 2013/14 and 2014/15. All patients attending the emergency department with a clinical compatible with ARI were taken a nasopharyngeal aspirate.

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(PO 82) Adeno, bioMerieux), although both EVIDENCE OF NOSOCOMIAL children were fully vaccinated with TRANSMISSION OF A ROTAVIRUS STRAIN RotaTeq (Merck Sharp & Dohme BV). There TO TWO PREVIOUSLY VACCINATED was no known contact between them. INFANTS A genotyping assay by conventional RT- P.F. SÁNCHEZ-LÓPEZ1,2, C. SALVADOR PCR, and subsequent semi-nested GARCÍA1, M. ITURRIZA-GÓMARA3, K. JERE3, multiplex PCR was performed in stool S. VILA VICENT4, J. BUESA GÓMEZ4, M. samples from both children, G and P SEGOVIA HERNÁNDEZ1 genotyping the strains, and partially 1. Hospital Universitario Virgen de la Arrixaca, amplifying 6 of the 11 fragments of the Murcia, Spain rotavirus genomic segments that encode 2. Servicio de Sanidad Ambiental. Dirección General the NSP1, NSP2, NSP4, VP4, VP6 and VP7 de Salud Pública y Drogodependencias. Consejería proteins. Forward and reverse amplicons de Sanidad y Política Social de la Región de Murcia, were sequenced with Applied Biosystems Spain BidDyeTM Fluorescent Terminator System, 3 . Institute of Infection and Global Health, University and the sequences were aligned using of Liverpool, UK ClustalW. The partial sequences of the 4. Universidad de Valencia, Spain NSP1 (936 bp), NSP2 (930 bp), NSP4 (692 Contact: [email protected] bp), VP4 (605 bp), VP6 (319 bp) and VP7 (836 bp) genomic fragments from both Nosocomial transmission of rotaviruses has strains were analyzed using MEGA version been extensively documented. We 6. Genotypes were confirmed using the describe the nosocomial transmission of a automated genotyping tool RotaC rotavirus strain to two previously (htpp://rotac.regatools.be). vaccinated infants. The genotype of both strains was G9P8. A The first child was admitted to the 100% similarity between both strains was University Hospital Virgen de la Arrixaca in observed, except for the VP7 fragment, Murcia, Spain, for asthma attack, and which differed in two nucleotides (99.76% developed loose stools five days after similarity). admission. The child was discharged on We conclude that both strains share a high eighth day post-admission. The second degree of identity, and that the virus was child was admitted to the hospital for eye probably transmitted within the hospital. swelling and fever, and was allocated the The incubation time in both cases was bed previously occupied by the first child relatively long, and rules out community two days ago. This second child started acquisition. This strain caused disease in developing loose stools and vomiting six both children, despite the fact that days post-admission, and was discharged RotaTeq contains the P8 type of the VP7 after eight days in the hospital. protein of the virus, suggesting a vaccine Both patients were positive for rotavirus failure. Further studies are being antigen in stools by an conducted for fully characterize these immunochromatographic test (VIKIA®Rota- strains, sequencing their whole genome.

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This report highlights the risks of a of24hours of evolution consisting on nosocomial rotavirus transmission, even postration and vomiting.Exploration did among vaccinated children. In the future, not show erythema or exanthema. studies that also include monitoring Blood tests showed metabolic asymptomatic shedding of rotavirus in the acidosis,leukocytosis and anemia,while hospital environment, may be useful in chest x-ray showed an increased order to assess the risk of transmission. retrocardiac density and cardiothoracic ratio.Finally, sinus tachycardia and other (PO 83) signs consistent with right ventricular FATAL MYOCARDITIS BY hypertrophy,together with Qwaves in DII IN CHILDREN were observed on the electrocardiogram. J.M. CAPELO MÍGUEZ1; J. FONTENLA After 6 hours,the patient started with a GARCÍA1; N. BALADO INSUNZA1; J. GARCÍA sharp respiratory deterioration with COSTA1; A. PÉREZ PEDROSA1; M. affectation of the overall CABRERIZA2; J.E. ECHEVARRÍA MAYO2. state,hepatomegaly and impaired distal perfusion. 1Complejo Hospitalario Universitario de Ourense, Ourense, Spain The echocardiogram showed a dilated left 2ISCIII Majadahonda, Madrid, Spain ventricle with impaired cardiac function,compatible with severe cardiogenic shock,followed by Introduction: Cardiomyopathies are cardiorespiratory arrest requiring defined as"an acute or chronic resuscitation maneuvers cardiopulmonary inflammatory process in the absence of advanced without success. ischemia produced by a variety of toxins,drugs or infectious agents, in Microscopic examination of the hearth association with myocardial tissue revealed intense areas of dysfunction,and confirmatory diagnosis inflammatory lymphocytic infiltrates,with established intense nuclear positivity.Parvovirus B19 histopathological,histochemical and DNA was amplified by PCR on myocardial molecular criteria”. tissue, as well as in ascetic, pleural and pericardial fluids.Inmunohistochemistry Incidence real of myocarditis in pediatric showed parvovirus B19antigens on age is unknown because of its broad endothelial cells of pericardial and spectrum of clinical presentation. epicardial vessels RT-PCR for human Clinical suspicion is a challenge for the enteroviruses and parechoviruses were clinician due to the absence of a validated negative.All the findings were interpreted gold standard test for diagnosis,its as indicative of multiorganic damage with insidious clinical presentation and its signs of viral process of myocarditis by variable evolution. parvovirus B19. Case Presentation Discussion and Conclusion: Myocarditis is A 16month infant was admitted in the a particularly important and Intensive Care Unit with symptoms heterogeneous entity in childhood. It is a

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major cause of morbidity and mortality in Background. It is not established if children, being the major cause of coinfections are more severe than single unexplained sudden death.Parvovirus viral respiratory infections. B19is currently the most common known Objective. Studying and comparing simple etiologic agent of pediatric viral infections and viral coinfections of myocarditis.It affecting the coronary respiratory syncytial virus (RSV) in endothelium and causing dysfunction and hospitalized children. myocardial ischemia. Patients & Methods. From September The initial clinical diagnosis of myocarditis 2005 to August 2013 a prospective study relied on the finding of lymphocytic was conducted on children under 14, infiltrate and myocardial admitted with respiratory infection to the degeneration,since there is no additional Pediatrics Department of the Severo test diagnostic of noninvasive Ochoa Hospital, in Spain. Specimens of myocarditis.The pathologic examination nasopharyngeal aspirate were taken for confirmed the diagnosis of suspicion.The virological study by using polymerase chain implication of B19DNA as the etiological reaction, and clinical data was recorded. agent was initially suggested by the Simple RSV infections were selected and positive PCR on myocardial tissue and compared with double infections of RSV fluids and confirmed by with rhinovirus (RV) or with human immunohistochemistry.The implication of bocavirus (HBoV). other viruses associated with cardiac Results. 2993 episodes corresponding to disease as enteroviruses was discarded.The 2525 children were analyzed. At least one initial clinical diagnosis is crucial to virus was detected in 77% (2312) of the optimize the treatment and transfer of episodes. Single infections (599 RSV, 513 patients to a referral hospital with heart RV and 81 HBoV) were compared with 122 transplant program. double infections RSV-RV and 61 RSV- HBoV. The RSV-RV coinfections had fever (PO 84) more often (63% vs. 43% p <0.001) (), and RESPIRATORY SYNCYTIAL VIRUS: hypoxia (70% vs. 43%; p <0.001) than RV COINFECTIONS WITH RHINOVIRUS AND infections. Hypoxia was similar beween HUMAN BOCAVIRUS IN HOSPITALIZED single or dual infectins (71%) . Bronchiolitis INFANTS were more frequent in RSV simple group C CALVO1, ML GARCÍA-GARCÍA 1, F POZO 2, (p<0.001). The PICU admission was more I CASAS2. common in RSV simple or RSV-RV group than in the RV monoinfection (p = 0.042). 1 Pediatrics Department.Severo Ochoa Hospital.Leganés. Madrid. Spain 2Respiratory Virus Hospitalization was longer for both the RSV and Influenza Unit. National Microbiology Center simple group and RSV-HBoV coinfection, (ISCIII), Madrid, Spain. about 1 day (4.7 vs 3.8 days, p <0.001) longer than in the simple HBoV infections. There were no differences in PICU

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admission. RSV single group were of associated to RSV A and RSV B were younger age than the other groups. selected and compared. Conclusions: Coinfections between RSV-RV Results. 3278 episodes of viral respiratory and RSV-HBoV are frequent. Overall viral infections were analyzed. RSV was coinfections do not provide greater detected in 1019 (31%); 619 (61%) cases severity, but have mixed clinical features. were RSV A, 244 (24%) RSV B and 156 could not be typed. Infections were (PO 85) present mainly in November-January, the mean each was 1 year (median 6 months), CLINICAL COMPARISON OF RESPIRATORY and the most frequent diagnosis was SYNCYTIAL VIRUS INFECTIONS SUBTYPE A bronchiolitis (57.4%). 586 (77%) of children VS SUBTYPE B IN HOSPITALIZED had fever, and 611(70%) hypoxia. Infiltrate CHILDREN. in Rx was present in 340 patients (49%). 1 1 ML GARCÍA-GARCÍA , C CALVO , C 35% of cases had a coinfection (mainly 3 3 2 2 LLORENTE , S DE BLÁS , F POZO , I CASAS . with rhinovirus). The hospitalization was of 1 Pediatrics Department.Severo Ochoa 4.7 + 2.5 days. Only 23 (2.7%) infants 2 Hospital.Leganés. Madrid. Spain Respiratory Virus needed PICU admission. We compared the and Influenza Unit. National Microbiology Center 3 clinical data of the total group, and also, (ISCIII), Madrid, Spain. Estudiante de 6º Curso de Medicina. Universidad Alfonso X el Sabio. Madrid. the patients diagnosed of bronchiolitis, the patients diagnosed of recurrent wheezing and of pneumonia and we did not find any Background. Although respiratory syncytial difference between the patients with RSV virus (RSV) infections are the most A or B. Single infections; 396 RSV A and important cause of hospitalization in 160 RSV B were also compared and no infants and has been extensively studied, is differences amongst them were detected. not well established if subtype A or B are associated to different severity. Some Conclusions: Respiratory viral infections authors consider than RSV B is more due to RSA virus in hospitalized children serious. have no different clinical characteristics associated to type B or A. RSV B infections Objective. To evaluate the relationship have not more severity. between the severity of bronchiolitis and RSV subtype in hospitalized children. Patients & Methods. From September 2005 to August 2014 a prospective study was conducted on children, admitted with respiratory infection to the Pediatrics Department of the Severo Ochoa Hospital, in Spain. Specimens of nasopharyngeal aspirate were taken for virological study by using polymerase chain reaction, and clinical data were recorded. Infections

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(PO 86) was detected in 76.5% (2504) of the HUMAN BOCAVIRUS IN HOSPITALIZED episodes, 727 coinfections (22%). RSV and CHILDREN AND COMPARISON WITH RV were the most frequent (31% each OTHER RESPIRATORY VIRUSES one). A total of 320 episodes were C CALVO1, ML GARCÍA-GARCÍA 1, D. associated to hBoV (9.8%), 80 single CARBALLO3, E MATÍNEZ-MONTESERÍN3, F infections, and 240 coinfections. Single POZO 2, I CASAS2. hBoV infections were mainly in November and December (50%), in children of 1 Pediatrics Department.Severo Ochoa Hospital.Leganés. Madrid. Spain 2Respiratory Virus 24.7+24 months of age. 69% had fever, and Influenza Unit. National Microbiology Center 52% hypoxia and 47% infiltrate in X-ray. (ISCIII), Madrid, Spain. 3Estudiante de 6º Curso de Recurrent wheezing (60%) and pneumonia Medicina. Universidad Alfonso X el Sabio. Madrid. (22%) were the most common diagnoses. Coinfections of hBoV with other viruses Background. The clinical characteristics of had higher proportion of bronchiolitis human bocavirus (hBoV) infections and its 27.5% vs 15.5%, p<0.0001 and less role are not yet well established. pneumonia). Objective. Prospective study of infections Single infections (665 RSV, 555 RV and 108 associated to hBoV in hospitalized children hMPV) were compared with single hBoV and comparing simple infections with viral infections (80). RSV single infections infections caused by the most frequent affected younger children (9 vs 24 months, viruses; respiratory syncytial virus (RSV), p<0,001), had more frequently hypoxia, rhinovirus (RV), and human (71% vs 52%, p<0.001), and the most metapneumovirus (hMPV) in the same frequent diagnosis was bronchiolitis (63%, population. vs 17%, p<0.001). Days of admission Patients & Methods. From September (0.002) at hospital and duration of hypoxia 2005 to August 2014 a prospective study (0.04) was longer in RSV group. hBoV was conducted on children under 14, patients had more frequent pneumonia, admitted with respiratory infection to the higher C-reactive protein (p<0.001) and Pediatrics Department of the Severo antibiotic treatment (41% vs 17%, Ochoa Hospital, in Spain. Specimens of p<0.001). nasopharyngeal aspirate were taken for RV children had less frequently fever (69% virological study by using polymerase chain vs 41%, p<0.001), and less pneumonia reaction, and clinical data were recorded. (11% vs 23%, p<0.001). hMPV children had Simple hBoV infections were selected and also less proportion of pneumonia (8.4% vs described. Single infections were 23%, p=0.14) and less CRP (35+41 vs 67 compared with hBoV coinfections with +79, p=0.004) and leukocytosis (p=0.019). other respiratory viruses and also with Conclusions: HBoV infections are frequent single RSV infections, RV infections and in hospitalized children and associate to hMPV infections. fever, pneumonia, increased CRP and Results. 3275 episodes of viral respiratory antibiotic treatment. There are infections were analyzed. At least one virus

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significantly differences with other with temperate climate, with epidemic respiratory virus infections. peaks occurring in winter and spring. Rotavirus vaccination may influence on the (PO 87) fluctuations of circulating rotavirus genotypes, especially in areas with low SURVEILLANCE OF HUMAN G12P[8] vaccine coverage. Significant annual ROTAVIRUS IN SPAIN changes in genotype distribution have 1 1 JAVIER BUESA , SUSANA VILA VICENT , been frequently detected even in the pre- 1 MANUEL FERNÁNDEZ JIMÉNEZ , CRISTINA vaccine era. 1 SANTISO BELLÓN , AINARA ARANA We participate in the European Rotavirus SALABERRÍA2, MILAGROS 2 2 Network, EuroRotaNet, which was MONTES ,GUSTAVO CILLA , JOSEP PRAT established in January 2007 to perform FORNELLS3, ROSA ESCOMS TRULLENQUE3, 4 epidemiological surveillance of rotavirus ROSA BARTOLOMÉ COMAS , RAUL ORTÍZ strains by characterizing their G and P DE LEJARAZU5, JOSÉ M. EIROS BOUZA5, 6 types. Uncommon strains of CRISTINA SERAL GARCÍA , F. JAVIER epidemiological importance are further CASTILLO GARCÍA6,PEDRO F. SÁNCHEZ- 7 8 characterized by analyzing the subgroup LÓPEZ , CARME SALVADOR GARCÍA , JESÚS (VP6) and NSP4 genotype or by whole RODRÍGUEZ DÍAZ1, MIREN ITURRIZA 9 genome sequencing. Overall, 6 genotypes GÓMARA circulate in Europe with a prevalence > 1% 1 Dpto. de Microbiología, Facultad de Medicina, and included G1P[8], G4P[8], G2P[4], Universidad de Valencia, Spain 2 G9P[8], G3P[8] and G12P[8], making up Servicio de Microbiología,Hospital Universitario these six genotypes to 91% of all Donostia, San Sebastián, Spain 3 characterized strains. Laboratorio de Microbiología, Hospital de Sagunto (Valencia), Spain A notorious emergence of G12P[8] strains 4 Servicio de Microbiología, Hospital Val d’Hebrón, was detected in the Basque Country Barcelona, Spain (Northern Spain) in 2004-05, being the 5 Depto. de Microbiología, Facultad de Medicina, predominant genotype in the 2010-11 Universidad de Valladolid, Spain (65% of all strains) and 2011-12 seasons 6 Servicio de Microbiología, Hospital Clínico (81.6%). Whereas the prevalence of this Universitario Lozano Blesa, Zaragoza, Spain genotype declined in the Basque Country 7 Servicio de Sanidad Ambiental. Consejería de to very low levels in 2012-13 (1.2%) and Sanidad y Política Social de Murcia, Spain 8 2013-14 (2.3%), an increase of G12P[8] Servicio de Microbiología. Hospital Clínico strains was detected during 2013-14 in Universitario Virgen de la Arrixaca, Murcia, Spain other Spanish regions (Castilla-León, 9Institute of Infection and Global Health, University of Liverpool, Liverpool, Inglaterra Aragón, Catalonia, Valencia and Murcia), accounting overall for 15.3% of 466 typed

strains. During the 2013-14 season, Group A rotaviruses are one of the leading G12P[8] strains were detected in Valencia causes of acute gastroenteritis in young in 27.5% of rotavirus-positive samples. children worldwide. Rotavirus displays a G12P[8] strains had also been detected in seasonal pattern of infection in countries the feces of pigs in farms of Aragón in

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2006. This finding is interesting, as it G4P[8] and G9P[8]. During the surveillance suggests G12 rotavirus transmission activity of RotaNet-Italy, three uncommon between humans and pigs. Phylogenetic G3P[6] RVA strains, designated as analyses of the VP7 and VP4 genes RVA/Human-wt/ITA/NA01/2009/G3P[6], demonstrated that they belong to lineages RVA/Human-wt/ITA/NA06/2009/G3P[6], III of both genotypes. These strains display and RVA/Human- the typical human Wa-like gene wt/ITA/NA19/2009/G3P[6], were identified constellation, and this may be the key to in stool specimens from children with their recent emergence and spread. diarrhea hospitalized in Southern Italy in Rotavirus G12[P8] should be considered as 2009. an emerging genotype in Spain causing After PCR genotyping following seasonal epidemics like the common standardized EuroRotaNet protocols, human rotavirus genotypes G1–G4 and G9. samples NA01, NA06 and NA19 showed After its emergence, G12[P8] genotype the G3P[6] genotype. Sequencing of the distribution fluctuates year to year and eleven genomic segments was planned and across different geographic regions. performed to characterize the three Continued surveillance of circulating uncommon RVA strains further and rotavirus strains will allow us to know the investigate their origin. RVA strains with a future evolution of this genotype. P[6] P-genotype in association with several G-genotypes have been isolated frequently (PO 88) in Africa, and sporadically also in DETECTION AND CHARACTERIZATION OF developed countries. P[6] RVA strains have UNCOMMON HUMAN G3P[6] ROTAVIRUS been detected in both patients with A STRAINS CAUSING DIARRHEA IN gastroenteritis and asymptomatic children, ITALIAN CHILDREN IN 2009 and P[6] has been established as a major P- genotype among porcine RVAs. G. IANIRO1, R. DELOGU2, L. FIORE2, F.M. RUGGERI1 G- and P- genotyping was performed by reverse transcription-nested polymerase 1.Dept. of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy chain reaction (RT-nPCR), using mixtures of 2.National Center for Immunobiologicals Research primers for either gene 9 or 4. For and Evaluation, Istituto Superiore di Sanità, Rome, sequence analysis, RT-PCR reactions Italy included primers specific for each gene investigated. Multiple sequence Group A rotaviruses (RVA) are the leading alignments and phylogenetic tree cause of acute gastroenteritis (AGE) in construction were performed with MEGA6, young children, causing up to 450.000 applying the Maximum-Likelihood (ML) deaths worldwide, mostly in developing method. countries. Most of RVA infections in NA01, NA06 and NA19 RVA strains were humans across developed areas of the found to possess the unusual genotype planet are related to five major G/P constellation G3-P[6]-I2-R2-C2-M2-A2-N2- combinations: G1P[8], G2P[4], G3P[8], T2-E2-H2. This study reports the first

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detection of uncommon G3P[6] RVA Rotavirus is the major cause of acute strains in human patients in continental gastroenteritis in infants worldwide, Italy. The phylogenetic analysis of the causing every year up to450.000 deaths, eleven genomic segments showed no mostly in developing countries. In Italy, the evidence of zoonosis or inter-species Istituto Superiore di Sanità has reassortment, revealing complete DS-1-like implemented a nationwide laboratory- genomic constellations previously based surveillance of acute rotavirus associated to human cases in Africa and gastroenteritis to investigate the diversity Europe. The analysis of the hypervariable of rotavirus strains circulating before the regions of VP7 and VP4 (VP8*) revealed introduction of large-scale vaccination. high amino acid identity between the RotaNet-Italy is linked to the EuroRotaNet G3P[6] RVA strains involved in this study. network, which includes 17 European The comparison of the G3 RVA strains diagnostic laboratories. investigated in this study and other G3 Since 2007, approximately 9390 rotavirus RVAs characterized previously in Italy positive stool samples were collected from reveals that a large variety of G3 genomic pediatric patients with acute variants have been reported throughout diarrheahospitalized in 14 Italian Regions, Italy, which might be partially related to and were genotyped, following the persisting massive immigration from EuroRotaNet protocols. Significant across the Mediterranean sea. The variation in the frequency of different detection of exotic RVA strains also in rotavirus genotypes was observed developed countries highlights the between different years and areas of Italy. importance of surveillance activity on Most strains belonged to genotypes G1-G4 rotaviruses, similar to other imported and and G9, and P[8] or P[4], commonly found emerging pathogens, in order to prepare in humans worldwide. and control public health threats. Overall, the predominant genotype detected during the seven rotavirus (PO 89) seasons was G1P[8](51%), followed by G9P[8] (16%), G4P[8] (9%), G2P[4] (8%) THE ITALIAN ROTANET SURVEILLANCE and G3P[8] (3%). However, unusual or PROGRAM, 2007-2014: DETECTION OF emerging strains, such as G3P[19], G6P[6], THE EMERGING GENOTYPE G12 G8P[4] and G12P[8], were also detected, R. DELOGU1, G. IANIRO2, F.M. RUGGERI2, L. 1 suggesting either gene reassortment FIORE , AND THE ROTANET-ITALY STUDY events between rotaviruses of different GROUP origin or importation of strains from other 1 . National Center for Immunobiologicals Research countries. In particular, during the 2012-13 and Evaluation, Istituto Superiore di Sanità, Rome, Italy surveillance the spread of the emerging 2 G12P[8] rotavirus genotype was . Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy unexpectedly detected in the Central Italian region of Umbria (75%), and in different regions during the following season 2013-14 (9%).All G12 strains were

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subjected to phylogenetic analysis. (PO 90) Sequence analysis showed a close INFLUENZA A VIRUS NS1 PROTEIN MIMICS nucleotide identity of both the VP4 and ONCOGENIC PI3K RESULTING IN ISOFORM VP7 genes among the G12P[8] strains. The SPECIFIC CELLULAR REDISTRIBUTION AND VP7 gene was similar to other G12 strains ACTIVATION circulating in different years and countries, J. AYLLON1, 4, B. G. HALE2, M. T. SÁNCHEZ- except for the Spanish G12 strains that APARICIO1, 4 AND A. GARCÍA-SASTRE1, 3, 4 showed a lower correlation with the Italian 1Department of Microbiology, 3Department of G12 strains, and clustered separately in the Medicine and 4Global Health and Emerging phylogenetic tree.The VP4 gene was Pathogens Institute, Icahn School of Medicine at closely related to other local and global Mount Sinai, New York, USA P[8] strains showing different G-types. 2Institute of Medical Virology, University of Zürich, Overall findings suggest the introduction Zürich, Switzerland and evolution of a single G12 VP7 gene into the local Wa-like rotavirus population. The non-structural protein 1 (NS1) of Most rotavirus infection occurred in influenza virus performs a broad variety of children <2 years of age, but cases were pro-viral activities in the infected cell, also reported in older subjects, identifying mostly mediating the evasion from the risks of infection through contact with host innate immune response by being the infected children and increased main viral interferon antagonist. However, susceptibility of the elderly population to among the multiple interactions described rotavirus. for this small, multifunctional protein there Data from 7-year RotaNet-Italy surveillance are several whose biological relevance confirm the genetic diversity of rotaviruses remains obscure, such as NS1 ability to circulating in Italy, and the existence of bind to and activate class IA phoinositide- remarkable differences between Regions 3-kinases (PI3K). PI3K are highly regulated and years. Continuous rotavirus strain lipid kinases that act as critical nodes in surveillance in different countries is multiple cell signaling networks that important to obtain a better understanding regulate cellular physiology, including of rotavirus genotype evolution, differentiation, growth, survival, trafficking particularly after vaccine introduction. and immune function. As such, PI3K are also important proto-oncogenes whose deregulation lies behind a great number of different human cancers. Structurally, class IA PI3K are heterodimers formed by a regulatory (p85) and catalytic (p110) subunits, of which there are several isotypes described, adding further layers of complexity to their activity.

In order to unravel the cellular relevance of NS1-activated PI3K, we have developed a

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bimolecular fluorescence CoV) and middle east respiratory syndome complementation (BiFC) assay to (MERS-CoV) cause high case fatality rates selectively track the different regulatory and remain as major human public health and catalytic isotypes of PI3K and their threats. No specific therapy for any human behavior upon activation. Using this coronavirus is available, making vaccine system we found that NS1 induces an development critical for protection against isotype-specific relocation and activation these viruses. Previously, we of the different PI3K heterodimers. demonstrated that a mouse-adapted SARS- However, the effects of other known CoV (SARS-CoV-MA15) lacking the activators of PI3K such as Ras, Src and envelope (E) protein (rSARS-CoV-MA15-E) receptor tyrosine kinases were different is attenuated in vivo. To identify E protein from those induced by NS1. By contrast, regions and host responses that contribute clinically relevant, oncogenic hyper- to rSARS-CoV-MA15-E attenuation, activating mutations in both catalytic and several mutants (rSARS-CoV-MA15-E*) regulatory subunits of PI3K recapitulate containing point mutations or deletions in the effect caused by NS1. We postulate the amino-terminal or the carboxy- that by mimicking an oncogenic terminal regions of the E protein were deregulation of the PI3K pathway influenza generated. We showed that small virus induces a transient, transformed-like deletions and modifications within E status in the infected cell to stimulate virus protein led to virus attenuation, causing replication. minimal lung injury, limited neutrophil influx to the lungs, reduced expression of (PO 91) proinflammatory cytokines, increased anti- inflammatory cytokine levels, and SEVERE ACUTE RESPIRATORY SYNDROME enhanced CD4+ and CD8+ T cell counts in CORONAVIRUS RECOMBINANT VACCINE vivo. These data suggests that the INCLUDING SAFETY GUARDS IN E AND described mutant phenotype contributed NSP1 GENES to virus attenuation. The attenuated 1 1 JA. REGLA NAVA , M. LOPEZ DEDIEGO , JL. mutants fully protected mice from 1 1 NIETO TORRES , JM. JIMENEZ GUARDEÑO , challenge with virulent virus. A major 1 C. CASTAÑO RODRÍGUEZ , R. FERNANDEZ problem of using live attenuated viruses as 1 2 DELGADO , CRAIG FETT , STANLEY vaccines is the possibility of reversion to 2 1 PERLMAN , AND LUIS ENJUANES . virulence. To overcome this limitation, we 1 Department of Molecular and Cell Biology, Centro introduced additional attenuating Nacional de Biotecnología (CNB-CSIC), Darwin 3, mutations into the nsp1 protein to Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain generate a safer vaccine candidate. Nsp1 2Department of Microbiology, University of Iowa, gene was selected as a target because it is Iowa City, Iowa, USA located at a distal position (>20kb) from that of E gene in the viral genome, making the generation of a virulent virusthrough a Coronavirus such as severe acute single recombination event with circulating respiratory syndrome coronavirus (SARS- coronaviruses highly unlikely. To identify

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nsp1 protein regions that contribute to Classical swine fever (CSF) causes major rSARS-CoV-MA15 attenuation, several losses in pig farming, with various degrees mutants (rSARS-CoV-MA15-nsp1*) of disease severity. Efficient live containing small deletions in of the nsp1 attenuated vaccines against Classical swine protein were generated. Deletion of 121 to fever virus (CSFV) are used routinely in 129 and 154 to 165 aminoacids in the endemic countries. However, despite carboxy terminal region of nsp1 protein led intensive vaccination programs in these to virus attenuation. Immunization with areas for more than 20 years, CSF has not single SARS-CoV mutants protected mice been eradicated. Molecular epidemiology against challenge with the lethal parental studies in these regions suggests that the virus. A recombinat virus including safety virus circulating in the field has evolved guards in E and nsp1 genes was generated. under the positive selection pressure This mutant virus was completely exerted by the immune response to the attenuated and protected mice against vaccine, leading to new attenuated viral challenge with the lethal parental virus, variants. Recent work by our group indicating that this virus is promising demonstrated that a high proportion of vaccine candidate. persistently infected piglets can be generated by early postnatal infection with a low and a moderate virulent CSFV (PO 92) strains. Here we studied the immune INEFFICACY OF A LIVE ATTENUATED response to a Hog Cholera Lapinized virus VACCINE IN CLASSICAL SWINE FEVER vaccine (HCLV), C-strain, in 6-week-old VIRUS POSTNATALLY PERSISTENTLY persistently infected pigs following post- INFECTED PIGS: IS THE CONTROL OF natal infection. CSFV-negative pigs were DISEASE ON THE LINE? vaccinated as control. The humoral and 1 1 S. MUÑOZ-GONZÁLEZ , M. PEREZ-SIMÓ , interferon gamma responses as well as the 1 1 M. MUÑOZ , J.A.BOHORQUEZ , R. CSFV RNA load were monitored during 21 1,2 3 ROSELL , A. SUMMERFIELD , M. days post vaccination. The experiments 1,4 3 1* DOMINGO , N. RUGGLI AND L. GANGES were approved by the Ethics Committee 1.Centre de Recerca en Sanitat Animal (CReSA), for Animal Experiments of the IRTA-Universitat Autònoma de Barcelona (UAB), Autonomous University of Barcelona Campus de la UAB, 08193 Bellaterra, Barcelona, (UAB), according to existing national and Spain 2. European regulations.A complete lack of Departament d'Agricultura, Ramaderia, Pesca, Alimentació i Medi Natural, (DAAM), Generalitat de detection of the vaccine viral RNA was Catalunya, Spain found in the serum samples and in the 3. Institute of Virology and immunology (IVI), tonsils from CSFV postnatal persistently Mittelhäusern, Switzerland infected pigs during 21 days post 4. Departament de Sanitat i d’Anatomia Animals, vaccination. Furthermore, lack of response Facultat de Veterinària, UAB, 08193 Bellaterra- to E2 specific antibodies and absence of Barcelona, Spain neutralizing antibody titres were shown in CSFV persistently infected-vaccinated animals. Likewise, absence of IFN-gamma

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producing cells response against CSFV or reported to play either an antiviral or a PHA was also observed. To our knowledge, proviral activity, depending on the virus. this is the first report demonstrating the ISG15 can exert its function either through absence of response to vaccination in CSFV conjugation to target proteins in a process persistently infected pigs. termed ISGylation, or in a conjugation independent manner. Previous studies (PO 93) from our laboratory demonstrated a strong ISG15 up-regulation during in vitro RSV ISG15 INHIBITS RESPIRATORY SYNCYTIAL infection. In this work, a more detailed VIRUS INFECTION THROUGH PROTEIN analysis of the ISG15 role in RSV infection ISGYLATION AT EARLY STAGES OF THE is presented. ISG15 overexpression and VIRAL CYCLE siRNA silencing experiments, along with 1,2 2,3 R. GONZÁLEZ-SANZ , M. MATA , J. ISG15 knockout cells demonstrated an 4 1 BERMEJO-MARTÍN , A. ÁLVAREZ , J. anti-RSV effect of this molecule at early 2,3 2,5 CORTIJO , J.A. MELERO AND I. stages of the virus cycle. Conjugation 1,2 MARTÍNEZ inhibition assays revealed that ISG15 1 Unidad de Infección Viral e Inmunidad. Centro exerts its antiviral activity via protein Nacional de Microbiología, Instituto de Salud Carlos 2 ISGylation. However, this antiviral activity III. Madrid, Spain. Centro de Investigación occurs only when high levels of ISG15 are Biomédica en Red. Enfermedades Respiratorias (CIBERES). Instituto de Salud Carlos III. Madrid, present in cells before RSV infection, as in Spain. 3 Fundación Investigación Hospital General the case of cells previously stimulated with Universitario de Valencia. Valencia, Spain. 4 Hospital interferon. Finally, ISG15 is also up- Clínico Universitario de Valladolid. Valladolid, Spain. 5 regulated in human respiratory pseudo- Unidad de Biología Viral. Centro Nacional de stratified epithelia and in nasopharyngeal Microbiología, Instituto de Salud Carlos III. Madrid, Spain washes from infants infected with RSV, suggesting a possible antiviral role of this molecule in vivo. These results improve our Human Respiratory Syncytial Virus (RSV) is understanding of the innate immune the leading cause of severe lower response induced by RSV and demonstrate respiratory tract infections in children but the antiviral activity of ISG15 against this also a significant cause of morbidity and virus for the first time, thus opening new mortality in the elderly and possibilities for infection control. immunocompromised individuals. Despite an intense research, neither a vaccine nor an effective therapeutic treatment is currently available. A better understanding of the complex interactions between RSV and the host may help to find new therapeutic targets against this virus. Interferon stimulated gen 15 (ISG15) is an ubiquitin-like protein that is highly induced during viral infections and has been

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(PO 94) regulatory factory factor 3) and NF-κB MODULATION OF HOST INNATE IMMUNE (nuclear factor-kappa B). With a similar RESPONSE BY BERNE VIRUS M AND N approach we showed that the M and N STRUCTURAL PROTEINS proteins inhibit the IRF-3 activation. G. NIEVES MOLINA, S. PLAZUELO, D. Nonetheless, only the M protein is able to RODRÍGUEZ AGUIRRE suppress the NF-κB reporter activation mediated by the TNF-α. Then, we wanted Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, CSIC, Madrid, to determine at which step of the IFN-β Spain pathway the M and N proteins were interfering. For this, we stimulated IFN-β transcription with different proteins Type I IFN system acts as the first line of involved in the signaling, including the RIG- defense to control the replication and I and MDA5 helicases, the mitochondrial dissemination of invading viruses, and to adapter protein MAVS, the TBK1 and IKKε promote the adaptive immune responses. kinases, and the IRF-3 transcription factor. The ability of the viruses to evade this The results indicated that the M protein innate response is crucial to ensuring their inhibits the IFN-β induction mediated by progeny survival. are enteric RIG-I, MDA5, MAVS, TBK1, and IKKε, while positive-sense, single stranded RNA viruses the N protein blocks its induction only after that belong to the Coronaviridae family, MAVS stimulation. Neither of these two Torovirinae subfamily, of the Nidovirales BEV proteins suppresses the transcription order. Unlike the coronaviruses, the of IFN-β mediated by IRF-3, suggesting that toroviruses have been poorly studied, and the block in signaling is prior to IRF-3 many issues about their interactions with activation. Finally, we observed that the M the host remain unexplored. In this study and N proteins interact with a complex we demonstrated that the equine torovirus formed by TBK1/IKKε/IRF-3 proteins and Berne virus (BEV), prototype member of this association could be a mechanism for the subfamily, antagonizes the disrupting IFN-β expression. In summary, transcription and production of the IFN- these results indicate that M and N α/β cytokines. Also, BEV infection reduces proteins play a crucial role in the IFN the expression of the IFN-stimulated genes antagonism exerted by BEV, and probably MxA, ISG15, and ISG56 induced upon in its pathogenicity. stimulation with IFN, SeV, and poly I:C. Next we analyzed the implication of the membrane (M) and nucleocapsid (N) BEV structural proteins in this process. Using an IFN-β promoter-luciferase reporter assay we observed that both proteins block the induction of this gene mediated by SeV. The activation of the IFN-β promoter depends on both the activation of the transcription factors IRF-3 (interferon

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(PO 95) properties as vehicle due to its genetic RECOMBINANT ADENOVIRAL VECTOR stability, safety, thermostability and EXPRESSING IFN-TAU INDUCES activation of innate immune response. To PROTECTIVE IMMUNE RESPONSE IN MICE evaluate the potential of this approach as CHALLENGE WITH A LETHAL DOSE OF an antiviral treatment in vivo, we chose to INFLUENZA VIRUS use the murine influenza model. Thus, V. MARTIN1*, E. PASCUAL1, M. AVIA1, G. congenic B6.A2G-Mx1 mice, expressing the RANGEL1, A. DE MOLINA2, E. BLANCO1, A. murine Mx1 protein, were challenged with ALEJO1 AND N. SEVILLA1 the FLUAV strain, A/PR/8/34 (H1N1) (designated hv-PR8). This variant is closely 1Centro de Investigación en Sanidad Animal (CISA- INIA), Valdeolmos, Madrid, Spain related to but distinct from the Cambridge 2 strain of A/PR/8/34 and is highly virulent in Centro Nacional de Investigaciones +/+ Cardiovasculares (CNIC), Madrid, Spain Mx1 mice. We hypothesised that, in Mx1 +/+ mice, the expression of IFN-τ could induce a strong innate immune response Virus-infected cells secrete a broad range and a more robust resistance to influenza of interferon (IFN) subtypes, which in turn by activating the Mx1 gene in addition to initiate the expression of antiviral factors other antiviral genes. This animal model that confer host resistance. Type I IFNs was previously used to demonstrate that (IFN-, IFN-, IFN- -) signal act IFN- might be used to prevent disease through a common universally expressed induced by highly lethal human H5N1 cell surface receptor (IFNRI). Interferon-tau influenza viruses. Using this experimental (IFN-τ) constitutes a new class of type I IFN system we demonstrate here that a single identified in ruminants that is structurally dose of intranasal administration of a   related to IFN-α - and- and capable of recombinant adenovirus vector expressing inducing an antiviral activity in a similar IFN-τ could protect against a highly virulent manner, although, unlike the former it is influenza strain (hv-PR8) in B6.A2G-Mx1 not inducible by dsRNA. While usually type mice, making it a good antiviral candidate I IFNs are highly species-specific, IFN-τ for this and other similar viruses. displays high antiviral, anti-proliferative Moreover, the results show that IFN- and immunomodulatory activities across active in other, non-ruminant species species with a prominent lack of indicating its potential cross species cytotoxicity at high concentrations in cell applicability in vivo. Furthermore, no toxic culture and possibly in vivo. IFNs play a effects were noted throughout the course central role in the defence of vertebrates of treatment. In summary, we present a against viral infections leading the cells to useful tool for the administration of a non- an antiviral state that effectively limits toxic type I interferonwithpotential virus replication. applicabilityin the treatment of several We present here the expression of IFN-τ viral diseases. from a second-generation human recombinant adenovirus 5, a vector widely used for vaccine delivery with excellent

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(PO 96) recombinant proteins because they are AN IMPROVED BACULOVIRUS produced in insect cells with an increased EXPRESSION VECTOR INCREASES THE viability late after infection. This increase SECRETION OF THE RECOMBINANT in cell viability is due to a delay in the virus- HEMAGGLUTININ FROM INFLUENZA induced apoptosis produced after infection VIRUS with the vector. The prolonged cell E. GUIJARRO- PARDO1*, S. GÓMEZ- integrity significantly contributes to avoid SEBASTIÁN2*, S. MARTINEZ-PULGARIN 2, the typicalaberrant forms and proteolysis MIGUEL CID2, C. ALVARADO2 J.M. found in the BEVS. The present study 1 consisted in the determination of the level ESCRIBANO of preservation of the secretion pathway in 1 Departamento de Biotecnología, INIA, Madrid, Spain cells infected by a TopBac®-modified 2 Alternative Gene Expression S.L. (ALGENEX), baculovirus expressing the HA in Pozuelo de Alarcón, Madrid, Spain comparison to a conventional baculovirus. * E. Guijarro- Pardoand S. Gómez-Sebastiánhave For this purpose, we obtained 2 equally contributed to this work. recombinant baculoviruses (modify or not by the TopBac® cassette) expressing this protein fused to the signal peptide of To simplify the manufacturing process of melittin to facilitate the protein secretion. the influenza hemagglutinin (HA) and The recombinant baculoviruses obtained circumvent the weaknesses in were used to infect insect cells and conventional and reverse genetics based Trichoplusia nilarvae. The results obtained vaccines, the improvements on in insect cells showed that at optimal recombinant-based production production times (48-72 hpi), when cellular technologies are highly relevant. The viabilities are above 80%, a secretion baculovirus vector expression system increment approximately of 2 times was (BEVS) is one of the most reliable and obtained by using the TopBac®-modified effective production methodology which baculovirus in comparison to the has been selected by several companies conventional vector. This increment in producing new generation vaccines against secretion was accompanied by the absence influenza. However, the transient of tubulin in the cell media, as an indirect expression mediated by the baculovirus measurement of the cellular integrity. infection affects the cells secretion Similar results of secretion were obtained pathways due to the lytic nature of the in the analysis of the hemolymph of system, reducing the recovered yields of infected larvae, with increments of 2 times recombinant proteins correctly processed of HA secreted when using the TopBac®- from infected cells supernatants. Recently, modified baculovirus. This result it has been described the TopBac® constitutes a step forward in the scaling-up baculovirus expression cassette. production of the HA protein using the Baculovirus vectors modified by TopBac® BEVS, providing a broad-based strategy for have unprecedented production yields, the simplification of the production of increasing also the quality of recovered recombinant subunit vaccines against

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seasonal or pandemic influenza at high Nevertheless, the role of CDR3 is still yields, avoiding the cumbersome egg- unclear. based production conventionally used. The aim of the present work was tostudy the involvement of CRD3 in the binding (PO 97) specificity and biological activity of the vTNFRs. With this purpose, we have ROLE OF THE CYSTEINE-RICH DOMAINS OF expressed in the baculovirus system twelve POXVIRUS TNF RECEPTORS IN THEIR mutant proteins of CrmB, CrmC, CrmD and IMMUNOMODULATORY ACTIVITY CrmE exchanging CRD3 between them and L. MESTRE, SM. PONTEJO, S. BLANCO, P. analysed their ability to inhibit the LANUZA, E. PRIEGO, A. ALCAMÍ Centro de Biología Molecular “Severo Ochoa” show that the specificity of the interaction (CBMSO) Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid site depends on the vTNFR studied. While (UAM), Cantoblanco, 28049, Madrid, Spain both CRD3 from CrmC and CrmE are not responsible for blocking cytotoxic effect Viral tumour necrosis factor receptors (vTNFRs) are soluble proteins secreted induced-cell death. Moreover, CRD3-CrmB after poxvirus infection characterized by is involved in the inhibition of cytotoxicity mimicking the extracellular domain of TNF superfamily receptors. Thus, vTNFRs bind to and inhibit the signalling induced by the host TNF superfamily ligands as a This work contributes to the understanding mechanism of immune evasion. Up to four of strategies used by viruses to evade the different vTNFRs expressed by poxvirus immune response, which provide us with a have been described and named CrmB, potent immunomodulatory tool that could CrmC, CrmD and CrmE. It was reported have therapeutic potential. that while CrmC and CrmE only bind to Keywords: poxvirus, vTNFRs, immune system. inhibit in vitro the biological effects of

The N-terminal region of these proteins is related to the TNF-binding site of cellular TNFRs and is characterized by three cysteine-rich domains (CRDs) named CRD1, CRD2, CRD3. CRD1 contains a conserved preligand assembly domain critical for ligand binding and receptor trimerization, while CRD2 has been proved necessary for the TNF-vTNFR interaction. It has been suggested that the specificity of this interaction is provided by CRD3.

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(PO 98) redistributed from the nucleus to the cell THE PDZ-BINDING MOTIF OF SEVERE cytoplasm, colocalizing with E protein ACUTE RESPIRATORY SYNDROME during infection with SARS-CoV containing CORONAVIRUS ENVELOPE PROTEIN IS A the E protein PBM. The relocalization of DETERMINANT OF PATHOGENESIS AND syntenin activated p38 MAPK and led to VIRAL FITNESS the overexpression of inflammatory JM. JIMENEZ-GUARDEÑO, JL. NIETO- cytokines. In fact, silencing of syntenin TORRES, ML. DEDIEGO, JA. REGLA-NAVA, C. using siRNAs led to a decrease in p38 CASTAÑO-RODRIGUEZ, R. FERNANDEZ- MAPK activation in SARS-CoV infected cells, reinforcing their functional DELGADO AND L. ENJUANES relationship. Furthermore, active p38 Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, MAPK was reduced in the lungs of mice Campus Universidad Autónoma de Madrid, 28049 infected with SARS-CoVs lacking E protein Madrid, Spain PBM as compared with those infected with viruses containing this motif, leading to a Severe acute respiratory syndrome decreased expression of inflammatory coronavirus (SARS-CoV) is the etiological cytokines and to virus attenuation. agent of a worldwide epidemic that Interestingly, administration of a p38 appeared in China in 2002, infecting 8000 MAPK inhibitor increased mice survival up people worldwide with an average to 80% after infection with SARS-CoV, mortality of about 10%. Previously, we indicating the relevance of this signaling demonstrated that a recombinant SARS- pathway in SARS-CoV pathogenesis. The CoV lacking the multifunctional envelope impact of E protein PBM during SARS-CoV (E) protein generated in our laboratory was infection was supported by showing that attenuated in vivo in three different animal recombinant viruses lacking the E protein models, being a promising vaccine PBM incorporated novel PBMs after serial candidate. Here we report that the E passages. This data confirms that the PBM protein PDZ-binding motif (PBM), a domain of E protein is a virulence factor that potentially involved in the interaction with increases virus fitness. more than 400 cellular proteins, what highlights its relevance in modulating host- cell behavior, is a major determinant of SARS-CoV virulence. Removal of PBM in

SARS-CoV E protein using reverse genetics, drastically diminished lung damage, leading to virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies: yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. In addition, syntenin

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(PO 99) in cytokine sensitivity assays. The mutant EFFECT OF HERPES SIMPLEX VIRUS TYPE 1 viruses dl41GF (ICP0-, vhs-, EGFP+) and SHUT-OFF PROTEIN (VHS) AND dlTKGF (ICP0-, TK-, EGFP+) were INTERFERON ON THE GROWTH OF A constructed by homologous recombination HERPES SIMPLEX VIRUS DEFECTIVE IN using the parental virus dl1403 (ICP0-). In ICP0 addition, we also constructed FTKGF (TK-, R. GARCÍA UTRILLA, B. MORENO, S. EGFP+) using the HSV-1 parental strain F, GUERRA, E. TABARÉS to serve as a reference viruses for the all assay performed. These three recombinant Preventive Medicine and Public Health and Microbiology Department, Medicine Faculty viruses were characterized by PCR and Autonomous University of Madrid, Madrid Spain direct fluorescence microscopy. Once characterized, the growth kinetics, sensitivity to IFN and the influence of the Herpes Simplex Virus Type 1 (HSV-1) viral shut-off (vhs) protein on viral growth affects more than 80% of the human were assessed in Vero and U2OS cells. population causing both acute infections, Replication of dlTKGF and dl41GF was 15.7 characterized by a wide range of clinical and 12.2 fold higher in U2OS cells, manifestations, and latent infections. Due respectively, compared to Vero cells. These to their structural and genomic orders of magnitude were significantly characteristics as well as the HSV-1 viral higher than those observed for the life cycle, these viruses have been widely reference viruses HSV41GF (vhs-, EGFP+) used as genetic tools to study and treat and FTKGF, which we contribute to the many diseases with considerable social, absence of ICP0 protein in the first mutant economic and cultural impact, such as viruses. The virus dl41GF was twice as central nervous system disorders or sensitive to IFN and exhibited 7.5 fold cancer. While promising, the development increased viral replication compared to the of new recombinant HSV-1-based viruses is dlTKGF virus in U2OS cells. In conclusion, still warranted and needed, specifically the the results of this study suggest a possible development of viruses that are not cooperative action between HSV-1 ICP0 susceptible to host cytokine inhibition, a and shut-off proteins, both at the level of factor the limits the therapeutic use of viral growth and IFN sensitivity carried out many viruses. on U2OS cells. The primary goal of this study was to produce enhanced green fluorescent protein (EGFP) HSV-1 reporter viruses defective in the immediate early protein ICP0, which renders HSV-1 hypersensitive to treatment with interferon (IFN). The ability to visualize and track the virus throughout the entire infection process via EGFP fluorescence highlights the potential of these viruses as powerful genetic tools

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(PO 100) with PRV-HgD and PRV-HgDB. A survival ANALYSIS OF IMMUNOPROTECTION OF study was carried out over a 21-day period gD AND THE CHIMERIC GLYCOPROTEIN after mice were challenged with a lethal gD-gB (gDB2) OF HERPES SIMPLEX VIRUS dose of an HSV-2 clinical isolate by TYPE 2 EXPRESSED BY PSEUDORABIES intravaginal infection. The results of this VIRUS RECOMBINANTS AND AMPLICONS analysis were compared with groups of E. RUBIO FERNÁNDEZ1, L. VARELA1, A. L BALB/c mice immunized with plasmids MUÑOZ1, B. MORENO1, E. TABARÉS1, I. (PpD and PpDB) or amplicons (AmpgD2 GADEA2 and AmpgDB2) expressing the same HSV-2 glycoproteins constructions that PRV 1. Departamento de Medicina Preventiva y Salud Pública y Microbiología, Facultad de Medicina recombinants. All the groups studied Universidad Autónoma de Madrid, Madrid, Spain showedstatistically significant differences 2. Servicio de Microbiología Médica, Fundación in survival with the control group, except Jiménez Díaz, Madrid, Spain for the groups of mice immunized with PRV-HgDB and AmpgDB2. The best results More than 500 million people worldwide of survival were achieved with the plasmid are infected with herpes simplex virus type PpDB and the amplicon AmpgD2, whose 2 (HSV-2), and 23 million new HSV-2 survival percentages were 85% and 75%, infections are developed each year. HSV-2 respectively. can produce from recurrent genital ulcers to potentially lethal infections and the (PO 101) need for a vaccine is widely recognized. A CHIMERIC HIV-1 GP120 FUSED WITH Over the last decades, numerous efforts VACCINIA VIRUS 14K (A27) PROTEIN AS have been made to develop effective AN HIV IMMUNOGEN vaccines against HSV-2; however, most 1 ANEESH VIJAYAN , JUAN GARCÍA- studies have not given satisfactory results 1 1 ARRIAZA , SURESH C. RAMAN , JOSÉ in human clinical trials. The main goal of 2 JAVIER CONESA , FRANCISCO JAVIER this study was to analyze in BALB/c mice, 2 3 CHICHÓN , CÉSAR SANTIAGO , CARLOS the immunoprotection properties of gD 4 2 ÓSCAR S. SORZANO , JOSÉ L. CARRASCOSA (gD2) and a chimeric glycoprotein gD-gB 1§ AND MARIANO ESTEBAN (gDB2) of herpes simplex virus type 2 by 1Department of Molecular and Cellular Biology, using pseudorabies virus (PRV) 2Department of Structure of Macromolecules, 3X-ray recombinant and amplicons as expression Crystallization Unit, 4Biocomputing Unit, Centro vectors. Three groups of mice were Nacional de Biotecnología, Consejo Superior de immunized subcutaneously with Investigaciones Científicas (CNB-CSIC), 28049, recombinant virus PRV-HgD, PRV-HgDB Madrid, Spain. and PRV-BT90. PRV-BT90 was used as reference, which it does not express any In the HIV vaccine field, there is a need to HSV-2 glycoprotein constructions.Humoral produce, in considerable quantities, immune response was analyzed by ELISA soluble and stable forms of the Env protein test, which showed the presence of anti- with the capacity to trigger broad B and T- HSV-2 antibodies in the groups immunized

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cell responses. Here, we report the innate immune response in human moDCs, generation and characterization of a and enhancing HIV-1-specific adaptive and chimeric HIV-1 gp120 protein (termed memory T-cell immune responses, as well gp120-14K) by fusing gp120 from clade B as humoral responses, in immunized mice. with the vaccinia virus (VACV) 14K This novel HIV-1 gp120-14K immunogen oligomeric protein (derived from A27L might be considered as an HIV vaccine gene). Stable CHO cell lines expressing HIV- candidate for broad T and B-cell immune 1 gp120-14K fusion proteins were responses. generated, and characterized by size exclusion chromatography, electron (PO 102) microscopy and binding to conformational antibodies. In human monocyte-derived VIROLOGICAL AND IMMUNOLOGICAL dendritic cells (moDCs), gp120-14K protein CHARACTERIZATION OF NOVEL NYVAC- upregulates the levels of several BASED HIV/AIDS VACCINE CANDIDATES proinflammatory cytokines and EXPRESSING CLADE C TRIMERIC SOLUBLE chemokines associated with Th1 innate GP140(ZM96) AND GAG(ZM96)-POL- immune responses (IL-1β, IFN-γ, IL-6, IL-8, NEF(CN54) AS VIRUS-LIKE PARTICLES 1 1 IL-12, RANTES). Moreover, we showed in a B. PERDIGUERO ,C. E. GÓMEZ ,V. 1 1 murine model, that a heterologous CEPEDA ,L. SÁNCHEZ-SAMPEDRO ,J. 1 1 prime/boost immunization protocol GARCÍA-ARRIAZA ,E. MEJÍAS-PÉREZ , V. 1 1 consisting of a DNA prime with a plasmid JIMÉNEZ ,C. SÁNCHEZ ,C. O. S. 2 2 3 expressing the gp120-14K protein followed SORZANO ,J. C. OLIVEROS ,J. DELALOYE ,T. 3 3 4 by a boost with MVA-B [a recombinant ROGER , T. CALANDRA ,B. ASBACH ,R. 4 5 5 modified vaccinia virus Ankara (MVA) WAGNER ,K. V. KIBLER ,B. L. JACOBS ,G. 6 1 expressing HIV-1 gp120, Gag, Pol and Nef PANTALEO AND M. ESTEBAN antigens from clade B], generates stronger, 1 Department of Molecular and Cellular Biology and 2 more polyfunctional, and greater effector Biocomputing Unit and Computational Genomics, Centro Nacional de Biotecnología, Consejo Superior memory HIV-1-specific CD4+ and CD8+ T- 3 de Investigaciones Científicas (CSIC), Madrid, Spain. cell immune responses, than immunization Infectious Diseases Service, Department of with DNA-gp120/MVA-B. The DNA/MVA Medicine, Centre Hospitalier Universitaire Vaudois protocol was superior to immunization and University of Lausanne, Lausanne, Switzerland. 4 5 with the combination of protein/MVA and University of Regensburg, Regensburg, Germany. The Biodesign Institute at Arizona State University, the latter was superior to a prime/boost of 6 Tempe, Arizona, USA. Division of Immunology and MVA/MVA or protein/protein. In addition, Allergy, Department of Medicine, Centre Hospitalier all of these immunization protocols Universitaire Vaudois and University of Lausanne, enhanced antibody responses against Lausanne, Switzerland. gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune The generation of vaccines against response. These results demonstrate that HIV/AIDS able to induce long-lasting fusing VACV 14K with HIV-1 gp120, forms protective immunity remains a major goal an oligomeric protein with apparent in the HIV field. The modest efficacy native-like structure, triggering a Th1 (31.2%) against HIV infection observed in

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the RV144 phase III clinical trial highlighted against HIV in human clinical trials with the need for further improvement of HIV these vectors. vaccine candidates, formulation, and vaccine regimen. In this study, we have (PO 103) generated two novel NYVAC vectors, expressing HIV-1 clade C gp140(ZM96) HEAD-TO-HEAD COMPARISON OF (NYVAC-gp140) or Gag(ZM96)-Pol- VACCINIA VIRUS-BASED VECTORS NYVAC Nef(CN54) (NYVAC-Gag-Pol-Nef), and AND MVA EXPRESSING LEISHMANIA defined their virological and immunological ACTIVATED C-KINASE IN MICE 1¶ characteristics in cultured cells and in mice. E. MEJÍAS-PÉREZ ; L. SÁNCHEZ- 1¶ 2 The insertion of HIV genes does not affect SAMPEDRO ; C.O. S. SORZANO, ; M. 1 the replication capacity of NYVAC ESTEBAN recombinants in primary chicken embryo 1 Department of Molecular and Cellular Biology, fibroblast cells, HIV sequences remain Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain. stable after multiple passages, and HIV 2 antigens are correctly expressed and Biocomputing Unit, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones released from cells, with Env as a trimer Científicas (CSIC), Madrid, Spain. (NYVAC-gp140), while in NYVAC-Gag-Pol- ¶These authors contributed equally to this work. Nef infected cells Gag-induced virus-like particles (VLPs) are abundant. Electron microscopy revealed that VLPs The poxvirus NYVAC and MVA vectors are accumulated with time at the cell surface, used as vaccine candidates against HIV, but with no interference with NYVAC experimental information on a head-to- morphogenesis. Both vectors trigger head comparison of immunogenicity and specific innate responses in human cells efficacy against a human disease is limited. and show an attenuation profile in Here we compared in mice the immune immunocompromised adult BALB/c and responses elicited and protection induced newborn CD1 mice after intracranial by NYVAC vectors expressing the inoculation. Analysis of the immune leishmania activated C-kinase antigen responses elicited in mice after (LACK) versus the well-known MVA-LACK homologous vector. In a head-to-head comparison by DNA prime/virus boost protocols, we show NYVAC prime/NYVAC boost immunization that replication competent NYVAC-LACK shows that recombinant viruses induced expressing the C7L host range gene polyfunctional Env-specific CD4 or Gag- (NYVAC-LACK-C7L) induced the highest specific CD8 T cell responses. Antibody quality of CD4+ and CD8+ adaptive and responses against gp140 and p17/p24 effector memory T cell responses (IFN, were elicited. Our findings showed TNF, IL-2, CD107a) against LACK antigen. important insights into virus-host cell The CD8+ T cell population induced by interactions of NYVAC vectors expressing NYVAC-LACK-C7L also showed the highest HIV antigens, with the activation of specific proliferative capacity when stimulated immune parameters which will help to with the LACK antigen. T cell differences unravel potential correlates of protection

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with MVA-LACK and between replication Rabbit Hemorrhagic Disease virus (RHDV) competent and replication defective is the prototype strain of the genus NYVAC vectors were restricted to Lagovirus within the family Caliciviridae, a magnitude of the response. After group of nonenveloped, icosahedral subcutaneous L. major infection, challenge viruses which are composed of 180 copies groups vaccinated with NYVAC-LACK-C7L of a single capsid protein, terms VP60. showed higher protection than NYVAC- Our research group has identified two LACK group, and similar efficacy as those independent locations within the RHDV vaccinated with MVA-LACK. Our results capsid protein that can accomodate revealed that the type of immune response foreign of up to 42 aminoacids in length, and potency induced by NYVAC and MVA without affecting the ability of the vectors is largely restricted to quantitative resulting chimeric protein to self-assemble differences in T cells, with a replication into VLPs (2, 3). Our goal is to develop competent NYVAC with C7L gene triggering RHDV VLPs as a delivery system for the the highest T cell adaptive, memory and multimeric presentation of immunogenic proliferative immune responses. epitopes derived from pathogens relevant for animal health. (PO 104) Foot-and-mouth disease virus (FMDV) is a VIRUS-LIKE PARTICLES (VLPS) DERIVED highly infectious disease of cloven-hoofed FROM CALICIVIRUS AS A DELIVERY animals and probably the most important SYSTEM FOR THE PRESENTATION OF livestock disease in terms of economic FOOT-AND-MOUTH DISEASE VIRUS impact. The aim of the present study was EPITOPES to analyze the potential of chimeric VLPs to G.RANGEL1, A. ALEJO1, B.GUERRA1, J.R. induce specific immune responses against CASTON2 J.BARCENA1 AND E. BLANCO1 T- and B-cell epitopes from FMDV. To this end we generated chimeric VLPs 1. Centro de Investigación en Sanidad Animal (CISA- INIA), Valdeolmos, 28130 Madrid, Spain. harbouring, in different insertion sites, a 2. Dpto. Estructura de Macromoléculas, (CNB-CSIC), neutralizing B-cell epitope derived from Cantoblanco, 28049 Madrid, Spain. FMDV type O (currently the most widespread serotype), located around positions 140 to 160 of capsid protein VP1 Virus-like particles (VLPs) are appealing as (loop G-H), and a T-cell epitope highly vaccine candidates because their inherent conserved among FMDV serotypes from 3A properties (i.e., virus-sized, multimeric non-structural protein (4). antigens, highly organised and repetitive structure, not infectious) are suitable for Groups of mice were inoculated with the the induction of safe and efficient immune chimeric VLPs and we analyzed the responses. In particular, VLPs from rabbit humoral and cellular immune responses haemorrhagic disease virus (RHDV) have elicited. The results obtained indicated been shown to be good vaccine platforms that the chimeric RHDV VLPs are able to (1). induce potent antibody responses against FMDV B-cell epitope,especially when

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inserted at an exposed site within the VLP cold chain, periodic re-vaccinations , risk of structure. virus release during vaccine production, Based on the outcomes, the potential etc).In this context, peptide-based vaccines suitability of these chimeric VLPs for new are promising alternatives in the control of vaccine development against pig viral infectious diseases, offering several infections will be discussed. advantages, such as safety, accurate References molecular delineation, ease of synthesis 1. Bárcena and Blanco. (2013) Structure and Physics and scaleup or uncomplicated storage and of Viruses: An Integrated Textbook, transport. Subcellular Biochemistry 68 Synthetic peptides incorporating 2. Bárcena et al. (2004) Virology322 118-134. protective B- and T-cell epitopes are 3. Luque et al. (2012). Journal of Virology 86 6470- candidates for new safer FMD vaccines. 6480. We have reported that dendrimeric 4.Blanco et al. (2001) Journal ofVirology 75 (7)3164- peptides including four copies of a B- cell 3174. epitope (VP1 136 to 154) linked to a T-cell epitope (3A 21 to 35) of FMD virus (FMDV) (PO 105) elicit potent B- and T- cell specific EFFECT OF EPITOPE MULTIPLICITY AND responses and confer protection to type C CONNECTIVITY ON THE FMDV challenge (1), while juxtaposition of IMMUNOGENICITY AND PROTECTION OF these epitopes in a linear peptide induces DENDRIMERIC PEPTIDE VACCINES less efficient responses. In order to extend AGAINST FOOT-AND-MOUTH DISEASE this proof of concept to FMDV serotypes VIRUS epidemiologically relevant at present, we designed new dendrimeric peptides E. BLANCO1, B.GUERRA1, B. G DE LA harboring as B-cell epitopes sequences TORRE2, D. ANDREU2 AND F. SOBRINO 1,3 1. from FMDV serotype O (currently the most Centro de Investigación en Sanidad Animal (CISA- widespreaded serotype). To assess the INIA), Valdeolmos, 28130 Madrid, Spain. relevance of B-cell epitope multivalency, 2. Departament de Ciències Experimentals i de la Salut, Universitat Pompeu-Fabra, 08003 Barcelona, downsized versions of the dendrimeric Spain. constructions, with two copies of the B 3. Centro de Biología Molecular "Severo Ochoa" epitope (B2T) were tested and compared (CSIC-UAM), Cantoblanco, 28049 Madrid Spain. to dendrimers bearing four copies (B4T), in the mice model, and we found that B2T Foot-and-mouth disease (FMD) is a highly constructions elicited similar or even infectious disease of cloven-hoofed better B- and T-cell specific responses than animals, admittedly the most important B4T (2). Interestingly, we also found that livestock disease in terms of economic modifications on the conjugation chemistry impact. FMD control in endemic regions is used to attach B- and T-cell epitopes implemented mainly by using chemically influenced the immunogenicity of the inactivated whole-virus vaccines,that show dendrimers, which was highest for several disadvantages (requirement of a maleimide-based conjugates.

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In light of these results, in the present depth understanding of the cellular study we have compared the mechanisms involved in HSV-1 infections, immunogenicity and protection against including viral immune evasion viral challenge elicited by B4T (thioether mechanisms. For example, the double- conjugate) and B2T (thioether or stranded RNA-dependent protein kinase maleimide conjugates) in swine, an FMDV (PKR) is one of the core players involved in natural host. Efficient induction of the cellular innate response to viral neutralizing antibodies and optimal release infections as it inhibits protein synthesis, of IFNγ was elicited by the three thereby preventing disease progression. constructions, but in correlation with the Herpesviruses, however, have developed mice model results, differences in strategies to interfere with and evade immunogenicity and protection were again these antiviral cellular innate responses, found. Taken together, our results provide allowing them to replicate in cells even useful insights for a more accurate design when PKR is active. One of the strategies of FMD subunit vaccines. used by HSV-1 includes the viral “shut off” References protein (vhs), encoded by the viral gene 1. Cubillos et al., (2008) Journal of Virology UL41, which functions to induce the 82 (14) 7223-7230 degradation of cellular and viral mRNAs, leading to inhibition of protein synthesis. 2. Blanco et al. (2013) Clinical and Understanding the exact role of vhs as a Developmental Immunology Article ID viral immune evasion protein would 475960, 1-9 certainly increase our understanding of this virus and the mechanisms by which (PO 106) HSV-1 circumvents the innate intracellular ANALYSIS OF GROWTH OF HERPES immune response. SIMPLEX VIRUS TYPE 1 RECOMBINANT Towards this end, the virus HSV41GF, (HSV41GF) WITH EGFP EXPRESSION AND deficient in vhs, was constructed by DEFICIENT IN “SHUT OFF” PROTEIN (VHS) homologous recombination, using HSV-1 IN VERO AND HELA CELLS strain F as a parental virus and EGFP as L. LERMA, B. MARTÍN, D. RODRÍGUEZ, S. selection marker. The virus was GUERRA, E. TABARÉS. characterized by PCR and western blot Department of Preventive Medicine, Public Health analysis using mono-specific antibodies and Microbiology.School of Medicine.Autónoma obtained in rabbits by immunization with University of Madrid. Spain. the fusion protein beta-galactosidase-vhs produced in E. coli. The viral growth of this Herpes simplex virus type 1 (HSV-1) affects mutant virus was analysed in Vero and a significant percentage of the human HeLa cells and compared with the parental population and is responsible for a wide virus. While HSV-1 strain F showed similar variety of diseases. Due to the impact HSV- growth in both Vero and HeLa cells, the 1 has on the general population and in the growth of HSV41GF in HeLa was clinical setting, it is important to gain an in approximately 2 logs lower than in Vero cells. Analysis of the influence of PKR on

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HSV41GF infection was carried out by deletion of C6L vaccinia virus infecting HeLa, HeLa SC and HeLa PKR- cells immunomodulatory gene, which encodes with HSV41GF and comparing infections for an inhibitor of IFN-β. Deletion kinetics across all cell lines and with the of C6L had no effect on virus growth parental virus. Results showed that PKR is kinetics or on the expression of HCV the main kinase involved in HSV-1 infection antigens; hence, the C6L protein is not and vhs deficiency correlates with a essential for MVA-HCV replication. The decrease in eIEF2alpha factor innate immune responses triggered by phosphorylation levels, even in the MVA-HCV and MVA-HCV ΔC6L in human presence of PKR. macrophages and monocyte-derived dendritic cells showed an upregulation (PO 107) of IFN-β, proinflammatory cytokines and chemokines. Furthermore, we have CHARACTERIZATION OF A NOVEL analyzed the immunogenicity elicited by HEPATITIS C VIRUS (HCV) VACCINE MVA-HCV ΔC6L following either CANDIDATE BASED ON MVA EXPRESSING homologous or heterologous prime/boost THE NEARLY FULL-LENGTH HCV GENOME immunization protocols in C57BL/6 AND LACKING C6L VACCINIA VIRUS vaccinated mice. The results showed that IMMUNOMODULATORY GENE MVA-HCV and MVA-HCV ΔC6L induced 1 1 M. QUIRÓS , J. GARCÍA-ARRIAZA , C. E. high, broad and polyfunctional HCV- 1 2 GÓMEZ , C. O. S. SORZANO AND specific CD4+ and CD8+ T-cell adaptive and 1 MARIANO ESTEBAN memory immune responses. Most of the 1 Department of Molecular and Cellular Biology and vaccine-induced T-cell responses were 2 Biocomputing Unit.Centro Nacional de mainly mediated by CD8+ T cells, being Biotecnología, Consejo Superior de Investigaciones CD4+ T cells also induced but at a lower Científicas (CNB-CSIC), Madrid, Spain. magnitude. Homologous immunization + protocols elicited HCV-specific CD8 T cells Hepatitis C virus (HCV) remains a global mainly directed against p7+NS2 antigens, problem despite advances in treatment. whereas in heterologous immunization Thus, the development of a safe and protocols the main target was the NS3 efficacious vaccine against HCV is one of protein. Significantly, HCV-specific CD4+ T the main goals for prevention and control cells directed against E1 and E2 antigens of hepatitis C. In an effort to improve the were induced in the heterologous immunogenicity of the previously regimens. Moreover, in the memory phase, described HCV vaccine candidate (termed HCV-specific CD8+ T cells with an effector MVA-HCV), based on the poxvirus MVA phenotype were predominant. These vector expressing the nearly full-length findings highlight the relevance of vaccinia HCV genome from genotype 1a (Core, E1, virus immunomodulatory genes in HCV E2, p7, NS2, NS3, NS4A, NS4B, NS5A and a vaccine design. part of NS5B), we have generated a novel

optimized MVA-HCV vaccine candidate (termed MVA-HCV ΔC6L) containing a

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(PO 108) show that the FMDV ncRNAs are able to THE ANTIVIRAL AND trigger a broad innate immune response in IMMUNOSTIMULATORY EFFECTS OF swine PBMCs and provide evidence for the SYNTHETIC RNAs CORRESPONDING TO involvement of both Toll-like receptors THE FOOT-AND-MOUTH DISEASE VIRUS (TLRs) and cytosolic sensing (RIG-I-like (FMDV) NON-CODING REGIONS (ncRNAs) receptors) of the FMDV ncRNAs, RELY ON INNATE RESPONSES TRIGGERED accounting for the induced type-I IFN and BY RIG-I AND TLR ACTIVATION cytokine response. Altogether, our findings B. BORREGO1, M. RODRÍGUEZ-PULIDO2, C. suggest that these synthetic non-infectious 3 3 2 molecules may have an REVILLA , B. ÁLVAREZ , F. SOBRINO , J. DOMÍNGUEZ3, M. SÁIZ2 immunostimulatory activity in livestock, as well as a potential application as 1. Centro de Investigación en Sanidad Animal, CISA- INIA, Valdeolmos, Madrid, Spain immunomodulatory compounds in new 2. Centro de Biología Molecular Severo Ochoa (CISC- antiviral and vaccine formulations. UAM), Cantoblanco, Madrid, Spain 3. Dpto. de Biotecnología, Instituto Nacional de (PO 109) Investigación y Tecnología Agraria y Alimentaria (INIA), Madrid, Spain INTERFERENCE OF SINGLE AND DUAL BIOTIC STRESSES ON HOST DNA

METHYLATION PATHWAYS The innate immune system is the first line E.M. TORCHETTI1, M. PEGORARO2, B. of defense against viral infections. NAVARRO1, M. CATONI3, E. NORIS2, F. DI Exploiting innate responses for antiviral, SERIO1 therapeutic and vaccine adjuvation 1 strategies is being extensively explored. .Istituto per la Protezione Sostenibile delle Piante, Consiglio Nazionale delle Ricerche, UOS Bari, Italy We have previously described the ability of 2.Istituto per la Protezione Sostenibile delle Piante, small in vitro RNA transcripts, mimicking Consiglio Nazionale delle Ricerche, Torino, Italy the sequence and structure of different 3.Sainsbury Laboratory, University of Cambridge, domains in the non-coding regions of the Cambridge, UK foot-and-mouth disease virus (FMDV) genome (ncRNAs), to trigger a potent and DNA methylation (DM) pathways play rapid innate immune response. These major roles in preservation of genome synthetic non-infectious molecules have integrity, transposon stability and proved to have a broad-range antiviral regulation of gene expression. In plants, activity and to enhance the DM has also been involved in responses to immunogenicity of an FMD inactivated abiotic and biotic stresses, including vaccine in mice. Here, we have studied the defense against geminiviruses (GV), a large involvement of pattern-recognition group of viruses with a single-stranded receptors (PRRs) in the ncRNA-induced DNA genome that replicates in the nucleus innate response and analyzed the antiviral forming minichromosomes associated with and cytokine profiles elicited in swine cellular histones. It is proposed that host cultured cells, as well as peripheral blood DM machinery impairs viral accumulation mononuclear cells (PBMCs). Our results

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in the infected tissues by targeting GV DNA (PO 110) for methylation. In contrast, whether DM COMPARISON OF THE IMMUNE is involved in the molecular interplay RESPONSES AND EFFICACY AFTER between plants and nuclear replicating CHALLENGE ELICITED BY RECOMBINANT viroids, which are infectious non-protein- MVAs EXPRESSING SINGLE RIFT VALLEY coding RNAs frequently inducing severe FEVER VIRUS GLYCOPROTEINS diseases in plants, is still unclear. Viroid ELENA LÓPEZ-GIL, GEMA LORENZO, RNAs are targeted by host enzymes SANDRA MORENO, ALEJANDRO MARÍN- involved in DM pathways, but whether the LÓPEZ, JAVIER ORTEGO AND ALEJANDRO genes implicated in this pathways are BRUN differentially regulated in response to Centro de Investigación en Sanidad Animal (INIA). viroid infection is unknown. In addition, Valdeolmos, Madrid, Spain whether DM pathways may differentially target host and GV DNA depending on the presence or absence of a nuclear infecting Rift Valley Fever virus (RVFV), a mosquito- viroid is also not known. To further explore borne bunyavirus widely distributed in the interference of single and dual Sub-Saharan countries, Egypt and the infections by nuclear replicating infectious Arabian Peninsula, causes disease in both agents, we have developed an human and livestock and is now experimental system based on tomato considered an emerging threat for non- plants infected by the geminivirus Tomato endemic countries due to the movement yellow leaf curl Sardinia virus (TYLCSV) of infected animals and insect vectors and/or the nuclear-replicating Potato including mosquitoes. The ample range of spindle tuber viroid (PSTVd). DNA competent mosquito vectors for RVFV in methylation profiles of TYLCSV DNA and of many areas of the Mediterranean basin two host genomic targets were tested as suggests that RVF outbreaks in non- molecular sensors of host DM under stress endemic areas could potentially end-up in conditions. Moreover, expression of genes establishment of enzootic infection cycles. involved in DM was investigated at If this happen it would cause serious transcriptional level by quantitative RT-PCR concern for both public and animal health. assays. Our data show that both TYLCSV It is therefore desirable to develop control and PSTVd interfere with the regulation of tools as well as enhance our knowledge most host genes involved in DM pathways about the immune mechanisms that and, interestingly, that the plant response correlate with the protection elicited by to a single stress strongly differs from that RVFV vaccines. In this work we have to dual stresses, with synergistic effects. characterized the efficacy and immune response of recombinant MVA viruses

expressing RVFV glycoproteins Gn and Gc. Previous data obtained in our laboratory showed that a single inoculation of MVA expressing both Gn and Gc was sufficient to induce a protective immune response in

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mice after a lethal challenge with RVFV. (PO 111) The protection elicited by the MVA ANALYSIS OF THE ANTIVIRAL ACTIVITY OF vaccination was related to the presence of SILVER NANOPARTICLES AGAINST RIFT glycoprotein specific CD8+ cells, in the VALLEY FEVER VIRUS IN VITRO AND IN absence of a consistent detection of VIVO neutralizing antibodies in vitro. To study B. BORREGO1, G. LORENZO1, H. ALMANZA2, the contribution of each glycoprotein F. MATEOS1, E. LÓPEZ-GIL1, N. DE LA LOSA1, antigen to protection a similar approach V. A. BURMISTROV3, A. N. PESTRYAKOV4, A. was extended to vaccines expressing only a BRUN1, N.BOGDANCHIKOVA5 single RVFV glycoprotein (either Gn or Gc). 1 Centro de Investigación en Sanidad Animal, INIA, Our results suggest that protection of Valdeolmos, Spain. BALB/c mice upon RVFV challenge can be 2 FMyP- Universidad Autónoma Baja California, mediated by the activation of a strong Tijuana, Mexico cellular response (mainly against Gc 3Vector-Vita Ltd, Novosibirsk, Russia epitopes) in the absence of a clear 4 Tomsk Polytechnic University, Tomsk, Russia induction of neutralizing antibodies. 5 Centro de Nanociencias y Nanotecnología, UNAM, However, this protection may be restricted Ensenada, Mexico to specific genetic backgrounds determining susceptibility to infection as Rift Valley Fever virus (RVFV) is a mosquito shown by the lack of survival upon borne pathogen causing an important challenge of 129SvEv mice immunized with disease in ruminants often transmitted to the same vaccines (MVAGn or MVAGc). humans after epizootic outbreaks, thus Our data also point out that the expression becoming a very relevant pathogen for of both glycoproteins enhances humoral animal health due to the economic losses immunogenicity perhaps explaining the associated, and also for human health. higher protection rates in MVAGnGc Currently there is no available treatment vaccinated 129 SvEv mice. The detection of or licensed Rift Valley fever vaccine for IL-2 and IL-6 supports the induction of human use, therefore the development of cellular responses since both cytokines new approaches able to inhibit viral play a role in T-cell survival and activation. replication and transmission allowing an Thus, the identified Gc specific CD8+ T-cell efficient control of the disease is a must. population may act as a key component in Silver nanoparticles have been described the protection after challenge observed in to exert some inhibitory effect against the MVA immunized mice, contributing to some enveloped viruses belonging to the elimination of infected cells and different families. Compared to the reducing morbidity and mortality. classical antiviral approaches, the use of metal nanoparticles poses many advantages, mainly the non-emergence of resistant variants, as well as their safety and low cost.

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In this work we have tested the antiviral Additionally, CrmD contains a structurally potential against RVFV infection, both in distinct domain termed, the SECRET cell culture and in animal models, of silver domain, that can bind and block the nanoparticles formulated as Argovit. activity of a reduced set of chemokines. Though the ability of silver nanoparticles to This domain was identified in several control an ongoing RVFV infection in the other poxviral secreted proteins. To conditions tested seems to be limited, the address the possible concerted anti- incubation of virus with Argovit before the inflammatory role of both domains, we infection leads to a reduction of viral generated recombinant ectromelia viruses infectivity both in vitro and in vivo. Our lacking CrmD or expressing a results reveal the potential application of truncated version that blocked TNF but the microbicidal properties of silver not chemokine activity. We found CrmD to nanoparticles to control the infectivity of block the inflammatory footpad swelling this important zoonotic pathogen. reaction in vivo, with the SECRET domain contributing significantly to this (PO 112) activity. We next tested the ability ANTI-INFLAMMATORY PROPERTIES OF of recombinant CrmD or a truncated THE SECRET DOMAIN FROM POXVIRUS version lacking the SECRET domain to TNF RECEPTORS block inflammation in a murine model of C. SÁNCHEZ1, A. ALEJO2, S.J. MARTIN rheumatoid arthritis. Both PONTEJO1, P. FALLON3 AND A. ALCAMI1. approaches confirmed that the presence 1 Centro Biología Molecular Severo Ochoa, (CBMSO). of a chemokine binding domain enhanced Madrid. Spain. the anti-inflammatory potential of a 2 Centro de Investigación en Sanidad Animal (INIA). vTNFR in vivo. Because secreted Madrid. Spain human TNFRs are currently used in the 3 Trinity College. Dublín. Ireland. clinic for the treatment of several inflammatory conditions, we Poxviruses encode numerous proteins reasoned that addition of a chemokine devoted to the control of the binding domain to such a protein might host’s immune response, including a set of enhance is activity. Therefore, we secreted, cytokine binding proteins that generated a set of secreted hTNFRs fused act mainly as competitive inhibitors of to SECRET domains derived from their ligands. Amongst these, a family of different viral proteins and screened them virally encoded TNF receptors (vTNFRs) for correct TNF and chemokine with homology to their cellular inhibitory activity. One selected construct counterparts are thought to have was further purified and its binding important roles during and inhibitory activity characterized in infection. Ectromelia virus, the causative vitro and in cell culture. Finally, agent of mousepox, encodes a single we determined the anti-inflammatory active vTNFR named CrmD which is known activity of this recombinant hTNFR- to block effectively TNFa. SECRET protein in vivo, showing its ability

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to block the development of a rheumatoid salivary and serum IgA and IgG antibody arthiritis like disease. titers and their capacity to block virus (PO 113) binding to their receptors were assayed. SECRETOR STATUS DETERMINES The results showed that the outbreak was SUSCEPTIBILITY TO DIARRHEA BUT NOT caused by a GII.4-New Orleans- 2009 TO INFECTION IN A FAMILY NOROVIRUS variant. The most relevant finding was that OUTBREAK an asymptomatic non-secretor individual also shed NoVs in his stools. N. CARMONA VICENTE, M. FERNÁNDEZ JIMÉNEZ, S. VILA VICENT, J. RODRÍGUEZ We observed that different NoV VLPs DÍAZ, J. BUESA showed different binding patterns Departamento de Microbiología, Facultad de determined by HBGAs and that the non- Medicina, Universidad de Valencia, Avda. Blasco secretors saliva was poorly recognized by Ibáñez 17, 46010 Valencia, España any of the VLPs GII.4 variants. Interestingly, anti-NoV IgA antibody levels both in saliva Human noroviruses (NoVs) are the main and in serum samples, from secretor and cause of non-bacterial gastroenteritis non-secretor individuals, showed no worldwide. Several studies have associated differences, while only high norovirus- NoV susceptibility to human histo-blood specific IgG antibody titers were found in group antigens (HBGAs), namely to the both convalescent sera (collected 14 days secretor status (FUT2 gene expression), post-infection) and in memory sera and to Lewis antigens (Lea and Leb) (collected 1 year post-infection) in secretor determined by the FUT3 gene. Earlier positive individuals. volunteer and outbreak studies showed It was examined the capability of the that only secretor-positive individuals were different sera (both convalescent and infected, either symptomatically or memory sera) to block the binding of VLPs asymptomatically. However, more recent to the saliva of a secretor-positive O blood studies have demonstrated that secretor- type donor, using GII.4-2006b variant VLPs. negative individuals may also be infected As expected, the binding to receptors by NoVs, and that the susceptibility to present in the saliva was blocked efficiently infection can be genotype-specific. A NoV by secretor positive sera, up to 68%. In gastroenteritis outbreak occurred in a contrast, non-secretor sera did not block household of 9 family members early the binding at all. January 2010, giving us the opportunity to This results reinforce the idea that study the susceptibility to NoVs of the susceptibility to human NoVs is both different individuals involved. To reach our dependent on HBGAs profile of the aim we recruited 22 volunteers including 8 individuals, as well as on the NoV genotype members of the family affected by the and variant. We also show that the outbreak and their secretor status, ABO immunity to NoV lasts for at least one year and Lewis antigens were analyzed. The after infection, showing that symptomatic binding of different NoV VLPs to their infection strongly stimulates the immune saliva samples and the NoV-specific system.

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(PO 114) efficiently in cells pretreated with IFN-I, THE INTERFERON-INDUCED ANTIVIRAL that express high levels of MxA, suggesting FACTOR MxA DOES NOT INHIBIT that virus replication is not blocked by VACCINIA VIRUS REPLICATION these, or other, IFN-induced antiviral M M. LORENZO, J.M. SANCHEZ-PUIG, AND proteins. R. BLASCO We have constructed a cell line inducibly Departamento de Biotecnología, INIA. Carretera de expressing human MxA, and showed that la Coruña km 7.5, 28040 Madrid, Spain MxA expression was able to block replication of RNA viruses like Vesicular Stomatitis virus (VSV). Vaccinia virus was Mx proteins contribute to the antiviral able to replicate unabated in those cells, in response induced by type I and type III conditions in which VSV replication was interferons. They belong to the dynamin severely inhibited, indicating that Vaccinia family of large GTPases, but their virus was not being affected by the molecular mecanism of action is currently presence of MxA. Further, a Vaccinia virus unknown. Interestingly, different Mx recombinant overexpressing MxA from proteins display widely different ranges of viral promoters was able to grow to wild- activities when assayed on unrelated type levels and form wild-type sized virus viruses. For instance, human MxA has plaques, further demonstrating the lack of antiviral activity against many RNA viruses. effect of MxA on Vaccinia virus replication. The activity of different Mx proteins seems to depend on the subcellular localization of We considered the possibility that Vaccinia the protein. Nuclear forms (like mouse virus resistance might be the result of Mx1) protect against viruses that replicate virus- encoded MxA counteracting in the cell nucleus, while cytoplasmic forms factor(s). To test this hypothesis, we (like mouse Mx2) inhibit replication of VSV carried out coinfections of Vaccinia virus and some other viruses that replicate in and VSV in conditions of MxA blockage of the cytoplasm. Remarkably, the human VSV replication. Consistently, we failed to MxA protein, which is localized to the detect cross protection of VSV from the cytoplasm, has a broad antiviral spectrum action of MxA by the coinfecting Vaccinia irrespective of the virus replication virus, suggesting that Vaccinia virus compartment. resistance to Mx is due to lack of susceptibility of the virus and not to Interestingly MxA has been reported to counteraction by trans-acting factors have inhibitory activity against some large expressed by vaccinia virus. DNA viruses like African Swine Fever virus and Monkeypox virus (Netherton et al 2009 Inhibition of a large double-stranded DNA virus by MxA protein. J Virol. 83:2310- 20; Johnston et al. 2012 In vitro inhibition of monkeypox virus production and spread by Interferon-β. Virol J. 2012, 9:5). However, Vaccinia virus (VV) can grow

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(PO 115) ruminants virus (PPRV), using the IMMUNE RESPONSE ELICITED BY multimerisation strategy. Five dendrimeric DENDRIMERIC PEPTIDES AGAINST CSFV IN constructs in different conformations were DOMESTIC PIGS. A NEW PATH TO DIVA formulated and inoculated in five groups STRATEGY (four animals each) of six-week old pigs J. A. BOHÓRQUEZ1, S. MUÑOZ-GONZÁLEZ1, (Landrace x Large White), while another S. DEFAUS2, B.G. DE LA TORRE2, M. PEREZ- group (control group) was inoculated with SIMÓ1, R. ROSELL1,3, M. DOMINGO1,4, D. NaCl 0.9%. Two doses of 2 mg each of the ANDREU2, L. FRAILE5 AND L. GANGES1* corresponding construct, mixed with Montanide v206 adjuvant (Seppic), were 1Centre de Recerca en Sanitat Animal (CReSA), Institut de Recerca i Tecnologia Agroalimentàries administered at days 0 and 21 of the trial. (IRTA), Campus de la UAB, Bellaterra, Barcelona, An experimental challenge with CSFV was 5 Spain performed with 10 TCID50 of CSFV (strain 2Departament de Ciències Experimentals i de la Margarita) 15 days after the second Salut, Universitat Pompeu Fabra, 08003 Barcelona immunisation. Additionally, four pigs 3 Departament d'Agricultura, Ramaderia, Pesca, (vaccination controls) were immunized Alimentació i Medi Natural, (DAAM), Generalitat de with one dose of a commercial live- Catalunya, Spain 4 attenuated vaccine (C-strain) and were Departamento de Sanitat i d’Anatomia Animals, challenged with the same inoculum at 16 Facultat de Veterinària, UAB, Bellaterra-Barcelona days post vaccination (dpv). Humoral as 5Departament de Producció Animal, ETSEA, Universidad de Lleida, 25198, Spain well as cellular immune response were evaluated in the 28 pigs at 8 different

dates after vaccination and challenge. Classical swine fever virus (CSFV) impairs Different levels of partial protection from the immune system of the host. The clinical signs were observed in the five degree of immune compromise is one of dendrimeric immunized groups. the determining aspects in the outcome of Interestingly, the best clinical protection the disease. Previous studies have shown was found in two groups inoculated with the existence of B and T cell epitopes into the same peptides in different CSFV, mainly in the E2 and NS3 proteins. conformations (B4T or B2T). In terms of Dendrimers represent a promising tool for CSFV specific humoral response analysed the multimeric presentation of epitopes in against the E2 protein, one of the candidate vaccines. This strategy can be dendrimeric peptides developed a faster useful for basic investigations of the humoral response at 8 days post challenge. mechanisms governing the induction and The cellular response was evaluated control of immunity. The aim of this work through INF-γ producing cells, with two was to evaluate the CSFV specific immune groups showing INF-γ levels after response generated by different epitopes stimulation with PHA on the day of within E2 and NS3 CSFV proteins, Challenge (0 DPI) and one of this groups combining them with a T helper epitopes increasing this response even further at 8 reported from Foot and mouth disease DPI. The specific humoral and cellular virus (FMDV) and Peste des petits response against every peptide will be

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presented. This results show the capacity accessory genes were not essential for of a previously reported epitope for a MERS-CoV replication in cell cultures. monoclonal antibody to induce immune Infection with rMERS-CoV-∆4ab deficient response in pigs. in 4a and 4b proteins significantly increased the expression of cytokines (PO 116) regulating the inflammatory and innate immune responses, suggesting their INTERFERENCE OF MERS-CoV ACCESSORY contribution to the inhibition of the NF- GENES WITH THE INNATE IMMUNE signaling pathway during infection. RESPONSE AND ITS CONTRIBUTION TO Consequently, these genes might modulate VIRULENCE pathogenesis. rMERS-CoV-∆4ab infection 1 1 I. SOLA , J. CANTON , F. J. GUTIERREZ- induced the formation of stress granules 1 1 1 ALVAREZ , L. MORALES , S. ZUÑIGA , M. T. (SG), suggesting the involvement of 4a-4b 2 2 SANCHEZ-APARICIO , A. GARCIA-SASTRE , proteins in the inhibition of the SG- 1 AND L. ENJUANES mediated antiviral response. In contrast to 1Department of Molecular and Cell Biology.National the deletion of accessory genes, an Center of Biotechnology (CNB-CSIC).Campus engineered virus lacking the structural Universidad Autonoma Madrid. Madrid. Spain 2 envelope, E, protein (rMERS-CoV-ΔE) was Department of Microbiology.Icahn School Medicine not successfully rescued, since viral at Mount Sinai. New York. USA infectivity was lost at early passages. Interestingly, rMERS-CoV-ΔE was rescued Middle East respiratory syndrome and propagated in cells transiently or coronavirus (MERS-CoV) is an emerging stably expressing E protein in trans, coronavirus infecting humans, associated indicating that rMERS-CoV-ΔE virus was with acute pneumonia, occasionally renal replication-competent and propagation- failure, and a high mortality rate (as of defective. Therefore, the rMERS-CoV-ΔE is th February 28 2015, 1030 laboratory- potentially a safe and promising vaccine confirmed cases have been reported, candidate. including at least 381 related deaths), which is considered a public health threat. A reverse genetics system for MERS-CoV has been developed by the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome, providing a tool to study the virus molecular biology and to develop attenuated viruses as vaccine candidates. A collection of recombinant MERS-CoVs deficient in the genus-specific genes 3, 4a, 4b and 5 was generated from cDNA clones. The growth kinetics of mutant viruses was similar to that of the wild-type virus, indicating that

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(PO 117) Moreover, release of Ebola VLPs seems to REGULATION OF EBOLA VIRUS MATRIX require the tyrosine phosphorylation of PROTEIN BY SUMO VP40 by the c-Abl1 tyrosine kinase. Here P. ALVARIÑO1, C.F. DE LA CRUZ-HERRERA2, we evaluated the regulation of VP40 by A. EL MOTIAM1, M. BAZ-MARTÍNEZ1, P. SUMO and analyzed the effect of VP40- RUIBAL3, J.V. PÉREZ-GIRÓN3, V. LANG4, SUMO interaction on the VP40 egress, M.S. RODRÍGUEZ4, C. MUÑOZ-FONTELA3, C. VLPs formation, and interaction with other RIVAS1,2 viral components. Our results reveal that SUMO plays an important role on VP40 1Centro de Investigación en Medicina Molecular (CIMUS), Universidade de Santiago de Compostela, functions. Instituto de Investigaciones Sanitarias (IDIS), Santiago de Compostela, Spain. (PO 118) 2Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología-CSIC, Madrid, PRESENCE OF PATHOGENIC ENTERIC Spain VIRUSES IN ILLEGALLY IMPORTED MEAT 3Heinrich Pette Institute, Leibniz Institute for AND MEAT PRODUCTS TO EU BY Experimental Virology, Hamburg, Germany INTERNATIONAL AIR TRAVELERS 4 Ubiquitylation and Cancer Molecular Biology D. RODRÍGUEZ-LÁZARO1,2,M. DIEZ- laboratory, Inbiomed, San Sebastián-Donostia, VALCARCE3, R. MONTES-BRIONES3, D. Spain GALLEGO4, M. HERNÁNDEZ1, J. ROVIRA3

1Instituto Tecnológico Agrario de Castilla y León, Ebola virus (EBOV) causes a severe and ITACyL, Valladolid, Spain often fatal febrile syndrome in humans. 2Área de Microibología, Facultad de Ciencias, The EBOV genome encodes seven genes, Universidad de Burgos, Burgos, Spain, the most abundantly expressed of which is 3Departamento de Biotecnología y Ciencia de los viral protein 40 (VP40), the major viral Alimentos, Facultad de Ciencias, Universidad de matrix protein. VP40 associates with Burgos, Burgos, Spain, 4 cellular membranes and coordinates Dependencia de Sanidad de Vizcaya, Delegación del Gobierno en el País Vasco, Bilbao, Spain numerous functions in the of EBOV, including regulation of viral transcription, morphogenesis, packaging One hundred and twenty two meat and budding of mature virions. In addition, samples confiscated from passengers on expression of VP40 is sufficient to generate flights from non-European countries at the virus-like particles (VLPs) that have similar International Airport of Bilbao (Spain) were characteristics to the actual infectious tested for the presence of the main viral virus. VP40 has been shown to interact pathogens (human norovirusesgenogroups with host cell factors such as the I and II, hepatitis A and E viruses) during endosomal sorting complex required for 2012 and 2013. A sample process control transport (ESCRT) machinery, COPII virus, murine norovirus, was used along proteins, and actin, which have been the whole process to evaluate the correct implicated in the budding, transport, and performance of the method. Overall, 67 movement of VP40, respectively. samples were positive for at least one

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enteric viruses, being 65 positive for neurons (SMN) complex that assembles Sm hepatitis E virus(53.3%), 3 for human proteins in splicesomal snRNPs. We have norovirus genogroup I (2.5%) and 1 for shown that, beyond its role in snRNPs human norovirus genogroup II (0.8%), biogenesis, Gemin5 acts as a down- whereas hepatitis A virus wasnot detected regulator of translation (1), competing out in any sample. The type of positive meat PTB from its binding site on the FMDV IRES samples was diverse, but mainly was pork (2). The minimal region of Gemin5 being meat products (64.2%). The geographical able to repress internal initiation of origin of the positive samples was wide translation in cells depleted of the and diverse; samples from 15 out 19 endogenous protein was mapped to the countries tested were positive for at least most C-terminal domain (G51383-1508) (3). one virus. However, the estimated virus However, deciphering the potential load was low, ranging from 55 to 9.0×104 partners of this factor influencing PDU per gram of product. The results translation control is a challenging obtained showed the potential unresolved question. To determine the introduction of viral agents in travelers’ mechanistic basis of the role of Gemin5 on luggage, which constitute a neglected translation control we have undertaken route of introduction and transmission. the analysis of a potential relation of Gemin5 with the ribosomal particles. In addition, we carried out tandem affinity (PO 119) purification (TAP) using different regions of DECIPHERING THE PARTNERS OF GEMIN5 the protein followed by mass spectrometry IMPACTING ON TRANSLATION CONTROL analysis to identify factors bound to R. FRANCISCO-VELILLA, J. RAMAJO, E. Gemin5. To get information about factors MARTÍNEZ-SALAS linked to Gemin5 by RNA bridges, we Centro de Biología Molecular Severo Ochoa, CSIC, conducted the TAP purification after Madrid, Spain exhaustive RNase A treatment during. Candidates were chosen based on both Initiation of translation of several RNA high score and biological function, aimed virus genomes is governed by internal at discovering new regulatory pathways ribosome entry sites (IRES) elements. IRES impacting on translation control. Promising function depends on the interaction with candidates have been produced as GST- eukaryotic initiation factors (eIFs) and fusions and used in pull-down assays with cellular RNA-binding proteins termed IRES different regions of Gemin5 to verify transacting factors (ITAFs). Mass whether the interaction was direct. Results spectrometry analysis of factors interacting on the most promising candidates with two viral IRES (foot-and-mouth influencing the role of Gemin5 on disease virus (FMDV) and hepatis C (HCV)) translation control will be presented. allowed the identification of proteins (1) Pacheco A, Lopez de Quinto S, Ramajo J, interacting with specific RNA domains. One Fernandez N, Martinez-Salas E. 2009. Nucleic of these factors is Gemin5, the RNA- Acids Res 37, 582-590. binding factor of the survival of motor

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(2) Pineiro D, Fernandez N, Ramajo J. Martinez- RNA molecules suggests that the Salas E. 2013. Nucleic Acids Res 41,1017-28. alternative frameshift product P3N+PIPO is (3) Fernandez-Chamorro J, Pineiro D, Gordon JM, produced, at least partially, through this Ramajo J, Francisco-Velilla R, Macias MJ, mechanism. Besides, we describe the Martinez-Salas E. 2014. Nucleic Acids Res 42, 5742-54. production of RNA molecules with an extra A inside the P1 coding sequence, which would be originated in a similar (PO 120) polymerase slippage event and yield a RNA POLYMERASE SLIPPAGE AS A novel ORF product, P1N+PISPO in the MECHANISM FOR THE PRODUCTION OF infection of diverse sweet potato¬infecting FRAMESHIFT GENE PRODUCTS IN PLANT potyviruses. Altogether, these findings VIRUSES OF THE POTYVIRIDAE FAMILY suggest that slippage might be a general DAVID SAN LEÓN *1, ADRIAN VALLI *2, property of viral RNA polymerases that can ARES MINGOT *3, DAVID BAULCOMBE 2, be exploited by viruses of different JUAN J. LÓPEZ-MOYA 3, JUAN A. GARCÍA 1, kingdoms to expand the coding capacity of BERNARDO RODAMILANS *1 their small genomes contributing to viral * These authors equally contributed to this work adaptation and evolution. 1 Centro Nacional de Biotecnología CNB, CSIC, Darwin 3, 28049-Madrid, Spain (PO 121) 2 Department of Plant Sciences, University of Cambridge, Downing street, Cambridge CB2 3EA, FREQUENCY OF HEPATITIS C VIRUS United Kingdom GENOTYPE 1A NS3, NS5A AND NS5B 3Centre for Research in Agricultural Genomics MUTATIONS ASSOCIATED TO ANTIVIRAL CRAG, CSIC-IRTA-UAB-UB, Campus UAB Bellaterra, RESISTANCE 08193¬Barcelona, Spain A. AVELLÓN CALVO, A. ZAPATA, M.C. GARCÍA GALERA, G. RODRÍGUEZ Viruses can use polymerase slippage to RODRÍGUEZ generate newly synthesized RNAs with Unidad de Hepatitis. Centro Nacional de nucleotide insertions or deletions, and thus Microbiología. Instituto de Salud Carlos III. Madrid produce alternative proteins relevant for infection in overlapping open reading Introduction:Routine resistance associated frames. This phenomenon, that is well variants (RAVs) analysis in hepatitis C virus described for some animal viruses (EBOV, (HCV) infection is currently limited to 80K MARV, HCV), has not been observed in any NS3 mutation pretreatment in genotype plant-infecting virus. The present work 1a, according to clinical guidelines. uses high-throughput sequencing to study However recent reports indicate that some RNA polymerase slippage during natural other NS3 or NS5A substitutions may infections of viruses from the Potyviridae predict the virologic failure (VF). family, the largest and economically most Objective:To describe the frequency of important group of plant RNA viruses, RAVs in NS3, NS5A and NS5B in HCV which is included in the Picorna¬like super genotype 1a infected patients, candidates group of viruses. The detection of modified for antiviral treatment.

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Material and methods:Samples: serum (PO 122) from: 219 patients (analysis of NS3), 62 EFFECTS OF VALPROIC ACID ON HSV-1 patients (NS5A) and 55 patients (NS5B); INFECTIONS Methods: in house developed nested PCR ANTONIO CRESPILLO1, BEATRIZ PRAENA1, systems. LUIS ÁNGEL DORADO1, LAURA LERMA1, Results: ENRIQUE TABARÉS1, FRANCISCO  NS3 RAVs: 80K (5%); 80R (0%); 122R SOBRINO2, RAQUEL BELLO-MORALES1, (0.9%); 155K (3.1%); 168AEV (0%); 170T JOSÉ ANTONIO LÓPEZ GUERRERO1. (0%) 1: Universidad Autónoma de Madrid, Madrid, Spain.  NS3 mutations in scored positions: 2: Centro de Biología Molecular Severo Ochoa, 122TGNC (10.3%), 170VP (4.5%) Madrid, Spain.

 NS5A RAVs: 30R (2.2%), 30H (2.2%), 30R+28L (1.6%), 31M+30C (1.6%) Valproic acid (VPA) is a small fatty acid used as drug in different neurologic  NS5A mutations in scored positions: diseases such as epilepsy, migraines or 28M (1.6%), 58P (6.4%), 58R+28L (1.6%) bipolar disorders. VPA acts by inhibiting  NS5B RAVs: none histone deacetylases (HDACs) in the cell  NS5B scored positions: 316NC: 5.4% nucleus, GABA pathways, Na+ channel in  NS3+NS5A RAVs: 3/62 (4.8%) cellular membranes, glycogen synthase kinase 3 (GSK3), protein kinase A (PKA) and (NS3:80K+NS5A:58L+28L;S3:80K+NS5A:28M; lipid metabolism. On the other hand, many NS3:122G+NS5A:30R) studies aim at the feasible role of VPA in Conclusions: demyelinating diseases (e.g. multiple NS3 RAVs other than 80K are almost as sclerosis) and its effect on the frequent as 80K (4% versus 5%). susceptibility of several cell types to the NS5A RAVs reached to 7.7%. infection of HIV, EBV and others viruses. NS5B RAVs were not detected in our Taken these data into account and the fact samples, in agreement with the low RAVs that HSV-1 has been involved in some rates previously reported. neuropathies, we have characterized the Almost a 5% of the studied samples effect of VPA on this herpesvirus infection combine both NS3 and NS5A RAVs of the human oligodendrocyte cell line mutations. HOG. First of all, the role of this compound in virus entry was tackled. Incubation with The role of routine testing of RAVs VPA induced a slight but reproducible previously to treatment is not defined yet. inhibition in the virus particles uptake. In addition, transcription and expression of viral proteins were significantly downregulated in the presence of VPA as well. Last but not least, the viral production was assessed with or without

the inhibitor of HDACs, measured by

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means of TCID50 assay. Surprisingly, and in protein was hyperphosphorylated at early comparison with the results shown above, times post-infection, suggesting that VPA dramatically blocked the detection of phosphorylation may modulate early infectious HSV-1 particles. events during viral infection. To study the In conclusion, VPA, a clinical compound role of CoV N protein phosphorylation currently used in convulsant and others during viral infection six recombinant neurologic disorders, could be considered TGEVs, each one including sets of grouped in a future as a therapeutic alternative sequential phosphorylable residues against viral infections such as several mutated to alanine were constructed. herpesvirus members. Overall, these residues accounted for 33 TGEV N protein predicted phosphorylation sites. Mutant viruses were recovered with (PO 123) peak titers similar to those of the wild type PHOSPHORYLATION OF CORONAVIRUS virus, suggesting that N protein phopho- NUCLEOCAPSID PROTEIN MODULATES mutants did not significantly affect virus VIRUS-HOST INTERACTIONS behavior in cell cultures, with the S. ZUÑIGA, M. BECARES, A. PASCUAL- exception of two mutants showing a IGLESIAS, I. SOLA, AND L. ENJUANES. delayed viral growth and reduced genomic Department of Molecular and Cell Biology.National RNA accumulation at early times post Center of Biotechnology (CNB-CSIC). Madrid. Spain. infection. Different patterns of interaction between phospho-mutant N proteins and Phosphorylation-based networks are host-cell proteins were observed. essential for cell proper function and According to these patterns we proposed phosphorylate viral proteins leading to a that N protein phosphorylation extent fine-tuning of virus-host interaction. affects its interaction with host cell Coronavirus (CoV) nucleocapsid (N) protein pathways, modulating CoV virulence. is a multifunctional phosphoprotein with key functions for both CoV life cycle and (PO 124) CoV-host interaction, as this protein affects IDENTIFICATION OF A GAIT-LIKE RNA multiple pathways in infected cells. N MOTIF AT THE 3’ END OF THE protein phosphorylated residues have TRANSMISSIBLE GASTROENTERITIS been identified in several CoV members CORONAVIRUS GENOME MODULATING from all genera. N protein phosphorylation INNATE IMMUNE RESPONSE during CoV infection was analyzed using S. MÁRQUEZ-JURADO1, S. ZÚÑIGA1, L. transmissible gastroenteritis virus (TGEV) ENJUANES1, F. ALMAZÁN1 as a model. A combination of OffGel and 1 Western-blot techniques was used. N . Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus protein phosphorylation pattern changed Universidad Autónoma de Madrid, Madrid, Spain during virus replication cycle. This protein was hypophosphorylated in the viral particle, as determined using mass- Coronavirus (CoV) replication and spectrometry analysis. In contrast, CoV N transcription are complex processes that

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require the specific recognition of RNA cis- motif during TGEV infection, a acting elements located at the ends of the recombinant virus harboring mutations in viral genome and are mediated by a huge this motif was engineered and protein complex encoded by the viral characterized. Mutation of the GAIT-like replicase gene together with cellular motif did not affect virus growth in cell proteins. In previous studies using cultures, indicating that the GAIT-like motif transmissible gastroenteritis CoV (TGEV) as was dispensable for TGEV replication in cell a model, nine cellular proteins interacting culture. However, an exacerbated innate with the 3’ end of the genome were immune response, mediated by the identified, from which a functional role on melanoma differentiation-associated gene CoV RNA synthesis was demonstrated for 5 pathway, was observed in cells infected heterogeneous nuclear ribonucleoprotein with the mutant virus compared with the (hnRNP) Q, glutamyl-prolyl-tRNA parental virus. Furthermore, the mutant synthetase (EPRS), arginyl-tRNA synthetase virus was more sensitive to interferon beta (RRS), and poly(A)-binding protein. In this than the parental virus. Altogether, these work, the RNA motifs interacting with data strongly suggest that the viral GAIT- these proteins were further analyzed to like RNA motif modulates the host innate study their mechanisms of action. A 32-nt immune response. RNA motif located at 410 nt from the 3’ end of the TGEV genome was found to (PO 125) specifically interact with aminoacil tRNA synthetases EPRS and RRS. This RNA motif RELEVANCE OF GENUS-α CORONAVIRUS has high homology in sequence and NSP14 DOMAINS IN VIRUS VIABILITY secondary structure with the gamma M. BECARES, A. PASCUAL-IGLESIAS, I. interferon activated inhibitor of translation SOLA, L. ENJUANES, AND S. ZUÑIGA. (GAIT) element, which is located at the 3’ Department of Molecular and Cell Biology.National end of several mRNAs encoding Center of Biotechnology (CNB-CSIC). Campus proinflammatory proteins. The GAIT Universidad Autónoma de Madrid. Darwin 3. Madrid, Spain. element is involved in the translation silencing of these mRNAs through its interaction with the GAIT complex (EPRS, The replication and maintenance of the hnRNP Q, ribosomal protein L13a, and largest RNA genome known is a hallmark of glyceraldehyde 3-phosphate coronaviruses (CoVs). As a consequence, dehydrogenase) to favor the resolution of these viruses encode a unique set of RNA inflammation. Similarly to the cellular GAIT modifying enzymes in the replicase gene. element, the viral RNA motif bound the One of them is non-structural protein 14 GAIT complex and inhibited the in vitro (nsp14) that is part of the CoV core translation of a chimeric mRNA containing replication-transcription complex, this RNA motif, suggesting that the viral providing proofreading activity during CoV RNA motif could constitute the first GAIT- RNA synthesis. Nsp14 is a bifunctional like motif described in a positive RNA virus. enzyme with 3'-5' exoribonuclease (ExoN) To test the functional role of the GAIT-like and guanine-N7-methyltranferase (N7-

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MTase) activities. ExoN hydrolyzes single recovered, despite their competence in and double-stranded RNAs and is part of a viral RNA and protein synthesis after mismatch repair system responsible for the transfection. The mutant lacking N7-MTase high fidelity of CoVs replication. activity was recovered, though it was Betacoronavirus mutants lacking ExoN highly prone to reversion. One of the activity exhibit a mutator phenotype, with mutants in the zinc finger domain was a 20 fold increase in the mutation rescued and characterized. Further frequency compared to wild-type viruses, analyses are in progress in order to clarify and enhanced sensitivity to RNA mutagens the interaction of the mutant viruses with such as 5-fluorouracil. ExoN mutant viruses the host cell. exhibit decreased virulence in mouse models. Nsp14 N7-MTase activity is critical (PO 126) for viral mRNAs capping. The cap structure allows efficient viral mRNA translation and EXPRESSION OF PSEUDORABIES VIRUS avoids their recognition as “non-self” by IE180 PROTEIN UNDER THE CONTROL OF the host cell. HUMAN TUMOR-SPECIFIC PROMOTERS (hTERT AND CEA): II.- EFFECT OF IE180 ON To elucidate the role of the different nsp14 APOPTOSIS INDUCTION domains in RNA synthesis and virus-host interaction, a set of mutants covering L. LERMA, S. ALCALÁ, B. MARTÍN, B. SAINZ different motifs of the transmissible JR., E. TABARÉS. gastroenteritis virus (TGEV) nsp14 protein Department of Preventive Medicine, Public Health and Microbiology.School of Medicine.Autónoma was engineered. The sequence changes University of Madrid. Spain. include mutations in: (i) the ExoN active site that, according to published information, abolish ExoN activity, (ii) the Pseudorabies virus (PRV) belongs to the zinc finger motif, which mediates nsp14 viral subfamily Alphaherpesvirinae. During binding to RNA, (iii) the N7-MTase activity, productive infection, genes are temporally and (iv) highly conserved regions in the N7- expressed in three ordered phases: MTase domain. The effect of these immediate early (IE), early (E) and late (L) mutations in CoV replication and genes. IE genes are essentially transcription was analyzed using replicons. transcription factors that induce E and L Six out of the ten mutants showed severe genes expression. PRV has a single IE gene, defects in RNA synthesis, with a moderate encoding the protein IE180, which has a decrease of the replication levels and a negative self-regulating function on its own strong to total reduction of transcription promoter, repressing its mRNA levels. This result confirmed the essential transcription. Like other herpes immediate role of nsp14 protein during CoV RNA early proteins, the role of PRV IE180 as a synthesis. Full-length infectious cDNA potent transactivator has been well clones of those mutants showing efficient studied and it has been shown that IE180 RNA synthesis were generated. In contrast can activate the transcription of various to betacoronaviruses, recombinant TGEV human cellular and viral promoters. viruses lacking ExoN activity were not Recently, it has been demonstrated that

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IE180 expression in transgenic mice induce apoptosis, a characteristic that produces neurological symptoms, could be beneficial for targeting specific suggesting a possible implication in the cell types, such as cancer cells. induction of apoptosis. In this study, we produced recombinant (PO 127) PRV viruses that express IE180 protein CELLULAR SENESCENCE LIMITS VIRAL under the control of the human tumor REPLICATION promoters: PRV-TER (human telomerase M. BAZ-MARTÍNEZ1,2, C.F. DE LA CRUZ- reverse transcriptase promoter, hTERT) 3 2 and PRV-CEA (carcinoembryonic antigen, HERRERA , S. DA SILVA-ÁLVAREZ , A. FERREIRÓS2, A. ELMOTIAM1,2, M. CEA) to better study the role of IE180 2 1,3 expression in vitro. The levels of IE180 COLLADO , C. RIVAS . 1 mRNAs expressed under the control of Centro de Investigación en Medicina Molecular (CIMUS), Universidade de Santiago de tumor-specific promoters in the Compostela, Instituto de Investigaciones recombinant viruses constructed was Sanitarias de Santiago de Compostela (IDIS), measured by qPCR and compared to the E15706 Santiago de Compostela, Spain parental virus vBecker2. PRV-TER- 2 Instituto de Investigación Sanitaria de Santiago de mediated IE180 mRNA expression levels Compostela (IDIS), Complexo Hospitalario were two-fold lower at 12 hpi but equal at Universitario de Santiago de Compostela (CHUS), SERGAS, E15706 Santiago de Compostela, Spain 24 hpi compared to vBecker2, while PRV- 3 CEA-mediated IE180 mRNA expression Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología-CSIC, Darwin 3, levels were 16- and 54-fold higher at 12 E28049 Madrid, Spain and 24 hpi, respectively. These results are consistent with those obtained previously in our laboratory by plasmid co- Cellular senescence is a stable proliferation transfection, where we showed that IE180 arrest triggered in the cell as a response to reduced the activity of the hTERT promoter stressful conditions that could endanger while an opposite activating effect was cell integrity. Although initially identified seen with the CEA promoter. The over- for cells after prolonged in vitro culture, expression of IE180 in U2OS cells infected the description of oncogene-induced with PRV-CEA produced a higher senescence was paradigmatic in defining cytopathic effect resulting in apoptosis this process as a tumor suppressor induction, which was confirmed by mechanism, and in general as a stress Annexin V staining. The percentage of late response. Indeed, many other situations apoptotic cells after PRV-CEA virus such as oxidative stress, fibrosis, DNA infection reached 3.47% compared to damage, etc, also result in senescence as a 0.31% in mock-noninfected cells and 0.52% protective response. Surprisingly though, and 0.46% in cells infected with vBecker2 little is known regarding the relationship or PRV-TER, respectively. In addition, the between cellular senescence and viruses. pro-apoptotic role of IE180 was Here, we evaluated cellular senescence as additionally confirmed in PK15-IE180 cells. a response triggered by viral infection and Thus, if sufficiently expressed, IE180 can addressed the putative protective effect it

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can have limiting viral replication. Our RNA structure-mediated patterns, we results demonstrate that whereas generated four transcripts (the entire oncogenic viruses inhibit senescence, domain 3, and subdomains C-rich, apical oncolytic viruses can stimulate senescence. region and stem-loop 3a, encompassing Furthermore, we show that cellular nts 86-299, 137-249, 151-227 and 159-194, senescence limits viral replication and respectively). RNAs were expressed using evaluate the different mechanisms and tRNA scaffold vectors, which preserve their pathways that may account for this native RNA structure. Besides, the antiviral activity. expressed RNAs are tagged with streptavidin aptamers that facilitate the (PO 128) capture of ribonucleoprotein complexes by using streptavidin-coated magnetic beads. IDENTIFICATION OF FACTORS Cellular factors associated to these RNAs INTERACTING DIFFERENTIALLY WITH were purified, and later identified by mass- STRUCTURAL RNA MOTIFS spectrometry. Silver staining of SDS-PAGE J. FERNÁNDEZ-CHAMORRO, E. MARTÍNEZ gels loaded with the RNA-interacting SALAS proteins revealed different pattern of Centro de Biología Molecular Severo Ochoa, CSIC- factors depending on the subdomain UAM, Madrid, Spain exposed to the cell lysate. Various groups of proteins, including eIFs, ITAFs, nucleic Internal Ribosome Entry Site (IRES) acid-binding proteins, trafficking factors, elements are highly structured RNA that cytoskeleton, signalling factors and control initiation of translation in metabolic enzymes were unequivocally picornaviruses, among other RNA viruses. identified by mass spectrometry. The The IRES element of foot-and mouth identified proteins were differentially disease virus (FMDV) is organized in five purified with the RNA transcripts, domains (1-5). Domains 2, 4, and 5 provide disclosing factors that bind preferentially binding sites for eukaryotic initiation to certain RNA structures. Proteins factors (eIFs) and RNA-binding proteins, belonging to different groups, ARF5, designated IRES trans-acting factors Rab1b, CELF-1 and RPS25, previously (ITAFs). However, little is known about unknown to interact with the FMDV IRES, proteins interacting with domain 3. This were selected to validate its ability to bind domain (nts 86-299) is self-folding RNA directly with the RNA structural that harbors conserved RNA motifs, subdomains by using in vitro approaches essential for IRES activity. The apical region (UV-crosslink and RNA mobility assay). Our (nts 151-227) consist of a cruciform data show that these factors behave as structure, of which the stem-loop 3a (nts IRES-binding proteins. 159-194) harbors the conserved GNRA motif. This motif has been proposed to participate in long-range interactions with the C-rich loop (located in transcript 137- 249). To identify proteins able to recognize

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(PO 129) to convert them into effective tools for REVERSE GENETICS FOR AVIAN REOVIRUS gene-deliver, epitope exposure, etc. Z. AFONSO PUERTA, L. K. BUSCH, C. DÍAZ However, such a system could not be JULLIEN, F. J. BENAVENTE MARTÍNEZ, J. M. develop for any of the Reoviridae members MARTÍNEZ COSTAS until only 2007 (2). The inherent difficulty to develop reverse-genetics for reoviruses Departamento de Bioquímica y Biología Molecular, Centro Singular de Química Biológica y Materiales can be partly explained by the presence of Moleculares (CIQUS), Universidad de Santiago de a segmented genome with an exquisite Compostela, 15782-Santiago de Compostela, Spain. selection method that picks up one (and only one) copy of each segment into every Avian reovirus (ARV) are the agents viral particle, together with the presence of responsible for avian arthritis (infectious precisely defined sequence ends in each tenosynovitis) and malabsorption genomic segment. In the last few years, syndrome among other avian diseases. several different methods have been Molecular virologists have devoted much described for the different members ot the more attention to the study of mammalian Reoviridae (2, 3). ARV present an aditional reovirus (MRV), considered the prototype difficulty when developing a method for of genus (1). Recently, reverse genetics, that is the need of using significant differences between the two primary chicken embryo fibroblasts (CEF) groups of viruses were made evident. for their in vitro culture. Here we describe More significantly, ARV but not MRV are the design and optimization of constructs able to induce the formation of syncitia in and methods aiming to develop a general the infected cells due to the presence of reverse genetics protocol for ARV. small FAST proteins (Fusion-Associated 1. Benavente, J., and Martinez-Costas, J. (2007). Virus Res. 123, 105-119. Transmembrane Protein) that cause membrane fusion. This characteristic lead 2. Kobayashi, T.; Antar, A.A.R.; Boehme, K.W.; Danthi, P.; Eby, E.A.; Guglielmi, K.M.; Holm, G.H.; to the division of the genus Orthoreovirus Johnson, E.M.; Maginnis, M.S.; Naik, S.; Skelton, into two differentiated groups: non- W.B.; Wetzel, J.D.; Wilson, G.J.; Chappell, J.D.; fusogenic reoviruses, being MRV their Dermody, T.S. (2007). Cell Host Microbe 1: 147– prototype, and the fusogenic reoviruses, 157. whose prototype virus is ARV. The most 3. Boyce, M.; Celma, C.C.P. and Roy, P. (2008). powerful technique in modern virology is Journal of Virology 82: 8339– 8348. reverse-genetics. Different systems have been developed for most virus families to produce infectious viruses from plasmid constructs. Reverse-genetics systems allow the researcher to develop viruses carrying designed mutations to make functional studies, with high reproducibility due to the absence of accumulated mutations because of serial virus passage. Reverse genetics also allows manipulating viruses

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(PO 130) observation to HCV using two viral STRONG REPLICATIVE SELECTIVE populations which differ about 2.2-fold in ADVANTAGE OF A MULTIPLY PASSAGED fitness, and also in resistance to antiviral HEPATITIS C VIRUS IN CELL CULTURE agents in particular to telaprevir and 1 1 1 ribavirin (Sheldon et al., J. Virol. 88:12098- G. BLANCO , E. MORENO , N. BEACH , I. 1 1 12111, 2014). The low fitness HCV is HCV GALLEGO , A.I. DE ÁVILA , E. p0, and the high fitness HCV is HCV p100 1,2 1,3 DOMINGO AND C. PERALES (fitness gain due to 100 serial passage in 1Centro de Biología Molecular “Severo Ochoa” human hepatoma cells). We reconstructed (CSIC-UAM), Campus de Cantoblanco, 28049, 5 different quasispecies containing a Madrid, Spain majority of low fitness HCV p0 and 2Centro de Investigación Biomédica en Red de decreasing proportions of the high fitness Enfermedades Hepáticas y Digestivas (CIBERehd), HCV p100. Infectivity in the course of 5 Barcelona, Spain 3 passages in human hepatoma cells in the Liver Unit, Internal Medicine, Laboratory of absence or presence of ribavirin and Malalties Hepàtiques, Vall d’Hebron Institut de Recerca-Hospital Universitari Vall d’Hebron, (VHIR- telaprevir was measured. Three HUVH), Universitat Autònoma de Barcelona, independent experiments in the absence Barcelona, Spain of drug confirmed a similar infectivity level for all the reconstructed quasispecies, The internal interactions within viral suggesting the lack of suppression of HCV quasispecies can dictate the behavior of p100 by excess HCV p0. A strain-selective mutant ensembles. The relevance of these qPCR confirmed the increasing dominance interactions is being increasingly of HCV p100 throughout the 5 passages. In recognized in natural infections in which the presence of telaprevir all the distinct mutant clouds converge in the reconstructed quasispecies which same environment. For example in the contained HCV p100 showed the competition between two hepatitis C virus characteristic antiviral resistance (HCV) populations following liver phenotype expected of HCV p100. Thus, a transplantation. Two major types of minority of high fitness HCV can drive an interaction have been observed among entire population to behave as the viral populations in general: minority component, without evidence of complementation among mutants of the suppressive effect by the majority same mutant spectrum, and interference population. The underlying molecular of replication of standard virus by mutated mechanisms are under investigation. mutant spectra. Previous results obtained with vesicular stomatitis virus showed that replication of a high fitness was suppressed by a majority of a low fitness cloud of mutants (de la

Torre and Holland, J. Virol. 64: 6278-6281, 1990). We aimed at extending this

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(PO 131) Del) in ENHII that modify HBx are INSERTIONS AND/OR DELETIONS IN THE associated with the severity of HBV MAIN REGULATORY REGION OF HEPATITIS infection. B VIRUS SUGGEST MULTICODING OF THE Aim:Evaluate the presence of insertions X PROTEIN and/or deletions in ENHII and the possible A. CABALLERO GARRALDA1, J. GREGORI effect of truncation or elongation of HBx FONT2, M. BUTI FERRET3,4, D. TABERNERO on the HBV quasispecies in untreated CAELLAS1,3,5, J. QUER SIVILA2,3, M. BLASI chronic hepatitis B patients. FORNAGUERA1,3,5, F. RODRIGUEZ- Patients and methods 6 2 ALGARRA , R. CASILLAS GOMEZ , C. Fifty samples from 50 antiviral-untreated 1 GONZÁLEZ FERNÁNDEZ , I. BELMONTE patients with chronic active hepatitis were 1 1 MULA , L. NIETO APONTE , R. ESTEBAN analyzed by ultra-deep pyrosequencing 3,4 1,3,5 MUR , M. HOMS RIBA , F. RODRIGUEZ- (454, Roche). FRIAS1,3,5 The region analyzed clustered positions 1 . Servicio de Bioquímica, Hospital Vall d'Hebron, 1596-1912, encompassing the 3’-end of X Barcelona, España ORF, the complete preCore and the 5’-end 2. Laboratorio Enfermedades Hepáticas, Institut de Recerca Vall d'Hebron, Barcelona, España of the Core gene, including the BCP and 3 ENII. . CIBEREHD, Instituto de Salud Carlos III, España 4. Departamento de Hepatología, Hospital Vall Insertion, deletion and insertion+deletion d'Hebron, Barcelona, España (Ins-Del) were studied, as well as their 5. Servicio de Microbiología, Hospital Vall d'Hebron, proportion in the total of sequences and Barcelona, España the different variants (haplotypes). 6 . Departamento de Telematica, Universidad Results:A total of 960921 sequences, Politécnica de Cataluña, Barcelona, España median 16734 sequences /patient (range 1905-57993) and 1039 haplotypes, median Background:The basic core promoter (BCP, 17 haplotypes/patient (range 4-55) were nt 1743-1849) of hepatitis B virus (HBV) analyzed. overlaps with the 3’-end of the X ORF (nt Overall, 128 different Ins-Del were 1374-1838), the 5’-end of the preCore detected in 7.1% of sequences and 27.5% region (nt 1814-2548) and the enhancer II of haplotypes. Ins-Del were observed in (ENHII). The ENH II regulates viral 47/50 samples (94%), a median of the 3.4% replication, encodes the preCore region sequences per patient (range, 0-74.5%) and the C-terminal region of the showed insertions and/or deletions. multifunctional transactivating protein X All these insertions and deletions changed (HBx), which most well-characterized the standard HBx stop codon (position binding partner is the protein DDB1. 155), leading to 49 altered codons and Naturally occurring sequence variation can resulting in a premature (truncated HBx) or be detected along the whole HBV genome, late (elongated HBx) stop codon. This especially relevant in the regions that occurred in 7.6% of sequences and 29.2% regulate the replication. In this regard of haplotypes. nucleotide insertions and deletions (Ins-

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Main Ins-Del included: (1) 8 nucleotide cytokines, growth factor deprivation, deletions between positions 1754-1777 oxidative stress, and DNA damage. PKR can causing truncated HBx, (2) duplications at phosphorylate the alpha subunit of the nt 1644-1670, that modified the HBx eukaryotic initiation factor (eIF)-2 complex interaction motif with DDB1 (3) one that results in inhibition of general insertion or (4) deletion of a T at position translation, plays a major role in the 1825, both encoding elongated HBx. activation and/or regulation of several Conclusions: The significant presence of transcription factors such as nuclear factor genomes that code for truncated or (NF)-kB and p53, and promotes apoptosis elongated HBx suggest a multicoding HBV in response to viral infection. PKR is mechanism with different HBx versions activated by binding to dsRNA, which with possible different functional roles. causes the homodimerization and The systematic presence of deletions autophosphorylation of the kinase. In and/or insertions could potentially affect addition, PKR can be activated by binding the transcriptional activity of HBV ENHII to cellular proteins ISG15 or PACT/RAX. and the clinical relevance should be Recently, our group demonstrated that evaluated in further studies. covalent attachment of small ubiquitin-like modifiers (SUMO) to PKR protein

promoted the activation of the kinase, (PO 132) potentiated the control of protein NON-COVALENT INTERACTION WITH synthesis by PKR and contributed to its SUMO IS NECESSARY FOR FULL antiviral activity. Here we demonstrate ACTIVATION OF PKR that PKR also interacts with SUMO in a C. F. DE LA CRUZ-HERRERA1, M. BAZ- non-covalent manner via SUMO MARTÍNEZ2, A. VIDAL2, V LANG3, A. E. interaction motifs (SIM). We also show MOTIAM2, M. S. RODRÍGUEZ3, M. that non-covalent interaction of PKR with ESTEBAN1, C. RIVAS1, 2 SUMO is required for full activation of PKR, 1 Departamento de Biología Molecular y Celular, inhibition of protein synthesis in response Centro Nacional de Biotecnología-CSIC, Madrid, to dsRNA, induction of apoptosis in Spain response to viral infection, and control of 2 Centro de Investigación en Medicina Molecular viral replication. In summary, these data (CIMUS), Universidade de Santiago de Compostela, highlight the relevance of SUMO in Instituto de Investigaciones Sanitarias (IDIS), Santiago de Compostela, Spain regulating the activity of this antiviral 3 Ubiquitylation and Cancer Molecular Biology protein. laboratory, Inbiomed, San Sebastian-Donostia, Gipuzkoa, Spain

The double-stranded RNA (dsRNA)- dependent serine/threonine kinase (PKR) is induced by type I interferon (α/β) and is activated in response to stress signals such as the presence of dsRNA, cytotoxic

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(PO 133) other gene product results in self-assembly INFLUENCE OF CARGO ON P22 CAGE of the procapsid (PC, 58 nm diameter, 3 STABILITY 58,000 nm ); heating at 65ºC for 10 min 1 2 1 causes irreversible expansion to yield the J.R. CASTÓN , A. LLAURÓ , D. LUQUE , B.L. 3 TRUS3, E. EDWARDS4, J. AVERA4, T. expanded capsid (EX, 64 nm, 113,000 nm ). DOUGLAS4, P.J. DE PABLO2 By heating PC or EX at 75ºC for 20 min, pentamers are released, and EX with 10 nm 1Department of Structure of Macromolecules, Centro Nacional de Biotecnología/CSIC, 28049 holes at 5-fold vertices are know as Madrid, Spain; 2Department of Physics of wiffleball (WB) particles. We used the SP Condensed Matter, Universidad Autónoma de fusion strategy to incorporate GFP or CelB 3 Madrid, 28049 Madrid, Spain; Imaging Sciences into the VLP interior when expressed in E. Laboratory, Center for Information Technology/NIH, coli together with CP; self-assembled Bethesda, MD 20892, USA; 4Department of Chemistry, Indiana University, Bloomington, IN chimeric PC mature into EX and WB. 47405 USA We used 3D cryo-electron microscopy (cryo-EM) and atomic force microscopy Virus capsids are used as protein cages to (AFM) to study how the nature of the cargo incorporate various types of materials at and its interplay with the capsid inner inner and/or outer capsid surfaces, or as surface might influence the properties of nanocontainers to encapsulate proteins or these nanocontainers. Cargo-loaded P22 other biomolecules with potential PC showed less deformation after application in nanomedicine and adsorption to a substrate than empty nanobiotechnology. Virus-like particles cages, regardless its nature. The rigidity (VLP) derived from the Salmonella increased for both GFP- and CelB-PC. There typhimurium bacteriophage P22 have been are nevertheless fewer SP-mediated CelB- used to encapsulate heterologous cargos, PC connections to cargo than for GFP-PC, - and CelB-PC were more fragilethan GFP-PC glucosidase (CelB) from the or empty PC. CelB-EX and CelB-WB hyperthermophile Pyrococcus furiosus. The particles were more rigid than empty-EX P22 capsid is built of 420 copies of a coat and -WB particles, although the cargo was protein (CP), which assemble into a T=7 detached from the shell, as shown by 3D icosahedral lattice with the aid of 100-300 cryo-EM. Rigidity is probably increased by copies of a scaffolding protein (SP); SP C- an electrostatic repulsion effect terminal residues interact with CP. (electrostatic potential surfaces of both inner capsid surface and CelB tetramers are P22 VLP undergoes a series of defined highly negative), although the elastic structural transitions after heating that collisions of the tetramers against the emulate bacteriophage P22 maturation. internal wall is also a plausible explanation. The P22 procapsid/capsid transition The detachment between cargo and capsid involves an increase in the internal volume, prevents an increase in fragility. CelB-EX as well as thinning and greater porosity of particles are heterogeneous; there are full the capsid shell. Heterologous expression capsids (48%), capsids with collapsed cargo of CP and N-terminal truncated SP fused to (28%), and empty, broken capsids (24%).

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Most CelB-WB particles are filled ello a pesar de los esfuerzos dedicados a homogeneously (75%) indicating that comprender los orígenes de la adaptación released pentamers reduced cargo-capsid y del fácil acceso a nuevas técnicas que tension, although the stabilizing cargo- permiten una exploración exhaustiva de la mediated effect is preserved. GFP-EX diversidad genotípica. Cualquier contain collapsed cargos and the diffusion aproximación teórica a la evolución effect appears to be dominant. Our results requiere un conocimiento suficiente de la show that the mechanical stability of the evidencia empírica a fin de diseñar protein cages depends on cargo-cargo and modelos realistas y falsables. Actualmente, cargo-capsid interactions. This influence is los virus representan el mejor sistema reciprocal, as P22 cages also modulate experimental para (i) estudiar la evolución rigidity of encapsulated cargo. en tiempo real, (ii) explorar estrategias adaptativas alternativas gracias al gran (PO 134) número de mecanismos moleculares que estos organismos despliegan, (iii) unir el ESTRATEGIAS ADAPTATIVAS EN nivel molecular al poblacional y (iv) diseñar POBLACIONES VIRALES. HACIA LA modelos evolutivos con capacidad IDENTIFICACIÓN DE COMPORTAMIENTOS predictiva. En esta contribución GENÉRICOS EN EVOLUCIÓN MOLECULAR presentaremos una serie de ejemplos de S. MANRUBIA dinámica de poblaciones virales que Centro Nacional de Biotecnología (CSIC), Madrid, indican que parece posible definir lo que España en física se denomina clases de universalidad. Estas clases dinámicas El desarrollo de teorías cuantitativas de la representan comportamientos genéricos evolución requiere sintetizar principios que caracterizarían, en particular, generales a partir de un gran número de transiciones adaptativas y transiciones a la observaciones. La dinámica adaptativa de extinción en poblaciones virales basadas una población implica muchos niveles únicamente en estrategias moleculares. Un distintos de descripción y selección, ingrediente esencial que permitiría una incluyendo mecanismos que causan clasificación de este tipo es la variabilidad genómica, los efectos de las independencia de la dinámica poblacional mutaciones en el fenotipo, las de ciertos detalles (como el tipo de interacciones entre individuos, la genoma o la tasa de mutación), pero no de competición inter-grupos o las la estrategia molecular precisa utilizada en restricciones impuestas por ambientes que la adaptación (como la existencia de varían en el tiempo. Como resultado, las recombinación o el uso de genomas aproximaciones teóricas solo han bipartitos). alcanzado un éxito parcial hasta el Este estudio se halla en un estadio momento. En parte, esto se debe a una incipiente y se completará en el futuro limitada comprensión de la relación entre mediante el desarrollo de modelos aspectos moleculares y los cambios dedicados a virus específicos, a fin de fenotípicos causados por las mutaciones, predecir las respuestas poblacionales y

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complementar conocimientos previos, y particles. We show that they lack core modelos de aplicabilidad general, capaces polypeptide V but do not lack the density de establecer una correspondencia entre corresponding to this protein in the X-ray mecanismos moleculares y las respuestas structure, therefore adding support to the adaptativas de las poblaciones. A medio adenovirus cryo-EM model. The two types plazo, el objetivo es contribuir al desarrollo of light particles present different degrees de una teoría evolutiva actualizada que of proteolytic processing, and their incluya la compleja estructura del espacio structure provides the first glimpse on the de genomas y las correspondientes organization of L1 52/55k protein inside estrategias adaptativas de poblaciones the capsid shell, and on how this naturales. organization changes upon partial maturation. (PO 135) STRUCTURE OF ADENOVIRUS c. 1. Liu, H., Jin, L., Koh, S. B., Atanasov, I., MATURATION INTERMEDIATES CLARIFIES Schein, S., Wu, L. and Zhou, Z. H., CAPSID ARCHITECTURE AND SHOWS Science329, 1038-1043 (2010). CHANGES RELATED TO SCAFFOLD d. 2. Reddy, V. S. and Nemerow, G. R., Proc PROCESSING Natl Acad Sci U S A111, 11715-11720 G. CONDEZO1, R. MARABINI2, M. CHILLÓN3, (2014). J. FLINT4 AND C. SAN MARTÍN1. e. 3. Pérez-Berná, A. J., Mangel, W. F., 1 Centro Nacional de Biotecnología (CNB-CSIC), McGrath, W. J., Graziano, V., Flint, J. and Madrid, Spain. San Martín, C., J Virol88, 1513-1524 2Escuela PolitécnicaSuperior, UAM, Madrid, Spain. (2014). 3 Centro de Biotecnología Animal y Terapia Génica (CBATEG). UAB, Bellaterra, Spain. (PO 136) 4Princeton University, New Jersey, USA. DIRECT VISUALIZATION OF BREATHING MOTIONS IN THE HIV CAPSID LATTICE, Adenovirus (AdV) is one of the most AND MODULATION OF ITS EQUILIBRIUM complex icosahedral, non-enveloped DYNAMICS AND MECHANICAL viruses. Although its structure was solved PROPERTIES by both cryo-EM and X-ray crystallography, the location of minor coat proteins is still A. VALBUENA, M. G. MATEU controversial (1, 2). Light density AdV Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and particles lack genome, contain the putative Universidad Autónoma de Madrid), Universidad scaffold L1 52/55k, and may represent Autónoma de Madrid, Madrid, Spain. assembly intermediates. L1 52/55k is required for packaging, and cleavages by The emerging area of Mechanical Virology the maturation protease facilitate its is providing novel insights into properties release from the nascent virion (3). Here of viruses such as elasticity, resistance to we present the molecular and structural rupture and susceptibility to material characterization of two different AdV light

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fatigue, unveiling the structural i) a unique, simple method to directly determinants of these properties, and quantify large breathing motions in a viral revealing biological adaptations based on capsid; ii) a description of the dynamics mechanical features of virus particles. In and mechanical features of a virus-based addition, virus particles and virus-derived bidimensional nanomaterial with materials are increasingly being explored applicability as a biological nanocoating; iii) as a source of novel biomaterials and proof-of principle that it is possible to nanodevices in Biomedicine and manipulate both breathing amplitude and Nanotechnology. However, viral particles mechanical strength of viral capsids and and other protein assemblies still present derived nanomaterials by binding small several issues for many intended molecules. applications. One basic issue is that they are "soft" materials, and may be too (PO 137) sensitive to degradation or disruption by chemical agents, temperature, and/or MOLECULAR EPIDEMIOLOGY OF MEASLES mechanical forces during their production, VIRUS IN SPAIN.GENOTYPE D4. 1 1,3 storage and/or use. Thus, it may be A.GÓMEZ-VECINO , J.E.ECHEVARRIA , 1,3 1,3 critically important to understand the M.M.MOSQUERA , A.CASTELLANOS , 1,3 1,3 1 structural basis of virus particle mechanics C.HOYAS , F.DE ORY , M.BANGERT , 2,3 2,3 and dynamics, and develop the know-how J.MASA , N.LÓPEZ-PEREA , 1,3 to manipulate their dynamic behaviour and A.FERNÁNDEZ-GARCÍA . mechanical features to improve their 1. Centro Nacional de Microbiología. Instituto de suitability for different technological Salud Carlos III. Majadahonda. Madrid. Spain. 2. applications. Centro Nacional de Epidemiología. Instituto de Salud Carlos III. Madrid. Spain. The protein capsid of the human 3. CIBER de Epidemiología y Salud Pública immunodeficiency virus (HIV) provides an (CIBERESP). Madrid. Spain. excellent model system to investigate and manipulate the mechanical properties and Introduction: Measles virus (MeV) causes a equilibrium dynamics of viral particles and highly contagious disease that is currently novel virus-derived biomaterials. In this targeted for a National Plan Elimination in study we have used atomic force our country since 2001. The MV microscopy to directly visualize and genotyping and phylogenetic analysis are quantify the large-scale equilibrium an essential part to trace the chains of dynamics ("breathing") and determine the transmission and to determine the mechanical behaviour (elasticity, elimination of the virus. The genotype D4 mechanical strength, and self-healing) of caused massive outbreaks in Europe (2010- the HIV capsid lattice in close to 2012). This study is focused on updating physiological conditions. We also the molecular epidemiology of this demonstrate that breathing amplitude and genotype in Spain from 2008 to 2012. mechanical features of the HIV capsid lattice can be manipulated by binding a Methods: The phylogenetic analysis was small ligand, betaine. The results provide: based on the450 nucleotides encoding the

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C-terminal end of the viral nucleoprotein during the 2012 but with lower number of (N-450). A total of 1640 sequences cases. obtained from GenBank and MeaNS Discussion: The pattern of measles databases were analyzed, 680 from Spain. genotype D4 circulation in Spain during the They were aligned (BioEdit v.7.0.5), period of studywas the same that in the haplotypes were described (DNAsp v.5) rest of Europe. Our results suggest the and their genetic distance analyzed (MEGA existence of different events of v.6). The phylogenetic trees were importation instead of continuous constructed by maximum likelihood circulation. In addition we described a new (RaxML v.7 y PhyML v.3). variant D4-Madrid that had a wide Results: We found 41 Spanish haplotypes, distribution in our country which caused grouped in 18 phylogenetic clusters, local outbreaks during years 2011 and belonged to prevalent European variants 2012 and was further detected in Rumania during these years: 11 haplotypes (182 and United Kingdom. The existence of 2 sequences) related to identical strains older than the reference MVs/Enfield.GBR/14.07/-variant (D4- strains D4-Enfield and D4-Manchester Enfield); 2 haplotypes (16 sequences) determine an older origin of the variants in related to the sub-variant India and France, respectively. MVs/Hamburg.DEU/03.09/-variant (D4- Hamburg) and 14 haplotypes (251 (PO 138) sequences) related to the sub-variant MVs/Manchester.GBR/10.09/-variant (D4- OCCURRENCE OF INTRA-OUBREAK Manchester). Furthermore there were 2 VARIABILITY IN SEMI-CLOSED sub-variants from D4-Machester INSTITUTIONS 1 (Mvs/Marmande.FRA/43.11/2/ and A. SABRIÀ, R. M. PINTÓ , A. BOSCH, N. 2,3 2,3 MVs/Maramures.ROU/3.11/) in minority- TORNER , A. MARTÍNEZ , A. 3, 4 1 way and we described a new one: DOMÍNGUEZ , S. GUIX and the Catalan MVs/Madrid.ESP/46.10-variant (D4- Viral Gastroenteritis Study Group of the Madrid) with 10 different haplotypes (126 Project PS09/02516. sequences). We denoted 2 strains older 1 Enteric Virus Group, Department of Microbiology, than the reference strains of the D4-Enfield University of Barcelona, Barcelona, Spain. 2 (Mv/Raichur.IND/38.06/1) and D4- Department of Health, Generalitat of Catalonia, Barcelona, Spain. Manchester (MVs/Lisieux.FRA/27.07/1) 3 variants. In 2008 the variant D4-Enfield CIBER Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III, Madrid, Spain. was detected in Spain in a low incidence 4Department of Public Health, University of rate as in the next year 2009. During 2010 Barcelona, Barcelona, Spain. the measles incidence increased and the variants D4-Hamburg and D4-Manchester replaced D4-Enfield. The highest incidence Human noroviruses (NoV) are the most of measles was in 2011, mainly due to the common cause of acute nonbacterial D4-Manchester variant and sub-variants gastroenteritis in humans, causing large related to this, which were also detected outbreaks worldwide. They are a highly

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diverse group of viruses with a single- was also analyzed. Sequence analysis stranded RNA genome encoding 3 major within the P2 domain showed a higher ORFs. ORF1 encodes the nonstructural degree of genetic diversity (0.14%-2.90%) proteins, while ORF2 and 3 encode the and allowed us to confirm that more than capsid proteins. The major structural one strain co-circulated in 4 out of the 5 protein (VP1) has a hypervariable domain outbreaks which showed variability within (P2 domain) which is the most exposed region C. part of the virion. Classification is In conclusion, sequence of the P2 domain established according to nucleotide proved useful for multi-strain tracking diversity in the full length VP1-encoding during outbreak investigations and results ORF2 gene, but genotyping is often indicated that multi-strain outbreaks, performed based on sequence information probably of a nosocomial origin, are very in the ORF1/2 junction region (region C). common within semi-closed institutions. If Genotype GII.4 is responsible for the made promptly available, P2 sequence majority of infections in healthcare analysis may help to make decisions settings. regarding control measures which are The aim of this work was to assess the especially important in healthcare settings occurrence and characteristics of genetic where nosocomial infections are common. variability within 13 acute gastroenteritis outbreaks caused by GII.4 NoV (New (PO 139) Orleans 2009 strain) in semi-closed institutions during a 2-year study period in EPIDEMIOLOGIC STUDY OF INFLUENZA Catalonia. Transmission route for all 13 VIRUSES IN THE 2009 POST-PANDEMIC studied outbreaks was person-to-person, PERIOD (2010-2015) 1, 2 1, 2 except for 2 outbreaks, which were I. SANZ MUÑOZ , S. ROJO RELLO , A. 2 foodborne and then were transmitted RODRÍGUEZ FERNÁNDEZ , R. ORTIZ DE 1, 2 interpersonally. The study analyzed 37 LEJARAZU LEONARDO individuals including patients and samples 1. Valladolid National Influenza Centre, Valladolid, from asymptomatic caregivers or food Spain handlers related to the outbreaks. Region 2. Microbiology and Immunology Service, Clinic C sequences for most of the outbreaks University Hospital of Valladolid, Valladolid, Spain were 100% identical, but nucleotide variations were found in 5 person-to- person outbreaks (38.5%). The degree of Introduction: Although influenza viruses nucleotide diversity within region C ranged have been widely studied and described, it between 0.35%-1.53%. To further has been done a high number of investigate whether variability was due to epidemiological and pathogenic scientific the co-circulation of multiple strains in the approaches regarding their circulation same setting, or whether nucleotide since 2009 influenza pandemic. One of the variations were due to the quasiespecies most relevant conclusions is the rate of distribution within infected individuals, hospitalizations that occur in the different sequence of the most variable P2 domain age groups, depending on the influenza

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type and subtype that circulate mainly. The However, out-patient increased till 60% aim of this study is to analyze the during 2012-13 influenza season. Average demographic and epidemiological age of hospitalized patients increased characteristics of the last 5 influenza constantly from 20.2 years in 2010-11 seasons after 2009 pandemic influenza influenza season till 56.1 years in 2014-15 emergence. influenza season. In the case of out- Material and methods: An observational patients, increasing of average age was retrospective study was conducted lower, from 22.1 years in 2010-11 including results from 1,356 respiratory influenza seasons till 28.3 years in 2014-15 samples from hospitalized patients from influenza season. Castilla y León Hospital Network (Spain) Conclusions: Epidemics with higher and out-patients from Sentinel Surveillance circulation of influenza A viruses Network of Castilla y León (RCSCyL-Spain). (H1N1pdm or H3) have been characterized These samples were obtained from 2010- by a higher proportion of hospitalized 11(n=151),2011-12(n=337),2012- cases. However, in the epidemic with 13(n=120),2013-14(n=320) and 2014- higher circulation of influenza B virus has 15(n=428) influenza seasons. Identification been detected a higher proportion of out- of influenza A and B viruses and their patients. Similarly, influenza A epidemics subtypes (A/H1N1pdm09; A/H3) was have produced a much greater absolute performed using different molecular number of cases in hospitalized and also in diagnostic techniques and platforms out-patients. The average age of (Luminex 200-XTAG RVPFAST-Luminex; hospitalized patients has grown steadily ABi7500Fast-CDC real time InfluenzaVirus due to a complex phenomenon linked to subtyping Panel-Applied Biosystems; “seasonal adjustment” of a pandemic virus LightCycler 2.0-Influenza A/H1N1 Detection after their emergence. Set Subtyping -Roche; Clondiag Array Mate-Influenza A Genotyping Panel-Alere), and also by cellular culture using MDCK and MDCK/Siat1 cells. Demographic and epidemiological characteristics were analyzed in influenza seasons included. Results: Influenza A were the most detected viruses during 2010-11(80.8%- A/H1N1pdm09), 2011-12(94.4%-A/H3),

2013-14(62.2%-A/H1N1pmd09;32.5-A/H3) y 2014-15(70.3%-A/H3) influenza seasons, while influenza B were the most detected virus only in 2012-13 (61.7%) influenza season. The proportion of hospitalized-out patients remained stable during the seasons in which mostly circulated influenza A viruses (65-35% respectively).

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(PO 140) at confirming their actual endogenous IN SILICO EVIDENCES OF ANCIENT nature and genetic features. A wide survey ENDOGENOUS PARVOVIRUS SEQUENCES of our findings will be presented, and some IN THE GENOME OF LIVING PRIMATES: A possible biological implications will be MOLECULAR AND PHYLOGENETIC discussed. ANALYSIS NOOSHIN BAYAT, CARLOS GALLEGO, AROA (PO 141) TATO, VIRGINIA SANDONÍS, DAVID ABIA*, LACKING OF RELATION BETWEEN HHV-6 AND JOSÉ M. ALMENDRAL* IMMUNE RESPONSE AND BRAIN DAMAGE Centro de Biología Molecular "Severo Ochoa" IN ALZHEIMER’S DISEASE PATIENTS (Consejo Superior de Investigaciones Científicas- S. AGOSTINI1, R. MANCUSO1, A. HERNIS1, F. Universidad Autónoma de Madrid), 28049 2 2 3 Cantoblanco, Madrid, Spain. *Corresponding BAGLIO , M. CABINIO , E. CALABRESE , M. 1,4 authors CLERICI 1 Laboratory of Molecular Medicine and Biotechnologies, Don Carlo Gnocchi Foundation – The genome of multiple host species has ONLUS, Milan, Italy. been evolutionary colonized by viruses, 2 Magnetic Resonance Laboratory, Don Carlo mainly by RNA viruses as Retroviruses and Gnocchi Foundation–ONLUS, Milan, Italy. Bornaviruses. Recently though, sequences 3 Department of Neurorehabilitation, Don C. and full genes of some virus members of Gnocchi Foundation – ONLUS, Milan, Italy. the ssDNA Parvoviridae have been 4 Department of Physiopathology and recognized as evolutionary inserted Transplantation, University of Milan, Milan, Italy. (endogenized) in a wide range of animal genomes, including mammals. In searching Introduction: Alzheimer’s disease (AD) is novel endogenous parvoviruses (EPAV), we the most common form of dementia in the have performed an extensive in silico world, and clinically is characterized by analysis of Parvovirus sequences in the progressive memory loss, impairment of genome of currently living primate species, other cognitive functions as well as for which assembled genomes are inability to performed daily living activities. becoming available in databases. Our The amnestic mild cognitive impairment preliminary obtained data, focused in (aMCI) subjects are individuals with stretches of amino acids homologous to impairment in memory but preserved the type species of the nowdays circulating functional abilities, and they often genera of the Parvoviridae, suggest that represent a borderline condition between EPAV sequences are more common in normal aging and dementia. Although the Primates than previously suspected. abnormal features and lesions of AD brain Genetic analyses of the putative EPAV are well studied and characterized, the indicate intricate and diverse genetic etiopathogenesis of the disease is still configurations, which may include part or unclear. In the last years our group studied full non-structural (NS) and structural (VP) the relation between HSV-1 antibodies and parvovirus genes. Molecular analyses of cortical grey matter (GM) volumes of AD these putative EPAV are underway aiming

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and aMCI patients, finding in patients a Conclusions: the lacking of any relation positive correlation between high antibody between humoral immune response levels anti-HSV-1, and the volumes of brain against HHV-6 (Ab titers and avidity) and regions typically affected in disease. The AD reinforces our hypothesis about a next important step is to understand if this pivotal role of HSV-1, and not of other relation is specific for HSV-1 or it is typical herpesviruses, in the pathogenesis of the of also other herpesvirus, like the HHV-6, a disease. neurotropic virus involved in other neurodegenerative disease and suspected (PO 142) that could related also with AD. POLIOVIRUS REINTRODUCTION Aims: HHV-6 humoral immune responses MONITORING THROUGH THE ACUTE were analyzed in patients with a diagnosis FLACCID PARALYSIS AND of either AD, aMCI and in healthy controls ENVIRONMENTAL SURVEILLANCE: RESULT (HC), to verify possible correlations OF THESE ACTIVITIES IN LOMBARDY, 2014. between titers and avidity of HHV-6- L. PELLEGRINELLI1, L. FIORE2, V.PRIMACHE2, specific IgG and cortical grey matter 1 2 1 volumes analyzed by MRI. L. BUBBA , S.FIORE AND S. BINDA 1.Department of Biomedical Sciences for Health; Material and Methods: 59 early AD, 60 University of Milan; Milan, Italy. aMCI and 61 age-matched HC were 2. National Center for Immunobiologicals Research enrolled in the study. Patients underwent and Evaluation; Istituto Superiore di Sanità; Rome, Mini Mental State Evaluation (MMSE). Italy. Serum HHV-6 IgG Ab levels and avidity index were tested by ELISA. Three Introduction. The WHO Strategic Plan of randomly selected subgroups of 44 AD, 23 the Global Polio Eradication Initiative aMCI and 22 HC HHV-6 seropositive indicates the Acute Flaccid Paralysis (AFP) patients underwent brain Magnetic surveillance and the Environmental Resonance Imaging (MRI) by 1,5 T scanner. Surveillance (ES) as a crucial activities in Results: HHV-6 seroprevalence was similar order to detect eventual poliovirus (PV) in the 3 groups (AD: 97.6 %; aMCI: 78.3 %; reintroduction in polio-free countries and HC: 75.4 %) and also the HHV-6 Ab titers to achieve PV worldwide eradication. were not different in AD (2.27±1.48 Nowadays, AFP surveillance is the gold- Positivity Index(PI) compared to aMCI tandard but ES is able to detect PV (2.36±2.44 PI) as well as to HC (2.22±1.59 reintroduction without polio clinical PI). No differences were found even for Ab manifestation. This study aimed at avidity (median AD: 93.2%; aMCI: 88.6%; describing the results of the AFP HC: 93.8%) and no associations were seen surveillance and ES in Lombardy, Northern- between Ab titers, avidity and MMSE. Italy, in 2014. Finally, no correlation was found between Methods: The surveillance activities were Ab titers/avidity and brain damage, neither carried out according to WHO guidelines regarding the region typically affected by (WHO/IVB/04.10;WHO/V&B/03.03). All disease. children < 15-years-old who fulfilled the

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WHO definition for AFP and placed in (PO 143) Lombardy were included in the study and DISSEMINATION OF VIRAL PATHOGENS IN their stool were collected. During ES, A MEDITERRANEAN CLIMATE REGION wastewater sample was collected regularly 1 1 M. RUSIÑOL , X. FERNANDEZ-CASSI , N. twice a month at the intel of 3 wastewater 1,2 2 1 TIMONEDA , J. ABRIL , S. BOFILL-MAS , R. treatment plants located in Milan. AFP 1 GIRONES . stool and wastewater sample were 1.Laboratory of Virus Contaminants of Water and investigated to detect PV and non-polio Food (Vircont), Microbiology Department, Biology Enterovirus (NPEV). faculty, Barcelona University, Catalonia, Spain. Result. 13 AFP cases were reported with a 2.Computational Genomics Lab (Compgen), Genetics incidence rate of 1/100 000 children < 15 Department, Biology faculty, Barcelona University, years of age. The sensitivity of the Catalonia, Spain. surveillance system was good. The major clinical diagnoses associated with AFP were Fecal contamination of water is closely Guillain-Barré Syndrome (GBS, 53.8%). related to human health. Microorganisms According to the virological results, none from intestinal tract may arrive to water AFP case was caused by PV infections and through sewerage overflows, wastewater NPEV was detected in one patient. During treatment plant effluents, surface runoff ES, 70 wastewater sample were collected and direct discharges into the receiving and no PV was isolated; in the other hand, waters. Understanding the environmental an high rate of NPEV was detected (49/70; fate of pathogens is useful for minimizing 70%). AFP surveillance and ES achieved the the present risks to humans and also WHO performance indicator. evaluate the future trends in relation to Conclusion. AFP surveillance and ES must climate change. be maintained until global PV eradication As part of the EU-FP7-funded VIROCLIME will be declared. Although AFP surveillance project, the present study developed a remains the gold-standard, ES is a surveillance program centered on a powerfully tool to detect PV in the absence typically Mediterranean climate region: the of polio cases; for this reason, ES in Llobregat River basin (Catalonia, northeast Lombardy, as well as in other Italian of Spain) in order to evaluate the Region, needs to be improve. dissemination of viruses in water. Sample matrices included river water, untreated and treated wastewater from a wastewater treatment plant within the catchment area, and seawater from potentially impacted bathing water. Five viruses were analyzed in the study: a) human adenovirus (HAdV) and b) JC polyomavirus (JCPyV) were analysed as

indicators of human faecal contamination of human pathogens and were reported in

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urban wastewater (mean values of 106 and (PO 144) 5 3 10 GC/L, respectively), river water (10 ANALYSIS OF THE INTERACTION BETWEEN 2 2 1 and 10 GC/L) and seawater (10 and 10 microRNA miR-122 AND THE HCV IRES GC/L), indicating that wastewater plays an ELEMENT BY ATOMIC FORCE important role in the transmission of viral MICROSCOPY AND BIOCHEMICAL pathogens in water; c) Merkel Cell TECHNIQUES polyomavirus (MCPyV), which is associated 1 2 M. MORENO , L. VÁZQUEZ , A. ARIZA- with Merkel Cell carcinoma an aggressive MATEOS3,4, A. GARCÍA-SACRISTÁN1,3, R.M. skin cancer, was detected in 75% of the JÁUDENES1, J.Á. MARTÍN-GAGO1,2, J. raw wastewater samples (31/37), 29% of 3,4 1,3 GÓMEZ , C. BRIONES . river water and 18% seawater samples, 1. Department of Molecular Evolution, Centro de and quantified by a newly developed Astrobiología (CSIC-INTA), Torrejón de Ardoz, quantitative polymerase chain reaction Madrid, Spain. 4 (qPCR) assay (wastewater mean values 10 2. Instituto de Ciencia de Materiales de Madrid GC/L). Seasonality was only observed for d) (CSIC), Cantoblanco, Madrid, Spain. norovirus genogroup II (NoV GGII), which 3. Centro de Investigación Biomédica en Red de was more abundant in cold months with Enfermedades Hepáticas y Digestivas. (CIBERehd), levels up to 104 GC/L in river water and 106 Spain. 4 GC/L in untreated wastewater; and e) . Laboratory of RNA Archaeology, Instituto de human hepatitis E virus (HEV) was Parasitología y Biomedicina “López-Neyra” (CSIC), Granada, Spain. detected in 13.5% of the wastewater samples when analysed by nested PCR (nPCR). Secondary biological treatment Both 5´ and 3´ untranslatable regions (UTR) (i.e., activated sludge) and tertiary of the single-stranded RNA genome of treatment using disinfection with hepatitis C virus (HCV) are highly chlorination, flocculation and UV radiation structured and include regulatory elements removed between 2.22 and 4.52 log10 of necessary for viral replication and the viral concentrations. translation. In particular, the 5´UTR Climate projections for the Mediterranean contains an internal ribosome entry site climate areas and the selected river (IRES) element responsible for the cap- catchment suggest general warming and independent translation initiation (1). The changes in precipitation distribution. ion-dependent tertiary fold of the minimal Persistent decreases in precipitation during HCV IRES element (containing domains II to summer can lead to a higher presence of IV) has been investigated (2), and human viruses because river and sea water significant progress has been made in present the highest viral concentrations determining the three-dimensional during warmer months. In a global context, structure of individual IRES domains and wastewater management will be the key to subdomains at high resolution (3). preventing environmental dispersion of Nevertheless, little information is still human fecal pathogens in future climate available on the tertiary structure of the change scenarios. whole functional HCV IRES element.

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Atomic force microscopy (AFM) is a 1. Lukavsky (2009). Virus Research 139, 166. powerful, nanotechnology-based tool for 2. Kieft et al. (1999). J. Mol. Biol. 292, 513. the structural analysis of a wide range of 3. Berry et al. (2011). Structure 19, 1456. biological entities. It provides a 3D surface 4. Hansma et al. (2004) Curr. Opin. Struct. Biol. 14, profile of the imaged sample without 380. requiring any staining or coating, and its 5. García-Sacristán et al. (2015). Nucleic Acids Res. nanometer resolution is optimal for the 43, 565. visualization of RNA and RNA-protein 6. Díaz-Toledano et al. (2009). Nucleic Acids Res. 37, complexes (4). Recently, we have used 5498. AFM to investigate the magnesium- dependent folding of the HCV IRES in a (PO 145) sequence context that includes its PHYLOGENETIC ANALYSIS OF AN structured, functionally relevant flanking EPIDEMIC OUTBREAK OF ACUTE regions (domains I, V and VI) (5). In the 574 HEPATITIS C IN HIV-INFECTED PATIENTS nt-long HCV genomic RNA molecule BY MASSIVE SEQUENCING analysed, a sharp structural switch has 1 2+ N. CARO PÉREZ , M. MARTINEZ- been monitored when Mg concentration REBOLLAR2, J. GREGORI3, 4, J.QUER3, P. increases from 2 to 4 mM. This effect has GONZÁLEZ1, M. GAMBATO1, H.VISSER2, J. I. been confirmed by classical techniques for ESTEBAN3, J. MALLOLAS2, X. FORNS1, M. RNA structural characterization such as 2 1 LAGUNO S. PÉREZ-DEL-PULGAR . gel-shift analysis and partial RNase T1 1 Liver Unit, Hospital Clínic, IDIBAPS, CIBERehd, cleavage. Barcelona, Spain. 2+ Such a Mg -induced conformational 2Infectious Diseases Unit, Hospital Clínic, IDIBAPS, rearrangement is partially similar to that Barcelona, Spain. caused by the liver-specific microRNA miR- 3Liver Unit, Vall d'Hebron Institut de Recerca- 122 that, as previously shown (6), interacts Hospital Universitari Vall d'Hebron, CIBERehd, Barcelona, Spain. with the I-II spacer region of the HCV IRES 4 and induces switching between ‘open’ and Roche Diagnostics SL. Sant Cugat del Vallès. Barcelona, Spain. ‘closed’ conformers. Herein, we present the AFM analysis of IRES-574/miR-122 complexes in buffers containing 100 mM Background and aims: The incidence of Na+ supplemented with 0 to 10 mM Mg2+. acute hepatitis C among HIV-infected men Our results, supported by gel-shift assays, who have sex with men (MSM) has show that the Mg2+-induced open/closed significantly increased in recent years. This switch in IRES-574 is hindered by the increase may be due to factors such as interaction of miR-122. The competing high HCV viral load in blood and semen, effects of Mg2+ and mir122 reinforce the sex with risk of mucosal damage, a higher previously suggested structural and number of sexual partners, presence of functional continuity among domains I-VI concomitant ulcerative sexually of HCV IRES in its natural sequence transmitted diseases and the use of context. recreational drugs. The aim of our study was to investigate the dynamics of HCV

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transmission in an outbreak of acute Conclusions: HCV infection spreads rapidly hepatitis C in HIV-infected MSM in among HIV-infected MSM through a local Barcelona. network in Barcelona. The implementation Methods: Between 2008 and 2013, 113 of public health campaigns and preventive cases of acute hepatitis C in HIV-infected measures, as well as treatment MSM were diagnosed in the Infectious interventions with the new direct-acting Diseases Unit, Hospital Clínic, Barcelona. antivirals will allow the development of Phylogenetic analysis of the HCV NS5B strategies to reduce the HCV transmission gene was performed in a total of 73 of HCV within these high-risk groups. patients. Viral RNA was extracted from serum samples collected from each patient (PO 146) at the time of diagnosis. Massive ULTRASTRUCTURAL ALTERATIONS sequencing was performed using the INDUCED BY HCV REPLICATION REVEALED Roche 454 GS Junior platform. To define BY CRYO SOFT X-RAY TOMOGRAPHY. possible transmission networks, a b phylogenetic trees and multidimensional PEREZ-BERNA AJ *, RODRÍGUEZ MJ *, CHICHON FJb, FRIESLAND Mb, SORRENTINO scaling maps were constructed from a b a genetic distance matrices (Da). A , CARRASCOSA JL , PEREIRO E AND GASTAMINZA Pb Results: At the time of diagnosis of acute aALBA Synchrotron Light Source, MISTRAL Beamline hepatitis C, 53 of the 73 (73%) patients – Experiments Division, 08290 Cerdanyola del included in the study were receiving Vallès, Barcelona, Spain antiretroviral therapy. HIV viral load was bCentro Nacional de Biotecnología-Consejo Superior undetectable in 48 patients (66%) and the de Investigaciones Científicas (CNB-CSIC), Campus mean CD4 cell count was 923 cells /ul. HCV Cantoblanco, 28049 Madrid, Spain viral load was 6.37 log IU/mL (range 3.73- 6.99). Thirty-five of 53 (66 %) patients Chronic hepatitis C virus (HCV) infection treated with pegIFN and ribavirin achieved causes severe liver disease and a sustained virological response. The hepatocellular carcinoma. The detailed prevalence of HCV genotypes was: 4d 48% mechanisms underlying HCV pathogenesis (n= 35), 1a 44% (n= 32), 1b 7% (n= 5) and are largely unknown, although immune 3a 1% (n= 1). Phylogenetic analysis showed response-mediated events are major the existence of at least 14 monophyletic players of the liver damage. In addition, groups: 5 of genotype 1a, 1 of genotype 1b HCV replication and protein expression and 8 genotype 4d. Molecular analysis causes alterations of the host cell showed that the genetic distances homeostasis by, among other mechanisms, between genotype 4d viruses were causing endoplasmic reticulum (ER) and significantly lower than those of the oxidative stress. This is reflected at the subtypes 1a (p< 2.2x10 e-16) and 1b ultrastructural level by a dilation of the ER (p<0.039) . This result may suggest the cisternae and mitochondrial abnormalities existence of a single source of infection for previously reported in liver biopsies from genotype 4d and different sources for infected patients. subtypes 1a and 1b.

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In this study we have performed full-field arecurrently investigating the nature of the cryo soft X-ray tomography (cryo-SXT) in ER-mitochondria contacts in HCV- the water window photon energy range to replicating cells using cryo-SXT to shed investigate in whole, unstained cells, the light on the mechanisms by which HCV ultrastructural alterations induced by HCV inflicts mitochondrial damage locally. replication under conditions that are close to the living physiological state. Using this (PO 147) technology and guided by parallel analyses of the samples by immunolabeling, ULTRADEEP PYROSEQUENCING REVEALS confocal and transmission electron POLYMORPHYSMS IN THE PROBE- microscopy, we haveobtained the first BINDING SITE OF HCV-3 ASSOCIATED native tridimensional mapsofcellular WITH INDETERMINATE RESULTS BY A modifications caused by a stable COMMERCIAL GENOTYPING ASSAY 1, 2 3, subgenomic HCV replicon in cell culture. V. SALUDES MONTORO , J. QUER SIVILA 4, 5, JOSEP GREGORI3,4,6, E. BASCUÑANA In contrast with control cells, which display 1 3, 4 continuous ER cisternae of relatively PRIETO , D. GARCÍA CEHIC , J.I. ESTEBAN MUR3, 4, 5, V. AUSINA RUIZ1, 5, 7, E. MARTRÓ constant diameter, HCV replicating cells * 1, 2 show enlarged blind-ended tubules with CATALÀ 1 prominent pseudospherical extrusions. As . Servicio de Microbiología, Hospital Universitari Germans Trias i Pujol (HUGTIP), Fundació Institut expected, these ER alterations are reverted d’Investigació en Ciències de la Salut Germans Trias to normal after elimination of the viral RNA i Pujol (IGTP), Badalona, Spain and proteins from the replicon cells by 2. Centro de Investigación Biomédica en Red (CIBER) treatment with DAAs. We have also de Epidemiología y Salud observed a profound alteration of the Pública (CIBERESP) del Instituto de Salud Carlos III, mitochondrial morphology that correlated Madrid, Spain with the extent with which the ER was 3. Liver Unit, Internal Medicine, Lab. Malalties modified, both in replicons and in a Hepàtiques, Vall d'Hebron Insititute of Research surrogate model of HCV infection. The 3D (VHIR-HUVH), Barcelona, Spain 4 maps indicate a topological relationship . Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) between altered, possibly dysfunctional del Instituto de Salud Carlos III, Madrid, Spain mitochondria, and highly modified ER 5 . Universitat Autònoma de Barcelona, Barcelona, tubules. Within the same infected cell, Spain structurally sound mitochondria are 6. Roche Diagnostics SL, Sant Cugat del Vallès, Spain observed in areas where ER is 7. Centro de Investigación Biomédica en Red (CIBER) indistinguishable from that of control cells, de Enfermedades Respiratorias (CIBERES) del while mitochondria within or juxtaposed to Instituto de Salud Carlos III, Madrid, Spain modified ER are clearly altered, indicating a short-range influence of the viral Background:HCV genotyping is required in machinery on the mitochondrial viability. clinical practice in order to determine the Given the strong relevance that has been type and duration of antiviral therapy. A given to HCV-induced mitochondrial commercial genotyping assay (Real-Time dysfunction on viral pathogenesis we

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HCV Genotype II, Abbott) is used at one major sequence was identified in 11 HUGTIP for this purpose. From 2009 to patients. In the rest of patients, 1-3 2014, an indeterminate genotype result additional minor sequences were found has been obtained for 26 out of 1338 (representing <6.5% of all sequences). HCV patients (1.94%). The reference genotyping genotyping based on 5’UTR UDPS was in method, based on Sanger sequencing and agreement with NS5B Sanger sequencing phylogenetic analysis of the NS5B region, in all cases, confirming the absence of classified 24 cases as genotype 3 (20 as 3a mixed infections and recombination events and 4 as 3k), representing 7.95% of all between different genotypes/subtypes. HCV-3 detected cases (N=302). One patient The alignment of the 5’UTR sequences was from Belarus, 15 (62.5%) from evidenced the presence of 1-3 Pakistan, and 8 (33.3%) from Spain. HCV-3 polymorphisms at the probe-binding site is the second most prevalent genotype differentiating indeterminate from worldwide (30%) and in Spain (19.6%), it correctly genotyped HCV-3 samples. has been associated with a higher risk for Conclusions:The sequences generated in liver disease progression, and has shown this study could help to improve the ability lower response rates to the latest to detect HCV-3 by this commercial assay. antivirals. This improvement requires a product Aim: To characterize the genetic diversity change, but would be relevant in Spain and of the HCV 5’UTR region by ultradeep in many other countries where HCV-3 is pyrosequencing (UDPS) in these highly prevalent (eg. inSoutheast Asia, indeterminate cases, in order to find out South Asia, and Eastern Europe)or receive whether these results were due to the significant immigration from these areas. presence of mutations in the binding site of the HCV-3-specific probe of the (PO 148) commercial assay. LONG NONCODING RNAs WITH PROVIRAL Methods: For the 24 indeterminate OR ANTIVIRAL PROPERTIES samples the 5’UTR region was amplified 1 1 and subjected to UDPS with the 454/GS- M. BARRIOCANAL* , E. CARNERO* , C. PRIOR, N. RAZQUIN, V. SEGURA2 AND P. Junior platform (Roche) following a 1 recently published methodology. For all FORTES 1 identified haplotypes within each sample, Department of gene therapy and hepatology.2Bioinformatic unit. the genotype/subtype was assigned by phylogenetic analysis. For comparison, Centro de investigación medica aplicada (CIMA/UNAV). Pamplona. Spain. three additional samples that were correctly identified as HCV-3 by the real- time assay (3a by the reference method) Few studies have analyzed the antiviral were also analyzed. role of long noncoding RNAs (lncRNAs). Therefore, we used HuH7 cells infected Results:A median UDPS coverage of 591× with Hepatitis C virus (HCV; JFH-1 strain) or (IQR,141-1830) was obtained per patient. control cells, and we treated them or not For the highly conserved 5’UTR region only with interferon (IFN). Their transcriptome

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was analyzed by microarray and RNASeq. disease that follows a chronic infection The results show that most of the validated with HCV. candidates are induced after IFN treatment or HCV infection. (PO 149) ISRs (IFN-stimulated RNAs) are also KEY ROLE OF RNA STRUCTURAL CONTEXT induced after infection with mutant viruses AND ITS DYNAMIC STRUCTURAL that do not block the IFN pathway and in TRANSFORMATIONS PROMOTED BY miR- cultured cells or patients infected with 122 IN THE HEPATITIS C VIRUS IRES-40S HCV. Induction results from direct PREINITIATION COMPLEX. activation of the JAK/STAT pathway or by 1,2 1,2 the effect of NFKB or downstream A. ARIZA-MATEOS AND J. GÓMEZ effectors of the IFN response. Genome- (1) Laboratory of RNA Archeology, Instituto de Parasitología y Biomedicina 'López-Neyra', CSIC, wide guilt-by-association studies predict Armilla, 18100 Granada, Spain. (2) Ciberehd. that ISRs may function in the antiviral response. In fact, the best ISRs are located in the genome close to IFN stimulated The hepatitis C virus (HCV) internal genes (ISGs) with well-known antiviral ribosome entry site (IRES) (domains II to properties, such as GBPs, IL6, IRF1, ISG15 IV) exists as a part of a much larger RNA or BST2. Further, ISRs expression levels structure which expands from domains I to correlate significantly with the expression VI and presents two alternative level of their neighbouring gene in cultured conformations: closed, “C”, and open, “O”, cells and patients. Inhibition experiments by miR-122 binding to domain I. An show that they regulate positively or additional miR-122 binding site, recently negatively the expression of their described in vitro, together with one found neighbouring gene or other ISGs, indicating in this work, form an unexplored tandem that they may have proviral or antiviral binding site at the 3’ end of the IRES. While properties. the HCV IRES binds 40S ribosomal subunits and seems not to be affected by miR-122, Similarly, CSRs (HCV-stimulated RNAs) may it is unknown whether the higher-order have proviral or antiviral potential as they structure surrounding the IRES and its are altered in cells selected to replicate dynamic structural transformation outlined HCV efficiently. Further, inhibition of some above, participate in and modulate 40S- of the candidates, leads to decreased viral binding. replication. Interestingly, some CSRs are induced after infection with other viruses, We found that RNA I-VI was more efficient (indicating that they may have a wider pro- in 40S binding than shortened forms or antiviral effect with therapeutic lacking either or both flanks. In addition, potential); but also in the liver of HCV- we found that these RNAs formed two pre- infected patients or in patients with liver initiation complexes that differentially cirrhosis or hepatocellular carcinoma, migrate in native gel electrophoresis: the suggesting that some CSRs may be fast and slow forms, corresponding to the involved in the development of the liver closed and open states of RNA I-VI, respectively. Only the slow form of RNA I-

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VI, but not shortened RNAs, was These include the 3’X-tail and the 5BSL3.2 progressively stimulated by miR-122, domain of the CRE (cis-acting replicating el indicating that IRES in its natural context ement) region. Interestingly, the same has a regulation level lacking in the elements that control HCV protein shortened forms. Mechanistically, we have synthesis are responsible for promoting an demonstrated that both IRES flanks enhanced replicative state in the advanced contribute to the tRNA-like structure which infection. Besides their role as binding includes the AUG start triplet, and that platforms for protein factors, functional miR-122 alters it either by quick and local RNA domains may act as organizing destabilization of domain IV, in the case of centers for the establishment of complex tandem binding to 3’-IRES sites, or through networks involving intramolecular RNA- slow and long-distance effects in the case RNA interactions. Thus, the apical loop of of tandem binding to the 5’site. the 5BSL3.2 domain is complementary to the apical loop of the 3’SLII within the 3’X- tail, while the 8-nts bulge may establish (PO 150) two different contacts: one with the apical HEPATITIS C VIRUS GENOMIC RNA loop of the subdomain IIId of the IRES DOMAINS. SEARCHING FOR THE SWITCH region; the second with the so-called Alt TRANSLATION-REPLICATION motif, placed upstream of the CRE region. 1 C. ROMERO-LÓPEZ , A. BARROSO- RNA elements are also involved in the 2 1 DELJESUS , P. RÍOS-MARCO , A. BERZAL- establishment of intermolecular contacts 1 HERRANZ by directing the genomic dimerization 1. Instituto de Parasitología y Biomedicina López- process, which is initiated at the 3’X-tail Neyra, IPBLN-CSIC, PTS Granada, Avda. del and relies on the exposition of the DLS Conocimiento s/n, 18016, Armilla, Granada, Spain. 2 (dimer linkage sequence) motif in the . Unidad de Genómica, IPBLN-CSIC. dimerizable isoform. We have demonstrated that the acquisition of this Hepatitis C virus (HCV) genome is a positive structural isoform in the 3’X-tail depends ssRNA molecule encoding for a single open on both the IRES and the CRE regions. In reading frame, which is flanked by this model, the CRE would act as an untranslated regions (UTRs). These regions enhancer element of the dimer formation, bear structurally conserved elements that whereas the IRES could play an inhibitory play essential roles for the consecution of role. This interference with the genomic the viral cycle. In the early stages of the dimerization could be feasible even in the infection, viral polyprotein synthesis is a presence of the CRE, thus pointing to a yet preferential event that is governed by an unknown CRE-independent mechanism for IRES element (internal ribosome entry controlling HCV RNA dimer formation. By site), mainly located at the 5’UTR and using a high-throughput structural spanning a short stretch of the coding mapping strategy, this work identifies the sequence. IRES-dependent translation is residues and specific domains involved in also regulated by additional RNA elements this regulatory event. The presented data placed in the 3’ end of the viral genome. validate the existence of a complex, long-

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range RNA-RNA interaction network that immunocytochemistry with anti-NS5a operates as regulatory partner in multiple antibody. Huh7.5 apoptosis viral processes and controls transitions (AnnexinV/ToproIII) and the expression of between different steps of the viral cycle. the death receptors, DR4/DR5, were analyzed by flow cytometry. (PO 151) Results. JCI infectivity was inhibited near PLASMOCYTOID DENDRITIC CELLS 70% by unstimulated pDCs, while in this CONTROL HCV INFECTION IN VITRO condition, only 42% reduction of the 1 2,4 infectivity was observed for IR and P100, B. DOMÍNGUEZ-MOLINA , C. PERALES , K. which is consistent with previous data of MACHMACH3, J.SHELDON4, M. LEAL1, E. 4 1 IFN-α resistance of these viruses. However, DOMINGO , E. RUÍZ-MATEOS after CpG pre-stimulation of pDCs, all virus 1 Instituto de Biomedicina de Sevilla/Hospital infectivities were completely inhibited, Universitario Virgen del Rocío. Seville. Spain. 2 probably due to the high amount of IFN-α Liver Unit, Internal Medicine Hospital Universitari released (>5 log pg/ml). Regarding HCV Vall d’Hebron,Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. RNA release, unstimulated pDCs inhibited 3 Institut de Reserches Cliniques de Montréal. the 58% of JCI, pointing out that not all the Montréal. Canada. produced virus were infective. Specific 4 Centro de Biología Molecular “Severo Ochoa” infectivity (TCID50/mL/HCV-RNA (CSIC/UAM), Madrid, Spain. copies/mL) of JCI was reduced a 49.4% by unstimulated pDCs while IR and P100 were Introduction. Plasmacytoid dendritic cells reduced 13.8% and 7.4% respectively. (pDCs) are the main IFN-α producing cells Again, CpG pre-stimulation of pDCs of the immune system, therefore they are induced a strong inhibition of the three key players against antiviral infections. It viruses (>90%). There was not apoptosis has been shown that pDCs inhibit RNA induction by pDCs (with or without CpG virus replication in vitro, such as HIV and previous stimulation) in JCI Huh7.5 Influenza virus. However, the anti-HCV infected cells, suggesting that the decrease activity of pDCs and the mechanisms of JCI infectivity was mainly due to IFN-α involved in this processes remain unclear. production. However, in IR Huh7.5 infected cells, pDCs induced cell apoptosis. Material and Methods. Human pDCs were Unstimulated and CpG pre-stimulated isolated from four healthy donors. pDCs pDCs induced two- and five-fold apoptosis (with or without CpG previous stimulation) compare to control, respectively. No were cocultured with HCV infected Huh7.5 apoptosis was induced in P100 infected cells. For infections, the wild type virus cells by unstimulated pDCs, however, 2.6 (JCI), a virus after 100 serial passages in fold increase apoptosis was observed in presence of IFN-α, IFN resistant (IR) and a CpG pre-stimulated pDCs. No apoptosis virus after 100 serial passages (P100), were induction in JCI infected cells by CpG-pre- used. Extracellular-RNA virus was stimulated pDCs were not due to measured by qPCR and HCV infectivity, differences in Huh7.5 TRAIL receptors expressed as TCID50/mL, by expression since DR5 were upregulated

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three-fold in all CpG-pre-stimulated naïve patients with chronic HCV genotype conditions. 1 infection in clinical trials. However, the Conclusions.pDCs inhibited HCV replication presence of substitution Q80K causes a and infectivity in vitro. IFN-mediated reduction of SVR rates to 58%, similar to infectivity inhibition was the main patients treated with pegylated-interferon mechanism in JCI virus while TRAIL- plus ribavirin. Therefore, detection of Q80K mediated apoptosis was the predominant before starting therapy with Simeprevir is mechanism in IR HCV inhibition. needed to avoid treatment-failure. Methods: 368 samples from genotype 1 (PO 152) HCV chronic infected patients were provided by Hospital Universitari Vall THE PREVALENCE OF HEPATITIS C VIRUS d'Hebron, Barcelona. A real time PCR NS3 Q80K SIMEPREVIR RESISTANCE based technique was developed to identify MUTATION IN SPANISH POPULATION the Q/K variants at NS3 80 position using a ANALYZED BY NEW REAL TIME FRET technology and melting curves TECHNOLOGY IS A USEFUL TOOL FOR analysis despitethe significant variants PERSONALIZED TREATMENT surrounding the specifically tested a b,c,e Q. CHEN , F. RODRÍGUEZ-FRIAS , I. position. e a,b,c BELMONTE MULA , M. BUTI FERRET , L. Results: Specific nucleotide probes were NIETO APONTEe, D. GARCIA CEHICa,b, J. a,b,d a,b,c able to differentiate between Q and K GREGORI I FONT , R. ESTEBAN MUR , variants showing a complete concordance J. IGNACIO ESTEBAN MURa,b,c, J. QUER a,b,c with Sanger direct sequencing in all SIVILA . compared samples (n=11). To test the Liver Unit, Internal Medicine, Lab. Malalties specificity and sensitivity of the new Hepàtiques, Vall d’Hebron Institut Recerca— Hospital Universitari Vall d’Hebron (VHIR-HUVH), technique, HCV strands carrying Q and K Barcelona, Spaina; Centro de Investigación variants were cloned and mixed in Biomédica en Red (CIBER) de Enfermedades different proportions. The prevalence of Hepáticas y Digestivas (CIBERehd) del Instituto de Q80K resistance mutation in G1 population b Salud Carlos III, Madrid, Spain ; Universitat was 6.5% (24/368). Moreover, among 128 Autònoma de Barcelona, Barcelona, Spainc; Roche Diagnostics SL, Barcelona, Spaind; Biochemistry high resolution HCV subtyped samples, the Unit, Virology Unit /Microbiology Department, prevalence was 17.5% in G1a patients HUVH, Barcelona, Spaine. (n=63) but no cases were detected in G1b samples (n=65). Background: Hepatitis C virus (HCV) Conclusion: A new diagnostic tool based polymorphism Q80K is associated with on real time technology using specific resistance to Simeprevir, a NS3 protease probe has been developed to detect single inhibitor. This direct-acting antiviral (DAA), Q80K resistant mutation in a highly approved in 2014, in combination with variable background. As previously pegylated-interferon and ribavirin as a reported, the frequency and outcome of triple therapy, achieve a sustained Q80K resistant mutation varied between virological response (SVR) rates of 85% in HVC subtypes and its presence is closely

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related to the treatment effectiveness. QIAamp MinElute Virus Spin Kit (QIAGEN). Therefore, optimal treatment regimen will NS3 gene amplification was carried out by be achieved by performing HVC subtyping reverse transcription PCR (QIAGEN) and and Q80K detection before starting any nested PCR (Roche). Next, NS3 gene was Simeprevir based treatment. sequenced by Sanger-based technology. Results: In total, 471 out of 1690samples (PO 153) analyzedcorresponded toHIV/HCVco- PREVALENCE OF THE HEPATITIS C VIRUS infected patients and 1033 to (HCV) POLYMORPHISM Q80K IN HCV HCVmonoinfectedpatients. Overall, INFECTED PATIENTS WITH GENOTYPE 1A 179samples hadQ80K polymorphism IN SPAIN (10.59%).The prevalence ofQ80K polymorphism in HIV/HCV SONIA VÁZQUEZ MORÓN, Mª ÁNGELES coinfectedpatients was12.31% (58/471) JIMENEZ SOUSA, MÓNICA GARCÍA and9.48% (98/1033) inHCVmonoinfected ÁLVAREZ, MÓNICA GUTIÉRREZ RIVAS, patients.The higher prevalence ofQ80K SARA GÓMEZ ROBLES, LAURA LÁZARO polymorphism was found inthe regions PÉREZ, AMPARO ALVAREZ FERRERO, ofCeuta (33%), Canary Islands (20.83%), SALVADOR RESINO GARCÍA Aragon (20%) and Madrid (19.6%). The Unidad de Infección Viral e Inmunidad; Centro Autonomous Communitieswith the lowest Nacional de Microbiología, Instituto de Salud Carlos III; Carretera Majadahonda- Pozuelo, Km 2,2; 28220 prevalencewereCastillaLa Mancha(0%), Majadahonda (Madrid). Valencia (6.09%), Andalucía(6.42%) and Cantabria (6.96%). Background and aim: The Q80K Conclusion: The global prevalence ofQ80K polymorphism is a naturally occurring polymorphism was similarto that found variation in the NS3/4A protease of inother European countries (France, Italy, hepatitis C virus (HCV) which substantially Germany);however the prevalence of Q80K reduces the efficacy of triple therapy with polymorphism in HIV/HCV coinfected simeprevir, interferon alpha, and ribavirin. patients was slightly higher. The prevalence of Q80K polymorphism variesamongdifferent regionsor countries.The aim of this study was to evaluate the prevalence of Q80K polymorphism in HCV infected patients with genotype 1a in Spain. Methods: We evaluated the sequence of HCV NS3 protease gene in 1690 samples collected from HCV infected patients with genotype 1a in 108 hospitals distributed geographically across Spain,between

October 2014 and April 2015. HCV-RNA was extracted from plasma by using the

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(PO 154) cellular LPIN1 expression levels are rate- STUDY OF THE ROLE OF PHOSPHATIDATE limiting for HCV infection. This dependence PHOSPHATASES LPIN1 AND LPIN2 IN appears to be specific for HCV, as infection HEPATITIS C VIRUS INFECTION of the same cell lines with human L. MINGORANCE1,2 , M.F.FRIESLAND1, V. coronavirus (CoV-229E) resulted in normal CASTRO1 AND P.GASTAMINZA1. infection efficiency regardless on the levels of LPIN1 expression. Subsequently, we 1Laboratorio de Infección por el Virus de la Hepatitis C, Centro Nacional de Biotecnología, Madrid, Spain. dissected the HCV life cycle in order to 2Liver Unit, Hospital Clinic, IDIBAPS, CIBERehd, determine which step/steps is/are Barcelona, Spain. dependent on LPIN1 function, using different surrogate models. While viral entry or primary translation are not Hepatitis C virus (HCV) is a major causative affected by LPIN1 downregulation, a step agent of acute and chronic liver disease leading to accumulation of intracellular worldwide. Compelling evidence indicates HCV RNA is strongly dependent on this that HCV infection relies on cell lipid cellular factor. In contrast, LPIN1 does not metabolism and causes profound changes appear to be rate-limiting for persistent in lipid and lipoprotein homeostasis. Thus, RNA replication nor for infectious virus host factors involved in lipid metabolism production. Altogether, these results are interesting targets for antiviral suggest that LPIN1 is rate-limiting for the intervention. In accordance with this, we establishment of HCV RNA replication in an focused our attention on lipins, a early state of infection, but not once phosphatidate phosphatase (PAP) enzyme infection has been established. We are family that plays a key role in glycerolipid currently conducting similar silencing biosynthesis. These phosphatases mediate experiments with a second member of the the conversion of phosphatidate to lipin family (LPIN2), which is expressed in diacylglycerol, the immediate precursor of the liver to higher levels than LPIN1 and triacylglycerol (TAG), which is a major mRNA expression of which is also component of Lipid Droplets (LD) and Very regulated during HCV infection. We will Low Density Lipoproteins (VLDL), both present the results describing the impact required for HCV assembly. Under certain of silencing LPIN2 alone or simultaneously metabolic situations, LPIN1 and LPIN2 may with LPIN1 on different aspects of HCV also translocate to the nucleus to act as infection. Furthermore, we will discuss the transcriptional regulators of lipo- and consequences that activation of these adipogenic genes. Interestingly, we have genes and subsequent unbalance of observed that LPIN1 and LPIN-2 mRNA important intracellular lipid messengers by abundance is modulated by acute HCV HCV infection may have on the infection. pathogenesis of this virus. In order to study the role of LPIN family members, we first tested the susceptibility of human hepatoma cell lines (Huh-7) deficient in LPIN1 and determined that

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(PO 155) using a vector derived from citrus leaf THE DEFENSIVE PATHWAY MEDIATED BY blotch virus (CLBV). In sour orange SALYCILIC ACID MAY BE INVOLVED IN seedlings where these genes were RESISTANCE OF SOUR ORANGE TO CITRUS silenced, CTV accumulated to higher levels TRISTEZA VIRUS and displayed a better distribution with N. GÓMEZ-MUÑOZ, K. VELÁZQUEZ, J. regards to non-silenced controls, with this AGÜERO, S. RUIZ-RUIZ, MC.VIVES, P. effect being particularly noticeable in MORENO, J. GUERRI* RdRp1-silenced plants. Centro de protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (PO 156) (IVIA). Ctra Moncada-Náquera KM 4,5 Moncada 46113 Valencia, España. DEVELOPMENT OF A METHOD TO INCREASE EFFICIENCY OF VIRUS-INDUCED

GENE SILENCING IN CITRUS Citrus tristeza virus (CTV) induces decline N. GÓMEZ-MUÑOZ, K. VELÁZQUEZ, J. and death of different citrus varieties AGÜERO, S. RUIZ-RUIZ, MC.VIVES, P. grafted on sour orange, one of the most MORENO, J. GUERRI* devastating citrus diseases that has killed Centro de protección Vegetal y Biotecnología, more than 85 million trees worldwide, 40 Instituto Valenciano de Investigaciones Agrarias of them in Spain. Citrus plants propagated (IVIA). Ctra Moncada-Náquera KM 4,5 Moncada on sour orange and infected by CTV show 46113 Valencia, España. necrosis in the sieve tubes resulting in the decline and death of the tree. A very Virus induced gene silencing (VIGS) is a attractive hypothesis to explain the helpful tool to evaluate necrosis observed in the grafted sour orange trees infected with CTV is the plant gene function by reverse genetics. induction in this rootstock of a This technology has advantages with hypersensitivity reaction (HR) triggering respect to traditional approaches like programmed cell death (PCD) that stops mutagenesis and genetic transformation, virus invasion.Even if the precise because it allows to study the function of mechanism by which HR is triggered genes in a short time. However, VIGS is remains unknown, it appears to be related affected in many plants by the position, to the routeofsalicylic acid. On the other length and orientation of the insert hand, the finding that CTV accumulates at designed to silence a specific gene, and lower levels in seedlings of sour orange in depends also on the accumulation in target comparison with seedlings of susceptible tissues of the viral vector at a level citrus hosts, like Mexican lime and sweet sufficient to trigger silencing, which orange, suggests the existence of certain otherwise is incomplete. A standardization resistance to CTV in the former. To study if of the VIGS protocol is required for each this partial resistance is related with the plant species. Recently, several vectors defense pathway mediated by salicylic based on citrus leaf blotch virus (CLBV) acid, two genes involved in this pathway have been developed for VIGS in citrus. (NPR1 and RdRp 1) have been silenced CLBV induces a symptomless infection and

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is not phloem-restricted. In this work we (PO 157) have analyzed the silencing of RNA- EVALUATION OF POTENTIAL SOURCES OF dependent RNA polymerase 1 (RdRp1), BIAS IN VIRAL METAGENOME PROTOCOLS which is involved in antiviral defense in M. PARRAS-MOLTÓ1, A. RODRÍGUEZ- plants, including restricting the GALET1, P. SUÁREZ-RODRÍGUEZ1, A. LÓPEZ- accumulation of citrus tristeza virus (CTV) BUENO1. in citrus. For this aim two CLBV-derived 1 Centro de Biología Molecular Severo Ochoa vectors were prepared containing: i) a 281- (Consejo Superior de Investigaciones Científicas- nt fragment of RdRp1, and ii) based on Universidad Autónoma de Madrid), Madrid, Spain. transitive RNA silencing, a 159-nt fragment of the sulfur gene (Su) coding for a subunit Metagenomic surveys of viruses are of the magnesium chelatase, which is subjected to a number of biases derived highly expressed in all plant tissues, fused from particle purification, genome to a 148-nt RdRp1 fragment. The vectors extraction, random DNA amplification and were subsequently agroinfiltratred in library preparation for next generation leaves of Nicotiana benthamiana and sequencing. However, these viral semipurified virion preparations were then enrichment steps are required to increase mechanically inoculated into rough lemon the sensitivity of detection for viruses in and from this intermediate citrus host by metagenomics. Only a few methodological grafting to sour orange, in which CTV studies have assessed the impact of accumulation is restricted. Once CLBV different protocols in the preservation of infection was established in sour orange, community composition during plants were re-inoculated with a CTV-GFP metagenomic approaches. Although these clone expressing the green fluorescent studies have identified ClCs gradient, protein. The levels of RdRp1 mRNA and chloroform treatment and multiple CTV accumulation were estimated by real displacement amplification (MDA) as major time RT-PCR and microscopy. Our results bias sources, an optimal standard protocol show that in plants carrying CLBV-Su- is not yet available. RdRp1 the levels of RdRp1 were decreased twice and CTV accumulation were Here we performed a quantitative increased four times regard to CLBV-RdRp1 approach to analyze biases introduced in inoculated plants. These results suggest every step of a simple purification protocol that constructions with two genes in using an artificial viral community. This tandem increase the silencing efficacies community consists of a balanced mixture through the mechanism of transitive RNA of seven DNA viruses with different silencing. genome and structure. The protocol included two consecutive low-speed

centrifugation steps, filtration through 0.22 µm or 0.45 µm filters, iodixanol cushion and nuclease treatment. Extracted genetic material was alternatively amplified by two commonly used procedures: MDA and

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sequence-independent, single-primer (PO 158) amplification(SISPA). Viral community HCV RNA-DEPENDENT RNA POLYMERASE composition was analyzed by quantitative INTERACTS WITH AKT/PKB INDUCING ITS real-time PCR (qPCR), in triplicates. The SUBCELLULAR RELOCALIZATION relative proportion of a large virus such as ROSARIO SABARIEGOS1,4,MARÍA LLANOS- poxvirus was affected when 0.22 µm filters VALERO1, FRANCISCO CIMAS2, CELIA and low-force centrifugation were used. PERALES5,6, ESTEBAN DOMINGO5,6,7, These two steps are normally employed in RICARDO SÁNCHEZ-PRIETO2,7,8, AND viral metagenome preparation. ANTONIO MAS1,3,7,* Furthermore, a recent report has shown 1 Laboratorio de Virología Molecular and 2 important bias introduced by CsCl Laboratorio de Oncología Molecular, Centro gradients, another frequently used Regional de Investigaciones Biomédicas (CRIB), technique. On the contrary, viral Universidad de Castilla-La Mancha, Albacete 02008- community remained invariable after 0.45 Spain. um filtration and iodixanol cushion. 3Facultad de Farmacia and 4Facultad de Medicina, Regarding to random amplification, we Universidad de Castilla-La Mancha, Albacete 02008- Spain. found that MDA results in an over- 5 representation of small circular ssDNA Centro de Biología Molecular “Severo Ochoa”, CSIC-UAM, Cantoblanco 28049-Madrid, Spain. viruses (M13 or PCV2a), as previously 6 Centro de Investigación Biomédica en Red de reported, and the consistent under- Enfermedades Hepáticas y Digestivas (CIBERehd), representation of linear ssDNA viruses Barcelona, Spain. (MVMp). Interestingly, SISPA amplification 7 Unidad de Biomedicina UCLM-CSIC. 8 Parque preserves the ratio of the original viral Científico y Tecnológico de Albacete (PCyTA), mixture better than MDA. In addition, we Albacete 02008-Spain. purified viruses from a natural complex sample derived from the oral cavity and HCV interacts with cellular components amplified their genomes by MDA or SISPA and modulates their activities for its own before Miseq-Illumina sequencing (~2 benefit. The cellular kinase Akt/PKB must million paired-end reads). Consistently be activated to increase the effectiveness with the qPCR results described above, of HCV entry, but is rapidly inactivated as some of the most abundant viral contigs the viral replication cycle progresses. Viral from these two viromes exhibit notable components have been postulated as differences in coverage. responsible of Akt/PKB inactivation but the This research will contribute to establish a underlying mechanism remained elusive. standard-gold protocol for preparation of In this study we demonstrate that HCV viromes. polymerase (NS5B) interacts with, is a substrate of, and changes the subcellular localization of Akt/PKB. Recombinant

Akt/PKB can phosphorylate HCV NS5B in vitro. The specific Akt/PKB inhibitor MK- 2206 prevents NS5B phosphorylation in vitro, and delays the cell culture

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propagation of HCV infectious particles, macrophages. In this study, we examined both in a dose-dependent manner. HCV the polarization of bone marrow derived NS5B expressed either ectopically or from macrophage following the infection with a replicating viral RNA co- vaccinia virus (VACV) infection and the immunoprecipitates with Akt/PKB. role of ISG15 in macrophage polarization Moreover, Akt/PKB in the presence of after viral infection. transiently expressed NS5B or in replicon- The major aim of this proposal is to dissect or virus-infected cells modifies its cellular the antiviral role and function of the ISG15- localization from cytoplasmic into conjugation machinery in poxviruses perinuclear region where HCV replication infection. Several reports have complexes are located. The NS5B-Akt/PKB demonstrated that the antiviral effects of interaction represents a new regulatory ISG15 are attributed to ISG15-modification step in the HCV infection cycle, opening of viral proteins. Our preliminary data new therapeutic options. show, for the first time, ISG15-modification of virion particules from a pannel of (PO 159) completely different virus. Particularly ISG15 MODULATE VACCINIA VIRUS when whe analyzed the virion of poxvirus INDUCED MACROPHAGE POLARIZATION we observed ISG15 residius in particles oft wild type Vaccinia virus (VACV) but not of MERCEDES FERNÁNDEZ ESCOBAR, BEATRIZ the NYVAC mutant virions, indicating that MARTÍN MORENO, SARA BALDANTA AND the ISGylated viral proteins candidates are SUSANA GUERRA. encoded by genes that are present in the Laboratorio D-9, Dto. De Medicina Preventiva, Salud wild type virus genome but absent in the Pública y Microbiología, Facultad de Medicina, Universidad Autónoma de Madrid, Spain mutant virus NYVAC genome (a total of 18 protein candidates) we will perform a

complementary but different approach. If Macrophage polarized to M1 or to M2 our hypothesis that viral proteins are also phenotypes in response to environmental targeted for ISGylation and that this signals. M1 macrophages characterize a modification impacts virulence, we want to proinflammatory phenotype, exhibiting study the biological characteristics increased phagocytic and antigen acquired by a virus that propagated processing activity as well as increased exclusively in ISG15-/- cells. A VACV stock production of proinflammatory to promote will be amplified and purified using ISG15- host defence and removal of damaged /- cells and as a control we will perform the tissue. In contrast, M2 macrophage same purification in ISG15+/+ cells (Fig. 8). represents a phenotype that is potentially We will be compare the viral properties of important in the promotion of wound both stocks, primarily the ISGylation levels healing and tissue remodelling as well as in both purified virion stocks will be the resolution of inflammation. Recently analyzed by western blot as previously we have demonstrated that the interferon shown. Also we will compare the in vitro stimulated gen 15 (ISG15) governs the infection of mouse fibroblasts ISG15 +/+ phagocytosis capacity of peritoneal and ISG15-/- with both viral stocks.

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*Flash presentations more recently, the movement protein of (PO 160) Tobacco mosaic virus (Peiró et al., 2014.J THE MOVEMENT PROTEINS (NSm) OF Virology, 88: 3016), the type member of DISTINCT TOSPOVIRUSES PERIPHERALLY the 30K family, suggesting that the ASSOCIATE WITH CELLULAR MEMBRANES membrane-associated topology could be a AND INTERACT WITH HOMOLOGOUS AND general property for all members of the HETEROLOGOUS NSm AND 30K family.BiFC analysis for protein-protein NUCLEOCAPSID PROTEINS interactions showed: i), dimer formation for all NSm and N proteins; ii), interaction M. O. LEASTRO1, V. PALLÁS2, R. O. 1 2 between NSm and the cognate N and iii), RESENDE , J. A. SÁNCHEZ-NAVARRO heterologous interactions between the 1 Departamento de Biologia Celular, Universidade de NSm and N proteins. However, the Brasília, 70910-900 Brasília, Brasil; 2 heterodimers formed between the NSm Instituto de Biología Molecular y Celular de proteins revealed compatible interaction Plantas, Universidad Politécnica de Valencia-CSIC, E- 46022 Valencia, Spain. only among TSWV, CSNV and TCSV. In contrast, BeNMV was unable to interact with the other heterologous NSm proteins. Tospovirus is the only genus containing Interesting, TSWV, CSNV and TCSV have virus species which infect plants in the been grouped in the same clade into ‘New Bunyaviridae family. In the present work World’ tospoviruses, meanwhile BeNMV we have analyzed the in vivo membrane belong to a complete new branch of the association of the movement protein American species. This observation (NSm) of the tospovirus species Bean supports the idea that compatible NSm necrotic mosaic virus (BeNMV), interaction occurs only amongst viruses Chrysanthemum stem necrosis virus that are phylogenetically related. In (CSNV), Tomato chlorotic spot virus (TCSV) contrast, the NSm proteins are able to and Tomato spotted wilt virus (TSWV) and interact with the three heterologous N the homologous and heterologous proteins assayed, indicating that the NSm interactions among NSm and nucleocapsid does not discriminate between virus protein (N). The results obtained by particles, including members of the ‘New bimolecular fluorescence world’ (TSWV, CSNV, TCSV) grouping in one complementation (BiFC) assay and clade and BeNMV placed in a complete chemical treatments after membrane new branch. This observation differs fractionation, revealed that the four NSm significantly from that reported for other proteins are associated with the biological movement proteins of the 30K family, in membranes with the N- and C-termini which the viral movement proteins interact oriented to the cytoplasm. Similar with the cognate CP but not with the membrane-associated pattern has been heterologous CPs. These results raise the reported for other members of the 30K question whether the capacity of the NSm family, including the movement protein of to interact with heterologous N proteins is Prunus necrotic ringsport virus (Martínez- a peculiarity of the Tospovirus genus or Gil et al., 2009. J Virology, 83: 5535) and, could be extended to other members of

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the 30K family and, by other hand, shed harbouring the tissue plasminogen light on the potential interactions in mixed activator (tPA) signal peptide with a Tp1 infections. construct without a signal peptide. Ten BoLA-1*01302-positive animals were (PO 161) inoculated intramuscularly with HAd5 and MVA vectors expressing either of the Tp1 COMPARISON OF THE IMMUNOGENICITY constructs, and cell-mediated immunity is OF THE THEILERIA PARVA CTL ANTIGEN currently being monitored by ELISpot, Tp1, WITH OR WITHOUT A LEADER proliferation and cytotoxicity assays, as SEQUENCE, USING HAd5 AND MVA well as by flow cytometry using newly VACCINE VECTORS generated bovine peptide-MHC class I 1 1 1 N. SVITEK , A. LACASTA , R. SAYA , E. tetramers. Initial findings indicate that 1 1 2 1 AWINO , R. PELLÉ , S. GILBERT , V. NENE , HAd5/MVA viral vectors containing the Tp1 1 AND L. STEINAA antigen without a signal peptide induce a 1International Livestock Research Institute, Nairobi, stronger Tp1-specific CD8 response than Kenya. the vectors expressing the Tp1 antigen 2 The Jenner Institute, Oxford, UK. harbouring the tPA leader sequence. This information will be valuable in our design Theileria parva is a tick-borne parasite able of a next-generation vaccine for the to transform bovine T lymphocytes control of T. parva. resulting in a lethal lymphoproliferative disorder in cattle. This pathogen claims the (PO 162) life of approximately one million cattle MECHANISM OF ACTION OF 4- each year and results in economic losses of DEOXYPHORBOL ON HIV-1 INFECTION. A more than 300 million US dollars per year. NEW MEMBER OF ANTI-LATENCY DRUGS Immune animals develop a lifelong 1 1 immunity based on a cytotoxic T cell (CTL) HE DE LA TORRE , M BELTRÁN , LF NOTHIAS 2, J PAOLINI 3, LM BEDOYA 1, M response against homologous strains, with 2 1 a strong immunodominance restricted by LITAUDON , J ALCAMÍ 1 the major histocompatibility complex AIDS Immunopathogenesis Unit, National Centre (MHC) class I molecules. Human of Microbiology, Instituto de Salud Carlos III, Madrid, Spain adenovirus serotype 5 (HAd5) and 2 Gif Research Center, Institute of Chemistry of Modified Vaccinia virus Ankara (MVA) are Natural Substances (ICSN), CNRS, Labex CEBA, Gif promising antigen delivery systems able to sur Yvette, France induce CTL responses against several 3 Laboratoire de Chimie de Produits Naturels, UMR intracellular pathogens. In this study, we CNRS SPE 6134, University of Corsica, Corte, France aimed at inducing CTL responses in cattle against the T. parva BoLA-1*01301/01302- Introduction: Antiretroviral therapy (ART) restricted CTL antigen Tp1 by using a cannot eliminate HIV infection mainly due heterologous HAd5 prime – MVA boost to the persistence of HIV in latently vaccination regimen. We compared the infected cells in the blood and organs of immunogenicity of a Tp1 construct

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infected patients. Viral reactivation has (PO 163) been proposed as ART adjuvant therapy to EXPRESSION OF ARTIFICIAL MIRNAS: AN eradicate viral reservoirs. In order to do ANTIVIRAL STRATEGY IN PLANT that, new drugs with different mechanisms BIOTECHNOLOGY of action and less toxicity are needed. F. MESEL-CASANOVA1, M. ZHAO1, J.A. Material and Methods: Anti-HIV-1 and GARCÍA1, C. SIMÓN-MATEO1 anti-latency effects of a 4-deoxyphorbol 1. Departamento de Genética Molecular de Plantas, isolated from Euphorbia Centro Nacional de Biotecnología-CSIC, Madrid, amygdaloideswere evaluated in vitro in Spain MT-2 cells and freshly isolated human PBMCs using recombinant HIV carrying MiRNAs are important regulators of gene luciferase-Renilla reporter genes. Receptor expression in both plants and animals. expression was evaluated by single-, They are short single-stranded RNAs double- or three-color generated from longer premiRNA immunophenotyping and performed with a precursors and recruited to RISC effector FACScalibur flow cytometer. complexes, which, in a sequence-specific Transcriptional activity was performed by manner, down regulate target mRNAs. HIV plasmid DNA cell transfection using an Sharka disease, caused by Plum pox virus Easyject plus Electroporator. (PPV), is a persistent threat to the Results: 4-deoxyphorbol showed antiviral production of stone fruit trees of the activity with IC50s of 3nM in MT-2 cells and Prunus genus, and novel approaches for 0.3nM in PBMCs, infected with protection are needed. Trying to explore recombinant HIV (NL4.3-Ren). Specificity new strategies to develop virus resistance index of the compound is >10000, and no and understand how viruses can evolve to long-term toxicity was observed in PBMCs. escape from antiviral pressure, we have Moreover, 4-deoxyphorbol induced the developed Nicotiana benthamiana internalization of the lymphocyte receptors transgenic plants expressing artificial CD4, CXCR4 and/or CCR5 in MT-2 cells and miRNAs (amiRNAs) amiR-C and amiR-D, IL-2 preactivated PBLs. 4-deoxyphorbol targeting NIb and CP PPV RNA regions, was able to reactivate viral transcription in respectively. Several transgenic lines HIV-1 transfected MT-2 cells and resting expressing either one of these amiRNAs or human PBMCs at concentrations as low as both (amiR-CD) showed complete 10nM. Finally, 4-deoxyphorbol increase protection against PPV-R. Other transgenic transcriptional activity of LTR and NF-kB lines were only partially resistant and a few regions in resting PBMCs. plants were infected. A large diversity of Conclusion: 4-deoxyphorbol represents a virus variants with different mutations in new member of anti-latency HIV agents the amiRNA targets emerged in the that could be further developed as ART infected plants. Several species, frequently adjuvants to eradicate latent HIV reservoirs with more than one mutation, used to or achieve a functional cure. accumulate in single plants. Sequence analysis of different escaping mutants

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suggests that targeting of the genomic RNA (PO 164) by the mature amiRNA and of the CLEAVAGE OF THE ADENOVIRUS complementary viral RNA by the amiRNA PACKAGING PROTEIN L1 52/55K BY THE star strand contribute to the viral VIRAL PROTEASE: IMPLICATIONS FOR resistance. Viral progeny from some VIRUS ASSEMBLY infected amiR-D plants was passaged in A. J. PÉREZ-BERNÁ1, G. N CONDEZO1, R. transgenic amiR-D lines with different MARABINI2, W. F. MANGEL3, J. FLINT4, M. levels of virus resistance and in wild type CHILLÓN5, C. SAN MARTÍN1 plants. Mutations selected in the partially 1Centro Nacional de Biotecnología, CNB-CSIC, resistant plants allowed the virus to infect Madrid, Spain; 2Escuela Politécnica Superior, UAM, the highly resistant plants and were stable Madrid, Spain; 3Brookhaven National Laboratory, in wild type plants. However, additional NY, USA; 4Princeton University, NJ, USA; 5CBATEG- mutations appeared to be necessary to UAB, Barcelona, Spain. facilitate PPV infection under strong antiviral pressure. Broadness of the Adenoviruses are among the most complex antiviral infection was assessed by non-enveloped icosahedral viruses, with a inoculating amiR-C, amiR-D and amiR-CD 95 nm icosahedral capsid composed of 9 lines with PPV isolates of the strains M and different proteins, plus a 35 kbp dsDNA C, which differ in 1 to 3 nt from PPV-R in genome condensed by multiple copies of 3 the C and D targets. Whereas one core proteins (1). Adenovirus maturation mismatch located in the seed of the star consists in proteolytic cleavage of several strand of amiR-D does not prevent antiviral capsid and core proteins by the adenovirus activity, mismatches in the seeds of both protease (AVP) (2). Immature particles lack the mature and star strands of amiR-C infectivity because of their inability to facilitated infection of transgenic plants. uncoat. We have previously shown how Interestingly, the amiRNA target sequence adenovirus maturation modulates virion of some PPV-R escaping mutants selected stability, and therefore its ability to uncoat under amiRNA pressure, mimicked the correctly for a successful infection (3-5). sequence of the natural PPV-PS (strain M) In this communication we focus on the and PPV-SwCM (strain C) isolates, which relationship between maturation and could suggest the paths by which viruses genome encapsidation in adenovirus, by evolve, by drift or under different selective characterizing the proteolytic processing of pressures, are limited. the packaging protein L1 52/55k by AVP. By treating immature particles with recombinant AVP we prove that L1 52/55k is a substrate for the maturation protease, and reveal multiple non-consensus

cleavage sites. Proteolytic processing of L1 52/55k disrupts its interactions with other capsid and core proteins, providing a mechanism for its removal during viral maturation (6). Cryo-electron microscopy

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of two maturation intermediates shows element into the liver and lung of mice. the location of L1 52/55k in genome- Specific expression of a transgene like lacking capsids and how this changes upon luciferase can be induced by different maturation. Immature, full length L1 stimuli like Poly I:C, CpG DNA, Imiquimod 52/55k is poised beneath the vertices to or recombinant IFN-β. Intravenous engage the viral genome. Upon proteolytic injection of Newcastle disease virus (NDV) processing, L1 52/55k disengages from the can induce luciferase in the liver numerous vertex region, liberating it for the initial times, despite the generation of NDV steps of sequential uncoating. neutralizing Ab. Intranasal instillation of 1. C. San Martín, Viruses4, 847 (2012). the AAV vector allows upper and lower 2. W. F. Mangel et al., Viruses6, 4536 (2014). respiratory tract allows a continuous 3. A. J. Pérez-Berná et al., J Mol Biol392, 547 (2009). monitorization of type-I IFN signature after 4. A. J. Pérez-Berná et al., J Biol Chem287, 31582 (2012). intranasal infection with Newcastle disease 5. A. Ortega-Esteban et al., Sci Rep3, art. no. 1434. doi: virus or influenza virus. The vector 10.1038/srep01434 (2013). presented here can be accommodated to 6. A. J. Pérez-Berná et al., J Virol88, 1513 (2014). study both the strength and the kinetics of

type I IFN signature in different animal *Flash presentations (PO 165) organs in response to viral infections.

A VERSATILE ADENO-ASSOCIATED VIRUS VECTOR TO MONITOR THE INDUCTION OF *Flash presentations (PO 166) TYPE I INTERFERON SIGNATURES IN VIVO IN VIVO DELIVERY OF IFN-Β INDU TION 2 E. NISTAL-VILLAN*, Y. POUTOU , E. PATHWAY ACTIVATING ELEMENTS USING 2 2 RODRÍGUEZ-GARCIA , J. PRIETO , R. ADENO ASSOCIATED VIRUS VECTORS TO 2 1 HERNANDEZ-ALCOCEBA , E. LARREA , G. GENERATE AN ANTIVIRAL STATE 2 GONZÁLEZ-ASEGUINOLAZA 1 1 1 E. NISTAL-VILLÁN, E. RODRÍGUEZ GARCÍA, Instituto de Salud Tropical, University of Navarra, 1M. DI SCALA, 1Á. VALES, 1R. FERRERO Pamplona, Spain LABORDA, 2E. LARREA, 1J. PRIETO, 1G. 2Gene Therapy and Regulation of Gene Expression Program. Center for Applied Medical Research GONZÁLEZ-ASEGUINOLAZA 1 (CIMA), University of Navarra, Pamplona, Spain Gene Therapy and Regulation of Gene Expression Program. Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain. Development of reporter systems to 2Instituto de Salud Tropical, University of Navarra, monitor type I interferon (IFN-I) induction Pamplona, Spain. in vivo is of great interest to characterize viral infections. We show here the RIG-I like receptors (RLRs) are cellular generation of a type I IFN induction sensor proteins that detect certain RNA sensitive system that can be triggered both species produced during viral infections. by the IFN-β induction and by the type I RLRs activate a signaling cascade that IFN signaling pathways. With the use of results in the production of interferon-beta adeno-associated virus vectors (AAV), we (IFN-β) as well as several other cytokines have delivered this type I IFN sensitive with antiviral and proinflammatory

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activities. The potential of different large family of secreted proteins that have constructs based on RLRs to induce the distinct functions in antiviral defence, cell IFN-β pathway and create an antiviral state growth regulation and immune activation. in type I IFN-unresponsive models was Upon viral infection, the innate immune analyzed. A chimeric construct composed response is initiated leading to the of RIG-I 2CARD and the first 200 amino activation of signalling cascades that acids of MAVS (2CARD-MAVS200) showed culminate in the production of IFN / . an enhanced ability to induce IFN-β as These signalling pathways are activated compared to other stimulatory constructs. upon recognition of pathogen-associated Furthermore, this human chimeric molecular patterns (PAMPs) such as dsRNA construct showed a superior ability to by a series of germ-line encoded pathogen activate IFN-β expression in cells from recognition receptors (PRRs). The family of various species. This construct was found cytosolic PRRs retinoic acid-inducible gene to overcome the restrictions of blocking I (RIG-I)-like receptors (RLRs) recognizes IFN-β induction or signaling by a number of viral RNA. After binding viral dsRNA, RLRs viral antagonist proteins. Additionally, the become activated, initiating a signalling antiviral activity of this chimera was cascade that culminates in the expression demonstrated in influenza virus and HBV of IFN / proteins. IFN / are secreted infection mouse models using adeno- and bind to target receptors on infected associated viral (AAV) vectors as a delivery and uninfected neighbouring cells to vehicle. We propose that AAV vectors initiate signalling cascades within them expressing 2CARD-MAVS200 chimeric that end with the expression of proteins protein can reconstitute IFN-β induction with antiviral activity. As this response is and recover a partial antiviral state in very effective in preventing the different models that do not respond to establishment of viral infection, all viruses recombinant IFN-β treatment. need ways to circumvent the IFN response in order to survive in nature. (PO 167) Paramyxoviruses are known to have very STUDY OF THE MECHANISMS INVOLVED effective mechanisms to counteract the IN THE ACTIVATION OF THE PRRs THAT IFN response. They are a large group of DRIVE THE IFN INDUCTION PATHWAY enveloped viruses with non-segmented, F. DOMINGUEZ, R. E. RANDALL, M. KILLIP, negative sense, single-stranded RNA D. YOUNG, L. ANDREJEVA. BIOMOLECULAR genome. Paramyxoviruses are generally SCIENCES UNIT, UNIVERSITY OF ST poor IFN inducers as they encode for ANDREWS, ST ANDREWS, SCOTLAND. proteins that antagonize IFN induction and signalling pathways. Nevertheless, preparations of paramyxoviruses rich in The interferon (IFN) response is a powerful defective interfering (DI) viruses have been tool of the immune system that inhibits shown to be good IFN. virus replication and spread, buying time for the activation of an appropriate Paramyxoviruses are known to generate DI adaptive response. The IFNs comprise a virus genomes as the result of spontaneous

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errors of the viral polymerase. DI viruses contain genomes that still possess the signals for packaging and replication, but lack critical genes that render them unable to complete a full replication cycle in the absence of a co-infecting, non-defective (ND) virus. DI viruses have been shown to be good activators of cytosolic PRRs and consequently can be used as potential tools to study the mechanisms involved in their activation. This research takes advantage of paramyxovirus DIs as good IFN inducers, with the aim of identifying the proteins involved in the complexes formed within the cell when RIG-I and Mda5 are activated, as well as their cellular localization. This will facilitate a better understanding of the mechanisms involved in the activation of PRRs that drive this essential IFN induction pathway.

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AUTHOR/SPEAKER INDEX

AFONSO, Zaira, PO 129 BLASCO, Rafael, PO 114

AGOSTINI, Simone, PO 141 BLASCO, Bernat, CO 89

ALCAMÍ, Antonio, CO 41, PO 30, PO 97, PO 112 BLÁZQUEZ, Ana Belén, CO 11, PO 31, PO 33, PO 48

ALCAMÍ, José, CO 4 BLÁZQUEZ, Daniel, CO 52

ALMAZÁN, Fernando, PO 124 BOHORQUEZ, José Alejandro, CO 15, PO 92, PO 115

ALMENDRAL , José María, CO 46, PO 14O BORREGO, Belen, PO 108, PO 111

ALONSO, Covadonga, CO 10, CO 43, CO 53, PO 77 BOSCH, Albert, P 7, CO 58, CO 60, PO 138

ALONSO, Graciela, CO 41 BRIONES, Carlos, CO 7, PO 23, PO 58, PO 72, PO 118, PO 144

AÑEZ, Rafael, CO 76, PO 7 BRUN, Alejandro, PO 110, PO 111

ARANDA, Miguel A., CO 25, CO 27, PO 64, PO 67 BUESA, Javier, PO 74, PO 82, PO 87, PO 113

ARGILAGUET, Jordi, CO 42 CABRERIZO, María, CO 51, PO 37

ARIZA-MATEOS, Mª Ascensión, PO 3, PO 144, PO 149 CALDERON, Ana Maria, PO 34

ARRIBAS, María, CO 86 CALDERÓN, katherine Ivette, PO 39

AVIA, Miguel, CO 37, PO 95 CALERO-MUÑOZ, Nieves, PO 3

ÁVILA, Ginés, CO 91 CALVO, Cristina, CO 51, PO 34, PO 84, PO 85, PO 86

AYLLON, Juan, CO 68, PO 90 CALVO-PINILLA, Eva, CO 33, CO 38, PO 43

BALDANTA, Sara, PO 159 CAMPO DEL, Rosa, CO 69

BALFAGÓN,Pilar, PO 36 CAMPO DEL, José A.,CO 38, PO 4

BALLANA, Ester, CO 19 CAPELO, Juan Manuel, PO 83

BALLESTEROS, Natalia, PO 6 CARIDI, Flavia, CO 56, PO 42

BÁRCENA, Juan, CO 67, PO 50 CARO, Noelia, PO 145

BARRADO, Lucía, CO 10, CO 43, CO 53, PO 77 CASAS, Inmaculada, PO 34, PO 84, PO 85, PO 86

BARREIRO, Natalia, CO 16 CASTAÑO, Carlos, P 5, CO 12, PO 91, PO 98

BARTENSCHLAGER, Ralf, P 1, PO 61 CASTELLANOS, Ana, PO 35, PO 137

BAYAT, Nooshin, PO 140 CASTELLARNAU DE, Montserrat, P 7, CO 60

BAZ, Maite, CO 87, PO 117, PO 127, PO 132 CASTRO, Victoria, PO 154

BÉCARES, Martina, PO 123, PO 125 CEÑA-DIEZ, Rafael, CO 47, CO 54, PO 53

BENHNIA RAFII-EL-IDRISSI, Mohamed, PO 16, PO 17 CHEN, Qian, PO 152

BERKHOU, Ben, P 12 CHICHÓN, Fco Javier, CO 84, CO 91, PO 26, PO 75, PO 101, PO 146

BERLANGA, Juan José, CO 3 CISCAR, Marina, CO 90

BERZAL, Alfredo, CO 25, CO 48, PO 150 COIRAS, Maria Teresa, CO 4

BLANC, Stéphane, P 15 COLOMA, Rocío, CO 92

BLANCO, Guillermo, PO 130 CONDEZO, Gabriela Nerida, PO 26, PO 135

BLANCO, Esther, PO 50, PO 95, PO 104, PO 105 CRESPILLO, Antonio Jesús, PO 122

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CRUZ DE LA, Carlos Felipe, CO 87, PO 117, PO 127, FRANCO, Sandra, CO 85 PO 132

CUBAS, Liliana, CO 59, CO 90 FRANCO, Myriam Leticia, CO 1, CO 2, CO 13, PO 25, PO 29, PO 36

DE SABATO, Luca, PO 51 GALÁN, Juan Carlos, PO 79

DELOGU, Roberto, PO 88, PO 89 GALINDO, Inmaculada, CO 10, CO 43, CO 53, PO 77

DI BARTOLO, Ilaria, PO 44, PO 45, PO 51 GALLO, Araiz, PO 66

DI PILATO, Mauro, CO 18, CO 66 GANGES, Llilianne, CO 15, PO 49, PO 92, PO 115

DÍAZ, Luis, PO 68 GARCÍA, Marga, PO 47, PO 69

DÍAZ-TOLEDANO, Rosa, PO 3, PO 22 GARCÍA-ÁLVAREZ, Juan Antonio, CO 26, PO 1, PO 65, PO 66, PO 120

DÍEZ, Juana, CO 89, CO 93, PO 60, PO 61 GARCÍA-ARRIAZA, Juan Francisco, CO 23, PO 52, PO 101 PO 102, PO 107

DOMENECH, Ana Maria, CO 75, PO 7 GARCÍA-BRONCANO, Pilar, CO 47, CO 54

DOMINGO, Esteban, P 2, PO 130, PO 151, PO 158 GARCÍA-COSTA, Juan, PO 83

DOMINGUEZ PALAO, Francisco, PO 167 GARCÍA-SÁNCHEZ, Elena, PO 41

DURÁN, Manuel D., CO 34 GARCÍA-SASTRE, Adolfo, CO 10, CO 68, PO 90, PO 116

EL MOTIAM, Ahmed, CO 87, PO 117, PO 127, PO 132 GARCIA-UTRILLA, Raquel, PO 99 ENJUANES, Luis, P 5, CO 12, PO 21, PO 91, PO 98, PO 116, GASTAMINZA, Pablo, P 17, CO 55, CO 81, CO 84, PO146 PO 123, PO 124, PO 125 PO 154

ESCRIBANO, Estela, PO 31, PO 33, PO 48 GIRONÉS, Rosina, CO 65, PO 12, PO 143

ESTÉ , José A., CO 19 GÓMEZ – CASTILLA, Jordi, PO 3, PO 144, PO 149

ESTEBAN, Mariano, CO 18, CO 23, CO 66, CO 87, PO 52, GÓMEZ-LOPEZ, Pedro , PO 64 PO 75, PO 101, PO 102, PO 103, PO 107, PO 132

FALCÓN, Ana María , CO 36, CO 94 GÓMEZ-LUCIA, Esperanza, CO 78, PO 7, PO 8

FERNÁNDEZ, Mercedes, PO 23, PO 159 GÓMEZ-MUÑOZ, Neus, PO 155, PO 156

FERNÁNDEZ-CASSI, Xavier, PO 12, PO143 GÓMEZ-SEBASTIÁN, Silvia, CO 67, PO 96

FERNÁNDEZ, Javier, CO 36, CO 91, PO 23, PO 128 GÓMEZ-VECINO, Aurora, PO 137

FERNÁNDEZ-GARCÍA, Aurora, PO 35, PO 137 GONDAR, Virgina, CO 55

FERNÁNDEZ-RETUERTO, Borja, PO 43 GONZÁLEZ, Rubén , PO 93

FERRIOL , Inmaculada , CO 31 GONZÁLEZ, Patricia, PO 145

FIALLO, Elvira, CO 29 GONZÁLEZ-MUÑOZ, Víctor Manuel, CO 82

FIORE, Lucia, PO 88, PO 89, PO 142 GRANDE, Ana, PO 32, PO 68

FLETA, Eric , PO 60 GUERRA, Susana, PO 99, PO 106, PO 159

FLORES, Ricardo, CO 28, PO 7, PO 11, PO 62, PO 72 GUERRERO-BELTRÁN, Carlos, PO 53

FONTENLA , Julio, PO 83 GUIX, Susana, P 7, CO 58, CO 60, PO 138

FORTES, Purificación, PO 148 GUTIÉRREZ, Francisco Javier, P 5, PO 116

FRANCISCO-VELILLA, Rosario, PO 119 IANIRO, Giovanni, PO 44, PO 88, PO 89

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INGLESE, Nadia, CO 35 MARTÍNEZ-GONZALEZ, Isidoro, PO 93

JIMÉNEZ - QUINTANA , Esther, CO 70 MARTÍNEZ-TURIÑO, Sandra, PO 1

JIMÉNEZ-CLAVERO, Miguel A., CO 9, CO 77, PO 24, PO 38 MARTÍNEZ-PICADO, Javier, PO 80

MARTINEZ-SALAS, Encarna, CO 63, CO 80, PO 22, PO 23, JIMÉNEZ-FUENTES, José Luis, CO 22, CO 81, PO 52,PO 53 PO 119, PO 128

JIMÉNEZ-GUARDEÑO, José Manuel, P 5, CO 12, PO 91, MEDINA, Javier, CO 73 PO 98

JUNGFLEISCH, Jennifer Sandra, CO 89, CO 93 MEDRANO , María, PO 20

KEKARAINEN, Tuija, CO 72 MEJÍAS, Ernesto, CO 18, CO 66, PO 102, PO 103

LACASTA, Anna, CO 39, PO 161 MELERO, José Antonio, CO 61, PO 10, PO 93

LÁZARO, Ester, CO 86 MÉNDEZ, Fernando, CO 59

LEAL, Manuel, CO 24, PO 16, PO 17, PO 151 MÉNDEZ, Francisco Eduardo, CO 27, PO 67

LERMA, Laura, CO 88, PO 106, PO 122, PO 126 MENÉNDEZ, Luis , CO 20, PO 56

LÓPEZ CARRASCO,Amparo, PO 62, PO 72 MERINO, Teresa, PO 31, PO 33

LOPEZ DE DIEGO, Marta, PO 91 MESEL-CASANOVA, Frida, PO 163

LÓPEZ GALINDEZ, Luis Cecilio, PO 59 MESTRE, Leyre, PO 97

LÓPEZ MONTEAGUDO, Paula, CO 39 MINGORANCE, Lidia, PO 154

LÓPEZ-ARGÜELLO, Silvia Daiana, CO 63 MINGOT, Ares, CO 26, PO 70, PO 120

LÓPEZ-BUENO, Alberto, CO 71, PO 30, PO 157 MINGUITO, Teodora, PO 36

LÓPEZ-GUERRERO, Jose Antonio , PO 122 MOLINA, Francisca, CO 55

LÓPEZ-‐LABRADOR, Xavier, CO 65 MORALES, Lucía, PO 21, PO 116

LÓPEZ-MOYA, Juan José, CO 26, PO 70, PO 120 MORENO, Ana Beatriz, PO 70

LORCA, Cristina, CO 21, PO 60 MORENO-FERNANDEZ, Sandra, PO 54, PO 55, PO 110

LORENZO, Maria del Mar, PO 114 MORENO-MOLINA, Miguel, PO 23, PO 72, PO 144

LOZANO, Gloria, CO 80, PO 22 MORTRÓ, Elisa, PO 147

MAJANO, Pedro, CO 55, CO 81 MUÑOZ, Sara, CO 15, PO 92, PO 115

MANRUBIA, Susanna, PO 134 MUÑOZ-FERNÁNDEZ, Mª Angeles, CO 17, CO 22, CO 47 CO 54, CO 62, CO 81, PO 52, PO 53, PO 54, PO 55, PO 80

MARIN, Alejandro, CO 33, CO 40, PO 43, PO 110 NAVAS, Jesús, CO 29, PO 32

MÁRQUEZ, Silvia, PO 124 NAVAS, María Jesús, CO 39

MARTIN GARCIA, Verónica, CO 14, CO 37, PO 95 NEGREDO, Ana Isabel, CO 1, CO2, CO 13, PO 9, PO 29

MARTIN-ACEBES, Miguel A., CO 56, PO 31, PO 33, PO 40, NEVOT, María, CO 85, PO 19 PO 42, PO 48

MARTÍN-DELGADO, Sara, CO 62 NIETO, Amelia, CO 36, CO 79, CO 82, CO 87, CO 94

MARTÍNEZ-AGUADO, Pablo, PO 2 NIEVES, Gliselle, PO 94

MARTÍNEZ DE LA SIERRA, Miguel A., CO 85, PO 19 NISTAL-VILLAN, Estanislao, PO 165, PO 166

MARTINEZ-ESCRIBANO, Jose Angel, CO 67 NÚÑEZ, Jose Ignacio, PO 14, PO 49

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NÚÑEZ-HERNANDEZ, Fernando, PO 14 RODAMILANS, Bernardo, CO 26, PO 65, PO

ORTEGO, Javier, CO 33, CO 38, CO 40, PO 43, PO 110 RODRIGUEZ, Miguel , PO 108

ORTÍN, Juan, CO 92 RODRÍGUEZ, María Josefa, CO 84, CO 90, PO 47, PO 75

ORTIZ DE LEJARAZU, Raul, PO 87, PO 139 RODRÍGUEZ, Rosa, CO 50, PO 10

ORY DE, Fernando, CO 1, CO 13, PO 35, PO 36, PO 137 RODRÍGUEZ, Dolores, CO 59, CO 90, CO 91, PO 94, PO 106 PO 118

PACHECO, Beatriz, CO 20 RODRÍGUEZ, José Francisco, CO 59

PACHECO, Yolanda, CO 24, PO 16, PO 17 RODRIGUEZ-SAINT-JEAN, Sylvia, PO 6

PALLÁS , Vicente, PO 71 RODRÍGUEZ-DÍAZ, Jesús, PO 74, PO 87, PO 113

PALÚ, Giorgio, P 14, CO 35 RODRÍGUEZ-DOMÍNGUEZ, Mario José, PO 79

PARRAS, Marcos, CO 71, PO 157 RODRÍGUEZ-FRÍAS, Francisco, P 9, PO 15, PO 131, PO 152

PASCUAL, Alejandro, PO 123, PO 125 RODRIGUEZ-GONZÁLEZ, Fernando, CO 39

PASCUAL, Elena, CO 14, CO 37, PO 95 RODRÍGUEZ-NEVADO , Cristina, PO 69, PO 150

PELLEGRINELLI, Laura, PO 142 RODRIGUEZ-RODRIGUEZ, Paloma, CO 82

PERALES, Celia B., P 2, PO 130, PO 151, PO 158 ROJAS, Aldo S., CO 83, PO 4, PO 25

PERDIGUERO, Beatriz, CO 18, CO 23, PO 52, PO 102 ROMERO, Cristina, CO 25, CO 48

PÉREZ, Carlos, PO 18 ROMERO, Javier, PO 7, PO 59

PÉREZ, Sara Isabel, PO 6 ROMERO, Manuel, P 8, CO 83, PO 4

PEREZ , Gemma, PO 61 ROSSI, Marcello, PO 9, PO 29

PEREZ DEL PULGAR, Sofia, P 10, PO 145 RUBINO, Luisa, P 16

PÉREZ-BERNÁ, Ana J., CO 84, PO 146 RUBIO, Enrique, PO 100

PÉREZ-NÚÑEZ, Daniel , CO 45 RUEDA, Paloma, PO 46

PINTÓ, Rosa M., P 7, CO 58, CO 60, PO 138 RUGGERI, Franco, P 6, PO 44, PO 45, PO 51, PO 88, PO 89

PION, Marjorie, CO 17, CO 62 RUIZ-MATEOS, Ezequiel, CO 5, PO 16, PO 17, PO 151

PONZ, Fernando, PO 63 RUIZ-RUIZ, Susana, CO 28, PO 155, PO 156

PRIETO, Luis M., CO 49 RUSIÑOL, Marta, CO 65, PO 12, PO 143

QUER, Josep, PO 7, PO 15, PO 131, PO 145, PO 147, PO 152 SABARIEGOS, Maria Del Rosario, PO 158

QUIRÓS, María, PO 1O7 SABRIÀ, Aurora, PO 138

RASTROJO, Alberto, CO 41, PO 30 SÁIZ, Juan C., CO 11, PO 31, PO 33, PO 48

REBOLLO, Belen, PO 13 SÁIZ, Margarita, PO 108

REGLA-NAVA, Jose Angel, P 5, CO 12, PO 91, PO 98 SALAS, Margarita, CO 6

REINA, Jorge, PO 78, PO 81 SALEH, Carla, P 18

RESINO, Salvador, PO 153 SALUDES, Verónica, PO 147

RÍOS-MARCO, Pablo, CO 48, PO 150 SAN LEÓN, David, CO 26, PO 120

RIVAS, Carmen, CO 87, PO 117, PO 127, PO 132 SAN MARTIN, Carmen, PO 26, PO 135, PO 164

282 Virología. Publicación Oficial de la Sociedad Española de Virología

SÁNCHEZ, Carolina, PO 102, PO 112 VIEIRA, Yuri Allende, PO 76

SÁNCHEZ –APARICIO, Maria Teresa, CO 68, PO 90, VIGNUZZI, Marco, P 13 PO 116

SÁNCHEZ -SAN PEDRO, Lucas, CO 66, PO 102, PO 103 VIGUERA, Enrrique, PO 68

SÁNCHEZ-CESPEDES, Javier, PO 2 VIJAYAN, Viji, CO 32, PO 101

SÁNCHEZ-LOPEZ, Pedro Francisco, PO 82 VILA, Susana, PO 74, PO 82, PO 87, PO 113

SÁNCHEZ-NAVARRO, Jesús Ángel, PO 160 ZUÑIGA, Sonia, P 5, PO 116, PO 123, PO 124, PO 125

SÁNCHEZ-PUIG, Juana María, PO 114 SÁNCHEZ-SECO, Mª Paz, CO 1, CO 2, CO 9, CO 13, PO 9 , PO 25, PO 27, PO 28, PO 29, PO 36

SASTRE-ANTORANZ, Patricia, PO 46

SERRA, Pedro, PO 11

SEVILLA, Noemi, CO 14, CO 37, PO 95

SHAN, Hongying, PO 65

SIMÓN, Carmen, PO 65

SMERDOU, Cristian, CO 57 SOBRINO, Francisco, CO 56, PO 31, PO 33, PO 39, PO 40, PO 42, PO 76, PO 105, PO 108, PO 122

SOLA, Isabel , P 5, PO 21, PO 116, PO 123, PO 125

SUZUKI, Nobuhiro, P 3, PO 18

TAMAYO, Miguel, CO 10

TARAVILLO, Irene, PO 37

TENORIO, Antonio, P 4, CO 1, CO 9, PO 9, PO 25, PO 29

THIEL, Volker, P 11

TORCHETTI, Enza Maria, PO 109

TORRE DE LA, Humberto Erick, PO 162

TRALLER, Gloria, PO 37

TRENTO, María Alfonsina, CO 61, PO 10

TRUNIGER, Verónica, CO 25

VALBUENA, Alejandro, PO 20, PO 136

VALLE, Mikel, CO 27, PO 67

VALLEJO, Alejandro, PO 5, PO 57

VASILIJEVIC, Jasmina, CO 94

VÁZQUEZ, Angela, CO 56, PO 39, PO 42

VÁZQUEZ-MORÓN, Sonia, PO 153

VÁZQUEZ, Ana, CO 1, CO 2, CO 9, PO 25, PO 27, PO 28

VENTEO, Angel, PO 13

Volumen 18 – Número 1/2015 - EXTRAORDINARIO 283

NOTES

284 Virología. Publicación Oficial de la Sociedad Española de Virología