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Estudo Metagenômico De Arbovírus E Outros Vírus De Rna De Vertebrados Em Roedores E Marsupiais Procedentes Do Município De Viseu, No Estado Do Pará-Brasil

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Estudo Metagenômico De Arbovírus E Outros Vírus De Rna De Vertebrados Em Roedores E Marsupiais Procedentes Do Município De Viseu, No Estado Do Pará-Brasil INSTITUTO EVANDRO CHAGAS NÚCLEO DE ENSINO E PÓS-GRADUAÇÃO PROGRAMA DE PÓS-GRADUAÇÃO EM VIROLOGIA NELIELMA GARCIA DE OLIVEIRA PRESTES ESTUDO METAGENÔMICO DE ARBOVÍRUS E OUTROS VÍRUS DE RNA DE VERTEBRADOS EM ROEDORES E MARSUPIAIS PROCEDENTES DO MUNICÍPIO DE VISEU, NO ESTADO DO PARÁ-BRASIL Ananindeua 2019 NELIELMA GARCIA DE OLIVEIRA PRESTES ESTUDO METAGENÔMICO DE ARBOVÍRUS E OUTROS VÍRUS DE RNA DE VERTEBRADOS EM ROEDORES E MARSUPIAIS PROCEDENTES DO MUNICÍPIO DE VISEU, NO ESTADO DO PARÁ-BRASIL Dissertação apresentada para obtenção de grau de Mestre em Virologia pelo Programa de Pós-Graduação em Virologia do Instituto Evandro Chagas Área de concentração: Biologia de Agravos por Agentes Virais na Amazônia Orientadora: Profª. Drª. Ana Cecilia Ribeiro Cruz Coorientador: Prof. Dr. Sandro Patroca da Silva Ananindeua 2019 Dados Internacionais de Catalogação na Publicação (CIP) Biblioteca do Instituto Evandro Chagas Prestes, Nelielma Garcia de Oliveira. Estudo metagenômico de arbovírus e outros vírus de RNA de vertebrados em roedores e marsupiais procedentes do município de Viseu no Estado do Pará- Brasil. / Nelielma Garcia de Oliveira Prestes. – Ananindeua, 2019. 88 f.: il.; 30 cm Orientadora: Dra. Ana Cecília Ribeiro Cruz Coorientador: Dr. Sandro Patroca da Silva Dissertação (Mestrado em Virologia) – Instituto Evandro Chagas, Programa de Pós-Graduação em Virologia, 2019. 1. Metagenômica viral. 2. Vírus de RNA. 3. Arbovírus. 4. Hepacivírus. I. Cruz, Ana Cecília Ribeiro, orient . II. Silva, Sandro Patroca da, coorient. III. Instituto Evandro Chagas. IV. Título. CDD: 579.2562 NELIELMA GARCIA DE OLIVEIRA PRESTES ESTUDO METAGENÔMICO DE ARBOVÍRUS E OUTROS VÍRUS DE RNA DE VERTEBRADOS EM ROEDORES E MARSUPIAIS PROCEDENTES DO MUNICÍPIO DE VISEU, NO ESTADO DO PARÁ-BRASIL Dissertação apresentada para obtenção de grau de Mestre em Virologia pelo Programa de Pós-Graduação em Virologia do Instituto Evandro Chagas Área de concentração: Biologia de Agravos por Agentes Virais na Amazônia Aprovado em: BANCA EXAMINADORA Dr. Rommel Thiago Juca Ramos Universidade Federal do Pará Drª. Lívia Carício Martins Instituto Evandro Chagas Drª. Flávia Barreto dos Santos Instituto Oswaldo Cruz Dr. Samir Mansour Mores Casseb (Suplente) Instituto Evandro Chagas Dedico este trabalho aos dois homens da minha vida. Meu esposo Edbruno Prestes por sua compreensão, amor e companheirismo e ao meu filho Benjamim Prestes que chegou a este mundo para completar a Graça de Deus em nossas vidas. AGRADECIMENTOS Em primeiro lugar ao Senhor Deus todo poderoso por permitir minha vivência neste mundo abençoado, por estar realizando grandes projetos em minha vida e por sempre estar conduzindo meus passos para crescer na fé. A minha orientadora Profa. Dra. Ana Cecília por todos os ensinamentos, paciência, orientação, apoio e por acreditar na minha capacidade profissional em conduzir esse projeto até o fim. Além de ser uma pessoa ótima, de grande coração e de vasto conhecimento. Ao meu co-orientador Dr. Sandro Patroca por sua incrível paciência, apoio e por seu vasto conhecimento transmitido com muita satisfação e de várias formas possíveis para melhor compreensão de minha parte e pela sua imensa dedicação em passar seus ensinamentos em biologia molecular e bioinformática. Ao meu esposo Edbruno Prestes pela sua paciência em mais essa etapa que passamos juntos, pelo seu companheirismo, cumplicidade, amorosidade e por sempre está ali para ouvir minhas queixas e me aconchegar em todos os momentos. Além de me apoiar e dar forças em tudo o que faço e ter sempre um sorriso e uma palavra confortadora nos momentos difíceis que passamos. Mesmo ainda muito bebê, ao meu filho Benjamim Prestes pelo momento único e abençoado que ele está me fazendo passar e sentir. Aos meus pais Leomário e Nelia por estarem ao meu lado nesse momento importante e incrível que estou vivendo e pelo apoio em minhas escolhas profissionais e a todos os meus irmãos que também nos bastidores torcem pela minha felicidade. As minhas colegas Andressa Aragão e Camila Braga por me deixarem acompanhar suas análises para aprender as técnicas necessárias. Além, é claro de muitos risos e papos descontraídos. Em especial a Andressa por ter separado um espaço em seu tempo para me ajudar em uma parte das análises. Ao Laboratório do Centro de Genômica e Biologia de Sistemas da Universidade Federal do Pará pela parceria e colaboração para esta pesquisa (Dr. Rommel Ramos). À CAPES e ao Instituto Evandro Chagas pelo fornecimento de subsídios financeiros necessários para realizar essa pesquisa; Ao Programa de Pós-Graduação em Virologia do Instituto Evandro Chagas “Paciência vale mais que valentia, e dominar a si mesmo vale mais que conquistar uma cidade” Provérbios 16, 32. RESUMO A metodologia metagenômica está em crescente desenvolvimento para a identificação de genomas virais, caracterizando as informações biológicas desses agentes sem necessidade de isolamento e cultivo em laboratório com o auxílio de tecnologias avançadas para a geração e processamento de milhões de leituras. Neste contexto propõe-se investigar a presença de arbovírus e outros vírus de RNA de vertebrados em roedores e marsupiais procedentes do município de Viseu, no Estado do Pará-Brasil. Um total de 10 amostras (fígado, vísceras e cérebro) de roedores e marsupiais foram submetidos a extração e purificação de RNA, síntese do DNA complementar (cDNA), sequenciamento através da tecnologia Illumina NextSeq e análise bioinformática. No geral os gêneros virais detectados neste trabalho foram: Ortobunyavirus, Hepacivirus, Gammaretrovirus, Betaretrovirus, Epsilonretrovirus, Alpharetrovirus, Pestivirus, Parapoxivirus, Amdoparvovirus , além de vírus de RNA não classificados em nenhum dos gêneros conhecidos. Este estudo apresentou diversidades de agentes virais, com predominância de gêneros da família Retroviridae e dessas detecções apenas o vírus Caraparu (CARV) membro do gênero Ortobunyavirus representa risco a saúde humana. A análise filogenética para os hepacivírus (Hepacivirus de roedor e Hepacivirus F ) detectados neste estudo, apresentaram similaridade com o Rodent Hepacivirus isolado do roedor da espécie Proechimys simispinosus , agrupando-se no mesmo clado. Os domínios da poliproteína apresentaram semelhança com os Hepacivirus nas regiões que codificam proteínas estruturais (Core e Env.), não estruturais (NS2, NS4b, NS5a_1a e NS5a_1b), com uma estrutura da peptidase (S29) responsável pela clivagem das proteínas estruturais e com um domínio que corresponde a família Flaviviriade (Flavi_DEAD). Diante disso, os achados deste estudo podem contribuir para o conhecimento científico sobre as comunidades virais que circulam em pequenos animais (roedores e marsupiais) nas proximidades da cidade de Viseu, mesmo que muitos deles não sejam nocivos para a saúde humana. Com isso, estudos contínuos nesses e em outros mamíferos devem ser realizados para obter informações das espécies virais circulantes e de possíveis novas descobertas de vírus e fontes de infecções zoonóticas, nessa e em outras regiões da Amazônia brasileira. Palavras-chave : Metagenômica viral; vírus de RNA; Arbovírus; Hepacivirus. ABSTRACT The metagenomics methodology is under development for the identification of viral genomes, characterizing the biological information of these agents without the need for isolation and cultivation in laboratory with the aid of advanced technologies for the generation and processing of millions of readings. In this context it is proposed to investigate the presence of arboviruses and other RNA viruses from vertebrates in rodents and marsupials coming from the city of Viseu, Para - Brazil. A total of 10 rodent and marsupial samples (liver, viscera and brain) were submitted to RNA extraction and purification, complementary DNA (cDNA) synthesis, sequencing by Illumina NextSeq and bioinformatic analysis. Primarily, the viral genera detected in this work were: Ortobunyavirus, Hepacivirus, Gammaretrovirus, Betaretrovirus, Epsilonretrovirus, Alpharetrovirus, Pestivirus, Parapoxivirus, Amdoparvovirus , and RNA viruses not classified in any of the known genera. This study presented viral agents diversity, with predominance of genera of the Retroviridae family and from these detections only the Caraparu virus (CARV ) member of the genus Ortobunyavirus poses a risk to human health. Phylogenetic analysis for hepacivirus (Rodent Hepacivirus and Hepacivirus F ) detected in this study, showed similarity with Rodent Hepacivirus isolated from rodent species Proechimys simispinosus , grouping in the same clade. The polyprotein domains were similar to Hepaciviruses in regions encoding structural (Core and Env), Nonstructural (NS2, NS4b, NS5a_1a and NS5a_1b) proteins, with a peptidase structure (S29) responsible for the cleavage of structural proteins and with a domain that corresponds to the Flaviviridae family (Flavi_DEAD). Thus, the findings of this study may contribute to the scientific knowledge about the viral communities that circulate in small animals (rodents and marsupials) near the city of Viseu, even though many of them are not harmful to human health. Therefore, ongoing studies on these and other mammals should be performed to obtain information on circulating viral species and possible new discoveries of viruses and sources of zoonotic infections in this and other regions of the Brazilian Amazon. Keywords : Viral metagenomics; RNA virus; Arbovirus; Hepacivirus. LISTA DE ABREVIATURAS E SIGLAS ASFV – vírus Africano Suíno SLEV – vírus da Encefalite Saint Loius BSQV – vírus Bussuquara SNG –
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