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Caracterização Genotípica Do Vírus Juruaçá Isolado De Morcego No Estado Do Pará

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Caracterização Genotípica Do Vírus Juruaçá Isolado De Morcego No Estado Do Pará INSTITUTO EVANDRO CHAGAS IVY TSUYA ESSASHIKA PRAZERES CARACTERIZAÇÃO GENÉTICA DO VÍRUS JURUAÇÁ ISOLADO DE MORCEGO NO ESTADO DO PARÁ ANANINDEUA 2016 IVY TSUYA ESSASHIKA PRAZERES CARACTERIZAÇÃO GENÉTICA DO VÍRUS JURUAÇÁ ISOLADO DE MORCEGO NO ESTADO DO PARÁ Dissertação de mestrado apresentada ao Programa de Pós-Graduação em Virologia do Instituto Evandro Chagas, como requisito para a obtenção do título de Mestre em Virologia. Orientadora: Dra. Daniele Barbosa de Almeida Medeiros ANANINDEUA 2016 Dados Internacionais de Catalogação na Publicação (CIP) Biblioteca do Instituto Evandro Chagas Prazeres, Ivy Tsuya Essashika. Caracterização genotípica do vírus juruaçá isolado de morcego no Estado do Pará. / Ivy Tsuya Essashika. – Ananindeua, 2016. 76 f.: il.; 30 cm Orientador: Dra. Daniele Barbosa de Almeida Medeiros Dissertação (Mestrado em Virologia) – Instituto Evandro Chagas, Programa de Pós-Graduação em Virologia, 2016. 1. Vírus Juruaçá. 2. Caracterização genética. 3. Sequenciamento next generation. 4. RNAss. 5. Tombusviridae I. Medeiros, Daniele Barbosa de Almeida, orient. II. Instituto Evandro Chagas. IV. Título. CDD: 579.2562 IVY TSUYA ESSASHIKA PRAZERES CARACTERIZAÇÃO GENÉTICA DO VÍRUS JURUAÇÁ ISOLADO DE MORCEGO NO ESTADO DO PARÁ Dissertação apresentada ao Programa de Pós- Graduação em Virologia do Instituto Evandro Chagas, para obtenção do título de mestre em Virologia Orientador (a): Dr.a Daniele Barbosa de Almeida Medeiros Aprovado em: 27/04/2016 BANCA EXAMINADORA Dr.a Adriana Ribeiro Carneiro Centro de Genômica e Biologia de Sistemas, Universidade Federal do Pará Dr.a Ana Cecília Ribeiro Cruz Seção de Arbovirologia e Febres Hemorrágicas, Instituto Evandro Chagas Dr. João Lídio da Silva Gonçalves Vianez Júnior Centro de Inovações Tecnológicas, Instituto Evandro Chagas Ao meu avô, Osamu Esashika. À minha avó, Julieta Prazeres. Que sempre seguirão comigo. AGRADECIMENTOS À Deus, que sempre esteve presente em minha vida, guiando meus passos e me mostrando o caminho, sempre me dando muita saúde para seguir em frente. Aos meus amados pais, Elizabete e Aroldo, por terem me ensinado o significado de uma família, e sempre me apoiarem em todas minhas decisões. Não há nada mais importante na vida do que a nossa base, é ela que nos apoia em momentos difíceis e é ela que nos levanta nas conquistas. Aos meus irmãos, Catarina, Italo, Ianie e Iasmin, pelo companheirismo e amor sempre presentes, obrigada apenas por vocês serem quem vocês são, minha vida não seria a mesma se não fossem essas quatro pessoinhas que sempre estiveram do meu lado. Ao meu namorado, Breno, por estar sempre comigo, dando apoio e solidariedade em todos os momentos da minha vida. Ao Programa de Pós Graduação em Virologia do Instituto Evandro Chagas por me habilitar para que esse projeto de mestrado fosse concluído. Ao Instituto Evandro Chagas, em especial a Seção de Arbovirologia e Febres Hemorrágicas, por permitirem que esse projeto fosse executado em suas dependências com todas as suas técnicas e materiais necessários. À minha querida mentora, educadora, orientadora, Dra. Daniele Barbosa, por todos os ensinamentos, todas as conversas, todas as explicações que foram dedicadas a mim durante a execução desse projeto, obrigada por me trazer de volta para a área da pesquisa, terei sempre em você um exemplo e um modelo de ser humano íntegro e que luta atrás dos seus sonhos. Ao Laboratório de Clonagem e Expressão de Proteínas, onde parte da pesquisa foi conduzida e executada, em especial à doutoranda Maria Helena e mestre Adriana pela amizade e pelos ensinamentos das técnicas essenciais para o projeto. Ao Laboratório de Biologia Molecular, Laboratório de Sorologia I e Laboratório de Cultura de Células pela disponibilidade do espaço, das técnicas e dos materiais utilizados durante a pesquisa, e obrigada também pela amizade e atenção de todos seus integrantes que sempre estavam dispostos a transmitir a informação e contribuir para o correto desenvolvimento deste projeto. Ao Centro de Inovações Tecnológicas pelo auxílio e desenvolvimentos das técnicas de bioinformática. A todos os funcionários, servidores e estagiários da Seção de Arbovirologia e Febres Hemorrágicas do Instituto Evandro Chagas que direta ou indiretamente estiveram envolvidos com o estudo, o meu muito obrigada à toda essa família do arbovírus que desde meus tempos de faculdade abrem os braços para me ajudar a percorrer esse caminho. “ A persistência é o menor caminho do êxito”. (Charles Chaplin) RESUMO Introdução: O vírus Juruaçá (VJUR) foi isolado em camundongos albinos suíços recém-nascidos a partir de fragmentos de vísceras de um morcego capturado na região de Porto de Trombetas, município de Oriximiná, Estado do Pará. Com base nas propriedades antigênicas, o VJUR é considerado como vírus não agrupado. Estudos iniciais mostraram que o VJUR apresenta morfologia semelhante aos integrantes da família Picornaviridae, no entanto são necessários estudos adicionais para confirmar tal classificação taxonômica. Ademais, o VJUR causa infecção persistente em cultivos primários de astrócitos e microglias e as alterações anatomopatológicas e reações imunohistoquímicas indicam como principal alvo da infecção o sistema nervoso central de camundongos albinos suíços, caracterizando uma patogenia imune inflamatória. Portanto, torna-se importante o desenvolvimento de um estudo de caracterização genética que proporcionará o melhor entendimento a respeito da organização genômica do vírus e sua posterior definição taxonômica. Objetivo: O objetivo do estudo é caracterizar geneticamente o VJUR. Material e Métodos: Para tentar o isolamento em cultivos celulares, foi utilizada linhagem derivadas de vertebrados: Vero E6. Foi realizado o sequenciamento completo do genoma do VJUR pela plataforma Ion Torrent. Posteriormente, foi feita a montagem do genoma utilizando os programas Newbler e Mira, definição das cadeias de leitura abertas (CALs) pelo programa ORFfinder, a análise de identidade pelo Blast, e análises de domínios protéicos utilizando o banco de dados INTERPRO, para a descrição da organização e características do genoma. As análises filogenéticas foram realizadas utilizando os métodos de Agrupamento de Vizinhos. Resultados: O vírus foi isolado com sucesso no cultivo celular de Vero E6, com resultado confirmado pela técnica de imunofluorescência direta. Seu genoma foi sequenciado, obtendo-se um total de 5.020 nt, no qual, preliminarmente, são reconhecidas três cadeias abertas de leituras. Apenas uma CAL foi reconhecida no Blastx apresentando 33% de identidade com o vírus Pelargonium line pattern, membro da família Tombusviridae, comumente associado a vírus que infectam plantas. A análise filogenética demonstrou uma relação do VJUR com os membros da família Tombusviridae (bootstrap 100%), porém em um ramo distinto. Conclusão: O VJUR pertence à uma nova família dentro dos vírus de RNAss. Palavras-Chave: Vírus Juruaçá; caracterização genética; sequenciamento next generation; RNAss, Tombusviridae. ABSTRAT Introduction: Vírus Juruaçá (VJUR) was isolated in newborn Swiss albino mice from viscera fragments of a bat captured in the region of Porto de Trombetas, municipality of Oriximiná, State of Pará. Based on the antigenic properties, VJUR it is considered as non-clustered virus. Initial studies showed that VJUR has similar morphology to the Picornaviridae family members, however additional studies are needed to confirm this taxonomic classification. Furthermore, VJUR cause persistent infection in primary cultures of astrocytes and microglia and pathological and immunohistochemical reactions show how the main target of central nervous system infection in Swiss albino mice characterized by an inflammatory autoimmune disease. Therefore, it becomes important to develop a study of genetic characterization will provide a better understanding about the genomic organization of the virus and its subsequent setting Taxonomy. Objective: The objective of the study is to characterize genetically the VJUR. Methods: To try isolation in cell culture, lines derived from vertebrates were used: Vero E6. Was performed the complete sequencing of the genome VJUR by Ion Torrent platforms. Subsequently, the genome assembly was made using the Newbler and Mira programs, definition of open reading frames (ORF) by ORFfinder program analysis identity by Blast, and analysis of protein domains using the InterPro database, to the description the organization and characteristics of the genome. Phylogenetic analyzes were performed using the Neighbor joing methods. Results: The virus was successfully isolated in cell culture Vero E6, with results confirmed by the technique of direct immunofluorescence. Its genome was sequenced, obtaining a genome of 5020 nt, in which, initially, are recognized three ORF’s. Only one ORF was recognized in Blastx presenting 33% identity with the Pelargonium line pattern virus, Tombusviridae family member, commonly associated with viruses that infect plants. Phylogenetic analysis showed a VJUR relationship with the members of the family Tombusviridae (bootstrap 100%), but in a different branch. Conclusion: VJUR belongs to a new family within the virus RNAss. Keywords: Juruaçá virus; genetic characterization; next generation sequencing; RNAss, Tombusviridae. SUMÁRIO 1 INTRODUÇÃO........................................................................................................12 2 OBJETIVOS............................................................................................................13 2.1 OBJETIVO GERAL..............................................................................................13
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