Microevolu????o in vitro do v??rus dengue, sorotipo-4: estudo de varia????es gen??ticas associadas ao aumento da compet??ncia viral

• Author(s): Nascimento, Valdinete Alves do
• Date: 2016
• Origin: Oasisbr
• Subject(s): Dengue; Arbov??rus; Cadeia da Polimerase; Citometria de Fluxo; CIENCIAS DA SAUDE
• Document Type: Master thesis
• Language: Portuguese

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CAPES - Coordena????o de Aperfei??oamento de Pessoal de N??vel Superior

Dengue virus (DENV) belongs to the family Flaviviridae and the genus Flavivirus, being recognized as four distinct serotypes termed DENV-1 to DENV-4. Like other RNA viruses, DENV displays variation in their sequences, with an error rate during replication cycle estimated at 10-3 to 10-5 errors / nucleotide. These variations are observed not only when samples from different individuals are analyzed, but also within the same host. Considering the need for a better understanding of related mechanisms of DENV evolutionary process and the emergence of viral subpopulations, this study aimed to evaluate the in vitro microevolution of dengue virus serotype-4, analyzing the existence of genetic variations associated with increased viral fitness. For this purpose, a sample of DENV-4 called BR005AM_2015 was inoculated in C6/36 cells and 25 serial passages were performed. After these passages, viral RNA extraction was performed and the cDNA was produced using a specificDENV-4 primer. The full-length genome of the sample BR005AM_2015 and passages 1, 5, 10, 15, 20 and 25 were obtained by the Sanger method using the ABI 3130 Genetic Analyzer and by Next-Generation Sequencing (NGS) using the Ion Torrent technology. The files generated after the sequencing reactions were analyzed using Geneious software version 7.1.7. The analysis of the data provided by the Sanger sequencing has shown that during the 25 passages, two nucleotide mutations occurred, one in the region which encodes for the envelope protein, resulting in the substitution of an amino acid residue T501P, and other silent mutation at NS1 gene. The results of NGS corroborate those obtained by Sanger sequencing. Moreover, seventeen other mutations not previously observed, being the same in the coding regions for proteins E, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. Of the total observed mutations, six of them led to residue substitution. Considering the observed amino acids substitutions, we conducted a study of viral kinetics by RT-qPCR using the ????Ct method which indicated a time-dependent increase in the number of copies of viral RNA present in both the supernatant or inside the infected cells, indicating a possible increase of the viral fitness. Other studies are necessary to confirm if the mutations described here also occurs in mammalian cells or in vivo systems, as well as to verify if the observed increase in viral fitness occurs in other systems.

O v??rus Dengue (DENV) pertence ?? fam??lia Flaviviridae e ao g??nero Flavivirus, sendo reconhecidos quatro sorotipos distintos denominados DENV-1 a DENV-4. Assim como os demais v??rus RNA, o DENV exibe varia????o em suas sequ??ncias, com uma taxa de erro durante o ciclo de replica????o estimada em 10-3 a 10-5 erros/nucleot??deo. Essas varia????es s??o observadas n??o s?? quando analisadas amostras de diferentes indiv??duos, mas tamb??m dentro de um mesmo hospedeiro. Considerando a necessidade de uma maior compreens??o dos mecanismos relacionados ao processo evolutivo do DENV e a emerg??ncia de subpopula????es virais, o presente trabalho teve por objetivo avaliar a microevolu????o in vitro do v??rus dengue, sorotipo-4, analisando a exist??ncia de varia????es gen??ticas associadas ao aumento da compet??ncia viral (viral fitness). Para tanto, uma amostra do DENV-4 denominada BR005AM_2015 foi inoculada em c??lulas C6/36, sendo realizadas passagens seriadas do v??rus nesta linhagem celular at?? a passagem 25. Finalizadas as passagens, foi realizada extra????o do RNA viral e o cDNA foi produzido utilizando iniciador espec??fico para DENV-4. O genoma completo da amostra BR005AM_2015 e das passagens 1, 5, 10, 15, 20 e 25 foi obtido pelo m??todo Sanger utilizando sequenciador autom??tico ABI 3130 genetic analyzer e por meio de sequenciamento de nova gera????o (NGS) utilizando a tecnologia Ion Torrent no equipamento Ion PGM??? System. Os arquivos gerados ap??s as rea????es de sequenciamento foram analisados utilizando o software Geneious vers??o 7.1.7. A an??lise dos dados fornecidos pelo sequenciamento de Sanger mostrou que no decorrer das passagens ocorreram duas substitui????es de nucleot??deos, sendo uma na regi??o codificante para a prote??na do envelope que resultou na substitui????o de um res??duo de amino??cido e outra na regi??o da prote??na NS1, por??m esta foi uma muta????o silenciosa. Os resultados do NGS corroboraram com os obtidos pelo sequenciamento de Sanger e apresentaram outras dezessete muta????es n??o observadas pelo m??todo anterior, sendo as mesmas nas regi??es codificantes para as prote??nas E, NS1, NS2A, NS2B, NS3, NS4A, NS4B e NS5. Do total de muta????es observadas, seis delas resultaram em substitui????o de res??duo de amino??cido. Considerando as muta????es observadas, realizamos o estudo de cin??tica viral por RT-qPCR utilizando o m??todo de ????Ct que indicou um aumento do n??mero de c??pias de RNA viral, dependente do tempo p??s-infec????o, tanto presente no sobrenadante de c??lulas infectadas quanto no interior das c??lulas, indicando um poss??vel aumento da compet??ncia viral. Outros estudos se tornam necess??rios para confirmar se as muta????es aqui encontradas tamb??m ocorrem em c??lulas de mam??feros e em sistemas in vivo, de maneira a verificar se o aumento do fitness viral observado neste estudo se repete em outros sistemas.