Estudo evolutivo do v??rus dengue predominante no estado do Amazonas (2011-2016), com foco na dispers??o viral e na falha no diagn??stico sorol??gico

• Autor(es): Monteiro, Dana Cristina da Silva ; https://orcid.org/0000-0003-0318-0564
• Data: 2019
• Origem: Oasisbr
• Assunto(s): Dengue; Evolu????o viral; Prote??na n??o estrutural 1; Amazonas; CI??NCIAS BIOL??GICAS
• Tipo de Documento: Dissertação de mestrado
• Idioma: Português

Descrição

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FAPEAM - Funda????o de Amparo ?? Pesquisa do Estado do Amazonas

Dengue is considered one of the most important arboviruses that affects man. In Brazil, in 2017, 252,054 probable cases of dengue were reported, of which only 4,000 cases of the disease were recorded in Amazonas. However, studies have shown that factors such as circulating serotype, geographic region of patients and secondary infections may be related to decreased sensitivity of the test when compared to the molecular methods. Therefore, in order to evaluate possible mutations that may be associated with dengue dispersal in Amazonas, as well as the failure to detect the NS1 antigen, we performed this study of the evolution of the predominant dengue virus serotype/genotype in the period from 2011 to 2016 in the municipalities of the interior and in the capital of the state. A total of 608 serum samples from dengue suspects were collected from 26 municipalities in Amazonas. The samples were submitted to viral RNA extraction and later to the real-time PCR method, in which 181 (29.7%) samples had a positive result. Subsequently using a semi-nested PCR protocol, we observed the predominance of DENV-4 (N = 80), followed by DENV-2 (N = 9) and DENV-1 (N = 7). For the DENV-1 and 2 serotypes, conventional PCR and sequencing reactions were conducted only for the ENV region, whereas for DENV-4 the coding region for NS1 was also sequenced. Phylogenetic analyzes were performed according to the maximum likelihood (ML) method in the PhyML program, using the model chosen with the help of the jModeltest v2.1.7 program and Bayesian Inference using the BEAST v1.8.4 program. 27 complete sequences of ENV and 25 NS1 of DENV-4 were obtained from seven municipalities of Amazonas, all belonging to genotype II. The DENV-1 (2016) and DENV-2 (2011) sequences collected in Manaus belonged to genotype V and Asian / American, respectively. During the analysis of the DENV-4 ENV sequences, 140 variable sites and eight amino acid exchanges were found, whereas in the NS1 sequences 91 variable sites and seven amino acid exchanges were observed. According to the phylogeographic analysis, there were at least two independent entries of DENV-4 in the state of Amazonas. In addition, our data also showed the spread of DENV-4 from Manaus to other municipalities within the state of Amazonas. In the seroprevalence tests, 45% (164/360) of the samples were reagents for IgG detection, and no significant difference was observed between samples NS1-Positive (43.9%) and NS1-Negative (47.2%). We believe that this study has an important role in arboviral surveillance in the state of Amazonas, reporting not only the serotypes and genotypes of DENV from different municipalities, but also how the DENV-4 GII spread in the State.

A dengue ?? considerada uma das mais importantes arboviroses que atingem o homem. No Brasil, em 2017, foram notificados 252.054 casos prov??veis de dengue, dos quais somente no Amazonas foram registrados quase 4 mil casos da doen??a. A detec????o do ant??geno NS1 ?? hoje uma importante ferramenta no diagn??stico r??pido da dengue, no entanto, estudos mostram que fatores como sorotipo circulante, regi??o geogr??fica dos pacientes e infec????es secund??rias podem estar relacionados com a diminui????o da sensibilidade do teste quando comparado com os m??todos moleculares. Portanto, com o intuito de avaliar poss??veis muta????es que possam estar associadas ?? dispers??o da dengue no Amazonas, bem como a falha na detec????o do ant??geno NS1, realizamos este estudo de evolu????o do sorotipo/gen??tipo do v??rus do dengue predominante no per??odo de 2011 a 2016 nos munic??pios do interior e na capital do estado. Ao todo foram analisadas 608 amostras de soro de pacientes com suspeita de dengue coletados em 26 munic??pios do Amazonas. As amostras foram submetidas ?? extra????o de RNA viral e posteriormente ao m??todo de PCR em tempo real, no qual 181 (29,7%) amostras tiveram resultado positivo. Posteriormente utilizando um protocolo de semi-nested PCR, observamos o predom??nio de DENV-4 (N=80), seguido de DENV-2 (N=9) e DENV-1 (N=7). Para os sorotipos de DENV-1 e 2 foram conduzidas rea????es de PCR convencional e sequenciamento somente para a regi??o do ENV, enquanto que para DENV-4 foi realizado tamb??m o sequenciamento da regi??o codificadora para NS1. As an??lises filogen??ticas seguiram de acordo com o m??todo de m??xima verossimilhan??a (ML) no programa PhyML, utilizando o modelo escolhido com a ajuda do programa jModeltest v2.1.7 e por Infer??ncia Bayesiana utilizando o programa BEAST v1.8.4. Foram obtidas 27 sequ??ncias completas de ENV e 25 de NS1 de DENV-4, provenientes de sete munic??pios do Amazonas, todas pertencentes ao gen??tipo II. As sequ??ncias de DENV-1 (2016) e DENV-2 (2011) coletadas em Manaus pertenciam ao gen??tipo V e Asi??tico/Americano, respectivamente. Durante a an??lise das sequ??ncias de ENV de DENV-4 foram encontrados 140 s??tios vari??veis e oito trocas de amino??cido, j?? nas sequ??ncias NS1 foram observados 91 s??tios vari??veis e sete trocas de amino??cidos. De acordo com a an??lise filogeogr??fica ocorreram pelo menos duas introdu????es independentes do DENV-4 no estado do Amazonas. Al??m disso, nossos dados tamb??m mostraram a propaga????o do DENV-4 a partir de Manaus para os outros munic??pios dentro do estado do Amazonas. Nos testes de soropreval??ncia, 45% (164/360) das amostras foram reagentes para detec????o de IgG, n??o sendo observado diferen??a significativa entre as amostras NS1-Positivas (43,9%) e NS1-Negativas (47,2%). Acreditamos que este estudo tenha importante papel na vigil??ncia arboviral no estado do Amazonas, relatando n??o apenas os sorotipos e gen??tipos de DENV de diferentes munic??pios, mas tamb??m como se deu a dissemina????o do DENV-4 GII no Estado.