Characterization and frequency of a newly identified HIV-1 BF1 intersubtype circulating recombinant form in São Paulo, Brazil

Background: HIV circulating recombinant forms (CRFs) play an important role in the global and regional HIV epidemics, particularly in regions where multiple subtypes are circulating. To date, several (>40) CRFs are recognized worldwide with five currently circulating in Brazil. Here, we report the characterization of near full-length genome sequences (NFLG) of six phylogenetically related HIV-1 BF1 intersubtype recombinants (five from this study and one from other published sequences) representing CRF46_BF1.Methods: Initially, we selected 36 samples from 888 adult patients residing in São Paulo who had previously been diagnosed as being infected with subclade F1 based on pol subgenomic fragment sequencing. Proviral DNA integrated in peripheral blood mononuclear cells (PBMC) was amplified from the purified genomic DNA of all 36-blood samples by five overlapping PCR fragments followed by direct sequencing. Sequence data were obtained from the five fragments that showed identical genomic structure and phylogenetic trees were constructed and compared with previously published sequences. Genuine subclade F1 sequences and any other sequences that exhibited unique mosaic structures were omitted from further analysisResults: of the 36 samples analyzed, only six sequences, inferred from the pol region as subclade F1, displayed BF1 identical mosaic genomes with a single intersubtype breakpoint identified at the nef-U3 overlap (HXB2 position 9347-9365; LTR region). Five of these isolates formed a rigid cluster in phylogentic trees from different subclade F1 fragment regions, which we can now designate as CRF46_BF1. According to our estimate, the new CRF accounts for 0.56% of the HIV-1 circulating strains in São Paulo. Comparison with previously published sequences revealed an additional five isolates that share an identical mosaic structure with those reported in our study. Despite sharing a similar recombinant structure, only one sequence appeared to originate from the same CRF46_BF1 ancestor.Conclusion: We identified a new circulating recombinant form with a single intersubtype breakpoint identified at the nef-LTR U3 overlap and designated CRF46_BF1. Given the biological importance of the LTR U3 region, intersubtype recombination in this region could play an important role in HIV evolution with critical consequences for the development of efficient genetic vaccines.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Hemoctr, Fundacao Prosangue, São Paulo, BrazilUniversidade Federal de São Paulo, Retrovirol Lab, São Paulo, BrazilUniversidade Federal de São Paulo, Retrovirol Lab, São Paulo, BrazilFAPESP: 06/50096-0FAPESP: 2004/15856-9FAPESP: 2007/04890-0Web of Scienc

Characterization of Partial and Near Full-Length Genomes of HIV-1 Strains Sampled from Recently Infected Individuals in São Paulo, Brazil

Background: Genetic variability is a major feature of human immunodeficiency virus type 1 (HIV-1) and is considered the key factor frustrating efforts to halt the HIV epidemic. A proper understanding of HIV-1 genomic diversity is a fundamental prerequisite for proper epidemiology, genetic diagnosis, and successful drugs and vaccines design. Here, we report on the partial and near full-length genomic (NFLG) variability of HIV-1 isolates from a well-characterized cohort of recently infected patients in Sao Paul, Brazil.Methodology: HIV-1 proviral DNA was extracted from the peripheral blood mononuclear cells of 113 participants. the NFLG and partial fragments were determined by overlapping nested PCR and direct sequencing. the data were phylogenetically analyzed.Results: of the 113 samples (90.3% male; median age 31 years; 79.6% homosexual men) studied, 77 (68.1%) NFLGs and 32 (29.3%) partial fragments were successfully subtyped. of the successfully subtyped sequences, 88 (80.7%) were subtype B sequences, 12 (11%) BF1 recombinants, 3 (2.8%) subtype C sequences, 2 (1.8%) BC recombinants and subclade F1 each, 1 (0.9%) CRF02 AG, and 1 (0.9%) CRF31 BC. Primary drug resistance mutations were observed in 14/101 (13.9%) of samples, with 5.9% being resistant to protease inhibitors and nucleoside reverse transcriptase inhibitors (NRTI) and 4.9% resistant to non-NRTIs. Predictions of viral tropism were determined for 86 individuals. X4 or X4 dual or mixed-tropic viruses (X4/DM) were seen in 26 (30.2%) of subjects. the proportion of X4 viruses in homosexuals was detected in 19/69 (27.5%).Conclusions: Our results confirm the existence of various HIV-1 subtypes circulating in São Paulo, and indicate that subtype B account for the majority of infections. Antiretroviral (ARV) drug resistance is relatively common among recently infected patients. the proportion of X4 viruses in homosexuals was significantly higher than the proportion seen in other study populations.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ São Paulo, Fac Med, Div Clin Immunol & Allergy, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilFundacao Pro Sangue, Blood Ctr Sau Paulo, São Paulo, BrazilUniv São Paulo, Dept Infect Dis, São Paulo, BrazilPubl Hlth Dept São Paulo, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilFAPESP: 04/15856-9FAPESP: 2006/50096-0Web of Scienc

Characterization and frequency of a newly identified HIV-1 BF1 intersubtype circulating recombinant form in São Paulo,Brazil

Formas recombinantes circulantes (CRFs) do HIV desempenham um importante papel na epidemia global e regional do HIV, principalmente em regioes onde diversos subtipos estao circulando. Atualmente 5 CRFs estao em circulacao no Brasil. Nesse artigo, nos reportamos a caracterizacao do genoma semicompleto (NFLGs) de 6 recombinantes intersubtipos HIV-1 BF1 filogeneticamente relacionados (5 desse estudo e 1 de outra sequencia publicada). Inicialmente, nos selecionamos 36 amostras de 888 pacientes adultos, residentes em São Paulo que haviam sido previamente diagnosticados como sendo infectado com o HIV-1 subtipo F1 baseado no sequenciamento do fragmento subgenomico pol. O DNA pro-viral integrado as celulas mononucleares do sangue periferico (PBMC) dessas 36 amostras foi amplificado por PCR de sobreposicao com 5 fragmentos, seguido pelo sequenciamento direto. Foram obtidos dados das sequencias apenas dos 5 fragmentos que mostraram estrutura genomica identica. Alem disso, foram construidas arvores filogeneticas, comparando nossas sequencias com sequencias previamente publicadas. Como resultados, obtivemos que das 36 amostras analisadas, apenas 6 sequencias, inferidas como subtipo F1 pela analise da regiao pol, mostraram estrutura genomica identica BF1, com um unico breakpoint identificado na sobreposicao nef-U3 (posicao 9347-9365 no HXB2; regiao LTR). Cinco desses isolados formaram um grupo coeso na arvore filogenetica de diferentes fragmentos do subtipo F1, os quais nos podemos agora designar como CRF46_BF1. Estimamos que a nova CRF representa 0,56% das cepas de HIV-1 circulantes 40 em São Paulo. Comparando com sequencias previamente publicadas observamos que mais 5 isolados compartilham uma estrutura de recombinacao identica a reportada em nosso estudo. Apesar de compartilharem uma estrutura recombinante similar, apenas 1 sequencia parece ser originaria da mesma CRF46_BF1 ancestral. Concluindo, nos identificamos uma nova CRF com um unico breakpoint intersubtipo na regiao de sobreposicao nef-U3 a qual foi designada CRF48_BF1. Dada a importancia biologica da regiao LTR U3, podemos inferir que recombinacoes intersubtipos nessas regioes devem desempenhar um importante papel na evolucao do HIV, gerando, concomitantemente, consequencias criticas para o desenvolvimento de vacinas geneticas eficientesBV UNIFESP: Teses e dissertaçõe