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

Prevalência, fatores de risco e caracterização genética dos vírus linfotrópico de células T humana tipo 1 e 2 em pacientes infectados pelo vírus da imunodeficiência humana tipo 1 nas Cidades de Ribeirão Preto e São Paulo

The aim of this study was to define the prevalence of human T cell lymphotropic virus types 1 and 2 in patients who were positive for human immunodeficiency virus type 1 in the State of Sao Paulo, Brazil. We evaluated 319 individuals infected with HIV type 1 who were attended at specialized clinics in two cities (Ribeirao Preto and Sao Paulo). The patients were interviewed and tested for antibodies against HTLV types 1 and 2 (Ortho HTLV-1/HTLV-2 Ab-Capture enzyme immunoassay). Direct DNA sequencing of polymerase chain reaction products from the tax region of HTLV type 2 and the long terminal repeat region of HTLV types 1 and 2 were performed to differentiate and determine the subtypes. The overall prevalence of anti-HTLV type 1 and 2 antibodies was 7.5% (24/319; 95% CI: 5.2-11.5). HTLV type 1 and 2 infection was associated with a history of injected drug use and with antibodies for hepatitis C virus (p 0.05). HTLV DNA was detected in 13 out of 24 samples, of which 12 were characterized as HTLV subtype 2c and one as HTLV subtype 1a. Among the 12 HTLV type 2 samples, seven were from injected drug users, thus indicating that this route is an important risk factor for HTLV type 2 transmission among our population infected with HIV type 1.Fundacao Pr6 Sangue, Hemoctr Sao Paulo, BR-05403000 Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Lab Retrovirol, Dept Doencas Infecciosas & Parasitarias, Sao Paulo, BrazilSecretaria Estado Saude Sao Paulo, Ctr Referencia & Treinamento DST Aids, Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Lab Retrovirol, Dept Doencas Infecciosas & Parasitarias, Sao Paulo, BrazilWeb of Scienc

Near full-length genome analysis of low prevalent human immunodeficiency virus type 1 subclade F1 in São Paulo, Brazil

Background: The genetic diversity of the human immunodeficiency virus type 1 (HIV-1) is critical to lay the groundwork for the design of successful drugs or vaccine. In this study we aimed to characterize and define the molecular prevalence of HIV-1 subclade F1 currently circulating in Sao Paulo, Brazil. Methods: A total of 36 samples were selected from 888 adult patients residing in Sao Paulo who had previously been diagnosed in two independent studies in our laboratory as being infected with subclade F1 based on pol subgenomic fragment sequencing. Proviral DNA was amplified from the purified genomic DNA of all 36 blood samples by 5 fragments overlapping PCR followed by direct sequencing. Sequence data were obtained from the 5 fragments of pure subclade F1 and phylogenetic trees were constructed and compared with previously published sequences. Subclades F1 that exhibited mosaic structure with other subtypes were omitted from any further analysis Results: Our methods of fragment amplification and sequencing confirmed that only 5 sequences inferred from pol region as subclade F1 also holds true for the genome as a whole and, thus, estimated the true prevalence at 0.56%. The results also showed a single phylogenetic cluster of the Brazilian subclade F1 along with non-Brazilian South American isolates in both subgenomic and the full-length genomes analysis with an overall intrasubtype nucleotide divergence of 6.9%. The nucleotide differences within the South American and Central African F1 strains, in the C2-C3 env, were 8.5% and 12.3%, respectively. Conclusion: All together, our findings showed a surprisingly low prevalence rate of subclade F1 in Brazil and suggest that these isolates originated in Central Africa and subsequently introduced to South America.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[06/50096-0

The frequency of CD127low expressing CD4+CD25high T regulatory cells is inversely correlated with human T lymphotrophic virus type-1 (HTLV-1) proviral load in HTLV-1-infection and HTLV-1-associated myelopathy/tropical spastic paraparesis

<p>Abstract</p> <p>Background</p> <p>CD4⁺CD25^highregulatory T (T_Reg) cells modulate antigen-specific T cell responses, and can suppress anti-viral immunity. In HTLV-1 infection, a selective decrease in the function of T_Regcell mediated HTLV-1-tax inhibition of FOXP3 expression has been described. The purpose of this study was to assess the frequency and phenotype of T_Regcells in HTLV-1 asymptomatic carriers and in HTLV-1-associated neurological disease (HAM/TSP) patients, and to correlate with measures of T cell activation.</p> <p>Results</p> <p>We were able to confirm that HTLV-I drives activation, spontaneous IFNγ production, and proliferation of CD4+ T cells. We also observed a significantly lower proportion of CTLA-4⁺T_Regcells (CD4⁺CD25^highT cells) in subjects with HAM/TSP patients compared to healthy controls. Ki-67 expression was negatively correlated to the frequency of CTLA-4⁺T_Regcells in HAM/TSP only, although Ki-67 expression was inversely correlated with the percentage of CD127^lowT_Regcells in healthy control subjects. Finally, the proportion of CD127^lowT_Regcells correlated inversely with HTLV-1 proviral load.</p> <p>Conclusion</p> <p>Taken together, the results suggest that T_Regcells may be subverted in HAM/TSP patients, which could explain the marked cellular activation, spontaneous cytokine production, and proliferation of CD4⁺T cells, in particular those expressing the CD25^highCD127^lowphenotype. T_Regcells represent a potential target for therapeutic intervention for patients with HTLV-1-related neurological diseases.</p

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

Prevalence and Risk factors of HTLV-I/II in a cohort of patients with HIV-1 infection in two cities from the state of São Paulo, Brazil

A prevalência e a subtipagem molecular foram usadas para identificar a epidemiologia de HTLV-I/II entre indivíduos infectados por HIV-1 no Estado de São Paulo. Além disso, foi feita a correlação da infecção por HTLV-I/II com os fatores de riscos relacionados a infecção por HIV-1. Este estudo de corte transversal retrospectivo incluiu 319 indivíduos infectados com HIV-1, atendidos em clínicas especializadas de duas cidades do Estado de São Paulo, Brasil (Ribeirão Preto & São Paulo, Capital). Os pacientes foram entrevistados e testados para anticorpos contra HTLV-I/II (ORTHO® HTLVI/ HTLV-II Ab-Capture enzyme immunoassay). Foi realizado o sequenciamento direto de DNA dos produtos de PCR da região pXTax de HTLV-II e LTR de HTLVI/ II para diferenciação e determinação do subtipo. A prevalência geral de anticorpos anti-HTLV-I/II foi de 7,5% (24/319; 95% CI: 5.2-11.5) e não diferiu significativamente por idade (p= 0.2), sexo (p=0.9), ou local de residência (p=0.7). A infecção por HTLV-I/II foi fortemente associada com histórico de uso de drogas injetáveis e anticorpos para o vírus da Hepatite tipo C (p0.05) between the presence of antibodies against HTLV and sexual behaviours or serological markers for sexual transmitted diseases (anti T.palliduim, Anti HHV8, or Anti HBV antibodies). HTLV-II DNA was detected in 50% (12/24; 95% CI: 28.3-68.2) of the subjects tested seropositive and they were all identified as subtype IIc by phylogenetic analysis. Of them, 58% (7/12) were injecting drug users. However, only 4,2% (1/24) of HTLV-I had detectable DNA and identified as subtype Ia. Our results indicate that i.v drug injection is an important risk factor for HTLV-II transmission, but not for HTLV-I, among our HIV-1 infected population and that HTLV-II-subtype c is a useful marker of HTLV-II transmission via i.v drug injection

Correlation between LTR point mutations and proviral load levels among human T cell lymphotropic virus type 1 (HTLV-1) asymptomatic carriers

Introdução: Estudos in vitro demonstram que delecoes ou mutacoes pontuais inseridas em cada repeticao imperfeita de 21 pb do Elemento Responsivo a Tax (TRE) nos promotores virais do Virus Linfotropico de celulas T humanas Tipo 1 (HTLV-1) selvagens aboliram a inducao de Tax. A partir deste dado, supomos que mutacoes similares poderiam afetar a proliferacao de celulas infectadas por HTLV-1 e alterar a carga proviral (CPv). Para testar esta hipotese, conduzimos uma analise genetica de corte transversal para comparar sequencias nucleotidicas completas de LTR que cobrem a regiao TRE-1 em uma amostragem de portadores assintomaticos de HTLV-1 com diferentes niveis de carga proviral. Metodos: Foi avaliado um total de 94 portadores assintomaticos de HTLV-1 com as sequencias de longa repeticao terminal (LTR) extremidade 5` determinadas e carga proviral de DNA de Tax definida usando-se uma sensivel tecnica de PCR em Tempo Real com SYBR Green. Os 94 individuos foram divididos em tres grupos baseados na medida da carga proviral: 31 com baixa, 29 com intermediaria e 34 com alta CPv. Alem disso, cada grupo foi comparado levando-se em conta sexo, idade e genotipo viral. Em outra analise, foi comparada a mediana da carga proviral entre individuos infectados com o virus mutante e virus o tipo selvagem. Resultados: Usando-se uma analise categorica, a substituicao G232A, localizada no dominio A do motivo TRE-1, foi detectada em 38,7% (12/31), 27.5% (8/29), e 61.8% (21/34) dos individuos com baixa, intermediaria e alta CPv, respectivamente. Uma diferenca significativa na deteccao desta mutacao foi encontrada entre sujeitos com alta e baixa CPv e entre aqueles com alta e intermediaria CPv (ambos p 0,05). Estes resultados foram confirmados por uma analise nao-parametrica que mostrou forte evidencia de aumento da CPv dentre individuos HTLV-1 positivos com a mutacao G232A em relacao aqueles sem esta mutacao (p 0,05). Conclusao: Os dados descritos aqui mostram que alteracoes no dominio A do motivo TRE-1 do HTLV-1, em especial a mutacao G232A pode aumentar a replicacao do HTLV-1 na maioria dos pacientes infectadosBackground: In vitro studies have demonstrated that deletions and point mutations introduced into each 21 bp imperfect repeat of Tax-responsive element (TRE) of the genuine human T-cell leukemia virus type I (HTLV-1) viral promoter abolishes Tax induction. Given these data, we hypothesized that similar mutations may affect the proliferation of HTLV-1 infected cells and alter the proviral load (PvL). To test this hypothesis, we conducted a crosssectional genetic analysis to compare the near-complete LTR nucleotide sequences that cover the TRE1 region in a sample of HTLV-1 asymptomatic carriers with different PvL burden. Methods: A total of 94 asymptomatic HTLV-1 carriers with both sequence from the 5’ long terminal repeat (LTR) and a PvL for Tax DNA measured using a sensitive SYBR Green realtime PCR were studied. The 94 subjects were divided into three groups based on PvL measurement: 31 low, 29 intermediate, and 34 high. In addition, each group was compared based on sex, age, and viral genotypes. In another analysis, the median PvLs between individuals infected with mutant and wild-type viruses were compared. Results: Using a categorical analysis, a G232A substitution, located in domain A of the TRE1 motif, was detected in 38.7% (12/31), 27.5% (8/29), and 61.8% (21/34) of subjects with low, intermediate, or high PvLs, respectively. A significant difference in the detection of this mutation was found between subjects with a high or low PvL and between those with a high or intermediate PvL (both p 0.05). This result was confirmed by a non-parametric analysis that showed strong evidence for higher PvLs among HTLV-1 positive individuals with the G232A mutation than those without this mutation (p 0.05). Conclusion: The data described here show that changes in domain A of the HTLV-1 TRE-1 motif resulting in the G232A mutation may increase HTLV-1 replication in a majority of infected subjects.BV UNIFESP: Teses e dissertaçõe

Sequence Variability of Human Erythroviruses Present in Bone Marrow of Brazilian Patients with Various Parvovirus B19-Related Hematological Symptoms

The presence of erythrovirus infections was investigated by PCR with bone marrow samples of patients with various parvovirus B19-related hematological symptoms. Erythrovirus DNA was found in 17.3% (12/69) of patients. Phylogenetic analysis revealed that five strains cluster with genotype 1, one clusters with genotype 2, and six cluster with genotype 3. Our study is the first to document the presence of the three erythrovirus genotypes in Brazil