Bernardo Galvão-Castro

A standardized framework for accurate, high-throughput genotyping of recombinant and non-recombinant viral sequences

Human immunodeficiency virus type-1 (HIV-1), hepatitis B and C and other rapidly evolving viruses are characterized by extremely high levels of genetic diversity. To facilitate diagnosis and the development of prevention and treatment strategies that efficiently target the diversity of these viruses, and other pathogens such as human T-lymphotropic virus type-1 (HTLV-1), human herpes virus type-8 (HHV8) and human papillomavirus (HPV), we developed a rapid high-throughput-genotyping system. The method involves the alignment of a query sequence with a carefully selected set of pre-defined reference strains, followed by phylogenetic analysis of multiple overlapping segments of the alignment using a sliding window. Each segment of the query sequence is assigned the genotype and sub-genotype of the reference strain with the highest bootstrap (>70%) and bootscanning (>90%) scores. Results from all windows are combined and displayed graphically using color-coded genotypes. The new Virus-Genotyping Tools provide accurate classification of recombinant and non-recombinant viruses and are currently being assessed for their diagnostic utility. They have incorporated into several HIV drug resistance algorithms including the Stanford (http://hivdb.stanford.edu) and two European databases (http://www.umcutrecht.nl/subsite/spread-programme/ and http://www.hivrdb.org.uk/) and have been successfully used to genotype a large number of sequences in these and other databases. The tools are a PHP/JAVA web application and are freely accessible on a number of servers including: http://bioafrica.mrc.ac.za/rega-genotype/html/ http://lasp.cpqgm.fiocruz.br/virus-genotype/html/ http://jose.med.kuleuven.be/genotypetool/html/.

Retrovirus infections in a sample of injecting drug users in Rio de Janeiro City, Brazil: prevalence of HIV-1 subtypes, and co-infection with HTLV-I/II.

BACKGROUNDnRetrovirus infections among injecting drug users (IDUs), a core at-risk population for both HIV-1 and HTLV-I/II infections in Brazil, were assessed within an ongoing cooperative research.nnnOBJECTIVEnThe study assessed the seroprevalences of HIV-1 and HTLV-I/II infections, as well as the prevalence of HIV-1 subtypes in a sample of IDUs from Rio de Janeiro, Brazil. An attempt to evaluate HIV incidence was carried out using a dual sensitive/less sensitive testing strategy.nnnSTUDY DESIGNnCross-sectional evaluation of 175 IDUs. Serostatus for HIV-1 and HTLV-I/II were established by enzyme-linked immunosorbent assays, and confirmed by western blot. The dual testing strategy aimed to estimate HIV-1 incidence rates. Differentiation between HTLV-I and -II was performed by western blot. DNA samples were polymerase chain reaction amplified by a nested protocol, and HIV-1 subtyping was determined by heteroduplex mobility assay.nnnRESULTSnForty-six and 29 samples were found to be, respectively, positive for HIV-1 and HTLV-I/II, 15 of them co-infected by both viruses. Among HTLV-I/II-infected patients, 75.9% were infected by HTLV-I. Thirty-one HIV samples were identified as B subtype, with seven of them showing the typical Brazilian B pattern in the gp120 V3 loop, and ten were identified as F subtype. The use of less sensitive assays for HIV infection wrongly identified a deeply immunocompromised patient as an incident case.nnnCONCLUSIONnModerately high seroprevalences were found for both HIV-1 and HTLV-I/II infections, HIV-1/HTLV-I co-infections being of special concern. A non-statistically significant higher prevalence of F subtype was observed, when compared with the distribution of F/B subtypes among Brazilian patients from other exposure categories. No recent HIV-1 infections were detected, but a limitation of the sensitive/less-sensitive testing strategy was made evident.

Globin haplotypes of human T-cell lymphotropic virus type I-infected individuals in Salvador Bahia Brazil suggest a post-Columbian African origin of this virus.

The city of Salvador, Bahia, Brazil, has sociodemographic characteristics similar to some African cities. Up to now, it has had the highest prevalence of human T-cell lymphotropic virus type I (HTLV-I) infection (1.74%) in the country. To investigate which strains of HTLV-I are circulating in Salvador, we studied isolates from 82 patients infected with HTLV-I: 19 from the general population, 21 from pregnant women, 16 from intravenous drug users, and 26 from patients and their family attending a neurologic clinic. Phylogenetic analysis from part of the LTR fragments showed that most of these isolates belonged to the Transcontinental subgroup of the Cosmopolitan subtype (HTLV-Ia). Only one sample from a pregnant woman was closely related to the Japanese subgroup, suggesting recent introduction of a Japanese HTLV-I lineage into Salvador. betaA-Globin haplotypes were examined in 34 infected individuals and found to be atypical, confirming the racial heterogeneity of this population. A total of 20 chromosomes were characterized as Central African Republic (CAR) haplotype (29.4%), 31 (45.6%) were characterized as Benin (BEN) haplotype, and 17 (25%) were characterized as Senegal (SEN) haplotype. Five patients genotypes (14.7%) were CAR/CAR; 10 (29,4%), BEN/BEN; 9 (26.5%), CAR/BEN; 2 (5.9%), BEN/SEN; and 7 (20.6%), SEN/SEN. One patients genotype (2.9%) was CAR/SEN. The betaA-globin haplotype distribution in Salvador is unusual compared with other Brazilian states. Our data support the hypothesis of multiple post-Columbian introductions of African HTLV-Ia strains in Salvador, Bahia, Brazil.

Detection of distinct human immunodeficiency virus Type 1 circulating recombinant forms in Northeast Brazil

The extraordinary genetic diversity and immune evasion of human immunodeficiency virus (HIV) pose significant challenges for vaccine development and antiretroviral therapy efficacy. The objective of this study was to characterize the molecular profile of HIV‐1 epidemic in Salvador, Bahia, Brazil, determining the genetic subtypes and the presence of antiretroviral resistance mutations. HIV‐1 pol DNA sequences from 57 individuals infected with HIV were obtained by PCR, followed by sequencing. The subtypes were determined by phylogenetic analyses and the intersubtype recombination was investigated by bootscanning. The pol subtypes were compared with gag and env subtypes. Antiretroviral susceptibility was evaluated through the Stanford HIV resistance Database. The subtypes frequencies were: 77.2% of subtype B, 1.8% of subtype F1, and 21.0% of BF recombinant forms. Two intergenic and three intragenic BF recombinant patterns were observed. Six (10.5%) viruses were related to CRF28/CRF29, two were related to CRF12 (3.5%), and one (1.8%) was CRF39. Fourteen (24.6%) strains carried one or more mutations associated with at least intermediate resistance: 24.6% had resistance to nucleoside reverse transcriptase inhibitors, 21.0% to non‐nucleoside reverse transcriptase inhibitors, and 7% to protease inhibitors. The substitutions I54V (7.0%), M184V (14.0%), and K103N (10.5%) were the most frequent within each class of drugs. The results show a high diversity of BF genotypes and a lower prevalence of major reverse transcriptase and protease drug resistance mutations in Salvador, compared with other regions of Brazil. These findings may contribute to improve treatment strategies of patients infected with HIV‐1 from this Brazilian region. J. Med. Virol. 83:2066–2072, 2011.

Do African and European ancestry and polymorphism of HLA class I play an important role in controlling HTLV-1 proviral load in admixed cohorts from Salvador, Brazil?

HAM/TSP is one of the most prevalent clinical manifestations of HTLV- 1 infection, likely related to high proviral load (PVL). We investigated HLA-A, -B and –C polymorphisms and determined the individual ancestry proportion of European, African and Amerindian in 209 HTLV-1 infected individuals in order to identify genetic factors that associate with HAM/TSP. The ancestry estimation using the STRUCTURE program considered the genotyping of 47 SNP ancestry markers. Using rigorous clinical and laboratory criteria, we defined two subsets: HAM/TSP (41 patients) and asymptomatic (168). No associations were found between HLA polymorphisms and HAM/TSP. The European ancestry in the HAM/TSP sample (43%) was 7% lower than in the asymptomatic group (50%), while the African ancestry was 6% higher among HAM/TSP patients (45%) than among asymptomatics (39%). The Amerindian ancestry was quite similar for both HAM/TSP (12%) and asymptomatic (11%) samples. Although these differences did not reached statistical significance, they provide interesting clues to be more deeply explored. There was no correlation between PVL and individual European, African and Amerindian ancestry estimates when considering the entire sample. However, considering only the HAM/TSP subsample, our results suggest that European ancestry may predispose to higher PVL (Spearman´s correlation; rs= 0.3611; p=0,0422), while African ancestry associates with lower PVL (rs= -0.3875; p=0,0283). These results suggest that once HAM/TSP develops, people of African ancestry may control PVL to a greater extent. More efficient PVL control among people of African descent may be the result of selection pressure occurring over the long history of co-evolution of HTLV-1 and its human host in Africa.