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HIV-1 CRF02_AG is the most prevalent intersubtype recombinant form worldwide. Six HIV-1 samples from patients living in Rio de Janeiro, Brazil, were subtyped as CRF02_AG at the pol gene between 2004 and 2011. To trace the origin of these viruses, they were compared with 793 CRF02_AG pol sequences of African origin and another four Brazilian CRF02_AG pol sequences previously described. Phylogenetic analysis reveals that there have been at least four introductions of the CRF02_AG clade in Brazil, as signified by the presence of four phylogenetically distinct lineages, probably originated from western African countries (Benin, Ghana, and Guinea-Bissau). At least two CRF02_AG Brazilian lineages were successful in getting established and disseminated throughout the Rio de Janeiro state, with evidence of both horizontal and vertical transmission. Continuous epidemiological surveillance of HIV-1 strains circulating in Brazil is of paramount importance to the early detection of newly emerging viral lineages.

Concerns have been raised that universal availability of antiretroviral agents in resource-limited settings might lead to the emergence and spread of resistant strains. We present the largest survey on human immunodeficiency virus type 1 (HIV-1) resistance among treatment-naïve and experienced patients followed in small, relatively underprivileged cities in Brazil with universal availability to standard of care antiretroviral combinations. Samples were collected between 2004 and 2006 from 95 patients followed in the cities of Saquarema and Santo Antonio de Pádua, state of Rio de Janeiro. A proviral fragment encompassing protease and reverse transcriptase (RT) regions was generated and drug susceptibility level was inferred. Among 50 strains from drug-naïve subjects, one (2%) had intermediate-level resistance to RT inhibitors. Among 38 patients on therapy as of sampling, 28 (73.7%) had plasma viral load (PVL) below detection limit (26 of whom without evidence of resistance mutations) and 11 (28.9%) harbored strains with reduced susceptibility. Only two strains harbored both protease and RT inhibitor mutations. Among seven patients who were off-treatment as of sampling, two (28.5%) harbored strains with reduced susceptibility to RT inhibitors. The relatively high frequency of undetectable PVL among patients on treatment and the overall low prevalence of resistance-associated mutations are reassuring. Continued surveillance, however, is necessary.

As part of an ongoing study on the features of AIDS spread towards small cities and rural areas, we present a molecular survey of human immunodeficiency virus type 1 (HIV-1) polymerase sequences recovered between 2004 and 2006 from 71 patients receiving care in the city of Saquarema, inner state of Rio de Janeiro. Phylogenetic reconstructions found the two prevalent lineages in the state (subtypes B [59 strains, 83.1%], F1 [6 strains; 8.4%], and BF1 recombinants [four strains; 5.6%]), as well as two (2.8%) CRF02_AG strains, which seems to be an emerging lineage in the capital. These CRF02_AG sequences were recovered from a married heterosexual couple who never traveled abroad, thus providing the first molecular evidence of autochthonous horizontal transmission of this lineage of major global importance. Also, three phylogenetic clusters of strains recovered from a total of 18.3% of the cohort were uncovered. Their close genetic relatedness suggests they were recovered from patients who probably took part in the same chain of viral spread. In conjunction with our previous surveys from inner Rio de Janeiro, these results suggest that although small cities harbor unique molecular features of HIV-1 infection, they also clearly reflect and may rapidly absorb the diversity recorded in large urban centers.

It has been postulated that the non-synonymous divergence (distance to the subtype consensus sequence) observed in several HIV-1 subtype populations during 1990s attained the maximum limit that is compatible with viral fitness or survival, at least in the V3 env gene domain. To test this hypothesis, 145 subtype B and 64 subtype F env V3 sequences isolated from Brazilian HIV-1 positive patients between 1989 and 2004 were analyzed. HIV-1 env V3 sequences were grouped by year of collection and the mean intra-subtype diversity and divergence were examined at synonymous, non-synonymous, and amino acid level. The analyses clearly show that the mean intra-subtype divergence constantly increases in both subtype populations in the last 15 years, and more importantly, this trend was not only driven by a significant increase of the synonymous distance but also by a significant increase of the non-synonymous and amino acid distances between Brazilian circulating viruses and subtype consensus sequences. These results clearly disagree with the notion that the non-synonymous distance to the HIV-1 subtype consensus observed at population level had already attained the maximum limit, and suggest that the likelihood for success of vaccines based on "central" immunogens, as those based on any other empirically selected viral sequence, could be continuously diminishing over time.

We recently performed a molecular epidemiology survey of human immunodeficiency virus type 1 (HIV-1) infection in Miracema, a small city in Southeast Brazil, and found multiple monophyletic clusters, consistent with independent introductions and spread of different viral lineages in the city. Here we apply Bayesian coalescent-based methods to the two largest subtype B clusters and estimate that the most recent common ancestors that gave rise to these two transmission chains were in circulation around 1991-1992. The finding that HIV-1 spread in this Brazilian small city was already taking place at a time Aids was considered a problem restricted to large urban centers may have important public health implications.

The reconstruction of the epidemic history of several HIV populations, by using methods that infer the population history from sampled gene sequence data, has revealed important subtype-specific and regional-specific differences in patterns of epidemic growth. Here, we employ Bayesian coalescent-based methods to compare the population history of the HIV-1 subtype B and F1 epidemics in Brazil from non-contemporary env and pol gene sequences. Our results suggest that after the introduction of the subtypes B and F1 into Brazilian population, around mid to late 1960s and late 1970s, respectively, these subtypes experienced an initial period of exponential growth with similar epidemic growth rates ( approximately 0.5-0.6year(-1)). Later, the spreading rate of both subtypes seems to have slowed-down since mid to late 1980s. This demographic pattern is very similar to that reported for the subtype B epidemics in high-income countries where HIV was initially transmitted through homosexual intercourse and injecting drug use, as in Brazil; suggesting that the characteristics of transmission networks may be a key determinant of the HIV epidemic growth pattern. It is important to note that most of the subtype B and F1 sequences used in this study come from the Southeast region that has been the most affected by the AIDS epidemic in Brazil, being responsible for around 63% of all AIDS cases reported since the early eighties; but may not represent the demographic trend of the HIV-1 epidemic in other Brazilian regions.

In the State of Rio de Janeiro, Brazil, HIV-1 diversity surveys have been performed mainly in the capital, and little is known about HIV-1 molecular epidemiology in small, inner cities. We studied 87 polymerase sequences recovered from 2 different studies conducted in inner cities of Rio de Janeiro State: a cross-sectional survey of patients receiving care in the small city of Santo Antonio de Pádua, and a case series of patients who were failing HAART in 26 additional inner cities throughout the State. Subtypes B (83.9%), F1 (8%), D (2.3%), and unique BF1 recombinants (5.7%) were demonstrated. The cross-sectional study found that 41.6% of the patients harbored genetically related strains that fell within clusters and probably took part in the same chain of viral spread. These clusters were phylogenetically unrelated to previously reported clusters from a neighboring city, suggesting that each small city harbors its own set of microepidemic lineages.

Objective: The AIDS epidemic in Brazil is spreading from the large urban centers to small counties and the innermost parts of the country; however, data on the features of HIV-1 infection in these places are scarce. To study the routes of HIV-1 spread and assess the existence of transmission networks in such a setting, we performed a genetic analysis of viral sequences from the small county of Miracema, Rio de Janeiro State., Methods: HIV-1 envelope and polymerase sequences recovered from 63 adult patients (from a cohort of 78 patients) were phylogenetically analyzed., Results: A polyphyletic pattern ensued. Six clusters of sequences sharing close genetic relatedness were also recovered from 29 (46%) patients. Envelope and polymerase phylogenies yielded essentially the same results., Conclusions: The polyphyletic pattern suggests multiple viral introductions in the region. Intracluster cases, including those with no known direct epidemiologic link, probably took part in the same chain of viral transmission. Such a pattern suggests the existence of sexual networks and the emergence of multiple new infections within a relatively short period, a potential molecular marker of high incidence.

HIV-1 Subtype D occurs mainly in East and Central African countries, especially Uganda, where the prevalence of HIV-1 infection is among the highest in the world. We present the phylogenetic analysis of one nonautochthonous and four autochthonous (including a near full-length genome) Brazilian HIV-1 subtype D strains identified in Rio de Janeiro State, where subtypes B, F1, and BF1 recombinants predominate. Phylogenetic inferences using maximum likelihood were applied on a near-full length genome and on concatenated gag, protease, reverse transcriptase, integrase, C2V3/env, gp41, and nef segments. Sequences from an Angolan immigrant showed close genetic similarity with a strain described in Finland, from an HIV patient of African origin, whereas all four autochthonous Brazilian sequences clustered with South African strains, where subtype D occurs only in isolated cases. Our results suggest the successful introduction and circulation in Brazil of closely related HIV-1 subtype D strains, possibly of South African origin.

In Brazil, surveillance studies on antiretroviral drug resistance among drug-naïve and treatment-experienced patients have focused primarily on patients living in large urban centers. As the epidemic spreads towards small municipalities and the innermost parts of the country, it will be essential to monitor the prevalence of antiretroviral drug resistance in these areas. We report the first survey on the prevalence of antiretroviral drug resistance in a small Brazilian municipality. Between July 1999 and March 2005, 72 adult human immunodeficiency virus type-1(HIV-1)-infected patients received care at the Municipal HIV/AIDS Program of the small, southeastern municipality of Miracema, state of Rio de Janeiro. A genotyping study of antiretroviral drug resistance was performed in 54 patients. Among 27 samples from treatment-experienced patients, 9 (33.3%) harbored strains with reduced drug susceptibility. Among these, 6 had reduced susceptibility to reverse transcriptase (RT) inhibitors and 3 to both RT and protease inhibitors. No primary antiretroviral drug resistance was recorded among 27 drug-naïve subjects. The relatively low prevalence of resistance mutations in the Miracema cohort argues against the concern that resource-poor settings should not implement widespread accessibility to standard of care antiretroviral combinations due to the possibility of sub-optimal adherence leading to the emergence and spread of drug-resistant strains.

Retroviral genomes with a high frequency of G-->A mutations are thought to originate during reverse transcription (RT). Here we present a case report of an AIDS patient infected with a subtype F variant where extensive G-->A hypermutation (G-->A Hypm) sequences were found in the protease gene. This patient was failing HAART at the time the hypermutation was found. These sequences were basically encountered in the proviral compartment on two occasions and were persistently absent in the plasma viral population. The patient's viral genotype showed several mutations related to antiretroviral drug resistance in RT (T69N, M184V, T215F, K219Q) and protease (M36I, G48V, I54V, T63L, V82A) genes. The drug regimen was changed and the viral load dropped 0.9 Logs and CD4 count increased by 200 cells/ml. The hypermutation was not found any more in a 1-year follow up. The patient's wife was infected with a similar virus strain and G-->A Hypm sequences were also detected in the RT gene. This is the first report of sexual transmitted G-->A Hypermutation in HIV-1 and suggest that this phenomenon can be genetically coded by the viral RT molecule.

A wide variety of disorders of diverse pathogenic mechanisms can trigger spinal cord dysfunction in HIV-1-infected patients. The most common such condition is HIV-1-associated myelopathy (HM) which characteristically complicates advanced HIV-1 disease in patients with low CD4 cell counts and previous AIDS-defining diagnoses. We describe an unusual presentation of HM in a previously asymptomatic patient with a relatively preserved CD4 cell count (458 cells/mm3) who was even unaware of his serological status. The patient presented with a clinically severe, slowly progressive myelopathy and could not walk unassisted. Significant neurological improvement could be obtained as rapidly as within 4 weeks after the institution of an antiretroviral combination of only two nucleoside analog HIV-1 reverse transcriptase inhibitors (zidovudine and didanosine). An HIV-1 protease inhibitor was also prescribed at that point but could only be added to intensify the regimen 3 months later, when significant neurological improvement had already been recorded. We also review the disorders reported to derange spinal cord function in previously asymptomatic HIV-1-infected patients.

We have investigated the phenotypic and genotypic susceptibility of 14 HIV-1 strains isolated from individuals failing HAART therapy to protease inhibitors (PI). Proviral and plasma viral pol gene fragment were amplified, sequenced and subtyped. Nine samples clustered with protease subtype B reference strains and the remaining samples were classified as non-B subtype corresponding to subtype F (n = 4) and subtype A (n = 1). Although all patients were treated with similar P1 drug regimen, the non-B subtype isolates did not present the L90M and 184V mutations and used mainly G48V and V82A/F to achieve drug resistance. A strong cross-resistance phenotype among all four PI was associated with the mutation L90M in the subtype-B isolates, and with G48V and V82A/F in the non-B counterparts. This observation revealed that the non-B viruses tested had specific genotypic characteristics contrasting with the subtype-B isolates.

Development of drug resistance is the inevitable consequence of incomplete suppression of virus plasma levels in HIV-1-infected patients treated with highly active antiretroviral therapy. Resistance mutations previously characterized have been found in B subtype viruses of developed countries. Moreover, mutation profiles for non-B and more divergent B subtype viruses found in developing countries shall be analyzed together with their ex vivo phenotyping in order to establish an exact correlation between the genotyping data and the clinical management counseling for those uncommon virus subtypes. In the present study, we evaluated the mutation profile for individuals infected with B subtype and non-B subtype viruses. Viral DNA fragments corresponding to the RT gene were amplified, sequenced, and subtyped. Phenotyping analysis for reverse transcriptase nucleoside (NRTI) and nonnucleoside inhibitor susceptibility was performed using the recombinant virus assay technology. Brazilian non-B subtypes (subtype F, n = 4, and subtype A, n = 1) isolates showed essentially the same B subtype mutation profile, presenting an NRTI drug resistance with similar MIC50% and MIC90% values for all drugs analyzed regardless of their subtypes. A strong cross-resistance phenotype among AZT, 3TC, and abacavir could be seen in all isolates analyzed. A novel result was that some RT sequences not only revealed the presence of G333D/E mutations but also correlated to the presence of mutation T386I that could abrogate the M184V-surpassing effect of L210W or L210W plus G333D/E. These findings suggest that Brazilian non-B subtype HIV-1 strains use an identical RT drug resistance mutation pattern when compared to B isolates and will contribute to the validation of the genotypic and phenotypic tests in these predominant worldwide-spread viral variants., (Copyright 2000 Academic Press.)