HIV-1 in Waraos Figure 1. Map of the Orinoco Delta showing the location of the Warao communities studied. doi:10.1371/journal.pone.0040626.g001 The subtype B sequences from Waraos formed a highly supported monophyletic clade in each genomic region analyzed (Figure 2). At the pol genomic region, the subtype B strains infecting Waraos displayed 97–99% identity between them and only 94–96% identity with the closely related sequence available at GenBank (accession number D10112). A sequence from a Venezuelan patient from Maracay, a city distant from the Orinoco Delta (HIV162, accession number FJ65968) was found to share an ancestor with Waraos subtype B clade, exhibiting 94– 95% identity with this clade at the pol genomic region. Phylogenetic analyses of the pol, env and vif genomic regions indicated that the Waraos clade was subdivided in two well supported subclades. Strains form individuals living in a same community were grouped in the same subclade (Figure 2). The only HIV-1 subtype C isolate found among Waraos was more closely related to the African and Asian strains (closely related sequences available at GenBank), than to the Brazilian subtype C clade. However, the grouping with these strains was not supported by a high bootstrap value (Figure 2). In order to confirm the hypothesis of a single introduction of the HIV-1 subtype B in the Warao Amerindians, Maximum Likelihood (ML) phylogenetic analysis was performed. HIV-1 sequences from Waraos were analyzed with a set of 141 pol and 112 env subtype B ‘‘background’’ sequences isolated from the general population in Venezuela. The ML tree confirmed the monophyletic clustering of the subtype B sequences from Waraos and also supported the relative close phylogenetic relationship between the Warao clade and the Venezuelan sequence HIV162, both when analyzing the pol and the env regions (Figure 3). Estimates of the time of the most recent common ancestor (Tmrca, years) of subtype B Warao clade were obtained using a Bayesian MCMC approach. A total of 156 pol sequences (collected between 2004 and 2011) and 104 env sequences from Venezuela (isolates collected between 1999 and 2009) were used for this analysis. The 95% HPD intervals of the estimated rate of evolution of both pol (1.6–2.561023 subst./site/year) and env (4.1–7.861023 subst./site/year) datasets almost coincide with the informative prior interval used in our analyses, indicating that not much temporal information was added by the data. Meanwhile, the coefficients of rate variation of both pol (0.27; 95% HPD: 0.22– 0.33) and env (0.30; 95% HPD: 0.22–0.40) genomic regions were higher than zero, thus demonstrating a significant variation of substitution rate among branches and supporting the use of a relaxed molecular clock model. The Tmrca of the Warao subtype B clade was estimated at 1999 (95% HPD: 1993–2003) when analyzing both pol and env genomic regions; while the Tmrca of the Waraos and Venezuelan patient HIV162 was 10 years older (Figure 4). The Tmrca of all subtype B sequences from the general population in Venezuela was estimated at 1977 (95% HPD: 1968– 1984) and 1979 (95% HPD: 1968–1986), according to the analysis of the pol and env regions, respectively. Six out of 15 subtype B strains from the Waraos clade exhibited drug resistance mutations to NRTIs or NNRTIs. The NRTI resistance mutation V75IV and the NNRTI resistance mutations P225H and K238KT were found in one strain each, while the NNRTI resistance mutation K103N was detected in five strains, and found as a single mutation in the Guayo-Usidu subclade (Figure 2). Not all the individuals infected with drug-resistant viruses received treatment before this study. The subtype C strain did not harbor any resistance mutation. Discussion HIV-1 infection in Waraos was first reported by the Venezuelan Red Cross and local health authorities, who diagnosed the first cases in 2007 in symptomatic patients. Our study two years later indicates that at least two independent introductions of HIV-1 occurred in the Venezuelan Warao population: a single introduc- tion of a subtype B strain and a HIV-1 subtype C introduction, documented in an infected woman. The Warao subtype B clade split into two subclusters indicating two independent networks of viral dissemination within the communities. One of the subclades consisted of patients from the communities of San Francisco de Guayo and Usidu, and the other subclade included patients of a community called Jeukubaka and one isolated patient from a community called Isla de Jobure. All the subtype B cases were PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40626