HIV-1 in Waraos infecting Waraos. The locality of origin of each Warao individual. RT mutations are shown in the name when present in the sequence. HIV162 sequence, which appears to share a common ancestor with the Amerindian subtype B clade, is shown in blue. Subtype C sequences are shown in grey. Phylogenetic analysis was performed by Neighbor Joining and distance calculated with Kimura 2 parameters. Bootstrap values over 50% are shown in the trees. Letters in bold in the tree indicate subtype. doi:10.1371/journal.pone.0040626.g002 found among men who refered sex with men. Sex practices between men have been documented among Warao Amerindians in Venezuela [12,13]. HIV-1 subtype B is highly predominant in Venezuela [4]. Indeed, the sequence from another Venezuelan patient, HIV162, was found to share a common ancestor with the Waraos clade. This man, with heterosexual habits, who lives far away from the Orinoco Delta, and denied having visited this region, might have been in contact with the person or partners of the person that might have transmitted HIV-1 subtype B to the first Warao(s) infected with this subtype. Bayesian coalescence analyses suggested that subtype B clade probably started to disseminate among Waraos at around 1999, a date compatible with the appearance of immunodeficiency symptoms in some Warao individuals in 2007. The Venezuelan patient HIV162 share a relative distant common ancestor with the Waraos subtype B clade estimated at around 1989–1990. Thus, despite patient HIV162 and Waraos could be part of the same network of HIV transmission in Venezuela, a direct epidemiological link between that Venezuelan patient and the Warao community was discarded. The Bayesian Coalescence analyses estimated the Tmrca of subtype B strains circulating in Venezuela between the mid and the late 1970s, a date compatible with the identification of the first AIDS case in the country in 1985. However, whether this estimated Tmrca represents a good approximation to the onset date of the subtype B epidemic in Venezuela is unclear because the monophyletic or polyphyletic origin of Venezuelan subtype B epidemic has not been formally tested until now. In contrast to subtype B, HIV-1 subtype C is still very rare in Venezuela [8]. The woman infected with HIV-1 subtype C denied any sexual contact apart from her husband, who died one year ago with cachexia and chronic diarrhea, both symptoms characteristic of the CDC staging system for AIDS [14]. The serum sample of her husband was not available for HIV molecular testing, but was found seropositive before this study. The presence of HIV-1 subtype C infection in a Warao might be due to a contact with a person not living in Venezuela. The subtype C isolate infecting Waraos did not group with Brazilian clade but rather with Nepalese and Chinese subtype C isolates. Foreign strains of Mycobacterium tuberculosis, circulating in India and Philippines, have been detected earlier in the Warao population [15], but have not been found in other localities in Venezuela [16], suggesting a contact with this part of the world. We speculate that the presence of this viral subtype in this geographical area might be related to a trade route of Asian ships that travel through the Orinoco Delta to transport metals from upstream mining industries. A Guyanese origin (whose border is adjacent to Orinoco Delta) cannot be ruled out either. The few HIV-1 Guyanese isolates that have been analyzed belong to subtype B [17]. The Guyanese subtype B isolates were not closely related to the Warao subtype B clade (data not shown). Guyana has the highest prevalence of HIV in South Figure 3. Maximum likelihood tree of the pol (approximately 1400 nt) and env (approximately 280 nt) genes of HIV-1 subtype B strains circulating in Venezuela. HIV-1 subtype B sequences infecting Waraos are shown in red while sequence from patient 162 is shown in blue. The aLRT support values are indicated only at key nodes. The tree was rooted using subtype C reference strains (grey branches) as outgroups. Horizontal branch lengths are drawn to scale with the bar at the bottom indicating nucleotide substitutions per site. doi:10.1371/journal.pone.0040626.g003 PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e40626