HIV Subtypes in Caribbean Gene gag pol rev env Table 1. Amino acid sites that differ between the Caribbean B strains and the subtype B reference strains. Location Mutation Caribbean B Reference B p matrix capsid V3 loop C5 C5 signal peptide N21TDA protease D102E I159V K97R V102I T319- N474D K476RM (n = 25) 23 (n = 14) (n = 14) (n = 14) 9 8 1 2 5 5 5 (n = 19) 11 15 18 13 13 0 15 17 0.01 0.005 0.03 0.0004 0.002 0.008 0.01 0.002 Figure 2. Phylogenetic analysis of 14 nearly full genome sequences of HIV-1 Subtype B from Caribbean countries: Trinidad and Tobago (TT), Dominican Republic (DO), Haiti (HT) and Jamaica (JM). Reference subtype B sequences are indicated by the country of origin; they are: US: MN, JRCSF, SF2, WR27, P896, NY5, BCSG3C, YU2, RF, U23487; DE: D31, HAN; GB: CAM1, study sequences (bold) are identified by name. A neighbor-joining phylogenetic tree was built using the Kimura 2-parameter model and significant parsimony bootstrap values (.70%) were placed next to the nodes. The genetic distance corresponding to the lengths of the branches is shown by the scale below the tree. doi:10.1371/journal.pone.0004814.g002 We analyzed the V3 loop of the samples and there was a deletion of threonine in the V3 loop in 40% of the Caribbean samples overall, and this was more common in samples from Trinidad and Tobago (67%) than in the other countries, where it ranged from 20% (JM), to 33% (DO). Overall, this mutation is associated preferentially with the Caribbean strains (env T319-, Table 1). Discussion Nearly full-length genome sequencing has been tremendously helpful in understanding the HIV epidemics in many parts of the world; when inter-subtype recombinants are circulating, partial genome sequencing can be very misleading. To date, the only HIV viral genomes collected in the Caribbean that have been fully sequenced were from Cuba [14]. Partial genome sequencing by many has shown that outside of Cuba the epidemic was dominated by subtype B, the strain of HIV-1 common in the developed world [2,6,8,15,16]. It was to improve our knowledge about the strains in the Caribbean that nearly full genome amplification and sequencing was performed for samples from 4 countries: Trinidad and Tobago, Jamaica, Dominican Republic and Haiti. Based on the sequence of the protease and RT region of pol, the majority of strains (66/67, 98.5%) were subtype B, although one, from the Dominican Republic, was a B/C recombinant. Phylogenetic analysis of this region revealed no clustering of Caribbean sequences distinct from non-Caribbean, or between one country and another. Analysis of the nearly full genome sequences of the 14 subtype B strains demonstrated no evidence of inter-subtype recombination. doi:10.1371/journal.pone.0004814.t001 Of note, however, is the fact that the region of the genome that was sequenced did not include the parts of the RNA that form the LTR in the provirus, consequently it is still possible that the LTR could be from a non-B subtype, although not likely. Full genome analysis of the subtype B strains did support the distinct clustering of the subtype B Caribbean sequences separate from those from the pandemic, including South America. This is surprising because there is undoubtedly a great deal of human traffic between the U.S. and Europe, on the one hand, and the Caribbean, on the other. In an attempt to locate the part of the genome that might be contributing to the monophyletic clustering, a sliding window examination of 8.5 kb revealed no specific location on the genome that was responsible. There were, however, isolated sites that distinguished the Caribbean strains from others. Shown in Table 1, there were some amino acid mutations that significantly distinguished the two populations. In comparisons such as this, it is appropriate to consider the fact that these comparisons are being done for each of the .3000 amino acids in the HIV-1 genome, and thus the statistical significance should be adjusted. If that adjustment were done, no amino acid mutations would statistically distinguish the two populations. As an exploratory exercise, however, it is valuable to explore where the phylogenetic signal may be coming from. The first site, gag D102E, occurs in the matrix protein and is associated with Elite Controllers [17]. Another Caribbean-specific mutation has already been reported: the presence of a threonine deletion in the V3 loop of Trinidad samples (env T319-) [8]. Most of the other changes were conservative, and are not known to be associated with specific phenotypic differences. These sites and others were so uncommon that phylogenetic clustering only achieved statistical significance when the full genome was analyzed (data not shown). It is unclear what the biological importance of this phylogenetic distinction is but this question could be explored in vitro. There are two possible explanations for the phylogenetic clustering of the Caribbean strains. The first is that it can be the result of founder effects from when the epidemic first began that are still detectable 20 years later. There are several arguments against that theory: given the human traffic in and out of the Caribbean, it is extremely unlikely that a founder effect would continue so long. Another fact that argues against the founder effect is that there is no clustering of samples by country or language, as you would expect if there were different founder PLoS ONE | www.plosone.org 4 March 2009 | Volume 4 | Issue 3 | e4814