To characterize the recombination breakpoints of our CRF more precisely, we performed an informative site analysis comparing consensus sequences of the CB recombinants with those of subtypes B and C. That analysis is depicted in Fig. 4. By analysing signature patterns among the three consensus sequences, we were able to narrow the recombination breakpoints down to nucleotides 712–731 and to 925–938 of our fragment (2965–2984 and 3190–3203 of HXB2, respectively). At positions 711, 732 and 924, 100% of the subtype C strains used for the consensus generation presented an adenosine, whereas all subtype B presented a guanine (P = 0.001082 for all three cases). At position 939, the frequency of cytosine was 100% in subtype C and 16.7% in subtype B (P = 0.004079). In external borders the mosaic consensus showed the same signatures of subtype C, whereas in internal borders it showed the same signatures of subtype B.
We wanted to confirm further the epidemically circulating property of the CB recombinant strains. For this, we generated three sets of local sequences, one representing subtype B, another subtype C, and a third one with the CB recombinant strains (with the exception of the one queried). Each recombinant strain was compared against those three consensus sequences through similarity plots. The results of these analyses can be seen in Fig. 5. They revealed that all recombinant strains analysed were closer to their CB consensus throughout the fragment analysed, even when they belonged to subtypes B or C and were compared with subtype B and C strains from the same geographical region (Fig. 5 and data not shown). We used only the initial 950 bp fragment because we only had that information from the ‘pure’ local subtypes, but on the other hand that allowed us to test all 17 putative recombinant sequences available. All of them showed the same pattern of similarity (data not shown), indicating a common ancestry of all recombinant strains described.
Earlier studies of HIV-1 subtype distribution in Brazil have shown the prevalence of approximately 3% of subtype C , but we and others have revealed the southern region as an endemic site for subtype C [19,20,33–35], notably in the state of Rio Grande do Sul. The molecular profile of HIV-1 subtypes observed in this study corroborates those previous observations. We found 62 samples belonging to subtype C, which represented 41% of the total.
Phylogenetic analyses have shown that Brazilian HIV subtype C viruses clustered in a well-defined clade (Fig. 1), in agreement with previous studies [22,33]. Altogether, these data show that the introduction of subtype C viruses in Brazil was probably a single event. The maximum parsimony and interior branch test of phylogeny with the neighbour-joining distance method tree and Kimura two-parameter model confirmed such an hypothesis (data not shown).
We have observed a subcluster within the Brazilian subtype C clade in this study comprising 17 samples with high bootstrap value, including two samples, 04BR137 and 04BR142, which had previously been described as pure subtype C strains . A more detailed analysis revealed that all samples were CB recombinants and shared the same recombination breakpoint, suggesting that mosaic viruses shared a single ancestor. A 2242 bp fragment within the pol gene was confirmed by bootscanning analysis and further phylogenetic inference.
The probability of generating a CRF of HIV requires and is directly related to the co-circulation of distinct subtypes in a population. The high co-prevalence of subtypes B and C, which together made up 87% of the HIV viruses in the state of Rio Grande do Sul, is thus likely to generate CB recombinants. The presence of CB recombinants is also common in countries where these subtypes predominate, such as India [36,37], China [38–40], and in south American countries where subtype C has recently been introduced [41–43]. An informative site analysis that compared the CB recombinants with local subtype B and C viruses has also shown that the former shared signatures at the DNA level with the latter, suggesting a local origin (Fig. 4).
The subtype B genomic fragment incorporated into the recombinant form described here comprises codons 138–217 of RT, and therefore harbours several positions associated with resistance to nucleoside (codons 151, 184, 210 and 215) and non-nucleoside (codons 181, 188 and 190) RT inhibitors. None of the 15 mosaic isolates found here harboured drug resistance-associated mutations at those positions, and they were all drug naive at the time of sample collection (data not shown). The nature of the selective advantage in incorporating such an RT region into the CRF remains to be determined. One explanation could be that such a region is more prone to develop drug resistance in subtype B than in C, but there is no current evidence for this bias. Previous work from our group has failed to show any differences in genetic barrier for those two subtypes in developing RT drug resistance .
Our samples presented the same breakpoint structure, a common origin and nucleotide signatures that indicated a local origin from subtype B and C isolates. To the best of our knowledge, this is the first concrete evidence of the existence of a Brazilian HIV-1 CRF comprising subtypes B and C.
Despite the fact that subtype C is responsible for more than 56% of HIV-1 infections worldwide , until now only three out of 29 CRF comprising subtype C had been described, CRF07_BC , CRF08_BC  and CRF10_CD . Our study may provide a new exemplar of CRF comprising subtype C in its structure.
It is noteworthy that among all samples from Rio Grande do Sul analysed in this study, 24% of the initially assigned subtype C samples now represent a CRF_BC. An independent study recently published  found mosaic samples with this same breakpoint structure in 43% of samples previously described as subtype C, corresponding approximately to 25% of the total viruses circulating in newly infected individuals. Interestingly, as we have data on the time of diagnosis for our patients, we can trace this CRF back to at least 1990, which means that it has been circulating in southern Brazil for over 15 years. We are currently estimating the more precise time of generation of this CRF in the region using coalescence techniques. Additional studies are necessary to obtain a complete understanding of the role of this CRF in the Brazilian and ultimately in the Latin American HIV/AIDS epidemic.
The authors would like to thank the staff from both clinical sites at Rio Grande do Sul, the Hospital de Clínicas de Porto Alegre and University Hospital of Rio Grande (Drs C.S. Moss, D.R. Mendoza and M. Gonzaga), for their help with patient care and sample collection, as well as to all patients who agreed to participate in this study. The authors are deeply indebted to Professor Rodrigo Brindeiro, Universidade Federal do Rio de Janeiro (UFRJ) for his support for this study and to Ana Flávia Pires and Renan B. Lengruber (UFRJ) for their technical support. Finally, they would like to thank Dr Carlos G. Schrago for helping with the phylogenetic analyses. This study is part of the dissertation of A.F.A.S. for his masters degree from the Graduate Programme in Genetics, UFRJ.
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