Together, these analyses suggest that either the recombinants originated from independent recombination events or they had been present in the population for a prolonged period. This is supported by the lack of phylogenetic clustering and by the variability of the RIP patterns showing >90% certainty in sequences of the same subtype or recombinant structure (Figs. 1 and 2).
To further address whether a founder effect may have been involved in the distribution of transmitted recombinants, we also amplified and sequenced the long terminal repeat (LTR) from several samples with D(gag)-A(env), D(gag)-D/C/D(env), C(gag)-A(env) or D(gag)-D/A/D(env). The lack of identity between LTR, gag, and env in with D(gag)-A(env), C(gag)-A(env) and D(gag)-D/A/D(env) supports our previous findings that many of these recombinants could have originated via independent events.
Viruses isolated from adult individuals shortly after seroconversion and during the asymptomatic phase of HIV-1 infections are mainly MDM-tropic and non-syncytium-inducing (NSI). Viral isolates from perinatally infected children have likewise been described as MDM-tropic and NSI [34,36]. The env V3 region has been shown to be a major determinant influencing viral tropism for macrophages and the ability of a virus isolate to cause syncytia. More recently, the β-chemokine receptor CCR5 has been shown to be the major coreceptor for primary non-T cell line-adapted viruses, and the agr;-chemokine receptor CXCR4 to act as the co-receptor for primary T cell line-adapted viruses[37,38]. Differences in coreceptor usage between different HIV-1 subtypes have recently been described. All (15 of 15) HIV-1 subtype C isolates used the CCR5 co-receptor exclusively. Subtype A isolates preferentially used CCR5, but some isolates used CXCR4 or CCR5 and CXCR4. Since viral determinants for macrophage and T-cell tropism are located in the V3 region, several reports have shown that sequences in the V3 region also determine coreceptor usage of HIV-1 isolates[40-42]. It is plausible that recombinant D/A/D and D/C/D envelopes might have acquired an enhanced ability to infect MDM cells, providing them a selective advantage for transmission over non-recombinant subtype D viruses. It has been shown that HIV-1 subtypes A and C have the most similar V3 amino acid sequences among phylogenetically distinct subtypes, while HIV-1 subtype D viruses have the most divergent set of V3 regions. This selection of conserved V3 regions in subtypes A and C has been proposed as the result of selection for transmission[44,45]. The uneven representation of V3 sequences from subtypes A, C, and D in recombinant samples is compatible with the natural history of subtypes in Tanzania. HIV-1 subtype C appears to have been introduced after HIV-1 subtypes A and D were already established and has since quickly become one of the major subtypes, while HIV-1 subtype D infections appears to have declined in Dar es Salaam.
How and where these recombinants were generated and transmitted is still unknown. Contrary to the subtype E and subtype G subtypes for which parent sequences have not been found, parent genomes are currently circulating in the Dar es Salaam population[24,27]. Comparisons between recombinant sequences and sequences from non-recombinant viruses using phylogenetic and VESPA analysis showed that recombinant regions were indistinguishable from non-recombinant parent sequences. Since we found the same subtype in all clones from each infant, we could probably rule out that recombinant viruses were generated and preferentially expanded in the infant (Renjifo and Essex, unpublished results). Each of the major recombinant patterns could have originated as a single recombination event in one individual and expanded in the general population in Tanzania. Our analysis of samples with D(gag)-A(env) and D(gag)-D/C/D(env) did not support this possibility. However, it is still plausible that these recombinants originated a long time ago, allowing for saturation of mutations that made the recombinants indistinguishable from non-recombinant viruses. If this is the case, these recombinants appear to have gained an advantage for transmission in the general population, and the V3 region might represent an important region for this selective expansion.
We thank members of the Tanzania Vitamin Group. We are grateful to F. McCutchan and P. Kanki for providing comments on this manuscript and to R. Rawat for editorial assistance. This study was supported in part by NIH grants R35 CA39805 and RO1 HD32257. D.M. was supported by grant D43 TW00004 from the Fogarty International Center.
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