Significant differences in the V3 region were not found between recombinants and background sequences.
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.
If HIV-1 subtypes A, C, and D have the same intrinsic ability to be transmitted perinatally as well as to be represented in a recombinant virus, the genetic composition of a recombinant should not have any preferential representation of any particular HIV-1 subtype. We compared three groups of recombinants to determine whether different subtypes had the same representation in recombinants between subtypes A and D, A and C, or C and D. Despite the amplified envelope including C2 to C5 regions, we did not include in the comparison the C2, V4, or V5 regions. Their small size (C2) and frequent insertion/deletions (V4, V5) made it difficult to assign a particular subtype when subjected to phylogenetic analysis as individual nucleotide segments. The contribution of each subtype in V3, C3, C4, C5, and gag for A-D, A-C, and C-D recombinants are shown in Table 2. Subtype A was preferentially represented in the V3 region from A-D recombinants, with all 15 samples possessing a V3 from subtype A (exact binomial test P = 0.00003). Conversely, subtype D was preferentially represented in gag in A-D recombinants (exact binomial test P = 0.0003). A Fisher‚s exact test comparing the proportion A V3 to the proportion of A gag was highly significant (P = 0.00001). Similar results was obtained when we compared the proportion of subtype A C3-C5 to subtype D gag. This suggests that A-D recombinants with A V3, C3-C5 and D gag may hold an advantage for perinatal transmission. On the other hand, comparison the proportion of subtype A in V3 to the proportion of subtype A in C3-C5 was not significant (P= 0.22). Based on this result, it did not appear that a V3 region of subtype A was a determinant separate from the C3-C5 region of env. All (12 of 12) C-D recombinants had a V3 region from subtype C (P = 0.0002) whereas 0.5 had a gag from subtype C (P=0.002). The Fisher‚s exact test comparing proportions was highly significant (P = 0.0001). Unlike A-D recombinants, the proportion of C-D recombinants with V3 region of subtype C was larger than the proportion of C-D samples with C3-C5 region of subtype C (P = 0.0013), suggesting that the presence of subtype C in the V3 region could be a determinant separate from C3-C5. The proportion of subtype A or subtype C sequences in A-C recombinants did not reach statistical significance even though the number of subtype A sequences was higher than the number of subtype C sequences.
Mother-to-child-transmission of HIV-1 is a multifaceted phenomenon involving host resistance factors as well as the properties of the transmitted virus. It has been shown that viral samples taken from young infants are substantially homogeneous, suggesting that one or few infectious units infected the infant[15-19]. Phenotypic studies of transmitted viruses suggested that most early viral isolates from perinatally infected children are tropic for monocyte-derived macrophages (MDM). These results raised the possibility that sequences in the V3 region could be responsible for escaping maternal immune responses while preserving determinants specific for tropism of cells exposed around or during birth.
At the present time, the viral genetic determinants responsible for perinatal infection as well as whether genetic differences detected between HIV-1 subtypes have biological consequences are largely unknown. As such, intersubtype recombinants could represent useful molecular tools to map viral determinants associated with perinatal transmission in the context of HIV-1 subtypes. In this study, we found 22% of infants infected with HIV-1 subtype A, 22% infected with HIV-1 subtype C, 19% infected with HIV-1 subtype D, and 37% percent infected with recombinant viruses. These estimates do not indicate the subtype-specific transmission rates. It is necessary to identify the prevalence of HIV-1 subtypes in the mothers to determine whether HIV-1 subtypes A, C, D, and recombinants have different rates of transmission.
Analysis of genomic patterns showed that viruses with similar recombination patterns infected several infants. Despite the fact that these recombinant patterns clearly originated from independent events involving different subtypes, they all supported a selection bias for the V3 region. These recombinants could be grouped into two main categories _ D/A/D(env) and D/C/D(env). Alike D/A/D envelopes occurred with A(gag) or D/A(gag) sequences. Similarly, resembling D/C/D(env) occurred with D(gag) or A(gag) (Fig. 1). The fact that V3 regions from subtype D were never found in A-D or in C-D recombinants, suggests that the fitness of D-V3 for perinatal transmission is reduced with respect to V3 from subtype A and/or subtype C.
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.
In summary, we have shown that a variety of recombinant HIV-1 viruses have been generated and efficiently transmitted to infants in Tanzania. The recombination patterns in these samples suggest that the V3 region could be a key determinant for transmission. The elevated number of recombinants transmitted perinatally suggests that co-infection or super-infection by two HIV-1 subtypes is not uncommon in this population.
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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