Inflammatory genital infections mitigate a severe genetic bottleneck in heterosexual transmission of subtype A and C HIV-1

Abstract

The HIV-1 epidemic in sub-Saharan Africa is driven largely by heterosexual transmission of non-subtype B viruses, of which subtypes C and A are predominant. Previous studies of subtype B and subtype C transmission pairs have suggested that a single variant from the chronically infected partner can establish infection in their newly infected partner. However, in subtype A infected individuals from a sex worker cohort and subtype B individuals from STD clinics, infection was frequently established by multiple variants. This study examined over 1750 single-genome amplified viral sequences derived from epidemiologically linked subtype C and subtype A transmission pairs very early after infection. In 90% (18/20) of the pairs, HIV-1 infection is initiated by a single viral variant that is derived from the quasispecies of the transmitting partner. In addition, the virus initiating infection in individuals who were infected by someone other than their spouse was characterized to determine if genital infections mitigated the severe genetic bottleneck observed in a majority of epidemiologically linked heterosexual HIV-1 transmission events. In nearly 50% (3/7) of individuals infected by someone other than their spouse, multiple genetic variants from a single individual established infection. A statistically significant association was observed between infection by multiple genetic variants and an inflammatory genital infection in the newly infected individual. Thus, in the vast majority of HIV-1 transmission events in cohabiting heterosexual couples, a single genetic variant establishes infection. Nevertheless, this severe genetic bottleneck can be mitigated by the presence of inflammatory genital infections in the at risk partner, suggesting that this restriction on genetic diversity is imposed in large part by the mucosal barrier.

Figure 1. Phylogenetic analysis of heterosexual transmission pairs.

Nucleotide sequences were aligned for all linked donors (green lines) and linked recipients (blue lines) and neighbor-joining trees were drawn representing (A) subtype C transmission pairs and (B) subtype A transmission pairs. Recipients in red indicate infection by multiple variants. Horizontal branch lengths are drawn to scale with scale bar representing 2% divergence. Asterisks indicate branches with bootstrap values greater than 0.99. Black lines represent unrelated reference sequences.

Figure 2. Transmission of multiple variants.

Aligned nucleotide sequences for linked donors (green circles) and linked recipients (blue circles) were used to generate neighbor-joining trees for individual transmission pairs (A) RW57 and (B) ZM229. Horizontal branch lengths are drawn to scale with scale bar representing 1% divergence. Open circles represent sequences derived from uncultured PBMC DNA and closed circles represent sequences derived from plasma RNA. PBMC samples were unavailable for ZM229; therefore, only plasma sequences were derived for this transmission pair. Asterisks indicate branches with bootstrap values greater than 0.99.

Figure 3. Homogenous virus population in newly infected individuals.

Aligned linked recipient sequences were analyzed by the Highlighter tool (Los Alamos National Laboratory website - HIV Sequence Database), examples of output files are shown for (A) ZM243F and (B) RW19F. Tic marks indicate nucleotide differences from the indicated master sequences derived from the recipient. Nucleotide differences are color-coded and are marked according to their genetic location along the length of V1–V4. Colors are as follows: A: green, T: red, G: yellow, C: blue and gaps: gray. Tics highlighted by circles represent G to A changes in a sequence consistent with an APOBEC3G/F signature. (C) Box plots were generated using the pairwise distances calculated for individual linked recipients. Horizontal lines within box plots indicate median pairwise distance values for each linked recipient. Red boxes indicate individuals infected by multiple genetic variants.

Figure 4. Analysis of unlinked recipients.

(A) Aligned nucleotide sequences for 8 unlinked recipients were used to generate a neighbor-joining phylogenetic tree. Horizontal branch lengths are drawn to scale, with scale bar representing 2% divergence. Lines drawn in blue indicate individuals infected by a single variant, those in red indicate infection by multiple variants. Asterisks indicate branches with bootstrap values greater than 0.95. Black lines indicate unrelated reference sequences. (B) Aligned linked recipient sequences were analyzed by the Highlighter tool (Los Alamos National Laboratory website - HIV Sequence Database), an example of the output file is shown for ZM247F. Tic marks indicate nucleotide differences from the indicated master sequences derived from the recipient. Nucleotide differences are color-coded and are marked according to their genetic location along the length of V1–V4. Colors are as follows: A: green, T: red, G: yellow, C: blue and gaps: gray. (C) Box plots were generated using the pairwise distances calculated for individual unlinked recipients. Horizontal lines within box plots indicate median pairwise distance values for each unlinked recipient. Red boxes indicate individuals infected by multiple genetic variants.