A limited number of simian immunodeficiency virus (SIV) env variants are transmitted to rhesus macaques vaginally inoculated with SIVmac251

Abstract

Single-genome amplification (SGA) and sequencing of HIV-1 RNA in plasma of acutely infected humans allows the identification and enumeration of transmitted/founder viruses responsible for productive systemic infection. Use of this strategy as a means for identifying transmitted viruses suggested that intrarectal simian immunodeficiency virus (SIV) inoculation of macaques recapitulates key features of human rectal infection. However, no studies have used the SGA strategy to identify vaginally transmitted virus(es) in macaques or to determine how early SIV diversification in vaginally infected animals compares with HIV-1 in humans. We used SGA to amplify 227 partial env sequences from a SIVmac251 challenge stock and from seven rhesus macaques at the earliest plasma viral RNA-positive time point after low- and high-dose intravaginal inoculation. Sequences were analyzed phylogenetically to determine the relationship of transmitted/founder viruses within and between each animal and the challenge stock. In each animal, discrete low-diversity env sequence lineages were evident, and these coalesced phylogenetically to identical or near-identical env sequences in the challenge stock, thus confirming the validity of the SGA sequencing and modeling strategy for identifying vaginally transmitted SIV. Between 1 and 10 viruses were responsible for systemic infection, similar to humans infected by sexual contact, and the set of viruses transmitted to the seven animals studied represented the full genetic constellation of the challenge stock. These findings recapitulate many of the features of sexual HIV-1 transmission in women. Furthermore, the SIV rhesus macaque model can be used to understand the factors that influence the transmission of single versus multiple SIV variants.

FIG. 1.

Neighbor-joining tree and Highlighter plots of SGA-derived env sequences from the SIVmac251 challenge stock. Bar, 0.001 nucleotide substitutions per site. Nucleotide polymorphisms are indicated by a colored tic mark (thymine in red, guanine in orange, adenine in green, and cytosine in blue).

FIG. 2.

Analysis of SGA-derived env sequences from macaques productively infected with a single transmitted variant. Results shown include neighbor-joining trees of SGA-derived env sequences (left panel), Highlighter plots (center panel), and phylogenetic trees of the inoculum with each animal's consensus sequence highlighted (right panel). (A) Animal 25948; (B) animal 25479; (C) animal 29459. Bar, 0.001 nucleotide substitutions per site. Nucleotide polymorphisms are indicated by a colored tic mark (thymine in red, guanine in orange, adenine in green, and cytosine in blue). Pink filled circles denote APOBEC signatures, open diamonds represent G-to-A conversions, and deletions are shown by gray tics in the Highlighter plots. ABOBEC-mediated G-to-A mutations are brown in the neighbor-joining trees.

FIG. 2.

Analysis of SGA-derived env sequences from macaques productively infected with a single transmitted variant. Results shown include neighbor-joining trees of SGA-derived env sequences (left panel), Highlighter plots (center panel), and phylogenetic trees of the inoculum with each animal's consensus sequence highlighted (right panel). (A) Animal 25948; (B) animal 25479; (C) animal 29459. Bar, 0.001 nucleotide substitutions per site. Nucleotide polymorphisms are indicated by a colored tic mark (thymine in red, guanine in orange, adenine in green, and cytosine in blue). Pink filled circles denote APOBEC signatures, open diamonds represent G-to-A conversions, and deletions are shown by gray tics in the Highlighter plots. ABOBEC-mediated G-to-A mutations are brown in the neighbor-joining trees.

FIG. 3.

Analysis of SGA-derived env sequences from animal 31523, which was productively infected with at least seven distinct transmitted SIV variants. The neighbor-joining tree and Highlighter plot indicate the presence of multiple low-level diversity lineages, and two lineages are represented by a single viral sequence. Bar, 0.001 nucleotide substitutions per site. Nucleotide polymorphisms are indicated by a colored tic mark (thymine in red, guanine in orange, adenine in green, and cytosine in blue). Pink filled circles denote APOBEC signatures, open diamonds represent G-to-A conversions, and deletions are shown by gray tics in the Highlighter plot. ABOBEC-mediated G-to-A mutations are brown in the neighbor-joining tree.

FIG. 4.

Analysis of SGA-derived env sequences from animal 30991, which was productively infected with at least five distinct transmitted variants. The neighbor-joining tree and Highlighter plot indicate the presence of at least five low-level diversity lineages, and two lineages are represented by a single viral sequence. Bar, 0.001 nucleotide substitutions per site. Nucleotide polymorphisms are indicated by a colored tic mark (thymine in red, guanine in orange, adenine in green, and cytosine in blue). Pink filled circles denote APOBEC signatures, open diamonds represent G-to-A conversions, and deletions are shown by gray tics in the Highlighter plot. ABOBEC-mediated G-to-A mutations are brown in the neighbor-joining tree.

FIG. 5.

Analysis of SGA-derived env sequences from animal 27337, which was productively infected with at least seven distinct transmitted SIV variants. The neighbor-joining tree and Highlighter plot indicate the presence of multiple low-level diversity lineages. Bar, 0.001 nucleotide substitutions per site. Nucleotide polymorphisms are indicated by a colored tic mark (thymine in red, guanine in orange, adenine in green, and cytosine in blue). Pink filled circles denote APOBEC signatures, open diamonds represent G-to-A conversions, and deletions are shown by gray tics in the Highlighter plot. ABOBEC-mediated G-to-A mutations are brown in the neighbor-joining tree.

FIG. 6.

Analysis of SGA-derived env sequences from animal 29271, which was productively infected with at least six distinct transmitted variants. The neighbor-joining tree and Highlighter plot indicate the presence of at least six low-level diversity lineages, and five lineages are represented by a single viral sequence. Bar, 0.001 nucleotide substitutions per site. Nucleotide polymorphisms are indicated by a colored tic mark (thymine in red, guanine in orange, adenine in green, and cytosine in blue). Pink filled circles denote APOBEC signatures, open diamonds represent G-to-A conversions, and deletions are shown by gray tics in the Highlighter plot. ABOBEC-mediated G-to-A mutations are brown in the neighbor-joining tree.

FIG. 7.

Composite neighbor-joining analysis of env diversity in SIVmac251 stock and the earliest plasma vRNA⁺ samples from infected animals. Variants from individual animals are represented as colored ticks on the phenogram; the colors of the ticks correspond to the animal that was the source of the variant. The number of the transmitted variants over the number of transmitted lineages observed in each animal is noted. Black stars indicate the variants that were amplified from the SIVmac251 stock. Bar, 0.001 nucleotide substitutions per site.

FIG. 8.

Composite alignment of the SIVmac251 stock env sequences and plasma SIV env sequences. Results shown are the Highlighter plot of SIVmac251 stock (red lines) and earliest plasma vRNA⁺ sample from infected animals (black lines). Variants isolated from the individual animals are indicated by the different-colored tic marks to the right of each line. Individual nucleotide polymorphisms are indicated by green (adenine), red (thymine), yellow (guanine), or blue (cytosine) tics and are in comparison to master sequence 251st01. Gaps are indicated in gray. To simplify the figure, APOBEC signatures and G-to-A conversions are not specifically indicated. The blue box denotes the group of identical sequences found in the SIVmac251 stock inoculum and the plasma samples from three animals.