Quantitating the multiplicity of infection with human immunodeficiency virus type 1 subtype C reveals a non-poisson distribution of transmitted variants

Abstract

Identifying the specific genetic characteristics of successfully transmitted variants may prove central to the development of effective vaccine and microbicide interventions. Although human immunodeficiency virus transmission is associated with a population bottleneck, the extent to which different factors influence the diversity of transmitted viruses is unclear. We estimate here the number of transmitted variants in 69 heterosexual men and women with primary subtype C infections. From 1,505 env sequences obtained using a single genome amplification approach we show that 78% of infections involved single variant transmission and 22% involved multiple variant transmissions (median of 3). We found evidence for mutations selected for cytotoxic-T-lymphocyte or antibody escape and a high prevalence of recombination in individuals infected with multiple variants representing another potential escape pathway in these individuals. In a combined analysis of 171 subtype B and C transmission events, we found that infection with more than one variant does not follow a Poisson distribution, indicating that transmission of individual virions cannot be seen as independent events, each occurring with low probability. While most transmissions resulted from a single infectious unit, multiple variant transmissions represent a significant fraction of transmission events, suggesting that there may be important mechanistic differences between these groups that are not yet understood.

FIG. 1.

Log viral loads of 68 participants categorized into stage I/II (viral RNA positive, p24 antigen and EIA antibody negative, or RNA and p24 antigen positive but EIA antibody negative), stage III (EIA antibody positive but negative by Western blot), stage IV (EIA antibody positive with an indeterminate Western blot result), stage V (Western blot positive but without reactivity to the p31 integrase band), and stage VI (Western blot positive with a p31 band present) (8). Boxes represent the 70th percentile; horizontal bars represent the median, and whisker bars correspond to minimum and maximum values. p24 antigen tests, which differentiate stages I and II of infection, were not carried out for all participants; therefore, HIV RNA-positive, enzyme-linked immunosorbent assay-negative individuals were classified as stage I/II. One participant was classified as being in stage V or VI of infection and is thus not included in this figure. The mean number of days postinfection was determined by Fiebig et al. (8) and modified by Keele et al. (16).

FIG. 2.

Neighbor-joining tree of env sequences from each of the 69 study participants from South Africa and Malawi. To limit the size of the tree, only 626 sequences of the 1,505 sequences generated were analyzed: where individuals harbored viruses with multiple identical env sequences, only the unique sequences were included. Red branches represent sequences from participants infected with more than one variant. Diamonds represent sequences with >6% divergence from the participant sequence population. The sequences for participants 703010131 and 703010159, a donor-recipient pair, cluster together, as indicated.

FIG. 3.

env sequence diversity was visually determined by the structure of the phylogenetic tree (left), and the pattern of nucleotide base mutations within sequences was observed on a Highlighter plot (right). The Highlighter plots compare sequences from each participant's sequence set to an intraparticipant consensus (uppermost sequence) and illustrate the positions of nucleotide base transitions and transversions using short, color-coded bars. (A) Participant 1172 with a highly homogeneous env sequence population displaying limited structure on a tree and a few nucleotide changes from the intraparticipant consensus. (B) Participant 1176 infected with three closely related env populations (indicated in black, blue, and red, respectively) based on the clustering of sequences into individual clades on a tree and the shared patterns of mutations observed on a Highlighter plot. Both participants were viral RNA positive but ELISA negative (stage I/II of infection).

FIG. 4.

(A and B) Clustering of mutations within stretches of 10 amino acid residues associated with putative CTL pressure illustrated in participant 703010054 (stage V of infection) (A) and participant 705010015 (stage V of infection) (B). (C and D) Changes in the V1/V2 loop in env associated with putative antibody pressure illustrated in participant 704010017 (stage VI of infection) displaying clustered mutations within the V2 loop resulting in the gain of three N-linked glycosylation sites (C) and participant CAP269 displaying multiple amino acid insertions within the V1 loop resulting in the gain of one to four N-linked glycosylation sites (D). Highlighter plots (left) compare sequences from each participant's sequence set to an intraparticipant consensus (uppermost sequence) and illustrate the positions of nucleotide base transitions and transversions using short, color-coded bars. Amino acid identity alignments (right) illustrate regions of clustering of mutations in sequences aligned with an intraparticipant subtype C consensus sequence with corresponding HXB2 env protein locations indicated above. The number of sequences harboring a particular mutation is displayed alongside each sequence. N-linked glycosylation sites are indicated as red amino acid residues. Where significant mutational clustering was detected by a randomization test, P values are provided in parentheses.

FIG. 5.

Highly heterogeneous, multiple variant env sequence populations are visually represented by phylogenetic trees with extensive branch structure and discernible clades (left) and Highlighter plots with diverse patterns of nucleotide base mutations compared to the intraparticipant consensus (right). env variants resulting from recombination between clades are displayed below with RAP (for recombination analysis program) plots. Parental strains for each recombinant are color coded on the trees, with regions within each recombinant likewise color coded to correspond to respective parental strains. Recombination breakpoints are illustrated by empty boxes on RAP plots. (A) Participant CAP37 was infected with three distinct variants. Sequence 5998 differs by up to 6% from the remaining sequences from this participant and is a suspected dual infection. (B) Participant CAP69 was infected with at least five distinct viruses with extensive recombination between clades. Six recombinant strains are illustrated here.

FIG. 6.

Model of the rates of transmission of multiple variants using a Poisson distribution. Transmission rates, shown above each bar, are modeled to account for differences in the frequency of transmissions of one, two, or more than two variants using transmission probabilities of 0.1 (Poisson mean, 0.1), 0.25 (Poisson mean, 0.5), and 0.4 (Poisson mean, 0.5). Transmission probability in this setting is defined as the sum of the probabilities of all nonzero events in the Poisson distribution. The frequency of transmission of one, two, or more than two variants is shown for the subtype C cohort described in the present study and from the subtype B cohort described in Keele et al. (16) and indicated by a “C” or “B” above each bar. In modeling the Poisson distribution to fit the cohort data all data were used as counts, not percentages, and values greater than two variants were not pooled but rather modeled in total.