Timing and source of subtype-C HIV-1 superinfection in the newly infected partner of Zambian couples with disparate viruses

Abstract

Background: HIV-1 superinfection occurs at varying frequencies in different at risk populations. Though seroincidence is decreased, in the negative partner of HIV-discordant couples after joint testing and counseling in the Zambia Emory HIV Research Project (ZEHRP) cohort, the annual infection rate remains relatively high at 7-8%. Based on sequencing within the gp41 region of each partner's virus, 24% of new infections between 2004 and 2008 were the result of transmission from a non-spousal partner. Since these seroconvertors and their spouses have disparate epidemiologically-unlinked viruses, there is a risk of superinfection within the marriage. We have, therefore, investigated the incidence and viral origin of superinfection in these couples.

Results: Superinfection was detected by heteroduplex mobility assay (HMA), degenerate base counting of the gp41 sequence, or by phylogenetic analysis of the longitudinal sequences. It was confirmed by full-length env single genome amplification and phylogenetic analysis. In 22 couples (44 individuals), followed for up to five years, three of the newly infected (initially HIV uninfected) partners became superinfected. In each case superinfection occurred during the first 12 months following initial infection of the negative partner, and in each case the superinfecting virus was derived from a non-spousal partner. In addition, one probable case of intra-couple HIV-1 superinfection was observed in a chronically infected partner at the time of his seroconverting spouse's initial viremia. Extensive recombination within the env gene was observed following superinfection.

Conclusions: In this subtype-C discordant couple cohort, superinfection, during the first year after HIV-1 infection of the previously negative partner, occurred at a rate similar to primary infection (13.6% [95% CI 5.2-34.8] vs 7.8% [7.1-8.6]). While limited intra-couple superinfection may in part reflect continued condom usage within couples, this and our lack of detecting newly superinfected individuals after one year of primary infection raise the possibility that immunological resistance to intra-subtype superinfection may develop over time in subtype C infected individuals.

Figure 1

Sequence and Highlighter Analysis of Longitudinal Samples Provides Evidence for Superinfection. Comparison of maximum sequence divergence in gp41 versus the maximum number of degenerate bases at any time point within acutely infected individuals (A) and chronically infected individuals (B). The maximum percentage of degenerate bases is plotted on the y-axis; the maximum percentage of genetic distance is plotted along the x-axis. Black arrows indicate superinfected subjects; arrowhead indicates a subject co-infected with two variants from a single donor. Highlighter plots for gp41 sequences of ZM289M (C), ZM282M (D), ZM247F (E), and ZM211 F (F) sampled at 0 to 36 months post-seroconversion (shown on right of plot). The sequence at each time point is compared to the initial infecting HIV-1 gp41 sequence. Tick marks denote nucleotide changes from the seroconversion sequence (T = red, A = green, C = blue, G = yellow), with dark blue indicating degenerate bases (See Methods).

Figure 2

Neighbor-joining tree of full-length SGA env sequences for ZM282M. Blue and red sequences represent the initial infection and superinfection sequences, respectively, from the acutely infected subject ZM282M; black sequences are derived from the epidemiologically unlinked cohabiting partner ZM282F, who is chronically infected. The time points are indicated along with M or F for each sequence (i.e. M_8 is the acutely infected ZM282M at 8 months post seroconversion). The ZM282F "0" time point corresponds to seroconversion of ZM282M. The duration of infection for ZM282F is not known. Bootstrap values > 80 are considered statistically significant. Sequences denoted by circles indicate the parental sequences (blue, red circle) and those denoted by stars identify potential recombinant daughter sequences that were used for recombination analyses (below).

Figure 3

Neighbor-joining tree representing full-length SGA env sequences for ZM247F. Blue and red sequences represent the env SGA sequences from acutely infected ZM247F (blue) and superinfected (red) viral sequences. Black sequences are derived from ZM247M, the epidemiologically unlinked chronically infected partner. Bootstrap values > 80 are considered statistically significant. Sequences denoted by circles indicate the parental sequences (blue, red circle), and stars denote potential recombinant daughter sequences (purple stars) that were used for recombination analyses (below).

Figure 4

Neighbor-joining tree of full length SGA env sequences for ZM211M and ZM211F. (A) The chronically infected ZM211M sequences are depicted in blue, and the superinfecting ZM211F sequences are depicted in red, while those in purple represent potential recombinant sequences between the blue and red sequences. (B) An expanded phylogenetic tree showing time points 0-36 months for the acutely infected ZM211F initial infecting virus (blue) is distinct from ZM211M (black). Sequences denoted by circles indicate the parental sequences (blue, red circle) and stars denote potential recombinant daughter sequences (purple stars) that were used for recombination analyses described below.

Figure 5

Neighbor-joining trees of SGA env and population gp41 sequences for the cohort. (A) Neighbor-joining phylogenetic tree of single genome amplified env gene sequences from each superinfected individual in the context of Zambian sub-type C env sequences. The Zambian subtype C sequences include twelve of the non-superinfected, newly infected individuals from this study. Superinfected individuals are assigned discrete colors and the superinfecting quasispecies is denoted by SI. (B) Neighbor-joining tree of gp41 sequences for all 22 couples. Superinfected individuals are assigned discrete colors.

Figure 6

Highlighter recombination plots of full-length env for each individual who was superinfected. Highlighter recombination analysis of sequences from ZM282M (A), ZM247F (B), and ZM211M (C) and ZM211F (D). The parental virus sequence from the acutely infected or a representative parental sequence from the chronically infected partner (ZM211M) are shown in blue in the first bar of each panel. The superinfecting parental sequence shown in red is the second bar. The presumptive recombinant daughter sequences from three representative time points are shown below the parents for each case of superinfection.