This study is the first to provide a comprehensive assessment of the multiplicity of HIV-1 subtype C infection in the context of heterosexual transmission in men and women. Our findings mirror observations of subtype B transmissions via heterosexual or homosexual routes using the same methodology (16
). Together with the subtype B study, these data demonstrate that in 171 individuals a single virus is responsible for infection of 77% of individuals, with 23% of individuals infected with multiple variants. Based on the frequency of the transmission of multiple variants, we find that infection with more than one variant does not occur as independent events at low probability. This implies that transmission of the second variant is linked to transmission of the first variant. Understanding the frequency and cause of multivariant transmission is relevant since individuals infected with multiple variants would require a vaccine that protects against greater initial viral diversity instead of a single homogeneous virus population. In addition, it is clinically important since high diversity following transmission has been associated with faster disease progression (10
). In keeping with this observation, the present study found an association between multivariant transmission and disease progression.
Discrepant results due to differences in methodological approaches have hindered a clear understanding of multivariant transmission. A key advantage of our study is that we used the same methodological and analytical approaches to define the founder virus population that was used recently to study subtype B acute infection (16
), thus enabling us to clearly enumerate the infecting viruses and also directly compare results. Despite different infecting subtypes and routes of transmission, the frequencies of multivariant transmission were strikingly similar: we report 22% in subtype C heterosexually infected men and women compared to the 24% of participants infected with subtype B via homosexual and heterosexual transmission reported by Keele et al. (16
). Phylogenetic analysis indicated that the multiple variants came from a single donor in 87% of the cases (13 of 15 subjects), and the time to the MRCA demonstrates that the variants diverged at times significantly before the transmission event.
This estimated frequency of multivariant transmissions should, however, be considered a minimum. Many infections in highly epidemic regions have been attributed to transmissions during the acute stage of infection (30
). Since this stage is generally associated with a highly homogeneous viral population, multiple variant transmissions in these instances could be missed. In addition, we may miss variants present at a low frequency (with a sample size of 20 sequenced amplicons, there is 95% confidence of detecting sequences present at frequencies greater than 15%) (16
Although we used a model which assumes neutral evolution (16
), deleterious mutations will be lost through purifying selection, and early innate and adaptive host responses are likely to impact the apparent mutation rate, especially in participants sampled after peak viremia. We did in fact identify putative immune pressure in acute infection, with a third of the sequence sets containing evidence of putative CTL pressure (based on clustered mutations) or antibody pressure (based on changes in N-glycosylation sites or variable loop length). The rates of mutation were also influenced by APOBEC3G-mediated hypermutation observed in eight individuals with single variant infections. In addition, sequences were under purifying selection with a higher rate of synonymous (dS) compared to nonsynonymous (dN) substitutions (mean dN/dS ratio of 0.79; variance, 0.44). A mean dN/dS ratio of <1 suggests that the rate of diversification of the sequences could be slightly less than the rate estimated under a strict assumption of neutrality. However, the impact of a relatively small departure from neutrality on the estimated times to the last common ancestors of intrapatient sequence sets is likely to be minor.
The rate of HIV transmission is in the range of one transmission event per 1,000 exposures (34
; reviewed in Powers et al. [32
]), although two studies reported rates of 31 per 1,000 exposures (26
) and 97 per 1,000 exposures(3
). However, even rates as high as 0.03 to 0.1 cannot account for a frequency of multivariant transmission of 22 to 24%, if multiple variants are transmitted independently. This suggests that transmission of each variant is not an independent event in the context of a low transmission probability. One explanation of the frequency of multiple variant transmissions is that different cofactors transiently change the rate of multivariant transmission. The distribution of frequency of one, two, and more than two variants can be explained if two rates are incorporated: one rate would account for 70 to 75% of transmission events and have a low probability of transmission with only rare occurrences of the transmission of multiple variants; the second rate would account for 25 to 30% of transmission events. However, a probability of transmission of ~0.8 would be required to result in equal numbers of transmission events of two variants and more than two variants, which would approximate the observed data. It is likely that increased transmission occurs as a result of sexually transmitted infections (38
) or traumatic breaks in the epithelium. However, it is also possible that the transmission of multiple variants represents a linked event, i.e., infection by one particle (or genome) is in some way linked to an increased probability of infection with a second particle (or genome). If the infectious unit were an infected CD4+
T cell, which can be infected with multiple viruses (15
), this could account for at least some of the multivariant transmission events. A recent report has shown the potential for infected cells to penetrate a disturbed epithelium (45
), and the apparent need for infection via infected cells in the case of HTLV-1 provides further support for a cell-mediated mechanism of transmission (33
). Alternatively, virus particles could be aggregated by biological molecules such as SEVI (for semen-derived enhancer of virus infection) (27
) or tetherin during budding (28
), potentiating infection with two particles in a single, rare transmission event.
A previous study from Kenya showed women were generally infected with more heterogeneous virus populations compared to men (21
). Although in our study, we did not find that females were infected with higher-diversity viral populations compared to men (data not shown), there were differences in frequency of sexually transmitted infections, with genital ulcerative disease being much more common in men from our study than in women (82% versus 13%). Thus, since genital ulcerative disease has an impact on transmission, this could confound our analysis. In addition, uncircumcised men are more susceptible to infection (1
); thus, this difference in results between studies may be due to the fact that most of the men in our study were from Malawi where there is a very low frequency of circumcision, whereas the Kenyan study recruited from a cohort of individuals where 87% were circumcised (34
In conclusion, infection with a single virus in the majority of individuals demonstrates the severity of the genetic bottleneck at transmission. These data in conjunction with the subtype B analysis suggests a universal observation that mucosal HIV-1 infection most frequently originates from a single infectious unit. Less frequently, multiple viral variants are transmitted, which not only increases the genetic diversity, but this increased diversity also provides the virus with greater opportunity to escape early selective pressure through recombination. Although the biological basis for the transmission of multiple variants remains unknown, possible explanations include transiently high rates of transmission due to cofactors, transmission via a multiply infected cell, or transmission of viral aggregates. Since one in five individuals will become infected with multiple infectious variants, it is important to translate how this information impacts on the breadth and targeting needed for protective vaccination.