Background: A single viral variant is transmitted in the majority of HIV infections. However, about 20% of heterosexually transmitted HIV infections are caused by multiple viral variants. Detection of transmitted HIV variants is not trivial, as it involves analysis of multiple viral sequences representing intra-host HIV-1 quasispecies.

Methodology: We distinguish two types of multiple virus transmission in HIV infection: (1) HIV transmission from the same source, and (2) transmission from different sources. Viral sequences representing intra-host quasispecies in a longitudinally sampled cohort of 42 individuals with primary HIV-1C infection in Botswana were generated by single-genome amplification and sequencing and spanned the V1C5 region of HIV-1C env gp120. The Maximum Likelihood phylogeny and distribution of pairwise raw distances were assessed at each sampling time point (n = 217; 42 patients; median 5 (IQR: 4-6) time points per patient, range 2-12 time points per patient).

Results: Transmission of multiple viral variants from the same source (likely from the partner with established HIV infection) was found in 9 out of 42 individuals (21%; 95 CI 10-37%). HIV super-infection was identified in 2 patients (5%; 95% CI 1-17%) with an estimated rate of 3.9 per 100 person-years. Transmission of multiple viruses combined with HIV super-infection at a later time point was observed in one individual.

Conclusions: Multiple HIV lineages transmitted from the same source produce a monophyletic clade in the inferred phylogenetic tree. Such a clade has transiently distinct sub-clusters in the early stage of HIV infection, and follows a predictable evolutionary pathway. Over time, the gap between initially distinct viral lineages fills in and initially distinct sub-clusters converge. Identification of cases with transmission of multiple viral lineages from the same source needs to be taken into account in cross-sectional estimation of HIV recency in epidemiological and population studies.