Background:  Recombination is important in the evolution of HIV-1 with approximately 10% of circulating HIV-1 being intersubtype recombinants. This observation implies that intrasubtype recombination is also likely to be frequent, albeit more difficult to detect. To study the frequency of HIV-1 recombination in vivo we used single genome sequencing (SGS), which allows analysis of many individual genomes per plasma sample.

Methods:  From 25 to 50 single sequences were obtained from three patients with recent HIV-1 infection. The region sequenced comprised a 1.33 Kb fragment encompassing the p6 region of gag, pro and the first 900 nucleotides of pol. Genetic relationships among the individual sequences were revealed using phylogenetic analyses.

Results:  In the first patient studied, sequences obtained 3 months after infection showed both wildtype (11/34) and NNRTI-resistant (K103N: 14/34) viruses that differed overall by approximately 1%. Diversity within each of these two populations was less than 0.01%. This pattern of diversity implied recent infection with a mixture of two viruses from the same donor. The remaining 9/34 sequences were recombinants between these 2 populations, two of which contained 103N. Samples from the second patient studied also contained 2 distinct virus populations (7% difference) 1 wild type and 1 multidrug resistant. The appearance of the wild type virus at later times than the resistant virus suggested superinfection rather than coinfection. In contrast to the first patient, there was no evidence of recombination between the multidrug-resistant and wildtype populations in multiple longitudinal samples. Samples from a third untreated patient were collected 15 and 36 months after infection. The earlier sample showed very low (0.004%) diversity and little evidence of recombination. The population diversified (to 0.01%) over the 21 months of observation with recombination occurring even across short distances (4 to 12 bases).

Conclusions:  Intrasubtype recombination between closely related viruses can be detected using SGS. Viruses that give rise to recombinants can originate from coinfection with distinct viruses or diversification following one infection. However, recombinants are not always detected in mixed infections, implying that other factors, such as selection against recombinants or separation of virus populations into distinct compartments may limit the appearance of recombinants.

Keywords: recombination; coinfection; superinfection