Background:  The best circumstantial evidence for dual infection in humans is the presence of recombinant forms. Recently, recombination has been documented between clade A and clade C viruses following superinfection. We demonstrate intraclade recombination into 2 subjects after co-infection and superinfection with clade B virus.

Methods:  We retrospectively analyzed plasma samples from 2 individuals recently identified with clade B intraclade superinfection (patient 1) and co-infection (patient 2) to investigate the extent of recombination. Clonal sequencing of the gp160 region of env was performed on samples collected before and after superinfection (patient 1) and at 8 different time points (patient 2). Recombination was detected using a newly developed maximum likelihood method, capable of detecting conflicting phylogenetic signal and inferring the location of putative recombination breakpoints using an efficient parallel genetic algorithm.

Results:  Using 26 gp160 sequences from patient 1 (16 clones sampled before superinfection and 10 after) we identified at least 2 recombination events. Non-recombinant fragments had estimated lengths of 1621 bp, 500 bp and 495bp (±20 bp). Using 83 gp160 sequences from patient 2 (8 time points with 5 to 20 clones per time point) we identified at least 2 recombination events. Non-recombinant fragments had estimated lengths of 1440 bp, 991 bp, and 170bp (±5 bp). In both cases, recombination between fragments 1 and 2 could be attributed to a single clonal sequence shifting between 2 well-resolved sequence clusters, whereas recombination between fragments 2 and 3 involved multiple sequences (complex pattern in patient 1; 3 sequences in patient 2). We confirmed discordant phylogenetic signal using the Shimodaira-Hasegawa test (p value <0.01) for all appropriate pair-wise fragment comparisons.

Conclusions:  Robust detection of recombination by 2 viruses of the same clade requires specialized techniques, such as genetic algorithms, because of relative genetic similarity of viral sequences. Recombination allows for a more rapid increase in viral diversity than the accumulation of mutations through replication errors. This genetic heterogeneity can facilitate rapid adaptation to host immune responses, target cell availability and ART, and lead to increased viral pathogenicity and reduced antiretroviral susceptibility, both of which have been documented after superinfection.