Recombination Following Superinfection by HIV-1
Background: There is increasing recognition of recombinant HIV-1 strains globally, but it has been unclear whether recombination results from superinfection during untreated, chronic infection.
Objective: To search for evidence of recombination and superinfection in Africa, where multiple HIV-1 subtypes facilitate identification of strains.
Methods: Serial blood samples from highly exposed, chronically infected women in Nairobi's Pumwani sex workers cohort were examined. Serial, complete HIV-1 RNA sequence analyses were performed for seven untreated long-term survivors. Sequences were subjected to computational analysis.
Results: One woman had evidence of both superinfection and recombination. Complete HIV-1 RNA sequences were first derived from plasma obtained in 1986, when the woman had been HIV seropositive for at least 21 months; this sequence was entirely subtype A. The sequences obtained from plasma in 1995 and 1997, however, were subtype A/C recombinants with a SimPlot demonstrating that the subtype A fragment in 1995 and 1997 was derived from the original 1986 A sequence. Heteroduplex tracking assays demonstrated that the subtype C sequences were not detectable as minor species in 1986.
Conclusion: Intersubtype recombination took place between the original non-recombinant subtype A strain and the superinfecting subtype C strain in an untreated, chronically infected woman. This finding helps to explain the rising prevalence of recombinant HIV-1 worldwide. Recombination resulting from superinfection with diverse strains may pose problems for eliciting broad immune responses necessary for an effective vaccine.

Recombination among genetically distinct subtypes and strains of HIV-1 is now recognized with increasing frequency in the global pandemic. Recombination provides a mechanism to increase viral sequence diversity rapidly, unlike the slow accumulation of mutations that occurs through replication errors. For recombination to occur between distinct HIV-1 strains, a cell needs to be dually infected with different viruses. The progeny virions that result possess RNA genomes from each virus, permitting strand-switching to occur during the next round of reverse transcription. Therefore, recombination requires coinfection of viral strains in an individual. This dual infection may occur during the primary infection period, before the immune response is fully developed, or it may occur as a superinfection with a new viral strain after the initial strain has established a chronic infection. Both superinfection and recombination have the potential to complicate efforts to develop vaccines, and reinfection with a drug-resistant virus could jeopardize treatment.

Superinfection with a new strain may make recombination between the original and new strains possible. Although recombination following superinfection has been postulated, it has not been observed in individuals. Previous studies have identified fully formed recombinants in patients, but not recombination between the non-recombinant first and superinfecting strains occurring in the same individual. Some individuals have been infected by recognized circulating recombinant forms, which were transmitted in primary infection; in these cases, the recombination event presumably occurred in a previously infected patient. A small number of unique recombinants have also been detected in the setting of primary infection. With increasing recognition of recombinant strains globally, it was unclear whether recombination resulted from superinfection of chronically infected individuals. The distinct HIV-1 subtypes or clades seen in different regions of the world differ from one another by as much as 30% in the envelope gene (env) and up to 15% in the core proteins. Strains classified as different subtypes are genetically quite distinct, making it relatively easy to detect superinfection and recombination. To determine whether recombination has occurred at any site in a viral genome, it is best to analyze the complete genomic sequence of the virus. Detection of superinfection and recombination between strains of the same subtype can be challenging because of the difficulty of distinguishing different viral species of the same subtype from variation within a strain as a result of evolution.

Instances of cross-clade and intraclade coinfection and superinfection have been previously reported, particularly during and soon after primary infection and in the setting of treatment interruptions of chronic infections. The determination of superinfection in two of these cases was aided by epidemiological and immunological investigation. The possibility of dual transmission during the primary infection period, however, was not completely excluded by highly sensitive, virological analyses in these and other studies. It has been unclear whether recombination occurs only in the primary infection period or can result from superinfection during untreated, chronic infection.

The present study searches for evidence of superinfection and recombination in Africa, where multiple HIV-1 subtypes exist, by examining serial blood samples from women in Nairobi's Pumwani sex worker cohort. The dominant HIV-1 subtype in Kenya is clade A HIV-1, which accounts for ~70% of HIV-1 infections, the remainder consisting of clades D (~20%), C, and several minor species. The women in this cohort, like most infected individuals worldwide, have never received treatment and it was interesting to determine whether such seropositive, chronically infected individuals are susceptible to superinfection with a new strain and whether the superinfection could lead to recombination. Highly exposed persons, such as the women in the Pumwani cohort, may be particularly vulnerable to reinfection because their partners expose them to a range of different viral strains; this makes recombination more likely to be detected in this group. Full-length HIV-1 RNA sequences from seven long-term survivors have been analyzed for the appearance of new subtypes and recombinants.