In Thailand, the HIV-1 epidemic started abruptly in 1988 with the introduction of subtype B and subtype E, now called the circulating recombinant form (CRF), CRF01_AE. These two strains appeared independently in distinct high-risk populations  : subtype B among injecting drug users (IDU) and CRF01_AE among those who were heterosexually exposed [2,3]. HIV-1 subtype B is still common among infected Bangkok IDU, but CRF01_AE was found in 80% of IDU surveyed in 1995–1998 . Dual infection with HIV-1 subtype B and CRF01_AE was observed by 1994 , providing the opportunity for recombination between these two subtypes in the Thailand epidemic. Whereas recombination between CRF01_AE and subtype B has occurred in an experimental dual infection of a chimpanzee , such a recombinant in humans has not yet been described. Here, we identify an AE/B inter-subtype recombinant of HIV-1 found in a multiply exposed individual in Thailand. The full-length genome of this recombinant has been analysed and characterized.
In 1997, screening assays for the subtype of the virus of a 40-year-old Thai man (NP1623) provided evidence of subtype discordance in different parts of the genome. A V3 loop peptide enzyme immunoassay  classified the serum of NP1623 as CRF01_AE and an envelope heteroduplex mobility assay  confirmed that designation. A restriction fragment length polymorphism analysis from the gag leader region , however, indicated that NP1623 was infected with subtype B, as did a differential polymerase chain reaction (PCR) assay in gp41 . These results suggested discordance between the subtype of gp120 and the subtype of the rest of the virus.
Peripheral blood mononuclear cells (PBMC) were separated by Ficoll gradient and co-cultivated with phytohemagglutinin-stimulated donor PBMC. Full genomes of HIV-1 were amplified from the cultured PBMC DNA by nested PCR, with endpoint dilution of the DNA template in the first round. The DNA template was fully sequenced on both strands using BigDye terminator reaction kits and an ABI 373 DNA sequencer. A multiple alignment of the NP1623 full-length sequence with reference sequences of all HIV subtypes was generated.
Bootscan analysis of the full genome sequence revealed that the virus had a recombinant structure with three segments (Fig. 1). Neighbor-joining phylogenetic analyses with parsimony bootstrap were performed on the segments of the genome and are shown in the upper panel of Fig. 1. The structure of the virus from NP1623 is as follows: subtype B from the beginning of gag until mid-vpu, where the subtype shifts to CRF01_AE. It then changes back to subtype B in the C5 region of gp120 and remains subtype B through gp41 and nef. The subtype B segments of the genome cluster most closely with the ‘Thai B’ sample, RL42. These same breakpoints were also confirmed in an independent amplification and sequencing of envelope directly from patient PBMC (Fig. 1, lower diagram).
The initial separation of CRF01_AE and subtype B in different risk groups in Thailand may have delayed the onset of significant numbers of dual infections, each of which can potentially lead to recombination, for almost a decade. The most significant factor in this respect may not be individuals who are exposed both heterosexually and through injecting drug use, as is the case reported here, but may rather be the growing proportion of CRF01_AE among an IDU population that, initially, was almost exclusively infected with subtype B. Indeed, we cannot discern whether the patient studied here was dually exposed by injecting drug use, was exposed to each strain by a different route, or was singly infected with the recombinant strain itself.
The HIV-1 epidemic in southeast Asia is becoming more complex with respect to HIV-1 diversity. Recent reports include subtypes B, ‘Thai B', C, D, and CRF01_AE and a B/C recombinant in southern China. A CRF01_AE/subtype C recombinant has been detected in Thailand. The CRF01_AE/B recombinant reported here may be a harbinger of more recombinants. Intensified monitoring is particularly important in the light of the ongoing and projected HIV-1 vaccine trials. A significant fraction of recombinant strains among incident infections could necessitate an approach such as that used here, with multiple genetic regions analysed and discordances followed up with full genome sequencing, to evaluate the relative effectiveness of vaccines against different HIV-1 subtypes.
The authors would like to thank Puangmalee Buapunth and the staff of the Joint Clinical Research Center in Bangkok for their invaluable assistance. The views and opinions expressed herein do not necessarily reflect those of the US Army or of the Department of Defense.
Mark De Souzacd
Merlin R. Robbg
Deborah L. Birxg
Francine E. McCutchanb
Jean K. Carrb
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