Widespread circulation of a B/F intersubtype recombinant form among HIV-1-infected individuals in Buenos Aires, Argentina

Argentina has, after Brazil, the second largest HIV-1-infected population in South America, with 120 000 estimated cases of infection by the end of 1997 [1] and almost 15 000 reported AIDS cases by the end of 1998, with a high proportion (76%) from the Buenos Aires metropolitan area [2]. Previous phylogenetic studies in Argentina, based on V3 [3,4], on V3 and gag[5], and on env and protease [6] sequences indicated the presence of viruses grouping with B or F subtypes, as well as a minority of B/F recombinant viruses. In a recent study, based on env and protease sequences [6], it was reported that 55% of 58 individuals from Buenos Aires harboured B subtype viruses, 40% F subtype viruses, and 5% had either B/F mosaic or dual infections. Here we report the results of the phylogenetic analysis of HIV-1 reverse transcriptase (RT) sequences from 52 adult individuals from the Buenos Aires metropolitan area. DNA from peripheral blood mononuclear cells was used to amplify and directly sequence a segment of approximately 0.6 kb in RT, comprising most resistance-associated codons, using the polymerase chain reaction (PCR). The phylogenetic neighbour-joining tree, constructed using Kimura's two-parameter distances, showed clustering in two groups: the first, consisting of 31 samples (60%), clustered with the B subtype reference sequence, and the second, consisting of 21 samples (40%), clustered with a high bootstrap value (73%) as outliers of the F subtype branch. Analysis of the second group with the recombinant identification program (RIP) [7] suggested that all were intersubtype recombinants (5′F/B3′) with breakpoints at the same approximate location. To investigate the presence of additional breakpoints downstream in RT, the remaining portion of RT was PCR-amplified in 10 putative recombinant and in four B subtype samples, obtaining a near-full-length RT sequence (except 90 nucleotides at the 5′ end). RIP analysis indicated that all 10 recombinant samples had a second intersubtype breakpoint approximately 0.7 kb downstream from the first, delimiting a segment with high similarity scores to the B subtype consensus, flanked by sequences with high similarity scores to the F subtype consensus. Bootscan analysis [8] with SimPlot [9] confirmed the presence of the two breakpoints, with the 5′F/B/F3′ subtype pattern supported by high bootstrap values (up to 100%). Protease and the N-terminal portion of RT were amplified and sequenced in three recombinant samples, and the assembled protease–RT sequences were analysed by bootscanning (Fig. 1a), which supported the branching of protease with the F subtype consensus sequence, except for a short stretch across protease–RT, for which bootstrap values supported branching with the B subtype consensus.Fig. 1.: Analysis for intersubtype recombination of HIV-1 protease and reverse transcriptase (RT) sequences in individuals from Buenos Aires. (a) Bootscan analysis of protease and RT sequences from one individual. Consensus sequences of the presumed parental subtypes, B and F, were used, plus an A subtype sequence as the outgroup. Plots represent bootstrap values, based on 100 resamplings, supporting branching with the reference sequences within a 400 nucleotide window moving in steps of 20 bases. Values in the x axis represent the position of the centre of the window from nucleotide 1 of protease. (b) Neighbour-joining phylogentic trees of RT segments delimited by intersubtype breakpoints in 14 HIV-1-infected individuals. Bootstrap values (out of 100 replicates) supporting branching of the sample sequences with subtype reference sequences are shown. Branches corresponding to B/F recombinant viruses (10 samples) are labelled with filled circles, and branches corresponding to B subtype viruses (four samples) are labelled with unfilled circles.Informative site analysis using the maximum chi-squared method [10,11] was consistent with RIP and bootscanning, identifying two intersubtype breakpoints, the first between RT nucleotides 424 and 453, and the second between RT nucleotides 1152 and 1181, with significance at the level of P < 0.001, in eight of 10 samples analysed, with slight variations within the mentioned ranges. In two samples breakpoints were slightly displaced relative to the rest: in one, the first breakpoint was between nucleotides 498 and 507, and in another, the second breakpoint was between nucleotides 1128 and 1129. Separate phylogenetic trees of the three RT segments delimited by breakpoints (Fig. 1b) confirmed the grouping of recombinant samples with the F subtype reference in the 5′ and 3′ segments and with the B subtype reference in the middle segment, in all cases with high bootstrap values. Nucleotide distances in the middle segment were significantly lower between B/F recombinants (3.0 ± 0.7%) than between B subtype viruses (6.9 ± 1.1%), which is consistent with a more recent common ancestor of the former. The phylogenetic tree of the V3 loop and flanking sequences of five B subtypes and five B/F recombinant viruses showed that samples of subtype B in RT were also of subtype B in V3, whereas samples B/F recombinant in RT were of subtype F in V3, with bootstrap values of 100% in all cases. Nucleotide distances were 11.9 ± 2.1% for the B subtype viruses and 9.1 ± 3.2% for the B/F recombinants. The prevalence of HIV-1 genetic forms varied with risk groups and sex. All six injecting drug users (IDU) (three men and three women) harboured B/F recombinants, whereas four of five (80%) homosexual men harboured B subtype viruses. Among 28 individuals with heterosexually acquired infection (13 men and 15 women), there was a clear predominance of B subtype viruses among men (85%) and of B/F recombinants among women (67%). These results therefore indicate that B/F intersubtype recombinant viruses are widely circulating among HIV-1-infected individuals in Buenos Aires (40% of our cases), and are predominant among IDU and among women with sexually acquired infection. Virtually identical breakpoints in RT (with slight variations expected from mutations accumulated along the years) are strongly suggestive of a common origin. Failure to detect non-recombinant F subtype viruses and the grouping of V3 of the recombinant viruses with subtype F viruses suggest that most, if not all, HIV-1 isolates of Buenos Aires previously identified as being of subtype F are in fact B/F recombinants. Analysis of the homology of V3 sequences with Genebank sequences using BLAST [12] shows the highest similarity scores with F subtype viruses from Brazil. RT sequences from Brazil, analysed by us (unpublished data) and by others [13], however, show a low prevalence of B/F recombination, with those recombinants detected having breakpoints different from the viruses from Argentina. Taken together, these data are consistent with an individual dually infected with B and F subtype viruses, with the F subtype virus originating in Brazil, who transmitted the recombinant form in Argentina, as the most likely source of the B/F recombinant form of Buenos Aires. Although numerous HIV-1 intersubtype recombinant forms have been reported, only three, the A/E(CM240) of southeast Asia, the A/G(IbNG) of Africa, and the A/B(Kal153) of Kaliningrad, Russia, have spread to epidemic proportions comparable to the prevalent B/F recombinant form of Buenos Aires reported here [14,15]. Sequencing of the full-length genome of this virus is underway further to validate its recognition as a major HIV-1 circulating recombinant form. Michael M Thomsona María Luisa Villahermosaa Elena Vázquez-de-Pargaa María Teresa Cuevasa Elena Delgadoa Nuria Manjóna Leandro Medranoa Lucía Pérez-Álvareza Gerardo Contrerasa Manuel Gómez Carrillob Horacio Salomónb Rafael Nájeraa