Distinct human immunodeficiency virus type 1 subtype A virus circulating in West Africa: sub-subtype A3

Abstract

Phylogenetic analyses demonstrate significant diversity in worldwide circulating strains of human immunodeficiency virus type 1 (HIV-1). Detailed studies have revealed a complex pattern of intersubtype recombinations, as well as evidence of sub-subtypes circulating in various populations. In this study, we characterized an HIV-1 strain that had previously been identified as a distinct subcluster within the subtype A radiation based on partial sequence data. These viruses were of particular interest given that we recently found that their prevalence was significantly higher in dually infected individuals compared to women who were singly infected with HIV-1. Five viruses isolated from commercial sex workers in Dakar, Senegal, were full-length PCR amplified and sequenced. Phylogenetic analyses indicated that, whereas three of these viruses were closely related and clustered overall within the HIV-1 subtype A radiation, they were distinct from previously characterized sub-subtype A1 and A2 viruses. The clustering pattern was maintained in the individual gag, pol, and env regions of the genome. Distance calculations between these viruses, which we termed A3, and other reference sub-subtype A1 and A2 viruses fell in the range of distances between previously characterized sub-subtype groups. In addition, we found evidence of two A3-containing recombinants in our cohort. These recombinants are mosaics composed of sequence from both sub-subtype A3 and CRF02_AG, the major circulating recombinant form in this West African population. Based on phylogenetic analyses, we propose that the group of viruses found in the Dakar sex worker cohort, previously referred to as HIV-1 A subcluster 2, be referred to as HIV-1 sub-subtype A3.

Figures

FIG. 1.

Maximum-likelihood tree of full-length sequences including three A3 sequences (DDI579, DDJ360, and DDJ369) and reference sequences from all known subtypes and sub-subtypes (A1, A2, B, C, D, F1, F2, G, H, J, and K; Los Alamos HIV Sequence database [http://hiv-web.lanl.gov]). The alignments were gap stripped, and a transition/transversion ratio of 1.42 was used. The scale bar indicates 1% nucleotide sequence divergence. The A3 sequences are indicated in boldface.

FIG. 2.

Maximum-likelihood subregion trees. (a) gag; (b) pol; (c) env. Sequences maintain unique clustering patterns in individual gene regions. The alignments were gap stripped, and these trees were generated by using the DNAML program in the PHYLIP version 3.6 package (9). A transition/transversion ratio of 1.42 was used for all trees. The scale bar indicates 1% nucleotide sequence divergence. For these trees, reference sequences from all major subtypes and sub-subtypes (A1, A2, B, C, D, F1, F2, G, H, J, and K) were used. The A3 sequences are indicated in boldface.

FIG. 3.

Plots of intrasubtype, intersubtype, and inter-sub-subtype genetic distances for full-length sequences. To calculate these distances, a pairwise distance matrix was constructed by using DNADIST from the PHYLIP version 3.6 package (9), with the F84 model of evolution, a transition/transversion ratio of 1.42, and empirical base frequencies. All alignments were gap stripped. CRFs were excluded from these calculations. Subtypes B and D were evaluated separately from all of the other intersubtype comparisons. Inter-sub-subtype distances were computed between sub-subtypes A1 and A2, sub-subtypes F1 and F2, and subtypes B and D. The value 0.01 on the x axis indicates a 1% nucleotide sequence divergence. The y axis shows the percentage of pairwise comparisons that have the same nucleotide sequence divergence.

FIG. 4.

Recombination analyses for two sequences previously designated as containing A3 in the C2-V3 region of the env. An alignment of nearly full-length strains was used; long terminal repeats were not analyzed. Alignments were gap stripped prior to analysis. (a) Maximum-likelihood tree containing references from all subtypes and sub-subtypes (A1, A2, B, C, D, F1, F2, G, H, J, and K), A3 sequences (DDI579, DDJ360, and DDJ369) and the two sequences assumed to be recombinants (DDJ362 and DDJ364). (b) Maximum-likelihood tree containing references from all subtypes and sub-subtypes (A1, A2, B, C, D, F1, F2, G, H, J, and K), A3 sequences (DDI579, DDJ360, and DDJ369), CRF02 sequences and the two sequences assumed to be recombinants (DDJ362 and DDJ364); both maximum-likelihood trees were generated by using DNAML from the PHYLIP version 3.6 package (9), using a transition/transversion ratio of 1.42. (c and d) Recombination analyses for sequences from subjects DDJ362 and DDJ364, respectively. The recombinant regions and corresponding breakpoints were determined by using bootscan plots (28). Bootscans were generated on NJ trees of gap-stripped sequences, using a transition/transversion ratio of 1.42. Final plots for both recombinants contain reference sequences from sub-subtype A3 (DDI578, DDJ360, and DDJ369), CRF02_AG (DJ264, IbNG, SE7812, MP1211, and MP807), subtype G (G6165, HH8793, NG083, and DRC.BL), and subtype B (HXB2, RF, SF2, and RL42) as the outgroup. The reference sequences were grouped, and the final bootscan plot was generated by using the consensus for the reference sequence groups. Bootscans were performed with a moving window of 400 bp and a step size of 50 bp. Breakpoints were determined by using informative site analyses and by maximizing χ2 values. For both samples, bootscan plots are shown. Below the plots, are schematics of the predicted recombinant sequence patterns, as well as the NJ phylogenetic trees for each of the recombined subregions within the plot. For each of the sequences, the roman numerals on the bootscan correspond to the roman numerals above the phylogenetic tree. All NJ trees were generated by using the SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE programs in the PHYLIP version 3.6 package (9). For all NJ trees, the F84 model of evolution and a transition/transversion ratio of 1.42 was used.

FIG. 4.

Recombination analyses for two sequences previously designated as containing A3 in the C2-V3 region of the env. An alignment of nearly full-length strains was used; long terminal repeats were not analyzed. Alignments were gap stripped prior to analysis. (a) Maximum-likelihood tree containing references from all subtypes and sub-subtypes (A1, A2, B, C, D, F1, F2, G, H, J, and K), A3 sequences (DDI579, DDJ360, and DDJ369) and the two sequences assumed to be recombinants (DDJ362 and DDJ364). (b) Maximum-likelihood tree containing references from all subtypes and sub-subtypes (A1, A2, B, C, D, F1, F2, G, H, J, and K), A3 sequences (DDI579, DDJ360, and DDJ369), CRF02 sequences and the two sequences assumed to be recombinants (DDJ362 and DDJ364); both maximum-likelihood trees were generated by using DNAML from the PHYLIP version 3.6 package (9), using a transition/transversion ratio of 1.42. (c and d) Recombination analyses for sequences from subjects DDJ362 and DDJ364, respectively. The recombinant regions and corresponding breakpoints were determined by using bootscan plots (28). Bootscans were generated on NJ trees of gap-stripped sequences, using a transition/transversion ratio of 1.42. Final plots for both recombinants contain reference sequences from sub-subtype A3 (DDI578, DDJ360, and DDJ369), CRF02_AG (DJ264, IbNG, SE7812, MP1211, and MP807), subtype G (G6165, HH8793, NG083, and DRC.BL), and subtype B (HXB2, RF, SF2, and RL42) as the outgroup. The reference sequences were grouped, and the final bootscan plot was generated by using the consensus for the reference sequence groups. Bootscans were performed with a moving window of 400 bp and a step size of 50 bp. Breakpoints were determined by using informative site analyses and by maximizing χ2 values. For both samples, bootscan plots are shown. Below the plots, are schematics of the predicted recombinant sequence patterns, as well as the NJ phylogenetic trees for each of the recombined subregions within the plot. For each of the sequences, the roman numerals on the bootscan correspond to the roman numerals above the phylogenetic tree. All NJ trees were generated by using the SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE programs in the PHYLIP version 3.6 package (9). For all NJ trees, the F84 model of evolution and a transition/transversion ratio of 1.42 was used.

FIG. 4.

Recombination analyses for two sequences previously designated as containing A3 in the C2-V3 region of the env. An alignment of nearly full-length strains was used; long terminal repeats were not analyzed. Alignments were gap stripped prior to analysis. (a) Maximum-likelihood tree containing references from all subtypes and sub-subtypes (A1, A2, B, C, D, F1, F2, G, H, J, and K), A3 sequences (DDI579, DDJ360, and DDJ369) and the two sequences assumed to be recombinants (DDJ362 and DDJ364). (b) Maximum-likelihood tree containing references from all subtypes and sub-subtypes (A1, A2, B, C, D, F1, F2, G, H, J, and K), A3 sequences (DDI579, DDJ360, and DDJ369), CRF02 sequences and the two sequences assumed to be recombinants (DDJ362 and DDJ364); both maximum-likelihood trees were generated by using DNAML from the PHYLIP version 3.6 package (9), using a transition/transversion ratio of 1.42. (c and d) Recombination analyses for sequences from subjects DDJ362 and DDJ364, respectively. The recombinant regions and corresponding breakpoints were determined by using bootscan plots (28). Bootscans were generated on NJ trees of gap-stripped sequences, using a transition/transversion ratio of 1.42. Final plots for both recombinants contain reference sequences from sub-subtype A3 (DDI578, DDJ360, and DDJ369), CRF02_AG (DJ264, IbNG, SE7812, MP1211, and MP807), subtype G (G6165, HH8793, NG083, and DRC.BL), and subtype B (HXB2, RF, SF2, and RL42) as the outgroup. The reference sequences were grouped, and the final bootscan plot was generated by using the consensus for the reference sequence groups. Bootscans were performed with a moving window of 400 bp and a step size of 50 bp. Breakpoints were determined by using informative site analyses and by maximizing χ2 values. For both samples, bootscan plots are shown. Below the plots, are schematics of the predicted recombinant sequence patterns, as well as the NJ phylogenetic trees for each of the recombined subregions within the plot. For each of the sequences, the roman numerals on the bootscan correspond to the roman numerals above the phylogenetic tree. All NJ trees were generated by using the SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE programs in the PHYLIP version 3.6 package (9). For all NJ trees, the F84 model of evolution and a transition/transversion ratio of 1.42 was used.