Complex Subtype Diversity of HIV-1 Among Drug Users in Major Kenyan Cities

Abstract

Drug users are increasingly recognized as a key population driving human immunodeficiency virus (HIV) spread in sub-Saharan Africa. To determine HIV-1 subtypes circulating in this population group and explore possible geographic differences, we analyzed HIV-1 sequences among drug users from Nairobi, Mombasa, and Kisumu in Kenya. We sequenced gag and env from 55 drug users. Subtype analysis from 220 gag clonal sequences from 54 of 55 participants (median = 4/participant) showed that 44.4% were A, 16.7% were C, 3.7% were D, and 35.2% were intersubtype recombinants. Of 156 env clonal sequences from 48 of 55 subjects (median = 3/participant), 45.8% were subtype A, 14.6% were C, 6.3% were D, and 33.3% were recombinants. Comparative analysis of both genes showed that 30 (63.8%) participants had concordant subtypes, while 17 (36.2%) were discordant. We identified one genetically linked transmission pair and two cases of dual infection. These data are indicative of extensive HIV-1 intersubtype recombination in Kenya and suggest decline in subtype D prevalence.

Keywords: HIV-1 subtypes; Kenya; drug users; intersubtype recombinants.

FIG. 1.

Relative proportions of pure subtypes and intersubtype recombinants among drug users in Kenya. (A) Proportion of participants infected with pure and recombinant subtypes together with the breakdown of the relative recombinant percentages based on HIV-1 gag sequences. (B) Proportion of participants infected with pure and recombinant subtypes together with the breakdown of the relative recombinant percentages based on HIV-1 env sequences and (C) relative proportions of participants containing pure subtypes, recombinants, or a mixture of both pure and recombinant subtypes for either the gag or env region of the HIV-1 genome. HIV-1, human immunodeficiency virus type 1.

FIG. 2.

Distribution (%) of HIV-1 subtypes and intersubtype recombinants among drug users in Nairobi, Kisumu, and Mombasa based on the number of gag and env clonal sequences obtained for each region. The number of participants screened for each city in Kenya is shown within the individual subtype bars.

FIG. 3.

Schematic representation of concordant and discordant subtypes based on full-length clonal gag and env genes analyzed from drug users in Kenya. (A) gag and env gene structure of the 30 concordant subtypes. (B) Gene structures of full-length gag and env showing discordant subtype distribution in 17 drug user participants. Clonal sequencing revealed mixed subtypes (PID 426 had mixed subtypes in the gag region, while PID 575 had mixed subtypes in the env region). *Represents known injecting drug users. Subtype classification of each gene is indicated. Subtype distribution is shown in different colors (A, red; C, yellow; D, light blue; G, dark green, and H, light green). Note that the gene lengths are not drawn to scale.

FIG. 4.

Phylogenetic analysis of drug user participants from three major cities in Kenya. (A) Maximum likelihood trees were constructed using 220 HIV-1 full-length gag clonal sequences collected from drug user participants recruited from Nairobi (green circles), Kisumu (blue circles) and Mombasa (red circles). Possible linked participants (PIDs 552 and 527) are highlighted in green text and possible dual infected participant is highlighted in red text (PID 426). (B) Detailed phylogenetic analysis of possible dual infected drug user participant 426 (red) and epidemiologically-linked drug users (green) are shown. Both clonal sequences (solid red circles) together with single genome amplification (SGA) derived gag sequences (open red circles) for PID 426 show infection with subtypes A, D, AD and ADH. Phylogenetic analysis shows strong bootstrap support of epidemiological linkage between participants PID 552 and 527 (green circles) after sequences were downloaded and included in phylogenetic analysis after BLAST analysis of individual participant sequences. (C) Phylogenetic analysis of 156 HIV-1 full-length env sequences from drug user participants from Nairobi (green), Kisumu (blue) and Mombasa (red). Possible dual infected drug user participant 426 (red text) and epidemiologically-linked drug users PID 552 and 527 (green text) are shown. (D) Detailed phylogenetic analysis of the epidemiologically-linked participants PID 552 and 527 (green) after adding downloaded BLAST analysis sequences suggest strong linkage between the two participants. All HIV-1 reference sequences representing genetic subtypes were obtained from the Los Alamos National Laboratory HIV Sequence Database included: Ref.A1.RW.92, Ref.A1.UG.92, Ref.A1.AU.03, Ref.A2.CD.97, Ref.A2.CY.94, Ref.A2.CM.01, Ref.B.NL.00, Ref.B.US.98, Ref.B.FR.83, Ref.C.ET.86, Ref.C.BR.92, Ref.C.ZA.04, Ref.C.IN.95, Ref.D.CD.83, Ref.D.TZ.01, Ref.D.UG.94, and Ref.D.CM.01 for both gag and env (blue text). Bootstrap support ≥70 is indicated with an asterisk (*). The gray boxes highlight alternate individual participant sequence clusters.

FIG. 4.

Phylogenetic analysis of drug user participants from three major cities in Kenya. (A) Maximum likelihood trees were constructed using 220 HIV-1 full-length gag clonal sequences collected from drug user participants recruited from Nairobi (green circles), Kisumu (blue circles) and Mombasa (red circles). Possible linked participants (PIDs 552 and 527) are highlighted in green text and possible dual infected participant is highlighted in red text (PID 426). (B) Detailed phylogenetic analysis of possible dual infected drug user participant 426 (red) and epidemiologically-linked drug users (green) are shown. Both clonal sequences (solid red circles) together with single genome amplification (SGA) derived gag sequences (open red circles) for PID 426 show infection with subtypes A, D, AD and ADH. Phylogenetic analysis shows strong bootstrap support of epidemiological linkage between participants PID 552 and 527 (green circles) after sequences were downloaded and included in phylogenetic analysis after BLAST analysis of individual participant sequences. (C) Phylogenetic analysis of 156 HIV-1 full-length env sequences from drug user participants from Nairobi (green), Kisumu (blue) and Mombasa (red). Possible dual infected drug user participant 426 (red text) and epidemiologically-linked drug users PID 552 and 527 (green text) are shown. (D) Detailed phylogenetic analysis of the epidemiologically-linked participants PID 552 and 527 (green) after adding downloaded BLAST analysis sequences suggest strong linkage between the two participants. All HIV-1 reference sequences representing genetic subtypes were obtained from the Los Alamos National Laboratory HIV Sequence Database included: Ref.A1.RW.92, Ref.A1.UG.92, Ref.A1.AU.03, Ref.A2.CD.97, Ref.A2.CY.94, Ref.A2.CM.01, Ref.B.NL.00, Ref.B.US.98, Ref.B.FR.83, Ref.C.ET.86, Ref.C.BR.92, Ref.C.ZA.04, Ref.C.IN.95, Ref.D.CD.83, Ref.D.TZ.01, Ref.D.UG.94, and Ref.D.CM.01 for both gag and env (blue text). Bootstrap support ≥70 is indicated with an asterisk (*). The gray boxes highlight alternate individual participant sequence clusters.

FIG. 4.

Phylogenetic analysis of drug user participants from three major cities in Kenya. (A) Maximum likelihood trees were constructed using 220 HIV-1 full-length gag clonal sequences collected from drug user participants recruited from Nairobi (green circles), Kisumu (blue circles) and Mombasa (red circles). Possible linked participants (PIDs 552 and 527) are highlighted in green text and possible dual infected participant is highlighted in red text (PID 426). (B) Detailed phylogenetic analysis of possible dual infected drug user participant 426 (red) and epidemiologically-linked drug users (green) are shown. Both clonal sequences (solid red circles) together with single genome amplification (SGA) derived gag sequences (open red circles) for PID 426 show infection with subtypes A, D, AD and ADH. Phylogenetic analysis shows strong bootstrap support of epidemiological linkage between participants PID 552 and 527 (green circles) after sequences were downloaded and included in phylogenetic analysis after BLAST analysis of individual participant sequences. (C) Phylogenetic analysis of 156 HIV-1 full-length env sequences from drug user participants from Nairobi (green), Kisumu (blue) and Mombasa (red). Possible dual infected drug user participant 426 (red text) and epidemiologically-linked drug users PID 552 and 527 (green text) are shown. (D) Detailed phylogenetic analysis of the epidemiologically-linked participants PID 552 and 527 (green) after adding downloaded BLAST analysis sequences suggest strong linkage between the two participants. All HIV-1 reference sequences representing genetic subtypes were obtained from the Los Alamos National Laboratory HIV Sequence Database included: Ref.A1.RW.92, Ref.A1.UG.92, Ref.A1.AU.03, Ref.A2.CD.97, Ref.A2.CY.94, Ref.A2.CM.01, Ref.B.NL.00, Ref.B.US.98, Ref.B.FR.83, Ref.C.ET.86, Ref.C.BR.92, Ref.C.ZA.04, Ref.C.IN.95, Ref.D.CD.83, Ref.D.TZ.01, Ref.D.UG.94, and Ref.D.CM.01 for both gag and env (blue text). Bootstrap support ≥70 is indicated with an asterisk (*). The gray boxes highlight alternate individual participant sequence clusters.

FIG. 4.

Phylogenetic analysis of drug user participants from three major cities in Kenya. (A) Maximum likelihood trees were constructed using 220 HIV-1 full-length gag clonal sequences collected from drug user participants recruited from Nairobi (green circles), Kisumu (blue circles) and Mombasa (red circles). Possible linked participants (PIDs 552 and 527) are highlighted in green text and possible dual infected participant is highlighted in red text (PID 426). (B) Detailed phylogenetic analysis of possible dual infected drug user participant 426 (red) and epidemiologically-linked drug users (green) are shown. Both clonal sequences (solid red circles) together with single genome amplification (SGA) derived gag sequences (open red circles) for PID 426 show infection with subtypes A, D, AD and ADH. Phylogenetic analysis shows strong bootstrap support of epidemiological linkage between participants PID 552 and 527 (green circles) after sequences were downloaded and included in phylogenetic analysis after BLAST analysis of individual participant sequences. (C) Phylogenetic analysis of 156 HIV-1 full-length env sequences from drug user participants from Nairobi (green), Kisumu (blue) and Mombasa (red). Possible dual infected drug user participant 426 (red text) and epidemiologically-linked drug users PID 552 and 527 (green text) are shown. (D) Detailed phylogenetic analysis of the epidemiologically-linked participants PID 552 and 527 (green) after adding downloaded BLAST analysis sequences suggest strong linkage between the two participants. All HIV-1 reference sequences representing genetic subtypes were obtained from the Los Alamos National Laboratory HIV Sequence Database included: Ref.A1.RW.92, Ref.A1.UG.92, Ref.A1.AU.03, Ref.A2.CD.97, Ref.A2.CY.94, Ref.A2.CM.01, Ref.B.NL.00, Ref.B.US.98, Ref.B.FR.83, Ref.C.ET.86, Ref.C.BR.92, Ref.C.ZA.04, Ref.C.IN.95, Ref.D.CD.83, Ref.D.TZ.01, Ref.D.UG.94, and Ref.D.CM.01 for both gag and env (blue text). Bootstrap support ≥70 is indicated with an asterisk (*). The gray boxes highlight alternate individual participant sequence clusters.