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Nucleotide sequences in three hypervariable regions of the human immunodeficiency virus type 1 (HIV-1) env gene were obtained by sequencing provirus present in peripheral blood mononuclear cells of HIV-infected individuals. Single molecules of target sequences were isolated by limiting dilution and amplified in two stages by the polymerase chain reaction, using nested primers. The product was directly sequenced to avoid errors introduced by Taq polymerase during the amplification process. There was extensive variation between sequences from the same individual as well as between sequences from different individuals. Interpatient variability was markedly less in individuals infected from a common source. A high proportion of amino acid substitutions in the hypervariable regions altered the number and positions of potential N-linked glycosylation sites. Sequences in two hypervariable regions frequently contained short (3to 15-bp) duplications or deletions, and by amplifying peripheral blood mononuclear cell DNA containing 102 or 103 proviral molecules and

To prevent new infections with human immunodeficiency virus type 1 (HIV-1) in sub-Saharan Africa, UNAIDS recommends targeting interventions to populations that are at high risk of acquiring and passing on the virus. Yet it is often unclear who and where these ‘source’ populations are. Here we demonstrate how viral deep-sequencing can be used to reconstruct HIV-1 transmission networks and to infer the direction of transmission in these networks. We are able to deep-sequence virus from a large population-based sample of infected individuals in Rakai District, Uganda, reconstruct partial transmission networks, and infer the direction of transmission within them at an estimated error rate of 16.3% [8.8–28.3%]. With this error rate, deep-sequence phylogenetics cannot be used against individuals in legal contexts, but is sufficiently low for population-level inferences into the sources of epidemic spread. The technique presents new opportunities for characterizing source populations and for targeting of HIV-1 prevention interventions in Africa.

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Direct sequencing of segments of the envelope gene of human immunodeficiency virus type 1 proviruses in peripheral blood mononuclear cells has revealed that a cohort of hemophiliacs who were infected after exposure to a single common batch of factor VIII share closely related virus strains. Seventy-four sequences extending from hypervariable regions V4 through V5 from nine patients yielded a mean intrapatient nucleotide distance of 5.5%, while a mean of 4.2% was observed in 39 sequences of the V3 loop (six patients). Phylogenetic analysis revealed that sequences of six Edinburgh patients were particularly closely related and those from a patient infected in the United States were very distinct. The mean nucleotide distance among these six was 8.3%, while the mean distance from the U.S.-derived sequences was 25.5% in the V4-V5 region. The rate of sequence change across this patient group has been estimated to be 0.4% per year in the V4-V5 region and 0.5% per year in the V3 region, with at least a twofold range across patients. Only two inactivating nucleotide substitutions have been observed in a total of 42 kb of sequence obtained from the env and gag genes during this study.