Figure 4.
Figure 4. Full-genome analysis of isolates with identical env sequences. (a) Full-genome sequences of isolates with identical env sequences. Randomly selected isolates (arrows in Fig. 3) belonging to sets of isolates with identical env sequence were subjected to full-length sequencing. Sequencing was performed on single HIV-1 genomic RNA molecules present in the supernatants of p24+ cultures after reverse transcription and nested PCR amplification performed at limiting dilution. Two overlapping half-genome fragments were amplified. To minimize errors, PCR products were directly sequenced without cloning. For each isolate, 6–12 limiting 5′ sequences and 6–12 3′ sequences were obtained. Neighbor joining phylogenetic trees demonstrated that all sequences from a given set of isolates with identical env sequences co-clustered. The minimal differences (one to four nucleotides) likely reflect mutations expected to arise during the 3-wk outgrowth culture. Symbols indicate the PHA stimulation after which the isolates were detected, as in Fig. 3. Results are shown for the 5′ half-genome sequences. Similar results were obtained for the 3′ half-genome sequences. (b) Genetic distance (mean ± SD) between single-genome sequences from a single isolate (intra-isolate), between single-genome sequences from different isolates belonging to a set of isolates with identical env sequences (intra-set), between single-genome sequences from different sets from the same study subject (intra-subject), and between single genome sequences from different patients. For each set of two to three isolates from a given subject, 6–12 sequences were used for intra-isolate and intra-set comparisons. Then, all sequences were used for intra-subject and inter-subject comparisons. (c) Phylogenetic tree of full-genome consensus sequences. The 6–12 single half-genome sequences from each isolate were condensed to half-genome consensus sequences, which were joined to produce full-genome sequences. Set 2 from subject 2 is not depicted because of failure of the 3′ half-genome reaction as a result of primer mismatch.

Full-genome analysis of isolates with identical env sequences. (a) Full-genome sequences of isolates with identical env sequences. Randomly selected isolates (arrows in Fig. 3) belonging to sets of isolates with identical env sequence were subjected to full-length sequencing. Sequencing was performed on single HIV-1 genomic RNA molecules present in the supernatants of p24+ cultures after reverse transcription and nested PCR amplification performed at limiting dilution. Two overlapping half-genome fragments were amplified. To minimize errors, PCR products were directly sequenced without cloning. For each isolate, 6–12 limiting 5′ sequences and 6–12 3′ sequences were obtained. Neighbor joining phylogenetic trees demonstrated that all sequences from a given set of isolates with identical env sequences co-clustered. The minimal differences (one to four nucleotides) likely reflect mutations expected to arise during the 3-wk outgrowth culture. Symbols indicate the PHA stimulation after which the isolates were detected, as in Fig. 3. Results are shown for the 5′ half-genome sequences. Similar results were obtained for the 3′ half-genome sequences. (b) Genetic distance (mean ± SD) between single-genome sequences from a single isolate (intra-isolate), between single-genome sequences from different isolates belonging to a set of isolates with identical env sequences (intra-set), between single-genome sequences from different sets from the same study subject (intra-subject), and between single genome sequences from different patients. For each set of two to three isolates from a given subject, 6–12 sequences were used for intra-isolate and intra-set comparisons. Then, all sequences were used for intra-subject and inter-subject comparisons. (c) Phylogenetic tree of full-genome consensus sequences. The 6–12 single half-genome sequences from each isolate were condensed to half-genome consensus sequences, which were joined to produce full-genome sequences. Set 2 from subject 2 is not depicted because of failure of the 3′ half-genome reaction as a result of primer mismatch.

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