Figure 6

Detection of recombinant viruses generated in the viral fitness assay. Three viruses (T/F, T242N and NIA) were co-cultured and passaged six times. The virus in the supernatants was harvested after 3 or 4 days in each passage and subjected to PASS analysis. (A) The same amplicons in the PASS gel were first probed to determine the bases at position 242 and subsequently the bases at position 247. The linkage analysis of bases at both positions was performed to distinguish the three viruses and the recombinant. The T/F virus (arrow 1) has 242T (green) and 247V (green); the T242N virus (arrow 2) has 242N (red) and 247V (green); the NIA virus has (arrow 3) has 242N (red) and 247I (red); and the recombinant (rec; arrow 4) has 242T (green) and 247I (red). The result from the viruses harvested at passage 5 from one experiment is shown. (B) Frequency of the recombinant genomes during multiple passages. The recombinants between T/F and NIA were detected for each passage. The viral culture was carried out in triplicate. Means ± standard errors are plotted. (C) Comparison of frequencies of the recombinant virus and other three viruses (T/F, T242N and NIA) in the same sample determined by PASS (596 genomes) and SGA sequencing (47 genomes). (D) Detection of recombinant viral genomes between the T/F and TK viruses during four rounds of passages. Two recombinants (viruses with I64T or R355K mutation) were detected by linkage analysis of mutations at positions 64 in Tat and 355 in Env. The viral culture was carried out in triplicate. Means ± standard deviations are plotted.