e of successful viral subpopulations may require selective pressures not exclusively linked to evolution of V3 variants with altered coreceptor use. high-resolution phylogenetic analysis. ML trees inferred from V1-V2 or C2-V3 domains were identical for S1 and S3, indicating no obvious intra-patient recombination. The finding was 
confirmed by the PHI test for recombination. In contrast, six sequences from S2 and 47 from S4 clustered in different clades depending on the domain used to infer the trees. Significant evidence of recombination was detected by the PHI test in both S2 and S4 alignments. When putative recombinant sequences were removed from S2 and S4 alignments, the PHI test was no longer significant. Recombinant sequences of R5 or X4 phenotype were predominantly detected in tissue samples rather than peripheral lymphocytes. In subject S4 about two-thirds of the recombinant sequences were found in the brain. It is important to notice that the low rate of PCR-mediated recombination , and the significantly different distribution of recombinant sequences in different tissues makes highly unlikely that PCR-recombinants, if any, may have biased the results of the analysis. Recombination breakpoints were mapped by bootscanning. In all 53 recombinant sequences, putative breakpoints were localized within the C2 domain, while no recombination was found within V1-V2. Representative bootscannings of two R5 and two X4 sequences are shown in Tempo and mode of R5 and X4 variants evolution during infection Subjects S2 and S4 harbored X4 variants both in PBMCs and the thymus, and were selected for an in depth study of in vivo evolution of R5 and X4 quasispecies. Non-recombinant sequences in PBMCs over the course of infection and from terminal tissues were combined for a high-resolution phylodynamic analysis. The genealogy of HIV-1 V1-V3 sequences, sampled over two years of infection from subject S2, showed three main lineages, A, B, and C. Each lineage was well supported by.75% Bayesian posterior probability, p values#0.001 in the zero branch length test, and.70% bootstrap. Moreover, both ML and Bayesian-based methods inferred the same root for the tree. Lineage A including the R5 viral sequences from early and 1828342 late PBMCs displayed clear temporal structure. Strains from early PBMC samples passed through an initial population bottleneck, followed by a second bottleneck IC261 web leading to the emergence of a new monophyletic subcluster that included sequences only from late PBMC samples. HIV-1 sequences from post mortem lung, spleen, and lymph nodes were exclusively R5, clustered as a separate phylogenetic lineage within clade B, and included at least two bottlenecks. Temporal structure was also evident in clade C where initial populations of R5 and X4 strains isolated from early PBMCs were replaced through a bottleneck by a subclade containing only X4 variants from early and 16103101 late PBMCs and from the thymus. HIV-1 X4 strains isolated from late PBMCs emerged after a second bottleneck, while the last bottleneck gave rise to a subclade consisting exclusively of X4 viral strains from the thymus. The inferred phylogeny for subject S4 was based on evaluation of sequences over a period of about 6.5 years and indicated at least three statistically supported clusters: A, B, and C. All viral sequences from PBMCs at sampling time T1 and T2, as well as from the brain, displayed V3 loops predicted to use the R5 Analysis of recombinant sequences HIV-1 frequently recombin
