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Background:  Gut-associated lymphoid tissue (GALT) represents more than half of all body lymphoid tissue, and exists in a heightened state of activation, thus offering a favorable milieu for viral replication. Despite its importance to transmission and pathogenesis, HIV evolution within GALT has rarely been studied. We hypothesize that GALT is an important site for the evolution of pathogenic genotypes including drug resistance and CXCR4 co-receptor usage.

Methods:  We present preliminary data from an ongoing prospective longitudinal observational study of HIV-1 evolution within the lower GI tract. We sampled blood and rectosigmoid mucosal biopsy material at 1 or more time points in 5 subjects with chronic HIV-1 infection. Viral nucleic acids were extracted from plasma (RNA), PBMC (DNA), and GALT (DNA and RNA), reverse transcribed as necessary, PCR-amplified, cloned, and sequenced by standard methods.

Results:  A total of 393 HIV-1 env and 47 pol sequences were aligned using ClustalX, alignments were edited in MacClade 4.06, and neighbor-joining phylogenetic trees were reconstructed using Paup* 4.0. Drug resistance was determined using the Stanford Drug Resistance Database. Env gene co-receptor usage was deduced using a previously described position-specific scoring method (PSSM). All sequences were segregated according to patient by phylogenetic analysis, reflecting lack of cross contamination. Among 48 pol sequences isolated from 1 patient at a single time point, there was significant clustering between mucosal DNA and PBMC; 70% of PBMC sequences scored intermediate resistance to 1 or more nucleoside analog drugs (usually lamivudine), and the remainder scored as susceptible. Of the GALT sequences, 89% scored as intermediate or highly resistant (Wilcoxon rank test pf= 0.01). X4 variants were observed in an isolated clade in 1 patient, and occurred only within GALT and PBMC, but never in plasma. X4 clade branch lengths were significantly longer than other sequences present (p < 0.01) suggesting a diversifying selection in this clade.

Conclusions:  In this analysis of HIV-1 pol sequences, resistance to NRTI drugs appears to be more heavily represented in GALT than in blood. Appearance of the X4 co-receptor usage phenotype in GALT and PBMC may precede its appearance in plasma. X4-tropic viruses appear to evolve from rapidly replicating viral subpopulations under diversifying selective pressure. Further studies will be required to evaluate these observations.

Keywords: HIV-1; evolution; gut-associated lymphoid tissue