THE EVOLUTIONARY ANALYSIS OF EMERGING LOW FREQUENCY HIV-1 CXCR4–USING VARIANTS THROUGH TIME – AN ULTRA-DEEP APPROACH

Large-scale parallel pyrosequencing produces unprecedented quantities of sequence data. However, when generated from viral populations current mapping software is inadequate for dealing with the high levels of variation present, resulting in the potential for biased data loss. In order to apply the 454 Life Sciences’ pyrosequencing system to the study of viral populations, we have developed software for the efficient processing of variable sequence data. Here we demonstrate our software by analyzing two temporally sampled HIV-1 intra-patient datasets, from a clinical study of maraviroc. This drug binds the CCR5 co-receptor, thus, preventing HIV-1 infection of the cell. The objective here is to determine viral tropism (CCR5 versus CXCR4 usage) and track the evolution of minority CXCR4-using variants that may limit the response to a maraviroc-containing treatment regimen. Five time points (two prior to treatment) were available from each patient. Our software, in conjunction with genotypic algorithms that predict HIV tropism, was used to quantify the viral population structure pre- and post-treatment. In both cases low frequency CXCR4-using variants (2.5 – 15%) were detected prior to treatment. Following phylogenetic inference these variants were observed to exist as distinct statistically supported lineages that were maintained through time. Combined with viral load and CD4 cell count data our analysis confirms the role of pre-existing CXCR4-using virus in the emergence of maraviroc insensitive HIV. Additionally we have characterized the effects of divergence on initial read mapping and demonstrated the importance of utilizing population specific template sequences in relation to the analysis next generation sequence data. The software will have utility for the study of intra-host viral diversity and evolution of other fast evolving viruses, and is available from http://www.bioinf.manchester.ac.uk/segminator/.