SIV Genome-Wide Pyrosequencing Provides a Comprehensive and Unbiased View of Variation within and outside CD8 T Lymphocyte Epitopes |
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| Authors: | Austin L. Hughes Ericka A. Becker Michael Lauck Julie A. Karl Andrew T. Braasch David H. O’Connor Shelby L. O’Connor |
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| Affiliation: | 1. Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, United States of America.; 2. Wisconsin National Primate Research Center, University of Wisconsin, Madison, Wisconsin, United States of America.; 3. Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin, United States of America.; University of Pittsburgh Center for Vaccine Research, United States of America, |
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| Abstract: | Deep sequencing technology is revolutionizing our understanding of HIV/SIV evolution. It is known that acute SIV sequence variation within CD8 T lymphocyte (CD8-TL) epitopes is similar among MHC-identical animals, but we do not know whether this persists into the chronic phase. We now determine whether chronic viral variation in MHC-identical animals infected with clonal SIV is similar throughout the entire coding sequence when using a sensitive deep sequencing approach. We pyrosequenced the entire coding sequence of the SIV genome isolated from a unique cohort of four SIVmac239-infected, MHC-identical Mauritian cynomolgus macaques (MCM) 48 weeks after infection; one MCM in the cohort became an elite controller. Among the three non-controllers, we found that genome-wide sequences were similar between animals and we detected increased sequence complexity within 64% of CD8-TL epitopes when compared to Sanger sequencing methods. When we compared sequences between the MHC-matched controller and the three non-controllers, we found the viral population in the controller was less diverse and accumulated different variants than the viral populations in the non-controllers. Importantly, we found that initial PCR amplification of viral cDNA did not significantly affect the sequences detected, suggesting that data obtained by pyrosequencing PCR-amplified viral cDNA accurately represents the diversity of sequences replicating within an animal. This demonstrates that chronic sequence diversity across the entire SIV coding sequence is similar among MHC-identical animals with comparable viral loads when infected with the same clonal virus stock. Additionally, our approach to genome-wide SIV sequencing accurately reflects the diversity of sequences present in the replicating viral population. In sum, our study suggests that genome-wide pyrosequencing of immunodeficiency viruses captures a thorough and unbiased picture of sequence diversity, and may be a useful approach to employ when evaluating which sequences to include as part of a vaccine immunogen. |
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