SIV genome-wide pyrosequencing provides a comprehensive and unbiased view of variation within and outside CD8 T lymphocyte epitopes

• Published in: PloS one 2012-10, Vol.7 (10), p.e47818-e47818
• Main Authors: Hughes, Austin L, Becker, Ericka A, Lauck, Michael, Karl, Julie A, Braasch, Andrew T, O'Connor, David H, O'Connor, Shelby L
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Animals; Antigenic determinants; Biology; CD8 antigen; CD8-Positive T-Lymphocytes - immunology; CD8-Positive T-Lymphocytes - virology; Controllers; DNA sequencing; DNA, Viral - genetics; Drug resistance; Epitopes; Epitopes, T-Lymphocyte - chemistry; Epitopes, T-Lymphocyte - genetics; Epitopes, T-Lymphocyte - immunology; Evolution (Biology); Gene sequencing; Genome, Viral; Genomes; Genomics; High-Throughput Nucleotide Sequencing; HIV; Human immunodeficiency virus; Immunodeficiency; Infection; Infections; Laboratories; Lymphocytes; Lymphocytes T; Macaca fascicularis - genetics; Macaca fascicularis - immunology; Macaca fascicularis - virology; Major Histocompatibility Complex; Medicine; Monkeys & apes; Mutation; Nucleotide sequence; Pathology; Population; Population studies; Replicating; RNA, Viral - genetics; Simian Acquired Immunodeficiency Syndrome - genetics; Simian Acquired Immunodeficiency Syndrome - immunology; Simian Acquired Immunodeficiency Syndrome - virology; Simian immunodeficiency virus; Simian Immunodeficiency Virus - genetics; Simian Immunodeficiency Virus - immunology; Stainless steel; Studies; T cells; Variation; Viral Load; Viruses
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0047818

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.