title: SIV genome-wide pyrosequencing provides a comprehensive and unbiased view of variation within and outside CD8 T lymphocyte epitopes.
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.
authors.affiliation: Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, United States of America.
authors.forename: Austin L
authors.initials: AL
authors.lastname: Hughes
authors.forename: Ericka A
authors.initials: EA
authors.lastname: Becker
authors.forename: Michael
authors.initials: M
authors.lastname: Lauck
authors.forename: Julie A
authors.initials: JA
authors.lastname: Karl
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Braasch
authors.forename: David H
authors.initials: DH
authors.lastname: O'Connor
authors.forename: Shelby L
authors.initials: SL
authors.lastname: O'Connor
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000821:virology*; D004279:DNA, Viral / Q000235:genetics; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D016679:Genome, Viral; D059014:High-Throughput Nucleotide Sequencing; D008252:Macaca fascicularis / Q000235:genetics / Q000276:immunology / Q000821:virology*; D008285:Major Histocompatibility Complex; D012367:RNA, Viral / Q000235:genetics; D016097:Simian Acquired Immunodeficiency Syndrome / Q000235:genetics / Q000276:immunology / Q000821:virology*; D015302:Simian Immunodeficiency Virus / Q000235:genetics* / Q000276:immunology; D019562:Viral Load
chemical_list: D004279:DNA, Viral; D018984:Epitopes, T-Lymphocyte; D012367:RNA, Viral