Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, United States of America.
PLoS One. 2012;7(10):e47818. doi: 10.1371/journal.pone.0047818. Epub 2012 Oct 24.
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.
深度测序技术正在彻底改变我们对 HIV/SIV 进化的认识。已知在 MHC 相同的动物中，CD8 T 淋巴细胞（CD8-TL）表位内的急性 SIV 序列变异是相似的，但我们不知道这种情况是否会持续到慢性期。我们现在使用敏感的深度测序方法来确定感染克隆 SIV 的 MHC 相同动物在慢性期整个编码序列中病毒变异是否相似。我们对从感染 SIVmac239 的 4 只 MHC 相同的毛里求斯食蟹猴（MCM）独特队列中分离的 SIV 基因组的整个编码序列进行焦磷酸测序，在感染后 48 周，该队列中的 1 只 MCM 成为精英控制者。在 3 只非控制者中，我们发现动物之间的基因组序列相似，并且与 Sanger 测序方法相比，在 64%的 CD8-TL 表位中检测到序列复杂性增加。当我们比较 MHC 匹配的控制者与 3 只非控制者之间的序列时，我们发现控制者中的病毒群体多样性较低，积累的变异与非控制者中的病毒群体不同。重要的是，我们发现病毒 cDNA 的初始 PCR 扩增对检测到的序列没有显著影响，这表明通过焦磷酸测序 PCR 扩增的病毒 cDNA 获得的数据准确地代表了动物内复制的序列多样性。这表明，当感染相同的克隆病毒株时，具有相似病毒载量的 MHC 相同动物的整个 SIV 编码序列的慢性序列多样性是相似的。此外，我们对全基因组 SIV 测序的方法准确反映了复制病毒群体中存在的序列多样性。总之，我们的研究表明，对免疫缺陷病毒进行全基因组焦磷酸测序可以全面、公正地描绘序列多样性，并且在评估作为疫苗免疫原的哪些序列时可能是一种有用的方法。
