利用深度测序技术研究北京MSM人群HIV多样性

This study aims to investigate the HIV sequence diversity in 300 HIV-positive men who have sex with men (MSM) in Beijing. Study subjects were enrolled as part of a recently concluded National Science Foundation study, "Multi-component HIV Intervention Packages for Chinese MSM (MP3)". This study will be a prospective cohort study consisting of two follow-up surveys. Blood samples will be collected from Beijing MSM and HIV pol and env gene regions will be sequenced through RT-PCR. Phylogenetic analysis using MrBayes and BEAST under maximum likelihood and Bayesian phylogenetic models will be combined with demographic data to reconstruct the epidemic history of the main clusters of HIV transmission among Beijing MSM. Subsequently, deep sequencing will be performed using an Illumina Miseq deep sequencing platform. HIV gene specific fusion primers will be designed for mixing samples to reduce the cost of sequencing. We estimate that a minimum of 1000 reads per sample will be capable of discerning quasispecies diversity of up to 0.1%. The diversity and complexity of viruses will be measured by calculating the genetic distance between quasispecies sequences. Multiple infection or superinfection rates can also be evaluated by comparing data from deep sequencing and Sanger sequencing. These data will be used to clarify the mechanisms of HIV recombination, disease progression, and drug resistance emergence. Furthermore, combined analysis with epidemiological survey data on transmission networks will allow us to identify the social and behavioral factors influencing viral diversity in MSM. This study will provide an evidence base for HIV prevention and control measures that target the reduction of viral diversity as a means to reducing the generalized transmission of HIV among Beijing MSM and those in their risk behavior networks.

在已完成的1年期国家自然科学基金项目基础上，对已入组的300名HIV阳性MSM感染者开展2次前瞻性队列随访。通过桑格尔法获得pol和env基因序列，利用系统进化分析获得基因型。通过贝叶斯分析重建已知的标准型毒株的流行史。针对上述基因中适当的片段设计编码特异性的融合引物（用于混合多个样本同时进行测序，以降低测序成本），预计每个样本的最低序列数为1000条，可以鉴定0.1%的准种突变。对不同准种序列进行多样性分析，获得亚型、亚簇的鉴定结果。采用桑格尔测序和深度测序相结合的方法评估北京MSM人群病毒的复杂度、双重感染、新重组株的发生率及产生的机制；阐述HIV多样性对疾病进程和耐药性产生的影响；基于社会网络理论的流行病学调查，了解MSM人群HIV多样性发生的影响因素；进而解释HIV在我国MSM人群快速流行的原因。提出通过减少HIV多样性来达到切断HIV在MSM中二代快速传播的措施。

本研究对416名艾滋病阳性的北京MSM人群进行了队列随访研究，并建立了一种基于Illumina的二代测序平台的能检测HIV-1亚型和亚簇重组以及超感染的实验方法和分析方法，首次量化分析了MSM患者中的基因复杂程度，获得了北京 HIV-1阳性MSM人群高达9.28%超感染的数据。同时多因素Logistic回归分析结果显示，与HIV-1基因多样性相关的影响因素是无工作（P=0.03）,过去三个月使用新型毒品（P=0.03）,和通过互联网寻找男性性伴侣的方式（P=0.01）。同时随访研究发现MSM有较高比例的新发生的超感染，北京MSM人群中有较高的HIV-1基因多样性，反映了HIV阳性MSM人群复杂的高危行为。通过分子网络分析和卡方检验结果显示：感染非单一亚型多样性病毒患者入网率显著高于单一亚型感染者，其传播风险显著高于单一亚型感染者。48个月随访到的240个治疗患者中，看到的仅有的2例治疗效果差的患者，这2例患者虽然治疗时间超过36个月，但其最初感染的都是多样性病毒株，也许与治疗失败有关联性。