【作者】 辛若雷；

【导师】 邵一鸣； 何翔； 邢辉；

【作者基本信息】 中国疾病预防控制中心 ， 免疫学， 2009， 博士

【摘要】 HIV-1 CRF07_BC重组毒株是我国流行的主要HIV毒株之一,主要分布于静脉吸毒人群（intravenous drug users,IDUs）中。CRF07_BC最初在四川和新疆IDUs中发现。了解四川和新疆流行CRF07_BC毒株的个体和群体变异特征并分析其流行情况对于阐明该毒株免疫逃逸和适应性机制、分子进化等具有重要的科学意义和应用价值。本文选择四川和新疆HIV-1感染者,从个体和群体水平分析CRF07_BC毒株的变异特征,探索其遗传动态变化并重建CRF07_BC在四川和新疆的流行史。本文对四川和新疆22例CRF07_BC感染者进行gag基因和gp120基因单基因组扩增和序列测定,分析个体内序列变异特征;选取2007-2008年从四川和新疆采集195例HIV-1感染者分析群体水平的遗传多样性特征。利用1996-2008年采集样本（四川108份,新疆216份）的env基因序列,进行贝叶斯合并理论方法重建CRF07_BC在四川和新疆的流行史,并分析其遗传动态变化特征。对感染者体内病毒序列分析表明CRF07_BC感染者血浆内毒株呈现复杂的准种变异。序列中存在不同的点突变和插入/缺失模式、以及个体内毒株间重组等造成氨基酸组成、N糖基化程度（gp120糖蛋白,18-34个N糖基化位点）和功能区长度多态性改变,在进化树上聚集成复杂的群体结构。CRF07_BC毒株的高度变异性主要集中在gp120基因,尤其是4个高变环区（V1、V2、V4和V5）。总体上,V4/V5区平均两两比对基因距离（0.024-0.178）高于V1/V2区（0.012-0.117）,呈现出独立进化特征,但两者的氨基酸长度多态性存在微弱的正相关关系（r=0.359,P＜0.001）。个体内高度变异gp120基因中包含相对保守的功能区（V3区）,表现在长度多态性低、均使用CCR5辅助受体、顶端四肽为GPGQ;这可能是宿主微环境对CRF07_BC的功能性选择和病毒适应性的结果。CRF07_BC gag基因的遗传多样性主要集中在p17区和p2-p6区。43.6%流行毒株p6区存在7个氨基酸残基缺失突变;18%病例的p6区CTL表位存在3-12个氨基酸残基插入,脯氨酸（Pro）和丙氨酸（Ala）所占比例增加。这些插入/缺失突变造成蛋白结构和功能的改变,可能有助于病毒免疫逃逸和适应性增加。系统进化分析结果表明四川和新疆流行CRF07_BC具有较高的遗传同源性,并起源于共同的祖先,总体上两省获得的序列交错分布,没有与地域、民族或样本采集时间相关的聚集倾向,仅在某些氨基酸位点上表现出与地域和民族相关联的氨基酸组成偏性。运用贝叶斯合并理论推断CRF07_BC传入四川和新疆的时间分别是1994.0年（95%置信区间,1991.9-1995.7）和1995.0（95%置信区间,1993.7-1995.8）年。该结果符合CRF07_BC从我国西南（云南）经由四川沿贩毒路线传入新疆的传播路线假说。CRF07_Bc毒株进入四川和新疆后,大致分为三个流行阶段,包括病毒传入期（1994-1996年）、快速传播期（1997-2002年）和高水平流行期（2003-2008年）。在流行早期,CRF07_Bc毒株具有较低的遗传多样性;随着流行时间的延长,群体流行CRF07_BC毒株表现出较高的遗传多样性。现有的数据表明四川和新疆CRF07_BC传播速率降低,有效感染群体接近饱和,流行毒株表现出较高的遗传多样性（gp120基因距离接近0.09）和较快的进化速率(7-8×10^-3替换/位点/年);但CRF07_BC感染仍然维持较高的流行水平,加强HIV/AIDS的防控势在必行。更多还原

【Abstract】 HIV-1 CRF07_BC is one of major HIV strains prevailing in China, mainly distributed in intravenous drug users （IDUs）. The HIV-1 CRF07_BC infection was first identified among IDUs in Sichuan and Xinjiang in 1996. It is very important to understand the genetic variation of CRF07_BC in Sichuan and Xinjiang within an individual or population, for the purpose of delineating the mechanisms of immune escape, fitness and molecular evolution. We analyzed the gpl20 and gag genes amplified from CRF07_BC infected cases in Sichuan and Xinjiang to explore the genetic diversity in an individual or at a population level, then to reconstruct its epidemic history.HIV-1 infected cases were recruited from Sichuan and Xinjiang, and the gp120 and gag genes were obtained using single genome amplification and sequencing to determine the subtype and explore the genetic characteristics in an individual. Another 195 cases were recruited among IDUs in Sichuan and Xinjiang in 2007 and 2008. The C2-V4 fragments of env gene obtained in Sichuan （n=108） and Xinjiang （n=216） covering 1996-2008, were used to reconstruct the CRF07_BC epidemic history and delineate genetic dynamics of CRF07_BC there, by Bayesian coalescent inference.In CRF07_BC-infected individuals, there was complex variation forms, including different models of numerous substitutions, insertion/deletions, and interlineage genetic recombination among quasispecies, et al, which caused the shift of amino acid （aa） constitutes, the numbers of putative N-linked glycosation sites, and the length polymorphisms in domains. All these factors brought comprehensive genetic diversity into the CRF07_BC strains, and sequences thereby formed complex structure in phylogeny trees. The gp120 gene accounted for the major variation of CRF07_BC, especially in the four variable loops （V1, V2, V4 and V5）. Although the independent evolution with higher average pairwise distances （APD） in V1/V2 （0.012-0.117） than that those in V4/V5 （APD 0.024-0.178） were observed, the weak correlations were formed between the lengths of the V1/V2 and those of V4/V5 （r=0.359, P<0.001）. V3 loop of gp120 gene exhibited high degree of conservation, with the constant length of amino acid residues, the unanimous coreceptor usage （CCR5 tropism） and the conservative GPGQ tetra-peptide in the critical crown of V3 loop. These phenomena may be the consequence of high functional selection and virus fitness. The pl7 and p2p6 regions likewise expressed high genetic diversity in CRF07_BC gag gene. In Sichuan and Xinjiang, 43.6% of the gag genes harbored 7-aa deletion in the p6 overlapping coding regions, while another 18% cases had three to twelve amino acids insertion in p6 region of the gag gene, which enhances the weight of proline and alamine. These indels may facilitate CRF07_BC immune escape and fitness.Phylogenetic analysis indicated that CRF07_BC circulating in Sichuan and Xinjiang shared high homology and were presumed to originate from a common ancestor. All the sequences were intermingled in phylogenetic trees, irrespectively of region, nationality, or sampling year differences. The overall variation of CRF07_BC was indistinctive, except that the amino acid constitutes in partial sites showed the correlation to regions and nationality.By Bayesian coalescent inference, we reconstructed the epidemic history of CRF07_BC in Sichuan and Xinjiang. CRF07_BC strains were first introduced into Sichuan in 1994.0 （95% confidence interval, 1991.9-1995.7）, and Xinjiangin 1995.0, （95% confidence interval, 1993.7-1995.8）. Combined with the other literatures, our observations supported the hypothesis that the CRF07_BC was transmitted from southwest China （Yunnan, in 1993.3） into Xinjiang via Sichuan along the heroin trafficking route. After the introduction of CRF07BC into Sichuan and Xinjiang, the CRF07_BC epidemic was established and experienced similar epidemic history of three successive phases, including the introduction and establishment of CRF07_BC epidemic （1994-1996）, the fast transmission and spread （1997-2002）, and high prevalence of CRF07_BC （2003-2008）. During the early phase of CRF07_BC epidemic, the strains were transmitted among a small IDU population, with high genetic homogeneity and low diversity. With the progress of the epidemic, the CRF07_BC diversity was steadily increasing. The present data suggested that the transmission rate of CRF07_BC slowed down in Sichuan and Xinjiang nowadays, with effective population getting close to saturation. Correspondingly, the circulating strains showed greater genetic diversity （APD of gp120 gene, nearly 0.09） and faster evolutionary rate (7-8×10^-3 substitutions/site/year). A high prevalence of CRF07_BC infections was maintained, and more measures should be taken to control the HIV/AIDS epidemic in Sichuan and Xinjiang.更多还原