【作者】 苗文泉；

【导师】 李敬云；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2008， 硕士

【摘要】 分离我国流行的HIV毒株并进行相关基因序列特征的分析是建立我国HIV毒种库,认识我国HIV的生物学特征、分析其流行和变异重组规律的重要基础;全基因组的序列测定有助于阐明毒株的进化特征,可以为有关病毒流行特征等方面的研究提供一个完整、清楚的遗传学和分子生物学背景。本研究首先对我国部分地区流行的HIV-1毒株进行分离培养,对其中的部分毒株进行了gag, pol, env基因特征进行分析,对2株病毒进行了全基因组序列测定和研究。目的分离我国部分地区流行的HIV-1毒株,了解分离毒株的生物学及基因序列特征。方法用外周血单核细胞（PBMCs）体外共培养的方法从我国HIV-1感染者中分离原代HIV-1毒株,分析毒株的体外生长动力学特征和融合诱导性。提取病毒基因组RNA,反转录PCR扩增病毒的gag,pol,env区基因并进行序列测定,使用软件Clustal,BioEdit version,MEGA3.1对基因序列特征进行分析。提取NX2、SH52两个HIV-1毒株的前病毒基因组DNA,套式PCR方法扩增全长基因组并分段测序,使用SimPlot软件进行基因序列重组分析。结果将本室冻存的67株初步分离呈阳性反应的毒株复苏传代,得到37株稳定传代的病毒;从5个省市的270名HIV-1感染者外周血中分离出58株HIV-1原代毒株。分离株的体外生长动力学呈现快/高、慢/高、慢/低三种类型。病毒载量越高分离的阳性率(HC=78.959,χ²_（1）0.01=6.63.P<0.01)越高、同时病毒分离株呈快高型生长的比例增大。52株分离株的序列呈现B（欧美B、泰国B’）、CRF01_AE重组、CRF_BC重组和G 4种亚型;以B亚型分布最广,CRF01_AE重组株主要分布在广西,上海发现G亚型病毒。V3环顶端四肽GPGR广泛的分布于B亚型和CRF_AE亚型中。CRF01_AE亚型毒株的V3环平均净电荷数略高于B亚型;基因离散率分析显示,env基因离散率远高于gag和pol基因;CRF01_AE亚型病毒pol和env区基因离散率均在三种亚型中最高。表型预测显示多数为利用CCR5辅助受体、M嗜性的毒株。扩增得到NX2 08BC重组亚型和SH52 G亚型全长基因组序列。NX2 CRF08_BC重组亚型在3个位置发生重组,分别位于gag,pol和nef区,重组位点位于1000nt,2600nt和9200nt。结论传代分离出我国部分地区的HIV-1流行毒株95株;毒株呈现多种体外复制动力学特征,并与血浆HIV-1病毒载量水平相关;毒株呈现多种亚型（B、CRF01_AE重组、CRF_BC和G）,在不同传播途径和地区具有不同的分布;env基因离散率远高于gag和pol基因, HIV-1毒株多数是不致细胞病变的M嗜性、NSI表型;V3环序列变化较大。得到2株HIV-1毒株的全长基因组序列,亚型分别为CRF08_BC和G亚型。更多还原

【Abstract】 Isolations and sequences analyses of HIV-1 strains circulating in China afford a solid basis to build the HIV strains stocks, acquire their biological features and analyze the rules of pandemic, diversities and recombinations.Full-length genomes sequencing play a important role in clarifying evolving characteristics of different clades, and supply a complete, clear genetics and molecular background to the virus epidemiology characters.In the research descried this article, we isoalted and cultured several HIV-1 strains from partional regions in China and analyze their important structural genes sequences such as gag, pol, env, in particular, full-length genome sequences analyses were also performed on the rare circulating isolates .Objective To isolate HIV-1 strains circulating in some regions of China, then learn their biological and sequence feature.Methods Primary HIV-1 strains were isolated from HIV-1 infected persons using PBMCs coculture method, then their growth dynamics feature and capacity of inducing syncytium were identified. Meanwhile, RNA was prepared and gag、pol、env gene was amplified by RT-PCR. Then the sequence was determined and sequence was analyzed by Clustal,BioEdit version,MEGA3.1 .The DNA of NX2 and SH52 were prepared and the full-length sequence was amplified by nested-PCR, sequence was analyzed by SimPlot.Results In 67 HIV-1 strains, 37 virus were subculturied steadily. 58 primary strains were isolated from 270 HIV-1 infected persons. It showed us three types growth dynamics that were rapid/high、slow/high、slow/low. It suggested that strains in samples of higher viral loads get a higher isolating rat(eH_C=78.959,χ²_（1）0.01=6.63.P<0.01),and the proportion of rapid/high strains in samples of higher viral loads were bigger than that in samples of lower viral loads. The sequence of 52 strains turned out to be 4 subtypes of B、CRF01_AE、CRF_BC and G. The strains of subtype B existed in the widest area. The subtype CRF01_AE strains mostly existed in Guangxi.Subtype G was found in shanghai.V3 loop central motif-GPGR mostly existed in the subtype B and subtype CRF01_AE The number of net charge in V3 loop in the subtype CRF01_AE is the higher in the subtype B.Genic diversity in env is bigger than gag and pol. Genic diversity in pol and env in subtype CRF01_AE is the biggest in three subtypes.Majority of the isolated strains were predicted to be M-tropism and CCR5 using strains.The full-length sequence of NX2 （subtype CRF_BC） and SH52（subtype G）was amplified by PCR.NX2 CRF08_BC showed three breakpoints located at positions at 1000 in gag、2600 in pol and 9200 in nef.Conclusion 95 HIV-1 strains circulating in some regions of China were subculturied and isolated; The replication dynamics of the isolated strains varied and related to HIV-1 viral loads in plasma; There existed many kinds of subtype as B、CRF01_AE、CRF07_BC and G, and the distribution of the subtype varied according to different regions and different transmission path; Genic diversity in env is bigger than gag and pol. Majority of the isolated strains were predicted to be M-tropism and NSI strains; The sequence variation of V3 regions were apparent.Two full-length sequence of subtype 08BC and G were amplified.更多还原