【作者】 房恩贞；

【导师】 夏雪山；

【作者基本信息】 昆明理工大学 ， 生物化工， 2009， 硕士

【摘要】 艾滋病(AIDS),即获得性免疫缺陷综合症,是由人类免疫缺陷病毒(HⅣ)慢性感染而引起。自1981年发现第一例艾滋病病人以来,AIDS已发展成严重危害人类健康的全球性流行病。HIV-1一个重要的生物学特性是基因序列的高度变异性,使得HIV-1在传播过程中产生了许多亚型和重组型,并具有时间性和地区性的分布特征。目前,云南省静脉吸毒人群仍是我国艾滋病感染率最高的人群之一,并以此为中心向普通人群扩散。鉴于云南省在我国HIV-1流行病学中的特殊地位,研究现时段云南省HIV-1的分子流行病特点,不仅有助于了解HIV-1传播规律,预测将来的流行态势,对HIV-1感染的预防控制以及疫苗的研制也具有重要意义。本研究利用简单快速的RT-Nested-PCR方法对来自云南省大理和开远的42例HIV-1毒株分gag-(P17-P24)、gag-Prot、RT三个区段分别进行扩增,扩增长度分别为1160bp,885bp,1010bp。最终得到有效基因序列gag-(P17-P24)为31份(73.8%),gag-Prot为41份(97.6%),RT为30份(71.4%)。将有效基因序列分段或拼接得到P17、P24、Pr、RT、P17-P24、P17-Pr、Pr-RT和P17-RT8个区段序列,分别进行系统进化分析。比较分型结果显示：无论是单独利用P17、P24基因区段还是联合P17-P24基因区进行亚型鉴定都存在一定的局限性,尤其对同源相似性较高的目的基因序列缺乏有效的区分能力；基于Pr、RT、gag(P17-Pr)、gag-pol(Pr-RT)基因区的测序分型方法能够相对稳定有效的鉴定一般亚型和中国流行的两种主要B’/C重组型,但对于形成复杂重组结构的毒株却无法准确的鉴定。基于P17-RT长约2.6kb的基因区域进行测序分型则是全长测序较好的替代方法,因为这个基因区包括了我国主要流行的CRF07和CRF08-BC重组毒株截然不同的重组位点,对于实验室小工作量的研究中,足以确定所有HIV-1亚型和我国主要的重组型。本研究在建立P17-RT基因区有效分型的基础上,对42份样本进行基因亚型分析发现：HIV-1在云南省静脉吸毒人群中以CRF08-BC重组模式为主要的流行型(开远,50.0%；大理,92.3%),却很少发现CRF07-BC(7.1%),另有6例样品是介于CRF07和CRF08-BC重组模式之间,经不同基因区的分型结果均有所不同,暂时命名为URFs-BC,占样本总数的14.3%。为进一步研究云南省静脉吸毒人群中HIV-1重组毒株的断点分布情况,我们重点分析了27例样品高度保守的gag-pol (2.6kb)基因区,通过Simplot软件分析,得出以下结论：CRF08_BCs和CRF07_BCs重组毒株在高度保守的gag-pol区仍以C亚型为主要骨架,在其不同位置上插入了两段和三段来自中国B’亚型的重组片段。而6个独特重组型(URFs)各有不同的重组形式,并且出现了明显的CRF07和CRF08-BC重组的相对重组断点。毒株DL310在2448-2472nt、3109-3207 nt、3250-3372 nt分别出现了三个可能的重组断点。毒株KY104的两个相对重组断点出现在1534bp-1619 nt和1654-2134 nt。KY137毒株的两个重组断点,分别位于1612-1619 nt和1654-1968 nt。毒株KY117的断点位于1612-1619 nt、2170-2188 nt、2617-2943 nt、3069-3255 nt; KY118的断点出现在1425-1507 nt、2067-2134 nt、2617-2712 nt、3207-3255 nt.毒株KY139相对重组断点位于2067-2092nt、2397-2511 nt和2601-2616 nt.进一步分段进化树和Information sites分析验证,绝大多数重组片段的Bootstrap值相对高于70%,并且Chi-Square (Pearson)检验均具有显著统计学意义,充分证实这6例URFs-BC毒株2.6kb的基因序列是在流行于中国的CRF07、CRF08-BC重组型的基础上,发生二次重组而成。综上所述,本研究建立了简便而有效测序分型方法,基于该方法我们对现阶段云南省静脉吸毒所致HIV-1感染人群的流行现状进行了较为系统的研究,为HIV-1感染者的药物治疗、预防控制措施的制定以及疫苗的研究提供了重要的分子流行病学资料。更多还原

【Abstract】 Acquired immune deficiency syndrome (AIDS) is a kind of immune deficiency disease which is caused by a chronic infection with HIV. Since the first case was identified in 1981, AIDS has become a serious global epidemic disease that has bad effect on the health of humankind. One of the significant characteristics of HIV is its high genetic variability, which makes HIV-1 generate a lot of subtypes and recombinant types during its propagation process, and presents the time and regional distribution characteristics. At present, intravenous drug users (IDU) in Yunnan Province of China are still the people who have the highest AIDS infection rate among the crowd. The epidemic has already begun to diffuse from the high-risk groups (IDU, as the center of diffusion) to general population. In view of the special status of Yunnan Province in Chinese HIV-1 epidemiology, it’s necessary to research the actual HIV-1 molecular epidemiological characteristics of Yunnan Province. The research will not only help to know HIV-1 disseminative laws, to predict the future trend of the epidemic, but also have great significance on HIV-1 infection prevention and control as well as the manufacture of vaccine.Using an easy-and-fast RT-Nested-PCR method to amplify three regions (respectively named:gag(P17-P24), gag-Prot, RT; length:1160bp,885bp, 1010bp) of forty-two HIV-1 samples from KaiYuan Prefecture and Dali Prefecture in Yunnan Province. Ultimately, we got 31 (73.8%)pieces gag(P17-P24),41 pieces (97.6%) Pr and 30 pieces (71.4%) RT effective gene sequences from the 42 samples respectively. By segmentalizing or jointing effective gene pieces, we obtained 8 regions--P27, P24, Pr, RT, P17-P24, P17-Pr, Pr-RT and P17-RT. Phylogenetic trees analysis shows that both the methods of using P17、P24 gene separately and of using P17-P24 jointed regions have their own limitation for determining the subtype, especially there is a lack of effective ability to distinguish the target gene fragments which have high homologous similarity. Genotyping assay based on Pr、RT、gag(P17-Pr)、gag-pol(Pr-RT) gene regions can effectively and steadily identify general subtypes and two kinds of B’/C recombinant strains mainly prevalent in China. But the method cannot identify the strains that have complex recombination structure precisely. Identification of genetic subtypes based on sequencing gene region P17-RT (2.6kb) is a good way to replace full-length sequencing, because the region has both completely different recombination sites of CRF07 and of CRF08-BC strains. Little workload in the lab will be enough to define all of subtypes and the recombinant types in China. Using this method, we found that the majority of IDUs were infected with CRF08-BC, (50.0% in Kaiyuan; 92.3% in Dali), whereas CRF07-BC was present in only low prevalence (3 of 42, 7.1%). Interestingly, there are six islates(14.3%), that branch out on the top of the phylogenic tree. These divergent viruses are probably derived from some novel B’/C recombinants, temporarily defined as URFs-BC.We had a focused analysis on 27 gag-pol (2.6kb) gene regions which are highly conservative to do some further study on the breakpoint distribution state of the HIV-1 recombinant strains among IDUs in Yunnan. By Simplot software analysis, we’ve got the result:CRF08_BCs and CRF07_BCs were comprised of subtype C throughout the majority of 2.6kb genome with two and three distinct B’subtype segments respectively. Moreover, these six URFs-BC appeared to be different recombinants, which showed structural similarity to both CRF07-BC and CRF08-BC in different regions of the gag-pol genome. The breakpoint distributions across the six HIV-12.6kb genome regions were scanned against a pair of parental strains of CRF07-BC and CRF08-BC. Three relative breakpoint regions were identified in sample DL310 at positions 2448-2472nt、3109-3207nt and 3250-3372nt(nucleotide numbering relative to HXB2). For the sample KY104, the first breakpoint is at approximately 1534-1619nt, while another breakpoint at position 1654-2134nt. Sample KY137 also presented two relative breakpoints at about 1612-1619nt and 1654-1968nt. Breakpoints of sample KY117 and KY118 are at 1612-1619nt, 2170-2188nt,2617-2943nt,3069-3255nt and 1425-1507nt,2067-2134nt,2617-2712nt, 3207-3255nt, respectively. Finally, sample KY139 has the breakpoints at positions 2067-2092nt,2397-2511nt and 2601-2616 nt.By further analysis of Explorotory trees and information sites, we have the finding that the Bootstrap value of most recombinants is more than 70% relatively, and the Chi-Square (Pearson) tests boast outstanding statistical significance. It can prove to be true that 6 samples belong to a new URFs-BC, which is likely to be generated by the recombinants between the CRF07-BC and CRF08-BC spreading in China.In summary, a simple and effective assay for genetic subtyping is established. Using this method, the systematic molecular epidemiology of HIV-1 infections among IDUs in Yunnan province have been investigated in our study, the observation will provide an important data for the therapy, prophylaxia and vaccine research of HIV-1.更多还原