我国深圳地区HIV分子流行病学调查及优势毒株感染性克隆的制备

发布时间：2018-05-18 12:19

本文选题：人类免疫缺陷病毒(HIV) + 分子流行病学调查　；参考：《安徽医科大学》2017年硕士论文

【摘要】：第一部分深圳地区HIV分子流行病学调查1992年,深圳发现首例HIV阳性感染者。随后深圳市报告的HIV/AIDS病例数呈现逐年上升趋势,年平均增长率高达24.7%;病例感染途径亦不断发生变化,由早期的IDU传播转变为以性传播为主,HIV感染呈现出从高危人群向普通人群快速扩散的态势。艾滋病的出现以及快速传播成为深圳市重要的公共卫生问题。鉴于深圳市存在大量非户籍人口,人员流动频繁、人口结构复杂,迫切需要对其进行艾滋病分子流行病学调查,以阐明其HIV流行模式。目的:对深圳地区传播的HIV进行分子流行病学调查,了解HIV流行趋势。方法:采用随机数字法挑选深圳地区2015年新确认HIV感染者作为研究对象,收集人口统计学信息;利用核酸提取试剂盒获取样本基因组,采用逆转录/巢式PCR的方法扩增HIVgag、pol基因片段,扩增片段纯化后测序并进行序列拼接;将各片段基因序列提交Los Alamos HIV Databases,分析亚型并构建系统进化树;扩增、测定URF病毒全长序列并进行重组分析;将深圳序列与全国序列比对后构建系统进化树,进行溯源分析;pol基因序列提交斯坦福大学耐药数据库检测耐药位点;利用HCV酶联免疫试剂盒检测样本中HCV抗体存在情况;将深圳地区2015年HIV流行状况与2013年进行比较,了解深圳HIV的流行变化;采用系统进化的方法对HES感染人群中的MSMs进行甄别研究。结果:随机法获得深圳市2015年新报告HIV感染者250例,其中88.4%为男性感染者;感染者年龄以青壮年为主,其中15~29岁人群高达49.2%;96.8%感染者传播方式为性传播,其中MSM占52%。主要传播亚型为CRF01_AE,CRE07_BC,CRF55_01B,分别占31.96%,37.90%,10.50%;URF比例较高,占7.76%。获得的样本中耐药率为2.8%,HIV与HCV共感染人数仅7人(2.8%)。溯源分析发现深圳样本中,CRF01_AE亚型中的HESs与MSMs分别形成单独的流行簇,与全国CRF01_AE的流行簇相重合,而CRF07_BC则无明显流行簇出现。与2013年本室获得的深圳市HIV分子流行病学数据进行比较时发现15~29岁感染者在2015年明显增加,差异性统计学检验显示P0.05,具有统计学意义。2013年和2015年流行亚型均为CRF01_AE、CRF07_BC、CRF55_01B,各亚型所占比例无明显变化。耐药率2013年与2015年分别为0.8%和2.8%;与HCV共感染人数分别为23人(9.2%)和7人(2.8%),主要人群均集中在IDU或HES中男性人群。溯源分析中2013年与2015年差别不大,CRF07_BC不聚集成簇,CRF01_AE存在单独的MSMs流行簇和HESs流行簇。利用系统进化树分析甄别了深圳感染CRF01_AE的HES男性感染者中约有11%的人是由于男男同性性行为感染HIV。结论:深圳地区HIV主要在男性性传播人群中扩散,感染者年龄呈现年轻化趋势;HIV感染亚型分布的明显多样,URF比例高,流行状况复杂;因为特殊的社会、文化背景,深圳地区部分MSM人群在现场调查中不愿暴露其男男性行为方式。第二部分深圳地区优势HIV毒株(CRF01_AE)感染性克隆的构建1994年我国发现第一例CRF01_AE毒株后,该毒株快速传播,目前已经成为我国大部分地区的(除了西南地区)主要流行亚型。在一些省份,超过80%的新报告病例为CRF01_AE感染。由于性传播造成的感染不断增加,CRF01_AE亚型在全国HIV感染者中所占比例也不断增加。在深圳地区,CRF01_AE也是主要流行亚型之一,因此对CRF01_AE流行病学和发病机理的研究刻不容缓。感染性克隆不仅是确定特定基因生物学效应的有力工具,更是研究病毒致病基因与人体免疫反应的有效手段。因此构建CRF01_AE病毒的感染性克隆对于深入研究CRF01_AE毒株的致病机制至关重要。目的:构建CRF01_AE毒株的感染性克隆,为深入研究其致病机制奠定基础。方法:采用半巢式PCR方法分两段扩增CRF01_AE毒株全长基因组,将扩增产物纯化后分别连接T载体构建半分子克隆;利用Aar I和Xhol I限制性内切酶酶切两个半分子克隆,将两个半分子片段进行连接,构建全长克隆;将获得的全长克隆转染HEK293T细胞,包装CRF01_AE感染性克隆的衍生病毒。将包装的病毒感染MT2细胞,观察细胞病变,测定获得的细胞培养上清的p24和TCID50;以p24的量为纵坐标,时间为横坐标,绘制衍生病毒在MT2上的复制动力学曲线,比较其与野生型病毒的复制能力差异;利用表达不同受体的U87细胞测定衍生病毒的嗜性。结果:经过PCR、酶切、连接、转化成功构建CRF01_AE全长克隆,获得长度13630bp的全长克隆质粒,其中病毒序列长为9702bp,具有15个独立的开放阅读框。将全长克隆质粒转染293T细胞,细胞培养上清的p24为0.375ug/ml;转染获得的衍生病毒再次感染MT2细胞,观察到明显的细胞病变,其培养上清的p24为19.1ug/ml,TCID50为15588;衍生病毒的复制动力学曲线与其野生株病毒没有明显差异,复制能力相近;利用U87测得CRF01_AE感染性克隆的衍生病毒为双嗜性病毒。结论:我们成功构建了一株完全来自野生株病毒并与其有相似复制能力的CRF01_AE感染性克隆,为研究病毒的致病机理以及疫苗研究提供了有力的工具。
[Abstract]:The first part of the Shenzhen area HIV molecular epidemiology survey in 1992, Shenzhen found the first case of HIV positive infection. Subsequently, the number of HIV/AIDS cases reported in Shenzhen showed an increasing trend year by year, the average annual growth rate was up to 24.7%; the infection route of the case also changed continuously from the early IDU transmission to sexual transmission, HIV infection showed The rapid spread of high risk population to the general population. The emergence and rapid spread of AIDS has become an important public health problem in Shenzhen. In view of the existence of a large number of non registered population in Shenzhen, the flow of people is frequent and the population structure is complex, so it is urgent to carry out an epidemiological survey on AIDS in order to clarify its HIV epidemic model. Objective: To investigate the molecular epidemiology of HIV spread in Shenzhen area and understand the trend of HIV epidemic. Methods: a random number method was used to select the newly confirmed HIV infected persons in Shenzhen area in 2015 as the research object and collect the demographic information; the DNA extraction kit was used to obtain the sample genome, and the HI was amplified by the reverse transcription / nested PCR method. Vgag, pol gene fragment, the amplified fragment was purified and sequenced and sequenced. The sequence of each fragment was submitted to Los Alamos HIV Databases, the subtype was analyzed and the phylogenetic tree was constructed; the full-length sequence of the URF virus was amplified and analyzed. The phylogenetic tree of the Shenzhen sequence was compared with the whole country sequence, and the source was traced and analyzed; P The ol gene sequence was submitted to the drug resistance database of Stanford University to detect the resistance loci, and the presence of HCV antibody in the samples was detected by HCV ELISA kit. The epidemic situation of HIV in 2015 in Shenzhen was compared with 2013, and the epidemic changes of HIV in Shenzhen were understood and the MSMs in the HES infected population was screened by systematic evolution. Results: 250 cases of HIV infection in Shenzhen in 2015 were obtained by random method, of which 88.4% were male infected persons; the age of the infected people was mainly young and young, among which the 15~29 years old was 49.2%. The transmission mode of 96.8% infected persons was sexually transmitted, and MSM accounted for the main transmission subtypes of 52%., CRF01_AE, CRE07_BC, CRF55_01B, respectively 31.96%, 37.90%, 10.50%. The ratio of URF to 7.76%. was 2.8%, and the number of HIV and HCV co infection was only 7 (2.8%). In the Shenzhen sample, HESs and MSMs formed a separate cluster in the CRF01_AE subtype, which coincided with the national CRF01_AE epidemic cluster, while CRF07_BC had no obvious cluster. The epidemiological data of HIV in Shenzhen were compared in 2015. The prevalence of 15~29 years was significantly increased in 2015. The difference statistical test showed P0.05. The statistical significance of.2013 and 2015 subtypes were CRF01_AE, CRF07_BC, CRF55_01B, and the proportion of each subtype had no obvious change. The rate of drug resistance in 2013 and 2015 was 0.8% and 2., respectively. 8%, the number of CO infection with HCV was 23 (9.2%) and 7 (2.8%). The main population was concentrated in the male population of IDU or HES. In the traceability analysis, the difference between 2013 and 2015 was not significant, CRF07_BC was not clustered, and CRF01_AE had a separate MSMs cluster and HESs cluster. The HES male infected with CRF01_AE in Shenzhen was identified by phylogenetic tree analysis. About 11% of the infected people are due to male sex sex infection HIV. conclusion: Shenzhen region HIV mainly spread among male sexually transmitted people, the age of the infected people is younger; the distribution of HIV infection subtypes is distinct, the proportion of URF is high and the epidemic situation is complex; because of special social, cultural background, and part of the MSM population in Shenzhen region The second part of the dominant HIV strain (CRF01_AE) infectious clones in Shenzhen area was constructed in 1994. After the first CRF01_AE strain was found in China, the virus spread rapidly in 1994, and it has become the main epidemic subtype in most regions of China (except in the southwest). In some provinces, more than one in China. 80% of the new reported cases are CRF01_AE infection. Because of the increasing infection caused by sexual transmission, the proportion of CRF01_AE subtypes is increasing in the national HIV infection. In Shenzhen, CRF01_AE is also one of the main epidemic subtypes. Therefore, it is urgent to study the epidemiology and pathogenesis of CRF01_AE. Infectious clones are not only determined. A powerful tool for specific gene biological effects is the effective means to study the virus pathogenic gene and the human immune response. Therefore, the construction of the infectious clone of the CRF01_AE virus is very important for the in-depth study of the pathogenesis of the CRF01_AE strain. Method: the full length genome of CRF01_AE strain was amplified in two segments by semi nested PCR, and the amplified products were purified to construct semi molecular clones with T vector respectively after purification. 2.5 molecular clones were cloned using Aar I and Xhol I restriction endonuclease enzyme, and 2.5 molecular fragments were connected to construct full-length clones, and the full-length clones were obtained to transfect HE. K293T cells, packing the derived virus of CRF01_AE infectious clones. Infect the packaged virus with MT2 cells, observe the cytopathic cells, determine the p24 and TCID50 of the cultured supernatant, use the p24 volume as the ordinate and the transverse coordinates, draw the replicative dynamic curve of the derived virus on MT2, and compare the replication capacity of the virus with the wild type virus. Difference; using U87 cells expressing different receptors to determine the eosinophilia of the derived virus. Results: after PCR, enzyme digestion, connection, and transformation, the full-length clone of CRF01_AE was successfully constructed and the full-length clone plasmid of length 13630bp was obtained. The virus sequence was 9702bp, with 15 independent open reading frames. The whole long cloned plasmid was transfected to 293T cells and cell culture. The p24 of the supernatant was 0.375ug/ml, and the transfected virus infected MT2 cells again and observed obvious cytopathic changes. The p24 of the culture supernatant was 19.1ug/ml and TCID50 15588, and the replication dynamics curve of the derived virus was not obviously different from the wild strain, and the replication ability was similar. The derivative of CRF01_AE was derived from the derivative of CRF01_AE. The virus is a double tropism virus. Conclusion: we have successfully constructed a CRF01_AE infectious clone which is completely from the wild strain and has similar replication ability. It provides a powerful tool to study the pathogenesis of the virus and the vaccine research.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.91

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