本文選題：人類免疫缺陷病毒(HIV) + 分子流行病學調(diào)查��； 參考：《安徽醫(yī)科大學》2017年碩士論文

【摘要】：第一部分深圳地區(qū)HIV分子流行病學調(diào)查1992年,深圳發(fā)現(xiàn)首例HIV陽性感染者。隨后深圳市報告的HIV/AIDS病例數(shù)呈現(xiàn)逐年上升趨勢,年平均增長率高達24.7%;病例感染途徑亦不斷發(fā)生變化,由早期的IDU傳播轉(zhuǎn)變?yōu)橐孕詡鞑橹?HIV感染呈現(xiàn)出從高危人群向普通人群快速擴散的態(tài)勢。艾滋病的出現(xiàn)以及快速傳播成為深圳市重要的公共衛(wèi)生問題。鑒于深圳市存在大量非戶籍人口,人員流動頻繁、人口結(jié)構(gòu)復雜,迫切需要對其進行艾滋病分子流行病學調(diào)查,以闡明其HIV流行模式。目的:對深圳地區(qū)傳播的HIV進行分子流行病學調(diào)查,了解HIV流行趨勢。方法:采用隨機數(shù)字法挑選深圳地區(qū)2015年新確認HIV感染者作為研究對象,收集人口統(tǒng)計學信息;利用核酸提取試劑盒獲取樣本基因組,采用逆轉(zhuǎn)錄/巢式PCR的方法擴增HIVgag、pol基因片段,擴增片段純化后測序并進行序列拼接;將各片段基因序列提交Los Alamos HIV Databases,分析亞型并構(gòu)建系統(tǒng)進化樹;擴增、測定URF病毒全長序列并進行重組分析;將深圳序列與全國序列比對后構(gòu)建系統(tǒng)進化樹,進行溯源分析;pol基因序列提交斯坦福大學耐藥數(shù)據(jù)庫檢測耐藥位點;利用HCV酶聯(lián)免疫試劑盒檢測樣本中HCV抗體存在情況;將深圳地區(qū)2015年HIV流行狀況與2013年進行比較,了解深圳HIV的流行變化;采用系統(tǒng)進化的方法對HES感染人群中的MSMs進行甄別研究。結(jié)果:隨機法獲得深圳市2015年新報告HIV感染者250例,其中88.4%為男性感染者;感染者年齡以青壯年為主,其中15~29歲人群高達49.2%;96.8%感染者傳播方式為性傳播,其中MSM占52%。主要傳播亞型為CRF01_AE,CRE07_BC,CRF55_01B,分別占31.96%,37.90%,10.50%;URF比例較高,占7.76%。獲得的樣本中耐藥率為2.8%,HIV與HCV共感染人數(shù)僅7人(2.8%)。溯源分析發(fā)現(xiàn)深圳樣本中,CRF01_AE亞型中的HESs與MSMs分別形成單獨的流行簇,與全國CRF01_AE的流行簇相重合,而CRF07_BC則無明顯流行簇出現(xiàn)。與2013年本室獲得的深圳市HIV分子流行病學數(shù)據(jù)進行比較時發(fā)現(xiàn)15~29歲感染者在2015年明顯增加,差異性統(tǒng)計學檢驗顯示P0.05,具有統(tǒng)計學意義。2013年和2015年流行亞型均為CRF01_AE、CRF07_BC、CRF55_01B,各亞型所占比例無明顯變化。耐藥率2013年與2015年分別為0.8%和2.8%;與HCV共感染人數(shù)分別為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年與2015年差別不大,CRF07_BC不聚集成簇,CRF01_AE存在單獨的MSMs流行簇和HESs流行簇。利用系統(tǒng)進化樹分析甄別了深圳感染CRF01_AE的HES男性感染者中約有11%的人是由于男男同性性行為感染HIV。結(jié)論:深圳地區(qū)HIV主要在男性性傳播人群中擴散,感染者年齡呈現(xiàn)年輕化趨勢;HIV感染亞型分布的明顯多樣,URF比例高,流行狀況復雜;因為特殊的社會、文化背景,深圳地區(qū)部分MSM人群在現(xiàn)場調(diào)查中不愿暴露其男男性行為方式。第二部分深圳地區(qū)優(yōu)勢HIV毒株(CRF01_AE)感染性克隆的構(gòu)建1994年我國發(fā)現(xiàn)第一例CRF01_AE毒株后,該毒株快速傳播,目前已經(jīng)成為我國大部分地區(qū)的(除了西南地區(qū))主要流行亞型。在一些省份,超過80%的新報告病例為CRF01_AE感染。由于性傳播造成的感染不斷增加,CRF01_AE亞型在全國HIV感染者中所占比例也不斷增加。在深圳地區(qū),CRF01_AE也是主要流行亞型之一,因此對CRF01_AE流行病學和發(fā)病機理的研究刻不容緩。感染性克隆不僅是確定特定基因生物學效應的有力工具,更是研究病毒致病基因與人體免疫反應的有效手段。因此構(gòu)建CRF01_AE病毒的感染性克隆對于深入研究CRF01_AE毒株的致病機制至關重要。目的:構(gòu)建CRF01_AE毒株的感染性克隆,為深入研究其致病機制奠定基礎。方法:采用半巢式PCR方法分兩段擴增CRF01_AE毒株全長基因組,將擴增產(chǎn)物純化后分別連接T載體構(gòu)建半分子克隆;利用Aar I和Xhol I限制性內(nèi)切酶酶切兩個半分子克隆,將兩個半分子片段進行連接,構(gòu)建全長克隆;將獲得的全長克隆轉(zhuǎn)染HEK293T細胞,包裝CRF01_AE感染性克隆的衍生病毒。將包裝的病毒感染MT2細胞,觀察細胞病變,測定獲得的細胞培養(yǎng)上清的p24和TCID50;以p24的量為縱坐標,時間為橫坐標,繪制衍生病毒在MT2上的復制動力學曲線,比較其與野生型病毒的復制能力差異;利用表達不同受體的U87細胞測定衍生病毒的嗜性。結(jié)果:經(jīng)過PCR、酶切、連接、轉(zhuǎn)化成功構(gòu)建CRF01_AE全長克隆,獲得長度13630bp的全長克隆質(zhì)粒,其中病毒序列長為9702bp,具有15個獨立的開放閱讀框。將全長克隆質(zhì)粒轉(zhuǎn)染293T細胞,細胞培養(yǎng)上清的p24為0.375ug/ml;轉(zhuǎn)染獲得的衍生病毒再次感染MT2細胞,觀察到明顯的細胞病變,其培養(yǎng)上清的p24為19.1ug/ml,TCID50為15588;衍生病毒的復制動力學曲線與其野生株病毒沒有明顯差異,復制能力相近;利用U87測得CRF01_AE感染性克隆的衍生病毒為雙嗜性病毒。結(jié)論:我們成功構(gòu)建了一株完全來自野生株病毒并與其有相似復制能力的CRF01_AE感染性克隆,為研究病毒的致病機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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R512.91

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