本文關(guān)鍵詞： 海南省 漢族 黎族 丙型肝炎病毒 基因型 基因亞型 全基因組序列 系統(tǒng)發(fā)育進(jìn)化分析 貝葉斯分析　出處：《南方醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：背景:目前,全球有近1.8億人感染丙型肝炎病毒(hepatitis C virus,HCV),而在中國約1000萬為抗-HCV陽性。HCV至今尚缺有效的疫苗。依據(jù)Simmond系統(tǒng),HCV可分為7個基因型和85個確定的以及20個待定的基因亞型。對HCV基因型分布及分子流行病學(xué)的研究,有助于控制HCV的播散。最新研究表明,中國HCV基因型以1b為主,占56.8%(582/997);其次為2型、3型和6型。但是,中國的南北地區(qū)HCV基因型組成和播散趨勢不盡一致。與北方不同,6a在華南地區(qū)已經(jīng)取代2a成為當(dāng)?shù)氐牡诙䝼�主要流行的HCV基因亞型,并曾在廣東河源發(fā)生快速流行。海南島毗鄰廣東省,主要居民為漢族(83.33%),其次是黎族(14.73%)。至今對海南島的HCV分子流行病學(xué)研究較少。此外,海南黎族族源研究顯示,其與南島語系的多個族群有明顯遺傳淵源關(guān)系,其被稱為“人類進(jìn)入東亞入口的古老活化石”。至今HCV基因型6的各個亞型主要發(fā)現(xiàn)在東南亞地區(qū)流行。HCV基因型6因其遺傳差異最大,亞型最復(fù)雜,已被視為HCV的最古老的病毒株,目前仍不斷地發(fā)現(xiàn)新的HCV基因型6特殊病毒株——指不能歸入已知的亞型的病毒株。HCV基因型6特殊病毒株由于病毒載量低、遺傳差異大和缺乏參照序列,因此對該類病毒進(jìn)行全長擴(kuò)增是世界難題。前期研究發(fā)現(xiàn)部分來源于海南黎族的HCV基因型6病毒起源古老且獨(dú)特。因此,對海南黎族的HCV基因型6特殊病毒株進(jìn)行全長擴(kuò)增,研究海南黎族的HCV基因型6與東南亞其他地區(qū)該型病毒的進(jìn)化關(guān)系,將有助于補(bǔ)充該型病毒的生活史研究。目的:1.本研究首先通過研究海南省漢族和黎族人群中的HCV基因型分布和臨床特點(diǎn)、高危因素來了解海南省HCV的感染概況。2.應(yīng)用系統(tǒng)發(fā)育樹分析(phylogenetic anlynasis)來了解海南漢族地區(qū)HCV的來源、傳播模式和規(guī)律。3.從黎族中的HCV基因型6特殊病毒株中,對兩株未知的病毒株HN1316和HN1350進(jìn)行了全基因組擴(kuò)增和測定。進(jìn)一步分析黎族“HCV基因型6特殊病毒株”與東南亞地區(qū)的HCV基因型6毒株的進(jìn)化關(guān)系。方法:1.樣品來源:納入2013.1～2014.6間收集的HCV-RNA陽性慢性HCV感染者血清標(biāo)本246例(其中漢族176例,黎族70例)�；虻臄U(kuò)增和測序:巢式PCR擴(kuò)增并測序Core-E1。數(shù)據(jù)分析:應(yīng)用系統(tǒng)發(fā)育樹分析海南漢族和黎族人群中HCV基因型的分布特點(diǎn)。2.應(yīng)用BEAST 1.6軟件和貝葉斯—馬爾科夫鏈—蒙特卡洛(Bayesian—MCMC)方法對海南漢族與中國其他地區(qū)漢族的HCV序列進(jìn)行種系地理學(xué)分析。最后,擴(kuò)大海南漢族的慢性HCV感染者的病例數(shù)至402例,檢測其基因分型,系統(tǒng)分析這些患者的流行病學(xué)特點(diǎn)和臨床特點(diǎn),Logistic回歸分析海南地區(qū)HCV感染的高危因素。3.設(shè)計簡并引物及特異性引物,通過“DNA walking on bridges and islands”策略進(jìn)行多片段PCR擴(kuò)增并測定兩株未知的黎族病毒株——HN1316和HN1350的全基因組序列。繼而采用生物信息學(xué)軟件,進(jìn)行核苷酸相似性分析,構(gòu)建ML(Maximun Likelihood)系統(tǒng)進(jìn)化樹以估算它們的系統(tǒng)進(jìn)化位置,進(jìn)行基因重組測定分析,來確定新的基因亞型。結(jié)果:1.海南省漢族人群的HCV流行概況:(1)海南省漢族的163例樣本基因型分布為:6a是最主要的亞型,其次是1b、3b、3a、2a和 1a:6a為 33.7%(55/163),1b為 33.1%(54/163),3b為 14.7%(24/163),2a 為 6.7%(11/163),3a 為 8.6%(14/163)和 1a 為 3%(5/163)。(2)種系地理學(xué)分析發(fā)現(xiàn):海南的HCV與中國其它地區(qū)漢族的HCV交替分布于每一簇中。進(jìn)而,擴(kuò)大至402例后的檢測基因型分布、流行病學(xué)特點(diǎn)和臨床特點(diǎn)如下:(3)6a仍是最主要的亞型,其次是1b、3b、3a、2a和1a。(4)Logistic分析表明:海南HCV傳播的高危因素分別是使用過污染的醫(yī)療器械(P0.05)、使用血制品(P0.05)和靜脈吸毒(P0.01);卡方檢驗(yàn)顯示6a亞型中靜脈吸毒明顯高于其他亞型(P0.01)。2.海南省黎族58例的基因型分布為:基因型6為94.8%(55/58),1b為1.72%(1/58)、2a 為 1.72%(1/58)、3b 為 1.72%(1/58)。進(jìn)一步對 55 例HCV 基因型 6 分析發(fā)現(xiàn):6a 為 3.5%(2/58),6xa 為 3.5%(2/58),6w 為 5.2%(3/58),6e 為 1.7%(1/58),6v 為 1.7%(1/58),6r 為 1.7%(1/58),特殊病毒株為77.6%(45/58)。系統(tǒng)發(fā)育樹分析顯示這45株病毒明顯有別于東南亞其他地區(qū)的病毒株而集聚為獨(dú)立的進(jìn)化簇。3.成功對HN1316和HN1350成功進(jìn)行全基因組擴(kuò)增。分析發(fā)現(xiàn):兩個病毒株與其他已知的6型全長序列的基因相似性在70～80%;接著構(gòu)建ML系統(tǒng)進(jìn)化樹發(fā)現(xiàn),HN1350與印度尼西亞、香港地區(qū)發(fā)現(xiàn)的6g、6w有著共同的祖先,HN1316與6a、6b、6xd和6_ZS202、老撾地區(qū)發(fā)現(xiàn)的6_L373有著共同祖先。進(jìn)一步分析發(fā)現(xiàn)無明顯流行病學(xué)關(guān)聯(lián)的三個病毒株:HN1350(全長)、HN1314和HN1411三個序列之間基因差異在7.6～14%。結(jié)論:1.6a是海南漢族中最主要的感染基因型,其次為1b,提示6a在我國可能已經(jīng)開始從廣東地區(qū)快速傳播開來。海南島雖然與大陸隔海相望,但是海南省漢族人群同屬于中國大陸漢族的HCV傳播網(wǎng)絡(luò)。2.海南黎族人群的HCV以基因6型為主,并發(fā)現(xiàn)多株基因6型的特殊病毒株且高度聚集,提示HCV基因型6病毒可能在黎族地區(qū)封閉地緩慢傳播了非常長的時期。3.首次在黎族中發(fā)現(xiàn)一個HCV基因型6的新亞型�；蛳嗨菩越Y(jié)果顯示HN1316和HN1350屬于HCV基因型6,但不能歸于已知亞型;這兩個病毒株與東南亞周邊地區(qū)來源的病毒有共同祖先,提示海南黎族可能是HCV基因型6起源的一部分;本研究中有三株病毒滿足新基因亞型命題條件,可以命名一個HCV基因型6新亞型(6xg?),聯(lián)系HCV命名委員確認(rèn)新命名的工作在進(jìn)行中。
[Abstract]:Background: at present, the world has nearly 180 million people infected with hepatitis C virus (hepatitis C, virus, HCV), and in Chinese about 10 million anti -HCV positive.HCV is still lack of effective vaccines. According to Simmond system, HCV can be divided into 7 genotypes and 85 identified and 20 undetermined subtype. Study on the distribution of HCV genotype and molecular epidemiology, contribute to the spread of HCV. The latest research shows that HCV gene Chinese dominated by 1b, accounting for 56.8% (582/997); followed by type 2, type 3 and type 6. However, the HCV genotype in the northern and southern composition and spread trend is not Chinese consistent with the north in the Southern China area, 6a has replaced 2a and become a local second main popular HCV subtypes, and worked in the Guangdong Heyuan fast fashion. Hainan Island is adjacent to Guangdong Province, mainly residents of Han nationality (83.33%), followed by Li (14.73%). Since the HCV molecule of Hainan Island flow Epidemiological research is less. In addition, the research of Li Nationality in Hainan shows the source, there is obvious genetic relationship with multiple populations of Austronesian languages, which is called "fossil" of ancient human live into the east entrance. Since many subtypes of HCV genotype 6 is found mainly in Southeast Asia for.HCV genotype 6 because of the genetic differences between the largest, most complex subtype, HCV has been regarded as the most ancient strains are still discovering new HCV gene type 6 virus strains that can not be classified as special known subtypes of the virus.HCV gene type 6 special strains due to low viral load, large genetic difference and the lack of reference sequence, so the length of amplification is the world's problems on the virus. Previous study found that the origin of HCV genotype in Hainan Li and part of the 6 sources of virus of ancient and unique. Therefore, HCV genotype of Hainan Li 6 special strains of Long was the evolutionary relationship of HCV virus genotypes of Li Nationality in Hainan and 6 other areas of Southeast Asia, will help replenish the life history of this virus. Objective: 1. this study first investigated the distribution of HCV genotypes in Hainan province Han and Li in the crowd and the clinical features, risk factors to understand analysis of phylogenetic tree of.2. infection in Hainan province application system HCV (phylogenetic anlynasis) to understand the source of the Han nationality in Hainan area HCV, HCV genotypes from the propagation pattern of Li and law of.3. 6 special strains, two strains of unknown virus strains HN1316 and HN1350 were amplified and determined. Further analysis of genome evolution the relationship between HCV gene type Li HCV genotype 6 special strains "and Southeast Asia 6 strains. Methods: 1. samples: a total of 2013.1 ~ 2014.6 collected HCV-RNA positive chronic HCV infection serum standard Of the 246 cases (including 176 cases of Han and Li 70). Gene amplification and sequencing: nested PCR amplification and sequence analysis of Core-E1. data: application of phylogenetic analysis of Hainan Han and Li population in HCV genotype distribution of.2. using BEAST 1.6 software and Bayesian Markov chain Monte Carlo Marco - (Bayesian - MCMC) the method of HCV sequence in Hainan Han Han and other areas China analyzed species geography. Finally, the expansion of the Hainan Han patients with chronic HCV infection cases to 402 cases, the detection of genotyping, analysis of epidemiological and clinical characteristics of these patients, Logistic regression analysis of risk factors of HCV infection in Hainan and the.3. design degenerate primers and specific primers, through the "DNA walking on bridges and islands" strategy of Li virus - HN1316 and HN strains of PCR fragments amplified and tested two strains of unknown 1350鐨勫叏鍩哄洜緇勫簭鍒，

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