海南省HCV分子分型、分子进化及黎族HCV基因型6特殊病毒株的全基因组研究

发布时间：2018-01-21 00:15

  本文关键词： 海南省 汉族 黎族 丙型肝炎病毒 基因型 基因亚型 全基因组序列 系统发育进化分析 贝叶斯分析　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：背景:目前,全球有近1.8亿人感染丙型肝炎病毒(hepatitis C virus,HCV),而在中国约1000万为抗-HCV阳性。HCV至今尚缺有效的疫苗。依据Simmond系统,HCV可分为7个基因型和85个确定的以及20个待定的基因亚型。对HCV基因型分布及分子流行病学的研究,有助于控制HCV的播散。最新研究表明,中国HCV基因型以1b为主,占56.8%(582/997);其次为2型、3型和6型。但是,中国的南北地区HCV基因型组成和播散趋势不尽一致。与北方不同,6a在华南地区已经取代2a成为当地的第二个主要流行的HCV基因亚型,并曾在广东河源发生快速流行。海南岛毗邻广东省,主要居民为汉族(83.33%),其次是黎族(14.73%)。至今对海南岛的HCV分子流行病学研究较少。此外,海南黎族族源研究显示,其与南岛语系的多个族群有明显遗传渊源关系,其被称为“人类进入东亚入口的古老活化石”。至今HCV基因型6的各个亚型主要发现在东南亚地区流行。HCV基因型6因其遗传差异最大,亚型最复杂,已被视为HCV的最古老的病毒株,目前仍不断地发现新的HCV基因型6特殊病毒株——指不能归入已知的亚型的病毒株。HCV基因型6特殊病毒株由于病毒载量低、遗传差异大和缺乏参照序列,因此对该类病毒进行全长扩增是世界难题。前期研究发现部分来源于海南黎族的HCV基因型6病毒起源古老且独特。因此,对海南黎族的HCV基因型6特殊病毒株进行全长扩增,研究海南黎族的HCV基因型6与东南亚其他地区该型病毒的进化关系,将有助于补充该型病毒的生活史研究。目的:1.本研究首先通过研究海南省汉族和黎族人群中的HCV基因型分布和临床特点、高危因素来了解海南省HCV的感染概况。2.应用系统发育树分析(phylogenetic anlynasis)来了解海南汉族地区HCV的来源、传播模式和规律。3.从黎族中的HCV基因型6特殊病毒株中,对两株未知的病毒株HN1316和HN1350进行了全基因组扩增和测定。进一步分析黎族“HCV基因型6特殊病毒株”与东南亚地区的HCV基因型6毒株的进化关系。方法:1.样品来源:纳入2013.1～2014.6间收集的HCV-RNA阳性慢性HCV感染者血清标本246例(其中汉族176例,黎族70例)。基因的扩增和测序:巢式PCR扩增并测序Core-E1。数据分析:应用系统发育树分析海南汉族和黎族人群中HCV基因型的分布特点。2.应用BEAST 1.6软件和贝叶斯—马尔科夫链—蒙特卡洛(Bayesian—MCMC)方法对海南汉族与中国其他地区汉族的HCV序列进行种系地理学分析。最后,扩大海南汉族的慢性HCV感染者的病例数至402例,检测其基因分型,系统分析这些患者的流行病学特点和临床特点,Logistic回归分析海南地区HCV感染的高危因素。3.设计简并引物及特异性引物,通过“DNA walking on bridges and islands”策略进行多片段PCR扩增并测定两株未知的黎族病毒株——HN1316和HN1350的全基因组序列。继而采用生物信息学软件,进行核苷酸相似性分析,构建ML(Maximun Likelihood)系统进化树以估算它们的系统进化位置,进行基因重组测定分析,来确定新的基因亚型。结果:1.海南省汉族人群的HCV流行概况:(1)海南省汉族的163例样本基因型分布为:6a是最主要的亚型,其次是1b、3b、3a、2a和 1a:6a为 33.7%(55/163),1b为 33.1%(54/163),3b为 14.7%(24/163),2a 为 6.7%(11/163),3a 为 8.6%(14/163)和 1a 为 3%(5/163)。(2)种系地理学分析发现:海南的HCV与中国其它地区汉族的HCV交替分布于每一簇中。进而,扩大至402例后的检测基因型分布、流行病学特点和临床特点如下:(3)6a仍是最主要的亚型,其次是1b、3b、3a、2a和1a。(4)Logistic分析表明:海南HCV传播的高危因素分别是使用过污染的医疗器械(P0.05)、使用血制品(P0.05)和静脉吸毒(P0.01);卡方检验显示6a亚型中静脉吸毒明显高于其他亚型(P0.01)。2.海南省黎族58例的基因型分布为:基因型6为94.8%(55/58),1b为1.72%(1/58)、2a 为 1.72%(1/58)、3b 为 1.72%(1/58)。进一步对 55 例HCV 基因型 6 分析发现:6a 为 3.5%(2/58),6xa 为 3.5%(2/58),6w 为 5.2%(3/58),6e 为 1.7%(1/58),6v 为 1.7%(1/58),6r 为 1.7%(1/58),特殊病毒株为77.6%(45/58)。系统发育树分析显示这45株病毒明显有别于东南亚其他地区的病毒株而集聚为独立的进化簇。3.成功对HN1316和HN1350成功进行全基因组扩增。分析发现:两个病毒株与其他已知的6型全长序列的基因相似性在70～80%;接着构建ML系统进化树发现,HN1350与印度尼西亚、香港地区发现的6g、6w有着共同的祖先,HN1316与6a、6b、6xd和6_ZS202、老挝地区发现的6_L373有着共同祖先。进一步分析发现无明显流行病学关联的三个病毒株:HN1350(全长)、HN1314和HN1411三个序列之间基因差异在7.6～14%。结论:1.6a是海南汉族中最主要的感染基因型,其次为1b,提示6a在我国可能已经开始从广东地区快速传播开来。海南岛虽然与大陆隔海相望,但是海南省汉族人群同属于中国大陆汉族的HCV传播网络。2.海南黎族人群的HCV以基因6型为主,并发现多株基因6型的特殊病毒株且高度聚集,提示HCV基因型6病毒可能在黎族地区封闭地缓慢传播了非常长的时期。3.首次在黎族中发现一个HCV基因型6的新亚型。基因相似性结果显示HN1316和HN1350属于HCV基因型6,但不能归于已知亚型;这两个病毒株与东南亚周边地区来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满足新基因亚型命题条件,可以命名一个HCV基因型6新亚型(6xg?),联系HCV命名委员确认新命名的工作在进行中。
[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鐨勫叏鍩哄洜缁勫簭鍒，

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