乙型脑炎病毒全基因组分子进化及基因重组研究

发布时间：2018-03-29 05:39

本文选题：乙型脑炎病毒　切入点：分子进化　出处：《中国疾病预防控制中心》2009年博士论文

【摘要】： 乙脑病毒(Japanese Encephalitis Virus,JEV)为黄病毒科黄病毒属单股正链RNA病毒。病毒基因组全长约11kb,编码十余个结构与非结构蛋白。该病毒感染可以引起人畜共患传染病,临床称为乙脑,病死率高,存活者留有神经系统后遗症。全世界数十亿人群生活在乙脑流行区,乙脑病毒感染已经成为世界关注的公共卫生问题。本研究对乙脑病毒全基因组开展基因重组及分子进化研究,研究结果对于解释该病毒致病机理、病毒分子流行病学特征、病毒疫苗开发研究以及乙脑病毒的分子诊断等均有重要意义。 1.乙型脑炎病毒的全基因组序列的测定本研究首先测定了121株我国分离的乙脑病毒全基因组核苷酸序列,毒株分离自我国20余种蚊虫标本、乙脑病例血清和脑脊液标本、以及蠓和蝙蝠标本;病毒分离时间自上世纪50年代至2007年;病毒分离地域覆盖我国乙脑主要疫区,北至我国黑龙江省,南至福建省,西至甘肃省,东至上海市。基因型包括基因Ⅲ型和基因Ⅰ型。测序质量分析统计显示:总平均覆盖率为7.41423927,平均质量值为40.34710744,即准确性值为99.99%,错误率为0.01%。接近于人类基因组计划测序质量标准,与相关研究中大规模病毒全基因组测定质量标准相当。 2.病毒数据库的建立本研究利用MySQL语言和phpMyAdmin图形化管理界面创建了病毒基因信息数据库。该数据库收集了目前国际基因库(GenBank)公布的所有黄病毒科病毒基因与基因组序列信息,以及本研究所测定的121株乙脑病毒全基因组序列信息,共同构成了黄病毒科病毒核酸序列信息库(共含159,179条病毒序列信息)用于进一步分析研究。 3.乙型脑炎病毒全基因组重组研究本研究利用12种基因重组分析软件对目前世界范围内178株乙脑病毒全基因组序列(57株乙脑病毒基因组序为GeneBank公布,121株乙脑病毒基因组序列为本研究测定)进行了乙脑病毒基因及基因组重组扫描(screen)。结果发现139株乙脑病毒存在大量基因型内和型间重组信号,占全部分析毒株78%(139/178)。基因Ⅱ型和基因Ⅳ型乙脑病毒无重组信号;通过生物信息学和统计学分析方法确定3株乙脑病毒发生基因Ⅰ/Ⅲ型间同源重组,其中中国蚊虫分离乙脑病毒(SC04-25株)存在9个重组断点,重组断点分布于除基因NS2b和3'非编码区之外的全基因各基因区域;另两株重组病毒分别来自我国黑龙江省和韩国蚊虫标本分离的乙脑病毒(HLJ02-144和K94P05株);精确计算了病毒重组区段位置,各重组区域最相似父本毒株分别分离自乙脑病人和蚊虫标本;本研究还比较了重组毒株与参考毒株间的相似性和遗传距离等。以上结果提示自然界已出现基因重组乙脑病毒,病毒重组事件可以发生在病毒单个基因以致全基因组水平。这是首次发现自然界存在基因Ⅰ/Ⅲ间的乙脑重组病毒。 4.乙型脑炎病毒分子进化研究本研究利用分子系统发育学与生物信息学技术,计算了乙脑病毒全基因组序列的碱基替换模型、分子钟模型、碱基替换速率以及进化速率,并分别依据遗传距离和进化时间构建了乙脑病毒基因及全基因组的进化树。结果显示,乙脑病毒结构蛋白和非结构蛋白编码基因的最佳碱基替换模型分别是HKY模式和GTR模式,而病毒全基因组序列数据最佳碱基替换模型为GTR模式;乙脑病毒碱基替换速率在10~(-4)数量级,生长进化速率趋近于0,说明乙脑病毒较其他RNA病毒,如流感病毒等生长进化速率缓慢。病毒基因组系统进化分析显示,基因Ⅲ型乙脑病毒的进化关系与宿主来源有关,而基因Ⅰ型乙脑病毒具有地域特征,该结果的发现为乙脑病毒基因型及基因亚型分类以及各地域和各宿主间乙脑病毒的分子差异研究提供了分子依据。本研究还从生物热动力学角度计算了乙脑病毒全基因组各位点生物熵值(0-1.08694),为分析乙脑病毒的保守序列和高变位点提供了分析数据;病毒进化研究结果还显示乙脑病毒起源自公元前801年,基因Ⅲ型和基因Ⅰ型乙脑病毒共进化祖先分别出现在1742年和1798年。以上研究是首次开展的乙脑病毒全基因组分子进化研究。
[Abstract]:Japanese encephalitis virus (Japanese Encephalitis, Virus, JEV) for flavivirus positive strand RNA virus. The virus genome is approximately 11KB in length, encoding ten structure and non structural protein. The virus infection can cause zoonotic diseases, clinical called JE, high fatality rate, survivors have neurological sequelae. Billions of stay people around the world live in endemic areas of Japanese encephalitis, Japanese encephalitis virus infection has become a public health problem of the world's attention. The study of gene recombination and molecular evolution of Japanese encephalitis virus genome, the results for the interpretation of the viral pathogenesis, molecular epidemiological characteristics of virus, virus vaccine development and significance of the research on the molecular diagnosis of Japanese encephalitis virus both.
Determination of the whole genome sequence of 1. encephalitis B virus
This study first examined 121 strains of JE virus isolated from China whole genome nucleotide sequence, more than 20 kinds of isolates isolated from mosquitoes collected in China, serum and cerebrospinal fluid specimens of JE cases, and midges and bats; virus isolation time since the last century, from 50s to 2007; China's JE virus isolation area covering the main epidemic areas in Heilongjiang Province, north to our country, to the south of Fujian Province, West Gansu Province, east of Shanghai city. The genotype and gene including type III genotype. Statistical analysis of sequencing quality shows that the average coverage rate is 7.41423927, the average quality value is 40.34710744, the accuracy was 99.99%, the error rate of 0.01%. is close to the human genome project sequencing the quality standards, and related research in large-scale virus genome determination of quality standards.
The establishment of 2. virus database
This study uses MySQL language and phpMyAdmin graphical management interface to create a virus gene information database. The database collection of the current international gene bank (GenBank) all Flaviviridae virus gene and genome sequence information released, and the determination of the 121 strains of B brain genome information, constitute the information base of Flaviviridae the viral nucleic acid sequence (containing 159179 virus sequences) used for further study.
Study on the whole genome recombinant of 3. encephalitis B virus
This study uses 12 recombinant analysis software in the world of 178 strains of Japanese encephalitis virus genome sequence (57 strains of Japanese encephalitis virus genome sequence for the GeneBank announcement, Japanese encephalitis virus genome sequences of 121 strains were measured in this study) of gene and genome of Japanese encephalitis virus scanning (screen). The results showed that 139 strains of Japanese encephalitis virus in the presence of a large number of genotype and type of recombinant signal, accounting for 78% of the total strains (139/178). Japanese encephalitis virus type and genotype II genes without recombination signal; through bioinformatics and statistical analysis method to determine 3 strains of Japanese encephalitis virus gene I / III type homologous recombination, which Chinese isolated from mosquitoes of Japanese encephalitis virus (SC04-25 strain) there are 9 recombination breakpoints, recombination breakpoints distributed throughout the gene NS2b gene and 3'gene region in non encoding region outside; the other two strains of recombinant virus were from Heilongjiang Province in China and South Korea In isolated from the mosquitoes of Japanese encephalitis virus (HLJ02-144 and K94P05 strain); recombinant virus segment position is calculated accurately, the recombination region most similar paternal strains are isolated from Japanese encephalitis patients and mosquito samples; this study also compared the recombinant strains and reference strains between similarity and genetic distance. These results suggest that nature has appeared in the gene recombinant Japanese encephalitis virus, virus recombination events can occur in a single virus gene or genomic level. This is the first time that there are gene I / III between the recombination of JEV.
Study on the molecular evolution of 4. encephalitis B virus
This study using molecular phylogeny and bioinformatics technology, whole genome sequence of Japanese encephalitis virus to calculate the nucleotide substitution model, molecular clock model, substitution rate and evolution rate, and were based on genetic distances and evolutionary time construction of JEV genes and whole genome phylogenetic tree. The results show that the optimal BP encoding gene structure Japanese encephalitis virus and non structural protein replacement models are the HKY model and GTR model, and the virus genome sequences of optimal nucleotide substitution model for GTR mode; Japanese encephalitis virus base for change rate in 10~ (-4) level, the growth rate of evolution tends to 0 and that of Japanese encephalitis virus than other RNA viruses, such as influenza rate virus growth slow evolution. The virus genome phylogenetic analysis showed that the relationship between the related host derived evolution of genotype III JEV, and gene type of Japanese encephalitis The virus has the regional characteristic, found the results of molecular and gene for gene of Japanese encephalitis virus subtype classification and the regional and the host of Japanese encephalitis virus differences provide a molecular basis. This study also from the perspective of biothermodynamic calculated JE virus genome to biological entropy (0-1.08694), provides data for analysis conserved sequence and hypervariable sites analysis of Japanese encephalitis virus; virus evolution results also show the origin of Japanese encephalitis virus from BC 801 years, Japanese encephalitis virus genotype III and genotype I co evolutionary ancestors respectively in 1742 and 1798. The above research is carried out for the first time the molecular genome of Japanese encephalitis virus evolution.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R373

【引证文献】