本文選題：乙型腦炎病毒　切入點：分子進化　出處：《中國疾病預防控制中心》2009年博士論文

【摘要】： 乙腦病毒(Japanese Encephalitis Virus,JEV)為黃病毒科黃病毒屬單股正鏈RNA病毒。病毒基因組全長約11kb,編碼十余個結(jié)構與非結(jié)構蛋白。該病毒感染可以引起人畜共患傳染病,臨床稱為乙腦,病死率高,存活者留有神經(jīng)系統(tǒng)后遺癥。全世界數(shù)十億人群生活在乙腦流行區(qū),乙腦病毒感染已經(jīng)成為世界關注的公共衛(wèi)生問題。本研究對乙腦病毒全基因組開展基因重組及分子進化研究,研究結(jié)果對于解釋該病毒致病機理、病毒分子流行病學特征、病毒疫苗開發(fā)研究以及乙腦病毒的分子診斷等均有重要意義。 1.乙型腦炎病毒的全基因組序列的測定 本研究首先測定了121株我國分離的乙腦病毒全基因組核苷酸序列,毒株分離自我國20余種蚊蟲標本、乙腦病例血清和腦脊液標本、以及蠓和蝙蝠標本;病毒分離時間自上世紀50年代至2007年;病毒分離地域覆蓋我國乙腦主要疫區(qū),北至我國黑龍江省,南至福建省,西至甘肅省,東至上海市�；蛐桶ɑ颌笮秃突颌裥�。測序質(zhì)量分析統(tǒng)計顯示:總平均覆蓋率為7.41423927,平均質(zhì)量值為40.34710744,即準確性值為99.99%,錯誤率為0.01%。接近于人類基因組計劃測序質(zhì)量標準,與相關研究中大規(guī)模病毒全基因組測定質(zhì)量標準相當。 2.病毒數(shù)據(jù)庫的建立 本研究利用MySQL語言和phpMyAdmin圖形化管理界面創(chuàng)建了病毒基因信息數(shù)據(jù)庫。該數(shù)據(jù)庫收集了目前國際基因庫(GenBank)公布的所有黃病毒科病毒基因與基因組序列信息,以及本研究所測定的121株乙腦病毒全基因組序列信息,共同構成了黃病毒科病毒核酸序列信息庫(共含159,179條病毒序列信息)用于進一步分析研究。 3.乙型腦炎病毒全基因組重組研究 本研究利用12種基因重組分析軟件對目前世界范圍內(nèi)178株乙腦病毒全基因組序列(57株乙腦病毒基因組序為GeneBank公布,121株乙腦病毒基因組序列為本研究測定)進行了乙腦病毒基因及基因組重組掃描(screen)。結(jié)果發(fā)現(xiàn)139株乙腦病毒存在大量基因型內(nèi)和型間重組信號,占全部分析毒株78%(139/178)�；颌蛐秃突颌粜鸵夷X病毒無重組信號;通過生物信息學和統(tǒng)計學分析方法確定3株乙腦病毒發(fā)生基因Ⅰ/Ⅲ型間同源重組,其中中國蚊蟲分離乙腦病毒(SC04-25株)存在9個重組斷點,重組斷點分布于除基因NS2b和3'非編碼區(qū)之外的全基因各基因區(qū)域;另兩株重組病毒分別來自我國黑龍江省和韓國蚊蟲標本分離的乙腦病毒(HLJ02-144和K94P05株);精確計算了病毒重組區(qū)段位置,各重組區(qū)域最相似父本毒株分別分離自乙腦病人和蚊蟲標本;本研究還比較了重組毒株與參考毒株間的相似性和遺傳距離等。以上結(jié)果提示自然界已出現(xiàn)基因重組乙腦病毒,病毒重組事件可以發(fā)生在病毒單個基因以致全基因組水平。這是首次發(fā)現(xiàn)自然界存在基因Ⅰ/Ⅲ間的乙腦重組病毒。 4.乙型腦炎病毒分子進化研究 本研究利用分子系統(tǒng)發(fā)育學與生物信息學技術,計算了乙腦病毒全基因組序列的堿基替換模型、分子鐘模型、堿基替換速率以及進化速率,并分別依據(jù)遺傳距離和進化時間構建了乙腦病毒基因及全基因組的進化樹。結(jié)果顯示,乙腦病毒結(jié)構蛋白和非結(jié)構蛋白編碼基因的最佳堿基替換模型分別是HKY模式和GTR模式,而病毒全基因組序列數(shù)據(jù)最佳堿基替換模型為GTR模式;乙腦病毒堿基替換速率在10~(-4)數(shù)量級,生長進化速率趨近于0,說明乙腦病毒較其他RNA病毒,如流感病毒等生長進化速率緩慢。病毒基因組系統(tǒng)進化分析顯示,基因Ⅲ型乙腦病毒的進化關系與宿主來源有關,而基因Ⅰ型乙腦病毒具有地域特征,該結(jié)果的發(fā)現(xiàn)為乙腦病毒基因型及基因亞型分類以及各地域和各宿主間乙腦病毒的分子差異研究提供了分子依據(jù)。本研究還從生物熱動力學角度計算了乙腦病毒全基因組各位點生物熵值(0-1.08694),為分析乙腦病毒的保守序列和高變位點提供了分析數(shù)據(jù);病毒進化研究結(jié)果還顯示乙腦病毒起源自公元前801年,基因Ⅲ型和基因Ⅰ型乙腦病毒共進化祖先分別出現(xiàn)在1742年和1798年。以上研究是首次開展的乙腦病毒全基因組分子進化研究。
[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.

【學位授予單位】：中國疾病預防控制中心
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R373

【引證文獻】

相關碩士學位論文 前1條

1 邵林;乙型腦炎病毒NS5和E蛋白單抗的制備及其特性分析[D];華中農(nóng)業(yè)大學;2013年

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本文編號：1679824