本文選題：乙型腦炎病毒　切入點(diǎn)：分子進(jìn)化　出處：《中國(guó)疾病預(yù)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(zhǎng)約11kb,編碼十余個(gè)結(jié)構(gòu)與非結(jié)構(gòu)蛋白。該病毒感染可以引起人畜共患傳染病,臨床稱(chēng)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(guān)注的公共衛(wèi)生問(wèn)題。本研究對(duì)乙腦病毒全基因組開(kāi)展基因重組及分子進(jìn)化研究,研究結(jié)果對(duì)于解釋該病毒致病機(jī)理、病毒分子流行病學(xué)特征、病毒疫苗開(kāi)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(cè)定 本研究首先測(cè)定了121株我國(guó)分離的乙腦病毒全基因組核苷酸序列,毒株分離自我國(guó)20余種蚊蟲(chóng)標(biāo)本、乙腦病例血清和腦脊液標(biāo)本、以及蠓和蝙蝠標(biāo)本;病毒分離時(shí)間自上世紀(jì)50年代至2007年;病毒分離地域覆蓋我國(guó)乙腦主要疫區(qū),北至我國(guó)黑龍江省,南至福建省,西至甘肅省,東至上海市。基因型包括基因Ⅲ型和基因Ⅰ型。測(cè)序質(zhì)量分析統(tǒng)計(jì)顯示:總平均覆蓋率為7.41423927,平均質(zhì)量值為40.34710744,即準(zhǔn)確性值為99.99%,錯(cuò)誤率為0.01%。接近于人類(lèi)基因組計(jì)劃測(cè)序質(zhì)量標(biāo)準(zhǔn),與相關(guān)研究中大規(guī)模病毒全基因組測(cè)定質(zhì)量標(biāo)準(zhǔn)相當(dāng)。 2.病毒數(shù)據(jù)庫(kù)的建立 本研究利用MySQL語(yǔ)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(kù)。該數(shù)據(jù)庫(kù)收集了目前國(guó)際基因庫(kù)(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(cè)定的121株乙腦病毒全基因組序列信息,共同構(gòu)成了黃病毒科病毒核酸序列信息庫(kù)(共含159,179條病毒序列信息)用于進(jìn)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(duì)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?yàn)镚eneBank公布,121株乙腦病毒基因組序列為本研究測(cè)定)進(jìn)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(hào),占全部分析毒株78%(139/178)。基因Ⅱ型和基因Ⅳ型乙腦病毒無(wú)重組信號(hào);通過(guò)生物信息學(xué)和統(tǒng)計(jì)學(xué)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(guó)蚊蟲(chóng)分離乙腦病毒(SC04-25株)存在9個(gè)重組斷點(diǎn),重組斷點(diǎn)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(lái)自我國(guó)黑龍江省和韓國(guó)蚊蟲(chóng)標(biāo)本分離的乙腦病毒(HLJ02-144和K94P05株);精確計(jì)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(chóng)標(biāo)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(gè)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(jìn)化研究 本研究利用分子系統(tǒng)發(fā)育學(xué)與生物信息學(xué)技術(shù),計(jì)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(jìn)化速率,并分別依據(jù)遺傳距離和進(jìn)化時(shí)間構(gòu)建了乙腦病毒基因及全基因組的進(jìn)化樹(shù)。結(jié)果顯示,乙腦病毒結(jié)構(gòu)蛋白和非結(jié)構(gòu)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(jí),生長(zhǎng)進(jìn)化速率趨近于0,說(shuō)明乙腦病毒較其他RNA病毒,如流感病毒等生長(zhǎng)進(jìn)化速率緩慢。病毒基因組系統(tǒng)進(jìn)化分析顯示,基因Ⅲ型乙腦病毒的進(jìn)化關(guān)系與宿主來(lái)源有關(guān),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(lèi)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(dòng)力學(xué)角度計(jì)算了乙腦病毒全基因組各位點(diǎn)生物熵值(0-1.08694),為分析乙腦病毒的保守序列和高變位點(diǎn)提供了分析數(shù)據(jù);病毒進(jìn)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(jìn)化祖先分別出現(xiàn)在1742年和1798年。以上研究是首次開(kāi)展的乙腦病毒全基因組分子進(jìn)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(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R373

【引證文獻(xiàn)】

相關(guān)碩士學(xué)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(xué);2013年

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本文編號(hào)：1679824