乙型脑炎病毒减毒活疫苗生产株SA14—14—2基因组全序列的测定

目的：对乙型脑炎减毒活疫苗SA14－14－2生产株进行全序列测定和分析,为进一步了解其基因组结构与疫苗减毒机制的关系及研究疫苗生产的遗传学质控标准奠定基础。方法：根据已发表的SA14－14－2株及SA14株的序列,设计6对相互重叠片段的引物,通过RT－PCR扩增出SA14－14－2疫苗生产株的cDNA片段,分别克隆到pGEM-T载体,转化至TG1受体菌中,挑取阳性克隆进行鉴定后测定全序列。结果：SA14－140－2生产株基因组全序列长10976个核苷酸,96到10394为一个长开放读码框,编码3432个氨基酸。与国外测定的SA14和SA14－14－2株的核苷酸序列和氨基酸序列相比,同源性均在99％以上,突变位点分散于各个区域,以往推测的7个可能与减毒相关的位点均未发生改变。1296－1506间有4个突变位点,有可能作为疫苗质控的遗传学标记。结论：乙脑减毒活疫苗生产株的基因组全序列基本类似于已发表的序列,若干不同位点产生的原因可能在于病毒株的不同传代。全序列的测定对于研究疫苗株的减毒机理及疫苗的遗传学质控标准具有一定意义。     

乙型脑炎病毒活疫苗生产株SA14-14-2的感染性克隆构建

目的　构建乙型脑炎病毒 (乙脑病毒 )活疫苗生产株SA1 4 14 2的感染性克隆。方法　在对SA1 4 14 2株全长基因组测序基础上 ,引入适当酶切位点和T7RNA聚合酶启动子 ,采用分部连接策略 ,获得全长基因组cDNA ,体外转录RNA后 ,转染细胞获得感染性克隆 ,病毒传代培养、抗体中和试验和乳鼠脑腔攻击试验鉴定病毒。结果　由疫苗株cDNA构建了病毒感染性克隆 ,经鉴定为乙脑病毒 ,且病毒毒力比野毒株弱。结论　获得了乙脑病毒活疫苗生产株SA1 4 14 2的感染性克隆 ,为进一步研究疫苗的减毒机理及开发新一代疫苗奠定了基础     

2008年辽宁省乙脑病毒分离株全基因组特征分析

目的 对2008年分离自辽宁蚊虫的乙脑病毒株进行全基因组序列测定和分析,了解其全基因组特征.方法 使用针对乙脑病毒全基因组测序引物,RT-PCR扩增片段,完成对病毒全基因组序列的测定.应用Chstal X(1.83)、ATGC(V4)、DNAStar、GENEDOC(3.2)、Mega(4.0)等生物学软件完成全基因组核苷酸和氨基酸序列分析及病毒的系统进化分析.结果 乙脑病毒LN0828株基因组全长10 965个核苷酸,其中从97位到10 392位为开放读码框,编码3432个氨基酸.与GenBank中的32株乙脑病毒在全基因组水平的核苷酸总体差异率为1.6%～16.4%,氨基酸总体差异率为0.3%～5.1%.与减毒活疫苗株SA14-14-2相比,编码区共存在1186个核苷酸差异,86个氨基酸差异.全基因组序列系统进化分析显示LN0828株属于基因Ⅰ型乙脑病毒.结论 与该地区2002年和2007年乙脑病毒分离株高度同源,关键位点氨基酸未见变异.     

从病毒性脑炎患者标本分离的基因I型乙脑病毒全基因组序列特征研究

目的通过现代分子生物学理论与技术测定和分析了从脑炎患者脑脊液标本分离的基因I型乙脑病毒（GZ56株）全基因组序列特征,以了解其致病性的分子基础。方法采用RT．PCR法和核酸序列测定法获得病毒基因组全序列,并利用DNASTAR、ClustalX version2．0．9及MEGAversion4．1等生物学软件分析该乙型脑炎病毒核苷酸序列、氨基酸序列及系统进化等。结果研究结果表明从病毒性脑炎患者脑脊液标本分离的基因I型乙脑病毒（GZ56株）基因组全长为10965nt,编码3432个氨基酸。病毒全基因组分子进化分析显示GZ56株全基因组与国际上第一株从蚊虫分离的基因I型乙脑病毒（M-28株）处于同一进化分支。GZ56株与其他基因I型乙脑病毒核昔酸和氨基酸序列同源性分别为96．2％～98．6％和98．2％～99．7％。病毒E基因与乙脑病毒灭活疫苗株P3相比存在11个氨基酸差异位点,而与乙脑病毒减毒活疫苗株SA14-14-2相比,在E蛋白上存在14个氨基酸差异位点。结论本研究提示,从病毒性脑炎患者标本分离的基因I型乙脑病毒全基因组未见明显变化,从基因组水平可以推测该病毒可以被现行的乙脑疫苗所保护。     

福建省新分离3株乙型脑炎病毒的分子特征鉴定

目的　确定福建省新分离乙型脑炎病毒 (JEV)的基因分型及其E区段氨基酸序列特征。方法　用逆转录聚合酶链反应 (RT PCR)扩增并克隆新分离JEV(0 2 4 1、0 2 4 3、0 2 10 2 )的PrM、E区段核苷酸序列 ,测序后应用ClustalX软件做碱基配对和比较分析 ,种系发生采用PHYLIP软件包分析。结果　新分离的 3株JEV属于基因Ⅲ型 ,E区段核苷酸和氨基酸与减毒活疫苗株SA 14 14 2株的同源性均在 96 %以上 ,在关键结构域有部分氨基酸差异。结论　福建省新分离的 3株病毒属于基因Ⅲ型JEV ,E蛋白与疫苗株相比有部分氨基酸差异     

中国基因3型乙型脑炎病毒E基因分子特征

目的 以减毒活疫苗(SA14-14-2株)为对照,分析我国分离的基因3型乙脑病毒E基因区段核苷酸及氨基酸序列分子特征.方法 从GenBank中获取相应乙脑病毒株E基因区段核苷酸序列,通过Clustal X(1.81)、DNAStar、GENEDOC(3.2)等生物学软件进行核苷酸和氨基酸位点差异分析.以蜱传脑炎病毒可溶性蛋白晶体结构为模板进行乙脑病毒E蛋白氨基酸位点分析.结果 我国不同地域、不同宿主分离的基因3型乙脑病毒与SA14-14-2株核苷酸同源性分别在96%和95%以上,氨基酸同源性在95%和94%以上.在同一地域、同一宿主类型分离的毒株之间核苷酸和氨基酸同源性非常高.在E基因区段存在10处共同的氨基酸位点差异,在结构域Ⅰ(E160)、结构域Ⅱ(E123和E227)和两个未在结构域中的氨基酸位点(E441和E487)等5个位点在部分基因3型乙脑病毒中存在差异.结论 我国分离的基因3型乙脑病毒与减毒活疫苗株(SA14-14-2株)E基因区段同源性高,存在5处基因3型乙脑病毒特异的氨基酸位点差异,但现行减毒活疫苗株理论上可以保护我国分离的基因3型乙脑病毒野毒株.     

山东省新分离乙脑病毒(SD08-10株)全基因组序列特征

目的 对山东省新分离乙脑病毒SD08-10株进行全基因组序列测定和分析,全面了解其基因组特征.方法 设计乙脑病毒全基因组序列扩增引物,RT-PCR扩增片段,PCR产物直接测序,拼接后获得全基因组序列.采用Clestal X(1.8)、DNAStar、GENEDOC(3.2)、Mega(4.0)等生物学软件进行核苷酸序列及氨基酸序列分析和病毒的系统进化分析.结果 新分离乙脑病毒SD08-10株基因组全长10 965个核苷酸,从97位到10 392位,共10 296个核苷酸编码一个开放阅读框,编码3432个氨基酸.与GenBank登录的所有59株乙脑病毒全基因组序列比较发现,其核苷酸总体差异率为0.7%～18.9%,氨基酸总体差异率为0.1%～5.2%.与目前使用的减毒活疫苗株SA-14-14-2株相比较,全基因组共存在1253个核苷酸差异,82个氨基酸差异.全基因组序列系统进化分析显示SD08-10属于基因Ⅰ型乙脑病毒.结论 新分离的乙脑病毒SD08-10株属于基因Ⅰ型,与2007年中国分离株SH17M-07进化关系最接近.     

汉坦病毒Z37株基因组全序列分析

目的 测定Z37株的全基因组序列，了解我国汉坦病毒疫苗生产株分子基因，分析其遗传学和抗原性差异等。方法 设计出针对各片段的特异性引物，用PCR方法从Z37病毒感染的细胞提取细胞总RNA，逆转录扩增、产物克隆T载体，纯化后测序，测定的序列应用DNASTAR软件比较分析。结果 Z37株全基因组由L片段6530nt、M3651nt、S1754nt组成，分别编码2151、1133、429个氨基酸，与同型病毒间同源率高达95．2％－99．5％，绘出了3个片段的核苷酸和氨基酸的系统发生树。结论 测定得到了Z37株的全基因组序列，为研究疫苗毒株的生物学特性、致病机理等提供了分子基础。     

甲肝减毒活疫苗L-A-1疫苗株的核酸序列测定与比较分析

目的 测定甲型肝炎(甲肝)减毒活疫苗龙甲-1株(L-A-1)核酸序列，了解其减毒和适应二倍体细胞的分子机制，并与其他甲肝病毒株进行比较，得出L-A-1株的一些特点。方法 通过抗原捕获聚合酶链反应(AC-PCR)扩增甲肝病毒减毒活疫苗L-A-1株23代的片段，产物纯化后克隆至T载体并进行序列测定，应用生物信息学软件分析比较。结果 得到了甲肝减毒活疫苗L-A-1株的基因序列(nt25-7418)。L-A-1株开放读码框架(ORF)长6675个核苷酸，编码2225个氨基酸。比较分析表明，该株与国际代表株MBB株和HM175野毒株在核苷酸水平上同源性分别为98．00％和94．00％，在氨基酸水平上同源性分别为98．51％和98．65％。通过与其他细胞适应株、细胞病变株以及减毒株的比较得出：L-A-1株5′非编码区(5′NTR)nt-152、591、646、687处的突变和180-181处插入11个核苷酸以及2B区的m3889(aa1052-Val)处的突变是病毒适应宿主细胞的分子基础；2C区的m4185(aa1152-Lys)处的突变可能参与病毒的减毒过程；由于3A区缺失6个核苷酸(nt5020-5025)从而导致两个氨基酸(Asn、Asp)缺失是病毒适应细胞快速增殖的分子基础。结论 分析了LA-1株的基因序列。得出其适应细胞和减毒的核苷酸位点。     

麻疹病毒CC-47株非编码区核苷酸序列测定与比较

目的 测定麻疹病毒疫苗株长－47（CC－47）的非编码区核苷酸序列,并和其他麻疹病毒比较,为利用疫苗株病毒进一步研究奠定基础。方法 用RT－PCR分段扩增并克隆麻病毒CC－47株基因组全序列,用基因特异引物测定非编码区的核苷酸序列。结果 CC－47疫苗株非编码区在基因级上为7个不连续的片段,总计1791个核苷酸。与其他野毒株及Edmonston衍生5株疫苗株非编码区序列比较发现,CC－47疫苗株具有Edmonston凤苗株系列相同的4个特殊性的核苷酸替代位点,分别位于基因组3′末端第26位（U→A）、42位（U→G）和M／F基因间隔区FmRNA5′非翻译区的4978位（A→G）、5349位（A→G）,这些位点的改变可能会影响病毒mRNA的合成、加工和翻译次效率以及基因组的复制和包装。结论 不同基因型麻疹病毒具有同样的位点改变可能与病毒在半许可细胞中适应或减毒有关,多区域的核苷酸改变参与病毒的减毒过程。     

流行性乙型脑炎病毒活疫苗株SA14-14-2基因稳定性研究

目的 通过研究流行性乙型脑炎活疫苗减毒株基因稳定性，从分子水平证实流行性乙型脑炎活疫苗的遗传稳定性。方法分析流行性乙型脑炎活疫苗主种子、工作种子及其相应的疫苗病毒E蛋白基因核苷酸和氨基酸序列，并与其强毒株和基因库中乙脑病毒减毒株(AF15119)比较。结果乙脑活疫苗主种子、工作种子及其相应的疫苗病毒的E蛋白基因核苷酸序列完全相同。这些病毒E蛋白的氨基酸序列与基因库中乙脑病毒弱毒株(AF315119)比较显示第E447位点氨基酸有差异。结论乙脑病毒活疫苗减毒株遗传学特性稳定。     

Nucleotide sequence of the virulent SA-14 strain of Japanese encephalitis virus and its attenuated vaccine derivative, SA-14-14-2

The attenuated SA-14-14-2 strain of Japanese encephalitis (JE) virus has been used to immunize people in the People's Republic of China. Oligonucleotide fingerprints of the parent SA-14 and vaccine strain indicate that multiple genetic changes occurred during attenuation of the virus. We have cloned and sequenced the genomes of both the virulent SA-14 and attenuated SA-14-14-2 viruses to define molecular differences in the genomes. Forty-five nucleotide differences, resulting in 15 amino acid substitutions, were found by comparing sequences of the SA-14 and SA-14-14-2 genomes. Transversion of U to A occurred at position 39 in the 5'-noncoding region of SA-14-14-2 and another SA-14 vaccine derivative SA-14-5-3. A single nucleotide change in the capsid gene of SA-14-14-2 altered a single amino acid which changed its predicted secondary structure. A silent nucleotide change was found in the prM gene sequence and the M-protein was unchanged. There are seven nucleotide differences, resulting in five amino acid changes, in the E glycoprotein sequence of the two viruses. Nine amino acid differences were found in the nonstructural proteins of SA-14 and SA-14-14-2: one in NS2A, two in NS2B, three in NS3, one in ns4a, and two in NS5. A single nucleotide change at position 10,428 in the 3'-noncoding region is vaccine virus-specific. The nucleotide and deduced amino acid sequences of the vaccine strain SA-14-14-2, the parent virus SA-14, and virulent strains JaOArS982 and Beijing-1 have been compared and are highly conserved.     

三带喙库蚊和致倦库蚊胸腔接种乙型脑炎病毒减毒活疫苗SA14-14-2株的研究

目的通过实验掌握SA14142乙脑减毒活疫苗株对蚊虫的敏感性,对该疫苗的特性和安全性进行确定。方法建立三带喙库蚊和致倦库蚊的实验室种群,将乙型脑炎SA14142疫苗株病毒和乙脑野毒株(Nak)悬液做10倍系列稀释,以100～109(共10个稀释滴度)胸腔接种两种库蚊,感染后分别于8天和12天每组取10只蚊虫,研磨制成悬液,应用空斑实验方法检测蚊虫体内的病毒存在情况和病毒滴度。结果接种疫苗株后两种库蚊在100～103稀释滴度下发生感染,在104～109稀释滴度时未发生感染;被感染的三带喙库蚊最高PFU为4.48logPFU/ml,致倦库蚊为5.63logPFU/ml。接种野毒株后,致倦库蚊在100～105稀释滴度时发生感染,最高PFU为6.59logPFU/ml;三带喙库蚊在100～104稀释滴度时发生感染,其最高PFU为5.74logPFU/ml。结论蚊虫经胸腔接种SA14142株后,该疫苗病毒能在三带喙库蚊和致倦库蚊体内繁殖。     

Characterization of Russian rabies virus vaccine strain RV-97

The RV-97 rabies virus vaccine strain is widely used in Russia as a component of the live attenuated oral anti-rabies vaccine "Sinrab". This vaccine has also been used in some other countries, such as Kazakhstan, Belarus, and Ukraine. Entire genome sequencing is an effective tool for studying the genetic properties of virus strains. In this study, a simple technique for obtaining the entire genome sequence of the rabies virus was used. The entire genome sequence and the deduced amino acid sequences of the major viral proteins were compared with those of other rabies vaccine virus strains. The RV-97 strain forms a separate phylogenetic branch and seems to be phylogenetically more related to the group of Japanese vaccine strains. It also contains several unique amino acid changes in known immunodominant sites of G and P proteins.     

Comparison of replication rates and pathogenicities between the SA 14 parent and SA 14-14-2 vaccine strains of Japanese encephalitis virus in mouse brain neurons

Summary The replication rates and pathogenicities of the SA 14 parent and SA 14-14-2 vaccine strains of Japanese encephalitis (JE) virus in neurons of the mouse brain following intracerebral inoculation were compared. All the mice inoculated with the SA 14 parent strain died within one week postinoculation (p.i.), whereas all the mice inoculated with the SA 14-14-2 vaccine strains survived without showing any signs of central nervous system (CNS) involvement. The virus titers of the mouse brains inoculated with the SA 14 strain reached progressively higher levels until day 5 when the animals died. On the other hand, the virus titers of the mouse brains inoculated with the SA 14-14-2 strain persisted at low levels for several days and could not be detected after 10 days. In the routine electron microscopical study, a majority of neurons in the mouse brains inoculated with the SA 14 strain contained virions and showed characteristic cytopathological changes in connection with viral replication. In the brains inoculated with the SA 14-14-2 strain, however, we failed to find neurons containing virions or showing characteristic cytopathological changes. In the alkaline phosphatase immunostaining of paraffin-embedded sections, a majority of neurons in the brains of mice inoculated with the SA 14 strain stained positively on day 5 p.i., but only a small number of neurons in scattered small foci stained positively in the brains inoculated with the SA 14-14-2 strain. The immunogold staining of Vibratome sections also revealed the identical patterns; moreover, electron microscopical examination of the immunopositive foci of the brain inoculated with the vaccine strain revealed neurons that contained virions in dilated cisternae of rough endoplasmic reticulum (RER), indicating that the SA 14-14-2 strain also replicated, albeit poorly, in neurons. The present results showed that upon intracerebral inoculation into mice the SA 14 parent strain of JE virus grew vigorously in a large number of neurons, killing the animals, while the SA 14-14-2 vaccine strain grew poorly only in a small number of neurons without causing mortality. Possible mechanisms involved in the alteration of pathogenicity between the SA 14 parent virus and the SA 14-14-2 vaccine virus are discussed.     

JE Nakayama/JE SA14-14-2 virus structural region intertypic viruses: biological properties in the mouse model of neuroinvasive disease

A molecular clone of Japanese encephalitis (JE) virus Nakayama strain was used to create intertypic viruses containing either the 5'-C-prM-E or the prM-E region of the attenuated JE SA14-14-2 virus in the JE Nakayama background. These two intertypic JE viruses, JE-X/5'CprME(S) and JE-X/prME(S), respectively, generally resembled the parental JE virus in cell culture properties. Similar to virus derived from the JE Nakayama molecular clone (JE-XJN), JE-X/prME(S) was highly neuroinvasive and neurovirulent for young adult mice, whereas JE-X/5'CprME(S) was attenuated for neuroinvasiveness and only partially attenuated for neurovirulence. Immunization of young mice with JE-X/5'CprME(S) virus elicited neutralizing antibodies against JE Nakayama virus and conferred protection against encephalitis following challenge with JE Nakayama virus. The sequence of the JE-X/5'CprME(S) virus differed from that of JE-X/prME(S) virus at two nucleotides in the 5' UTR, 3 amino acid positions in the capsid protein, 4 positions in the prM protein and 1 in the envelope protein. For JE-X/prME(S) virus, the 4 differences in prM and the single substitution in the envelope represented reversions to the sequence of JE Nakayama virus. Overall, this study reveals that molecular determinants associated with the prM-E region of the attenuated JE SA14-14-2 virus are insufficient by themselves to confer an attenuation phenotype upon JE Nakayama virus. This suggests a role for determinants in the 5' UTR and/or the capsid protein of the JE SA 14-14-2 virus genome in influencing the virulence properties of the JE Nakayama virus in the mouse model.     

The stability of E protein gene of the Japanese encephalitis live attenuated vaccine virus SA_(14)-14-2

Japanese encephalitis (JE) virusis one of the ma-jor causes of serious encephalitis throughout Eastand South Asia .Itistransmittedto humans mainlyby infected mosquitoes . Nowadays there are twokinds of Japanese encephalitis vaccines ,inactivat-ed vaccine and live attenuated vaccine , on themarket in China . The Japanese encephalitis wildstrain SA14wasisolatedforthefirsttimein Xi-an,China in1954 .Threelive attenuated vaccine can-didates,SA14-14-2 [1] ,SA14-5-3 [2] and SA14-2-8 [3] , wer…     

武汉市新分离2株乙型脑炎病毒E基因分型及序列分析

目的 对2009年武汉市新分离的2株乙型脑炎（简称乙脑）病毒进行基因分型和序列分析,了解本地乙脑病毒株的分子生物学特性。方法 将2009年从三带喙库蚊中分离的两株乙型脑炎病毒用RT-PCR法扩增E基因,将其进行测序,并用DNAstar and MegAlign软件与其他基因型代表株进行比对。结果 16组样品检出两株阳性（WHJX9-09、WHJX10-09）,这两株阳性均属于GI型。两株新分离JEV之间的核苷酸和氨基酸同源性分别为98.9％和100％。同目前在武汉市使用的疫苗株SA-14-14-2相比,核苷酸同源性分别为87.4％、87.9％,氨基酸同源性为96.9％。共有15个氨基酸发生变异分布在3个不同结构域,中和位点没有变异但是神经毒力位点仍然存在。结论 武汉市本地新分离乙脑病毒基因型为GI型,不同于1988年在武汉检出的GⅢ型的基因型,和疫苗株SA-14-14-2相比,其神经毒力并没有减弱,但疫苗产生的抗体对新出现的GI型乙脑病毒仍有中和作用。因此提高乙脑疫苗的接种率并配合防蚊灭蚊措施对控制乙脑疫情依然至关重要。同时有必要对本市蚊虫及乙脑患者进行长期的病原学监测工作,为乙脑预测预警体系的建立提供科学依据。