云南省乙型脑炎病毒基因分型研究

目的 对近30年来云南省分离的19株乙脑病毒进行PrM基因核苷酸序列测定,以确定病毒基因分型.方法乙脑病毒接种3日龄乳鼠,取发病濒死的乳鼠脑组织经研磨制成上清液,提取核酸,通过RT-PCR扩增PrM-C基因片段,用病毒PrM-C(456～695)区核苷酸序列与国内外不同年代分离的72株各基因型乙脑病毒相应序列作比较,用Woan-Ru Chen建立的方法进行基因型分析.结果 云南省分离的乙脑病毒均可引起3日龄乳鼠在78 h之内死亡;核酸序列分析结果显示,19株乙脑病毒中,17株属基因Ⅲ型,2株属基因Ⅰ型,其中M28分离自1977年;两株Ⅰ型病毒与17株Ⅲ型病毒问核苷酸序列的差异大于15%.分离自不同地区、不同时间、不同宿主的Ⅲ型乙脑病毒核苷酸间仅存在3.8%～5.2%的差异.结论云南省广泛存在基因Ⅲ型和Ⅰ型乙型脑炎病毒流行,Ⅲ型为主要流行型.     

四川省流行性乙型脑炎病毒优势基因型的研究

目的了解四川省乙脑主要流行区乙脑病毒的分子生物学特性,为防治提供依据。方法对2007-2010年间分离到的13株乙脑病毒进行PreM和E基因区扩增,采用MEGA5生物学软件完成氨基酸序列和病毒进化树分析。结果基因分型显示13株均属于基因I型。13株病毒之间比较,PreM基因核苷酸和氨基酸同源性为97％-100％和98．7％-100％,E基因核苷酸和氨基酸同源性为97．8％～99．9％和99．6％～100％,其同源性极高。13株病毒与2004年四川分离株比较E基因核苷酸同源性在97．7％～99．6％之间,氨基酸同源性在98．6％-100％之间;PreM基因的核苷酸同源性在96．2％-99．1％之间,氨基酸同源性在97．5％-98．7％之间;与疫苗株P3和SA14—14—2比较E基因核苷酸和氨基酸同源性分别为87．6％～88．3％和97％～97．8％;PreM基因核苷酸和氨基酸同源性分别为84．1％～85．8％和93．7％-96．2％。13株病毒E基因的8个氨基酸毒力位点均没有发生改变。结论四川省乙脑病毒已呈现基因I型为优势型别的态势,其PreM和E区核苷酸和氨基酸高度保守,关键的氨基酸毒力位点没有变化,提示目前使用的疫苗对流行株的感染具有保护作用。     

2010年福建省流行性乙型脑炎病毒监测

目的掌握福建省自然界蚊虫中乙型脑炎病毒感染率及基因型别特征。方法2010年在福建省三明市、建阳市和福州市采集蚊虫标本,研磨处理后采用乙脑病毒特异性检测引物进行分子筛查。PCR产物直接进行双向测序,应用ATGC、ClustalX（1．83）、MegAlign、GeneDoc3．2和Mega4等生物学软件完成全基因组序列拼接、比对和核苷酸与氨基酸同源性分析、系统进化分析。结果共采集6987只蚊虫标本,主要是三带喙库蚊和中华按蚊。3个监测点均检测到乙脑病毒核酸序列,三明市、建阳市和福州市蚊虫中乙脑病毒带毒率分别为1．25％、1．76％和0．65％。从建阳市采集的三带喙库蚊中扩增出1株乙脑病毒全基因序列,经鉴定所有检测到的乙脑病毒序列均属于基因I型。结论福建省自然界蚊虫中以基因I型乙脑病毒为主。     

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。     

A tripeptide (NSK) inhibits Japanese encephalitis virus infection in vitro and in vivo

Japanese encephalitis virus (JEV) is a major pathogen that can cause acute viral encephalitis in both humans and animals. Domain III of the viral envelope protein (EDIII) is involved in binding to host cell receptor(s) to facilitate virus entry. Our previous study showed that the loop3 peptide of EDIII possesses antiviral activity against JEV infection. In this paper, we demonstrate that three residues (NSK) in loop3 are responsible for the antiviral activity of loop3 peptide. In vitro experiments showed that the tripeptide NSK could inhibit JEV infection in both BHK-21 and Neuro-2A cells by inhibiting attachment of JEV to the cells, with IC₅₀ values of 8 μM and 6.5 μM, respectively. In vivo experiments showed that the tripeptide could increase the survival of mice challenged with JEV to 75 % when administrated intracerebrally. Therefore, this tripeptide may serve as the basis for the development of novel antiviral agents against Japanese encephalitis virus infection.     

四川省分离的基因1型乙型脑炎病毒分子特征分析

目的 从四川省巴中市采集的蚊虫标本中分离乙型脑炎(简称乙脑)病毒(JEV),确定其基因型别,并分析相关的基因1型乙脑病毒PrM和E基因区段氨基酸序列特征.方法 对2004年采集蚊虫标本进行病毒分离,对新分离的乙脑病毒进行生物学、血清学及分子生物学鉴定.逆转录聚合酶链反应(RT-PCR)扩增新分离JEV的PrM、E区段核苷酸序列,测序后应用Clustal X软件做碱基配对分析,MEGA4软件完成病毒进化分析,GENEDOC(3.2)软件完成氨基酸位点分析,根据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蛋白三维结构模拟预测分析.结果 共采集4668只蚊虫标本,主要是骚扰阿蚊和库蚊,分离到6株病毒,经鉴定均属于基因1型的乙脑病毒.将四川省分离的6个毒株结合我国新分离的基因1型乙脑病毒与减毒活疫苗株SA14-14-2株的PrM区段和E区段氨基酸比较,发现PrM区段在PrM2、64和65位存在基因1型乙脑病毒独有的氨基酸位点差异,E区段存在14处共同的氨基酸位点差异,其中在E129、222、327和366位点为中国目前分离到的基因1型乙脑病毒所特有的位点特征.结论 从四川省巴中市首次分离到基因1型的乙脑病毒,并发现基因1型乙脑病毒与减毒活疫苗株之间PrM、E基因区段存在氨基酸差异,但现行疫苗株理论上可以保护新分离的基因1型乙脑病毒.     

Cytolytic effector pathways and IFN‐γ help protect against Japanese encephalitis

Japanese encephalitis, caused by infection with the neurotropic flavivirus, Japanese encephalitis virus (JEV), is among the most important viral encephalitides in Asia. While previous studies established an essential role of Ab and type I IFN, it is still unclear if the cell‐mediated immune responses, through their direct antiviral effector functions, contribute to protection against the fatal disease. We report here that mice defective in both the granule exocytosis and death receptor pathways of cytotoxicity display increased susceptibility to JEV. The two cell contact‐dependent cytotoxic effector mechanisms act redundantly within the CNS to reduce disease severity. We also demonstrate that IFN‐γ is critical in recovery from primary infection with JEV by a mechanism involving suppression of virus growth in the CNS, and that T cells are the main source of the cytokine that promotes viral clearance from the brain. Finally, we show by in vivo depletion of NK cells that this innate immune cell population is dispensable for control of JEV infection in the periphery and in the CNS. Accordingly, cell contact‐dependent cytolytic and IFN‐γ‐dependent noncytolytic clearance of virus mediated by T cells trafficking into the CNS help in recovery from lethal infection in a mouse model of Japanese encephalitis.     

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。     

乙型脑炎病毒持续感染株在不同条件下病毒量的变化及分析

目的研究乙脑病毒持续感染株在不同条件下病毒量的变化及探讨影响病毒增殖的因素。方法乙脑病毒野生株JaGAr-01株和Nakayama株分别感染人肝癌细胞株KN73，建立乙脑病毒持续感染模型。采用空泡斑点实验进行病毒滴度测定。用持续感染病毒感染人神经纤维母细胞瘤细胞株IMR．32，在30℃和37℃下检测病毒的温度感受性；分别将两种野生株感染持续感染了病毒的KN73细胞，进行病毒重复感染实验；为探讨持续感染病毒的增殖性，将其感染KN73细胞和IMR．32细胞。结果两种持续感染病毒的增殖量均无温度差异性。重复感染实验结果：JaGAr-01株的病毒量是对照的1．3％和8．8％；Nakayama株的病毒量是对照的80．0％和1．7％。两种野生株及其持续感染株分别感染KN73和IMR．32细胞，前者中两种持续感染株的增殖性比野生株明显低下，而在后者中两株的增殖性与其野生株相近。结论两种持续感染的乙脑病毒均无温度变异株；乙脑病毒的持续感染系中可能存在含有DI粒子的变异病毒；在不同细胞中宿主因子对阻碍持续感染株的增殖存在差异。     

河南省唐河县分离到基因1型乙型脑炎病毒

目的 从河南省唐河县采集的蚊虫标本中分离乙型脑炎病毒(JEV)并确定其基因分型及E基因区段氨基酸序列特征.方法对2004年采集蚊虫标本进行病毒分离,对新分离的乙脑病毒进行生物学、血清学及分子生物学鉴定.逆转录聚合酶链反应(RT-PCR)扩增新分离JEV的PrM、E区段核苷酸序列,测序后应用Clustal X软件做碱基配对分析,MEGA3.1完成病毒进化分析,GENEDOS(3.2)软件完成氨基酸位点分析.结果共采集3722只蚊虫标本,包括:库蚊、骚扰阿蚊、伊蚊及按蚊.从库蚊标本中分离到3株属于基因1型的乙脑病毒,E区段核苷酸和氨基酸与减毒活疫苗株SA14-14-2株的同源性分别为86.9%～87.7%,氨基酸同源性为95.2%～97.0%,存在12处共同的氨基酸位点差异.结论从河南省唐河县首次分离到基因1型的乙脑病毒.E基因与疫苗株相比有部分氨基酸差异,但现行疫苗株理论上可以保护新分离乙脑病毒.     

四川省分离的基因1型乙型脑炎病毒分子特征分析

目的 从四川省巴中市采集的蚊虫标本中分离乙型脑炎(简称乙脑)病毒(JEV),确定其基因型别,并分析相关的基因1型乙脑病毒PrM和E基因区段氨基酸序列特征.方法 对2004年采集蚊虫标本进行病毒分离,对新分离的乙脑病毒进行生物学、血清学及分子生物学鉴定.逆转录聚合酶链反应(RT-PCR)扩增新分离JEV的PrM、E区段核苷酸序列,测序后应用Clustal X软件做碱基配对分析,MEGA4软件完成病毒进化分析,GENEDOC(3.2)软件完成氨基酸位点分析,根据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蛋白三维结构模拟预测分析.结果 共采集4668只蚊虫标本,主要是骚扰阿蚊和库蚊,分离到6株病毒,经鉴定均属于基因1型的乙脑病毒.将四川省分离的6个毒株结合我国新分离的基因1型乙脑病毒与减毒活疫苗株SA14-14-2株的PrM区段和E区段氨基酸比较,发现PrM区段在PrM2、64和65位存在基因1型乙脑病毒独有的氨基酸位点差异,E区段存在14处共同的氨基酸位点差异,其中在E129、222、327和366位点为中国目前分离到的基因1型乙脑病毒所特有的位点特征.结论 从四川省巴中市首次分离到基因1型的乙脑病毒,并发现基因1型乙脑病毒与减毒活疫苗株之间PrM、E基因区段存在氨基酸差异,但现行疫苗株理论上可以保护新分离的基因1型乙脑病毒.     

Japanese encephalitis and Japanese encephalitis virus in mainland China

Japanese encephalitis (JE), caused by Japanese encephalitis virus (JEV) infection, is the most important viral encephalitis in the world. Approximately 35,000–50,000 people suffer from JE every year, with a mortality rate of 10,000–15,000 people per year. Although the safety and efficacy of JE vaccines (inactivated and attenuated) have been demonstrated, China still accounts for 50% of the reported JE cases worldwide. In this review, we provide information about the burden of JE in mainland China and the corresponding epidemiology from 1949 to 2010, including the morbidity and mortality of JE; the age, gender, and vocational distribution of JE cases; its regional and seasonal distribution; and JE immunization. In addition, we discuss the relationships among vectors, hosts, and JEV isolates from mainland China; the dominant vector species for JEV transmission; the variety of JEV genotypes and the different biological characteristics of the different JEV genotypes; and the molecular evolution of JEV. Copyright © 2012 John Wiley & Sons, Ltd.     

从辽宁省再次分离到基因1型乙型脑炎病毒

目的 了解2007年在辽宁省分离的乙型脑炎病毒基因型别及其病毒E基因分子特征.方法2006年8月在辽宁省东港市采集蚊虫标本,利用组织培养细胞进行病毒分离,对病毒分离物进行血清学和分子生物学鉴定.结果从采集的30批,共1500只三带喙库蚊标本中分离到2株病毒,命名为LNDG07-02、LNDG07-16,经鉴定均为基因1型乙脑病毒.病毒E基因区段核苷酸和氨基酸序列与乙脑减毒活疫苗株(SA14-14-2株)的同源性分别为87.8%～88.0%和97.2%,新分离病毒E基因区段与疫苗株存在11处氨基酸位点差异,与2002年在辽宁省分离的乙脑病毒相比,未发现氨基酸位点变异.结论自2002年以来在东港市再次分离到基因1型乙脑病毒,与2002年在辽宁分离的基因1型乙脑病毒相比E基因区段氨基酸未发生变异.基因1型乙脑病毒在辽宁省东港市持续存在.     

Molecular characterization of Japanese encephalitis viruses circulating in pigs and mosquitoes on pig farms in the Chinese province of Henan

Since the first Chinese case report of Japanese encephalitis, Japanese encephalitis virus (JEV) has circulated in China for at least 60 years. Even though pigs play a critical role in the JEV transmission cycle information on the prevalence of JEV in pigs has not been investigated in China. As the central Chinese province of Henan has the largest human population in China, a history of serious JEV and is the largest pig producing province it was chosen for this study. We have found that currently natural infection with JEV in pigs and mosquitoes is prevalent and both genotypes 1 and 3 co-circulate in pigs and mosquitoes in central China. Phylogenetic analysis showed that all of the newly obtained pig-derived JEV isolates are more closely related to isolates from the 1950s to 1960s than to those recently isolated from humans and mosquitoes. Further analyses based on all the previous reported Chinese isolates indicates that presently genotype 3 JEV is the predominant genotype in pigs but genotype 1 JEV is emerging and spreading rapidly in recent years. Our study provides information for understanding the current epidemiology of JEV in China and suggests possible measures applicable to the further control of JEV.     

Griffithsin inhibits Japanese encephalitis virus infection in vitro and in vivo

Griffithsin (GRFT) is a broad-spectrum antiviral protein that is effective against several glycosylated viruses. Here, we have evaluated the in vitro and in vivo antiviral activities of GRFT against Japanese encephalitis virus (JEV) infection. In vitro experiments showed that treatment of JEV with GRFT before inoculation of BHK-21 cells inhibited infection in a dose-dependent manner, with 99 % inhibition at 100 μg/ml and a 50 % inhibitory concentration (IC₅₀) of 265 ng/ml (20 nM). Binding assays suggested that binding of GRFT to JEV virions inhibited JEV infection. In vivo experiment showed that GRFT (5 mg/kg) administered intraperitoneally before virus infection could completely prevent mortality in mice challenged intraperitoneally with a lethal dose of JEV. Our study also suggested that GRFT prevents JEV infection at the entry phase by targeting the virus. Collectively, our data demonstrate that GRFT is an antiviral agent with potential application in the development of therapeutics against JEV or other flavivirus infections.     

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.     

辽宁省乙脑病毒的分离与鉴定

目的 对2002年辽宁省采集的蚊虫标本进行病毒分离与鉴定。方法 从辽宁省采集蚊虫标本4927只，用细胞培养的方法分离病毒，对新分离的病毒进行血清学（ELISA和IFA方法）、分子生物学鉴定。结果 本实验共研磨蚊虫标本50批，分离到2株病毒，命名为LN02-102、LN02，104。这2株病毒均可致BHK-21细胞病变（CPE）（2—3d），致Vero细胞病变（2—4d），也可致C6／36细胞病变（2—4d）。对3日龄乳鼠2—3d可致死。这2株病毒可与标准乙脑病毒抗体反应。利用病毒PrM区段（基因组456-695核苷酸）进行基因分型分析。新分离的2株病毒属于基因Ⅰ型乙脑病毒。对这2株病毒的E基因区段进行分析，它们之间核苷酸和氨基酸的同源性为100％，与疫苗株SA14-14-2相比，核苷酸差异为4．11％，氨基酸差异在0．60％。结论 在辽宁省采集的蚊虫标本中分离到2株病毒，经血清学和分子生物学鉴定为基因Ⅰ型乙脑病毒，是近年来在辽宁省首次分离到基因Ⅰ型的乙脑病毒。     

Immunomodulatory cytokines determine the outcome of Japanese encephalitis virus infection in mice

Japanese encephalitis virus (JEV) induces an acute infection of the central nervous system, the pathogenic mechanism of which is not fully understood. To investigate host response to JEV infection, 14‐day‐old mice were infected via the extraneural route, which resulted in encephalitis and death. Mice that received JEV immune splenocyte transfer were protected from extraneural JEV infection. Pathology and gene expression profiles were then compared in brains of mice that either succumbed to JEV infection or were protected from infection by JEV immune cell transfer. Mice undergoing progressive JEV infection had increased expression of proinflammatory cytokines, chemokines, and signal transducers associated with the interferon (IFN) pathway. In contrast, mice receiving immune cell transfer had increased production of the Th2 cytokine IL‐4, and of IL‐10, with subdued expression of IFN‐γ. We observed IL‐10 to be an important factor in determining clinical outcome in JEV infection. Data obtained by microarray analysis were further confirmed by quantitative RT‐PCR. Together, these data suggest that JEV infection causes an unregulated inflammatory response that can be countered by the expression of immunomodulatory cytokines in mice that survive lethal infection. J. Med. Virol. 82:304–310, 2010. © 2009 Wiley‐Liss, Inc.     

Japanese encephalitis virus infection of mouse cell lines: ability to prime mice for generation of virus specific cytotoxic T lymphocytes and differences in CTL recognisable viral determinants

Summary Ten different mouse cell lines were examined for Japanese encephalitis virus (JEV) infection in vitro and then tested for their ability to generate virus specific cytotoxic T lymphocytes (CTL). Among all cell lines examined, Neuro 2a (a neuroblastoma) was readily infected with JEV as examined by immunofluorescence and viral replication. Among other cells, P388D1, RAW 264.7 (Macrophage origin), Sp2/0 (B-cell Hybridoma), YAC-1 (T-cell lymphoma), and L929 (Fibroblast) were semipermissive to JEV infection. The cytopathic effects caused by progressive JEV infection varied from cell line to cell line. In the case of YAC-1 cells long-term viral antigen expression was observed without significant alterations in cell viability. Intermediate degrees of cytopathicity are seen in RAW 264.7 and L929 cells while infection of PS, Neuro 2a, P388D1 and Sp2/0 caused major viability losses. All infected cell lines were able to prime adult BALB/c (H-2^d) mice for the generation of secondary JEV specific CTL. In contrast to YAC-1, the permissive neuroblastoma cell line Neuro 2a (H-2K^kD^d) was found to be least efficient in its ability to stimulate anti-viral CTL generation. Cold target competition studies demonstrated that both Neuro 2a and YAC-1 (H-2K^kD^d) cells expressed similar viral determinants that are recognised by CTL, suggesting that the reason for the lower ability of Neuro 2a to stimulate anti-viral CTL was not due to lack of viral CTL determinants. These findings demonstrate that a variety of mouse cell lines can be infected with Japanese encephalitis virus, and that these infected cells could be utilised to generate virus specific CTL in BALB/c mice.