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     

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。     

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。     

Molecular phylogenetic and positive selection analysis of Japanese encephalitis virus strains isolated from pigs in China

Japanese encephalitis virus (JEV) is one of the most important virus which causes encephalitis. This disease is most prevalent in the south, southeast and the east region of Asia. In this study, two JEV strains, named JEV/SW/GD/01/2009 and JEV/SW/GZ/09/2004, were isolated from aborted fetuses and seminal fluid of pigs in China. To determine the characteristic of these virus isolates, the virulence of two newly JEV isolates was investigated, the result evidenced that the JEV/SW/GD/01/2009 did not kill mice, while the JEV/SW/GZ/09/2004 displayed neurovirulence with 0.925 log₁₀ p.f.u./LD50. Additionally, the full genome sequences of JEV were determined and compared with other known JEV strains. Results demonstrated that the genome of two JEV isolates was 10,976 nucleotides (nt) in length. As compared to the Chinese vaccine strain SA14-14-2, the JEV/SW/GD/01/2009 and the JEV/SW/GZ/09/2004 showed 99.7% and 97.5% identity at the nucleotide level, 99.6% and 96.7% identity at the amino acid level, respectively. Phylogenetic analysis, based on the full-length genome revealed that two JEV isolates were all clustered into genotype III compared to the reference strains. Furthermore, selection analyses revealed that dominant selective pressure acting on the JEV genome was purifying selection. Four sites under positive selection were identified: codon 521 (amino acid E-227), 2296 (amino acid NS4b-24), 3048 (amino acid NS5-521) and 3055 (amino acid NS5-528). Amino acid E-227 was proved to be related to neurovirulence. Taken together, the molecular epidemiology and functional of positively selected amino acid sites of two newly JEV isolates were fully understood, which might be helpful to predict possible changes in virulence.     

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.     

Preparation of Japanese encephalitis virus nonstructural protein NS1 obtained from culture fluid of JEV-infected Vero cells

Summary The Japanese encephalitis virus (JEV) nonstructural protein NS1 was released efficiently into culture fluid of JEV-infected Vero cells. The JEV NS1 protein in the infected culture fluid was found almost as a high-molecular-weight form, probably a dimer form of NS1, and was converted to a monomer by boiling. Large amounts of NS1 protein were accumulated in the infected culture fluid. The NS1 protein, separated from JE virions by centrifugation through sucrose layer, could be obtained in large quantities.     

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.     

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.     

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。     

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.     

乙型脑炎野毒株和弱毒株（SA14-14-2）在豚鼠体内的病毒血症比较试验

目的 利用豚鼠病毒血症的产生强度对乙型脑炎野毒株和弱毒株病毒毒力进行评价.方法 以不同乙脑野毒株和减毒株及其母株病毒分别接种豚鼠,观察不同时间产生病毒血症的水平.结果 接种后1 d和3 d,不同乙脑野毒株均可检测到不同水平的病毒血症（1.00～3.40 lg pfu）,以相同滴度（104pfu）的病毒接种豚鼠时,疫苗株母株SA14产生较高水平的毒血症（2.41～3.40 lg pfu）并至少持续3 d,而SA14-14-2疫苗株未产生毒血症.结论 豚鼠病毒血症的产生强度可以作为一种新的方法对乙型脑炎病毒的减毒和疫苗株的毒力进行评价.     

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.     

Inhibition of Japanese Encephalitis Virus NS1 Protein Expression in Cell by Small Interfering RNAs

Japanese encephalitis virus (JEV), a serious mosquitoborne flavivirus, causes an acute infection of the central system resulting in encephalitis of humans and many kinds of animals. A high proportion of the survivors exhibit neurogical and psychiatric sequelae. NS1 is one of important non-structural proteins, which was found to be associated with viral RNA replication. To inhibit NS1 expression, four small interfering RNAs (siRNAs) expression plasmids (pS-NS1A, pS-NS1B, pS-NS1C and pS-NS1D) were generated to target four different coding regions of the NS1 gene, and were separately co-transfected into Vero cells with an NS1-EGFP fusion expression plasmid pNS1-EGFP. NS1 expression was evaluated by fluorescence microscope, flow cytometry assay, Western blot and RT-PCR. The results revealed that pS-NS1B, pS-NS1C and pS-NS1D could effectively and specifically inhibit NS1 expression in Vero cells. Our data suggested that these siRNAs could be used to inhibit JEV replication by silencing NS1 protein expression in further study.     

Genomic sequence of a Japanese encephalitis virus isolate from southern China

We determined the complete nucleotide sequence of a Japanese encephalitis virus (JEV) isolate (designated SH17M-2007) from a pool of Culex tritaeniorhynchus collected in southern China in 2007. The genome consisted of 10,965 nucleotides and included a single open reading frame (10,296 nucleotides) that encodes a 3,432-amino-acid polyprotein. The SH17M-2007 had 97.3 to 98.4% nucleotide identity with two Korean strains (KV1899, K94P05) and two Japanese strains (Ishikawa, JEV/sw/Mie/40/2004), but only 88.8% identity with the Chinese vaccine strain SA14-14-2. Five unique amino acid substitutions including one in the envelope (E) protein (Glu^E-306-Lys) were found in the SH17M-2007 strain. Phylogenetic relationships based on the full-length nucleotide sequences were similar to those based on the E gene.     

Pathogenic and vaccine strains of Japanese encephalitis virus elicit different levels of human macrophage effector functions

In India, Japanese encephalitis virus (JEV) remains one of the major causative agents of pediatric encephalitis. Macrophages support various neurotropic viruses and influence the immune response. However, the functional status of human macrophages during JEV infection remains unidentified. In this study, we examined the cytokine response and co-stimulatory marker levels in primary human monocyte derived macrophages (MDMs) infected with JE057434 (neurovirulent, primary clinical isolate) or SA14-14-2 (non-neurovirulent, live-attenuated vaccine) JEV strains. We also examined the differential susceptibility of these JEV strains to antiviral effects of interferon and nitric oxide. The results indicate that both JEV strains are capable of inducing various cytokines (type-I IFN, TNFα, IL6 and IL8) and co-stimulatory molecules (CD86 and CD80) in MDMs. However, they varied in replication potential and corresponding interferon sensitivity. SA14-14-2 was highly susceptible to interferon and nitric oxide when compared to JE057434. Thus, reduction in infectious virion production and increased sensitivity of SA14-14-2 towards interferon in MDMs could potentially play a role in limiting viral spread to additional target tissues.     

Activated CD56(+) lymphocytes (NK+NKT) mediate immunomodulatory and anti-viral effects during Japanese encephalitis virus infection of dendritic cells in-vitro

Japanese encephalitis virus (JEV) remains one of the major causative agents of pediatric encephalitis. Interaction of dendritic cells (DCs) with innate lymphocytes (NK and NKT) represents a crucial event during anti-viral innate immune response. In the current study, we have tried to understand the interaction between JEV, human monocyte derived DCs (MDDCs), and CD56⁺ cells (NK+NKT) in-vitro. We have used two JEV strains (i) JE057434 (neurovirulent, wild-type) and (ii) SA14-14-2 (non-neurovirulent, live-attenuated vaccine) to investigate the effect of viral virulence on the functional status of primary human MDDCs. Our preliminary results indicate that replicating JEV induces MDDCs maturation via PI3K and p38 pathways. We also show that the presence of IL2-activated CD56⁺ cells impart both immunomodulatory and anti-viral effects on DCs infected with JEV. Mechanistic studies illustrate that, IL2-activated CD56⁺ lymphocytes mediated immunomodulation occurs through direct cell-to-cell contact and TNFα, while the anti-viral effect is dependent on direct cell-to-cell contact.     

Genomic changes in an attenuated genotype I Japanese encephalitis virus and comparison with virulent parental strain

Genotype I Japanese encephalitis virus (JEV) strain SCYA201201 was previously isolated from brain tissues of aborted piglets. In this study, we obtained an attenuated SCYA201201-0901 strain by serial passage of strain SCYA201201-1 in Syrian baby hamster kidney cells, combined with multiple plaque purifications and selection for virulence in mice. We investigated the genetic changes associated with attenuation by comparing the entire genomes of SCYA201201-0901 and SCYA201201-1. Sequence comparisons identified 14 common amino acid substitutions in the coding region, with two nucleotide point mutations in the 5′-untranslated region (UTR) and another three in the 3′-UTR, which differed between the attenuated and virulent strains. In addition, a total of 13 silent nucleotide mutations were found after attenuation. These substitutions, alone or in combination, may be responsible for the attenuated phenotype of the SCYA201201-0901 strain in mice. This information will contribute to our understanding of attenuation and of the molecular basis of virulence in genotype I strains such as SCYA201201-0901, as well as aiding the development of safer JEV vaccines.     

An hsp70 fusion protein vaccine potentiates the immune response against Japanese encephalitis virus

Summary. To evaluate the possibility of developing an effective subunit vaccine against Japanese encephalitis virus (JEV), mice were intraperitoneally immunized with either a neutralizing epitope (a 27-amino-acid region of the JEV E protein), or with a fusion protein between this region and a Mycobacterium tuberculosis hsp70. Both antigens were heterologously expressed in Escherichia coli as fusion proteins with thioredoxin. The fusion protein antigen elicited a higher titer of anti-thioredoxin-neutralizing epitope antibodies and a stronger proliferation of lymphocytes than did either the neutralizing epitope (irrespective of the presence of mineral oil as an adjuvant), or the conventional JEV SA14-14-2 vaccine. Assays of antibody isotype and IFN-γ and IL-4 content in post-immunization serum showed that the fusion protein elicited a higher IgG2a titer and higher levels of IFN-γ suggesting a potentiation of the Th1 immune response. The fusion protein antigen elicited a long-lived immune response, and the antibodies were able to neutralize JEV in vitro more strongly than did those elicited by the JEV SA14-14-2 vaccine. Immunization with the fusion protein generated both humoral and cellular immune responses to JEV, and the fusion protein appeared to be a more efficient protectant than the JEV SA14-14-2 vaccine.     

Recombination and positive selection identified in complete genome sequences of Japanese encephalitis virus

The mosquito-borne Japanese encephalitis virus (JEV) causes encephalitis in man but not in pigs. Complete genomes of a human, mosquito and pig isolate from outbreaks in 1982 and 1985 in Thailand were sequenced with the aim of identifying determinants of virulence that may explain the differences in outcomes of JEV infection between pigs and man. Phylogenetic analysis revealed that five of these isolates belonged to genotype I, but the 1982 mosquito isolate belonged to genotype III. There was no evidence of recombination among the Thai isolates, but there were phylogenetic signals suggestive of recombination in a 1994 Korean isolate (K94P05). Two sites of the genome under positive selection were identified: codons 996 and 2296 (amino acids 175 of the non-structural protein NS1 and 24 of NS4B, respectively). A structurally significant substitution was seen at NS4B position 24 of the human isolate compared with the mosquito and pig isolates from the 1985 outbreak in Thailand. The potential importance of the two sites in the evolution and ecology of JEV merits further investigation.