Antigenic and Genetic Variations among Japanese Encephalitis Virus Strains Belonging to Genotype 1

Hyperimmune antisera against four Japanese encephalitis (JE) virus strains, ThCMAr4492 and ThCMAr6793 from Thailand and Nakayama and JaGAr01 from Japan, were used to analyze the antigenic relationships among 12 Thai strains belonging to genotype 1, and two Japanese strains and one Chinese strain belonging to genotype 3. The antiserum for ThCMAr6793 significantly neutralized nine of the 12 Thai strains, none of which was significantly neutralized by antisera for the Nakayama and JaGAr01 strains. The antiserum for ThCMAr4492 neutralized only its homologous strain; therefore, ThCMAr4492 was antigenically different from all other strains. Two Thai strains (Subin and KE-093/83) were significantly less neutralized by all four of the antisera tested. In the deduced amino-acid sequence of the E protein, the 12 Thai strains revealed 100 to 98.2% identity among them and 90.0 to 98.8% identity with the published strains, respectively. Among significant amino-acid substitutions, three residues at positions E-222, E-327 and E-366 were shared by all of the Thai strains, whereas residues at E-89, E-123, E-131, E-178, E-293, E-351 and E-373 seemed to be strain-specific. The amino acids at positions E-178, E-327, E-351, E-373 and E-366 are found either in the peptides with functional T-helper cell epitopes or in the ectodomain of the E protein of other flaviviruses. These amino acids may therefore be responsible for determining the antigenic heterogeneity of these strains.     

乙脑/寨卡病毒嵌合减毒活疫苗的构建与评价

为构建乙脑/寨卡嵌合病毒减毒活疫苗候选株并评估其安全性及免疫保护作用,将寨卡病毒FSS13025株和PRVABC59株的prM/E基因分别替换至乙脑病毒疫苗株的相应区域,构建乙脑寨卡嵌合病毒全长感染性克隆,体外转录获得病毒RNA,并电转染入BHK-21细胞拯救获得ChimZIKV(13025)和ChimZIKV(59)两株嵌合病毒。酶切、免疫荧光及测序结果显示,感染性克隆构建正确且嵌合病毒拯救成功;蚀斑实验发现与亲本株相比,嵌合病毒的蚀斑直径变小(P<0.01);生长曲线显示,嵌合病毒在BHK-21和C6/36细胞中的增殖能力弱于乙脑疫苗株;细胞毒性检测发现嵌合病毒对传代细胞的毒性弱于乙脑疫苗株(P<0.01);嵌合病毒对小鼠表现出低神经侵袭力与较高的脑内神经毒力;ChimZIKV(13025)和ChimZIKV(59)免疫小鼠可产生较高的中和抗体效价,免疫雌鼠后,新生乳鼠对寨卡野毒株PRVABC59的攻击保护率分别为95%和89%,且在乳鼠体内检测不到病毒血症。本研究结果表明嵌合病毒ChimZIKV(13025)和ChimZIKV(59)具有较好的免疫原性和较低的神经侵袭力,免疫雌鼠后可对新生乳鼠提供较强的保护力,但对小鼠仍有一定的脑内神经毒力。     

马西尼罗热病毒E蛋白部分基因在大肠杆菌中的表达

参考GenBank发表的西尼罗病毒(west nile virus,WNV)的E蛋白基因序列,自行设计合成一对引物,利用RT-PCR扩增出了WMV E基因318bp片段,将其克隆入pMD18-T-Vector载体中,阳性克隆命名pMD-E,并进行序列分析。进一步亚克隆入表达载体pET-32a( )。重组质粒pET32a-E转化BL21(DE3)感受态细胞中表达,表达产物经SDS-PAGE可检测到分子量约为32kD的目的蛋白带,经薄层扫描分析,目的蛋白占菌体总蛋白的33．1%。表达产物纯化后,Wester-blotting分析证明表达产物能被WNV的阳性血清所识别,为下一步建立以表达产物为包被抗原建立检测马的WNV的ELISA方法打下了基础。     

Localization and Functions of Japanese Encephalitis Virus Nonstructural Proteins NS3 and NS5 for Viral RNA Synthesis in the Infected Cells

Recently it has been reported that Japanese encephalitis virus (JEV)-specific RNAs can be synthesized in vitro in the subcellular fraction including outer-nuclear membrane (Takegami and Hotta, 1989). The results of Western blot analysis and indirect immunofluorescence test using two kinds of monospecific antisera against JEV nonstructural proteins NS3 and NS5 showed that NS3 and NS5 were membrane-associated proteins and formed the complex at the perinuclear site in the infected cells. Both antisera against NS3 and NS5 inhibited in vitro RNA synthesis. These results suggest that NS5 and NS3 play important role(s) in flavivirus RNA replication.     

Japanese Encephalitis Virus Infection in Vero Cells: The Involvement of Intracellular Acidic Vesicles in the Early Phase of Viral Infection Was Observed with the Treatment of a Specific Vacuolar Type H+-ATPase Inhibitor,Bafilomycin A1

The involvement of intracellular acidic vesicles in the early phase of Japanese encephalitis (JE) virus infection in Vero cells was observed by adding a specific vacuolar type H⁺ -ATPase (V-ATPase) inhibitor (bafilomycin A1) in the cell culture medium. Studies with the detection of viral envelope (E) protein suggested that bafilomycin A1 inhibited virus infection in the cells. Subcellular distribution of incoming biotinylated virions and ³H-uridine-labeled viral RNA were observed in fractions of a Percoll density gradient. At 10 min of the chasing period, virions and viral RNA were found mainly in fractions with a mean density of 1.04 g/ml corresponding to the endosome both in the control and bafilomycin A1-treated cells. At 60 min of the chasing period, the peak of biotin activity was detected in fractions with a mean density of 1.08 g/ml corresponding to the lysosome, whereas the peak of radioactivity did not run parallel with that of biotin and shifted to fractions with a mean density of 1.05 g/ml and higher than 1.084 g/ml, respectively. At 60 min of the chasing period in bafilomycin A1-treated cells, the peak of biotin and radioactivity were still found mainly in the fraction with a density of 1.04 g/ml, representing the endosome. Subcellular fractionation by a Percoll density gradient revealed that bafilomycin A1 treatment resulted in the accumulation of virions in the endosome fraction and suggested the prevention of intracellular translocation of the virions which occurs during the early entry process of an infecting virus to the cells.     

Construction of human Fab (gamma1/kappa) library and identification of human monoclonal Fab possessing neutralizing potency against Japanese encephalitis virus

A combinatorial human Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from Japanese encephalitis virus hyper-immune volunteers on pComb3H phagemid vector. The size of the constructed Fab library was 3.3x10(8) Escherichia coli transformants. The library was panned 3 times on the purified Japanese encephalitis virus (JEV) virion, and phage clones displaying JEV antigen-specific Fab were enriched. The enriched phage pool was then screened for clones producing Fab molecule with JEV neutralizing activity by the focus reduction-neutralizing test. Among 188 randomly selected clones, 9 Fab preparations revealed neutralizing activities against JEV strain Nakayama. An E. coli transformed with TJE12B02 clone, which produced human monoclonal Fab with the highest neutralizing activity was cultured in a large scale, and the Fab molecule was purified using affinity chromatography. The purified FabTJE12B02 showed the 50% focus reduction endpoint at the concentration of 50.2 microg/ml (ca. 1,000 nM) when JEV strain Nakayama was used. The FabTJE12B02 recognized E protein of JEV strain Nakayama, and the dissociation equilibrium constant (Kd) of the FabTJE12B02 against purified JEV antigen was calculated as 1.21x10(-8) M. Sequence analysis demonstrated that TJE12B02 used a VH sequence homologous to the VH3 family showing 88.8% homology to germline VH3-23, and used a Vkappa sequence homologous to the VkappaII subgroup showing 92.8% homology to germline A17.     

JEV-DNA实时荧光定量标准品的构建

利用TaqMan荧光定量PCR技术,建立JEV-DNA定量标准品的制备方法。通过处理JEV减毒活疫苗提取病毒RNA,进行RT-PCR扩增目的片段,与T载体连接,转化感受态细胞,T-A克隆,测序鉴定后定量。获得预期的重组质粒,建立的标准曲线有较大的线性范围。此法制备的重组质粒标准品可用于对病毒载量进行测定。     

Epidemiological and Ecological Studies of Japanese Encephalitis in Okinawa,Subtropical Area in Japan. II. Prevalence of Japanese Encephalitis Antibody in Residents in Okinawa,Miyako and Ishigaki Islands

During 1989 to 1990, human sera were collected by age groups in Okinawa (the northern, central and southern areas), Miyako and Ishigaki islands and examined for the neutralization (N) antibodies to two strains, Nakayama (vaccine strain) and C307 (Okinawan strain), of Japanese encephalitis (JE) virus. In Okinawa island, the N antibody positive rate to C307 was higher than that to Nakayama, while in Miyako and Ishigaki islands, the positive rate to Nakayama was higher than that to C307, suggesting that JE virus transmission rate was higher in Okinawa than in Miyako and Ishigaki islands. In Okinawa Prefecture, JE vaccine had not been administered to most of residents over 31 years of age at the time of serum collection. In residents over 31 years old, the positive rate to C307 was highest in the north of Okinawa (83.3%) and was lowest in Miyako (26.3%), with the second lowest in Ishigaki (33.3%). The distribution of N antibody titers to C307 gave hyperbolic patterns in the 0–5 age groups in Miyako and Ishigaki, and also in the 31–40, 41–50 age groups in Miyako and the 41–50 age group in Ishigaki, suggesting low rates of natural infection in these 4–5 decades in both islands. In residents of ages subjected to JE vaccine, a characteristic pattern was obtained, in which the curves to Nakayama shifted to higher titers than those to C307, suggesting that the first antigenic stimulation was caused by vaccine, not by natural infection of JE virus.     

乙型脑炎病毒及其疫苗的研究进展

流行性乙型脑炎是由乙型脑炎病毒引起的、经蚊虫传播的严重危害中枢神经系统的人畜共患急性传染病,其重症病死率高,易造成永久性的神经系统后遗症,严重威胁着人类的健康。目前尚无特效的治疗流行性乙型脑炎的方法,控制蚊虫传播和免疫接种是当前的主要防御手段。简要综述了乙型脑炎病毒的基因组结构、结构蛋白与非结构蛋白功能、基因分型,以及流行性乙型脑炎疫苗的研究进展。     

乙型脑炎疫苗研究进展

乙型脑炎（乙脑）的流行是世界性的公共卫生问题之一,其流行范围正逐步扩大。目前对于乙脑尚无特效治疗手段,因此预防乙脑的发生尤为重要。随着对日本脑炎病毒研究的深入,利用基因工程技术研制新型候选疫苗已成为预防乙脑新的发展方向。我们简要综述了乙型脑炎病毒的基因组结构及其蛋白功能,以及国内外乙型脑炎疫苗研发的新进展。     

中国流行性乙型脑炎病毒分子生物学特性研究

为了从分子水平了解中国流行性乙型脑炎(乙脑)病毒与国外分离株的差异,对1949年以来在中国乙脑主要流行地区分离的19株乙脑病毒的PrM-C区及E蛋白基因区的核苷酸序列进行了对比研究,以期了解不同时间不同地区在中国流行的乙脑病毒的分子差异.结果显示:病毒生物学初步鉴定显示,病毒感染BHK细胞后56h所有细胞均发生病变,约50%以上细胞脱落(CPE( ));3日龄乳鼠接种病毒72h之内死亡;所有病毒均与乙脑病毒(A2株)抗血清发生阳性反应,但反应强度不同,提示所有病毒均为乙脑病毒.病毒PrM-C(456～695)区核苷酸序列与各个国家及地区、各个基因型以及各个年代的73株乙脑病毒相应序列,用Woan-Ru Chen建立的方法进行基因分型分析,结果显示,所有19株中国分离的病毒均属于基因型Ⅲ型,与国外相关文献所报道的乙型脑炎病毒中国分离株的基因型相符.以乙型脑炎病毒疫苗株P3株为标准,对各病毒E蛋白500个氨基酸序列进行分析.各毒株核苷酸差异在0和4.2%之间,氨基酸差异在0和4.0%之间.所有核苷酸差异均为碱基的替换,无论核苷酸或氨基酸均无插入或丢失.大多数核苷酸变异在氨基酸编码的第三位,属于沉默突变,不引起氨基酸变化.18株病毒E蛋白活性结构域与疫苗株P3株相比共有247个氨基酸的差异,平均每株病毒有12.35个氨基酸的差异,有11株病毒(SA14、TLA、CZX、CBH、G35、GSS、LYZ、SH-3、YLG、YN、ZMT)在该区的差异数低于这个平均值.而在超过这一平均数的7个病毒株(A2、HA-3、47、CH-13、CTS、LFM、ZSZ)中,LFM、ZSZ、47与疫苗株相比较分别有16～20个氨基酸的差异,相对其它17株病毒而言,差异较为明显.以上线索提示,现有的乙型脑炎灭活疫苗在理论上可以覆盖包括现存病毒在内的毒株.