【作者】 王（韦华）；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2009， 博士

【摘要】 流行性乙型脑炎(Epidemic encephalitis B)也称日本脑炎(Japanese encephalitis),是由流行性乙型脑炎病毒(Japanese encephalitis virus,JEV,日本脑炎病毒,Epidemic encephalitis virus B)引起的一种重要的蚊媒性人兽共患传染病。乙型脑炎以三带喙库蚊为主要传播媒介,属于自然疫源性疾病,猪被认为是乙型脑炎病毒的重要贮存宿主与增殖宿主,是乙型脑炎的主要传染源,也是对人类威胁最大的扩散宿主。人感染后可引起脑炎症状,轻者留下后遗症,重者可导致死亡。JEV是引起母猪出现繁殖障碍的主要病原体之一,对猪的致死率不高,但可引起怀孕母猪流产、死胎或木乃伊胎,也可引起公猪的睾丸急性炎症反应或不育,给养猪业的持续高产发展造成巨大的经济损失。我国是乙型脑炎发病最多的国家之一。随着时间的变化,JEV在不同的地区存在着潜在的生物学特性变异,这就为乙型脑炎的监控和防制带来了一定的难度。近年来国内外许多学者从猪脑组织、流产的死胎及蚊虫体内分离到乙型脑炎病毒,并对部分毒株进行了基因组全序列的克隆与分析。为了解陕西省猪乙型脑炎的流行状况,本试验对部分地区进行了流行病学调查,从猪脑组织中分离到新的JEV毒株,对其进行全长基因组克隆和分析,并在大肠埃希菌中表达其主要抗原蛋白,建立ELISA检测方法,获得了以下结果:1.采用乳胶凝集试验和一步反转录套式PCR方法对陕西省部分地区进行猪乙型脑炎病毒的抗体检测及核酸检测,结果表明,已免疫猪场JEV抗体阳性率为94.80%,核酸阳性率为0.56%,可能存在免疫失败现象;未免疫的小型猪场和散养户JEV抗体阳性率为11.72%,核酸阳性率为10.73 %,确诊感染乙型脑炎病毒;该地三带喙库蚊和淡色库蚊为优势蚊种,可能为乙型脑炎传播的主要媒介。2.采用乳鼠接种试验和BHK-21细胞分离培养试验,从猪脑组织病料中分离获得了具有特异致病性和致细胞病变(CPE)的毒株。根据细胞病变特征、血凝试验结果和间接免疫荧光试验结果,证实分离毒株为乙型脑炎病毒,命名为SXBJ07株。采用RT-PCR法对分离株的PrM基因和E基因进行扩增测序,与SA14株和SA14-14-2株E基因核苷酸的同源性分别为88.4%和87.7%,氨基酸同源性分别为99.0%和97.0%,进一步从分子水平上证实了分离出的病毒为JEV。3.应用RT-PCR和RACE方法,扩增得到了全长为10 965 bp的SXBJ07株基因组全序列,并对其进行序列分析和同源性比较,结果表明,SXBJ07株5′端和3′端分别存在多个位点碱基的缺失和插入;在E蛋白活性结构域内有13个位点的氨基酸发生了变异;SXBJ07株全基因组序列与参考株比较,与基因Ⅰ型分离株核苷酸同源性为97.2%～99.0%,氨基酸同源性为98.1%～99.8%,其中与JEV/sw/Mie/40株亲缘关系最近;根据PrM基因和E基因进行基因型鉴定,SXBJ07株属于基因Ⅰ型,为陕西省境内首次分离到的基因Ⅰ型JEV毒株。4.将JEV E蛋白基因插入到原核表达载体pET-32α中,成功构建了E蛋白的重组表达载体;转化DH5α大肠埃希菌,成功表达了E蛋白。对表达的蛋白鉴定证明其与预测的目的蛋白大小一致。Western blot检测结果表明,原核系统表达的E蛋白可以与猪乙型脑炎阳性血清抗体特异性结合。以纯化的JEV E基因表达产物为包被抗原,经方阵滴定、阴阳性界限、特异性试验及批内、批间试验等成功的建立了一种特异、敏感的检测JEV血清抗体的间接ELISA方法,临床应用表明该方法具有很高的特异性和灵敏度。更多还原

【Abstract】 Epidemic encephalitis B (Japanese encephalitis), caused by Epidemic encephalitis virus B (Japanese encephalitis virus, JEV, Epidemic encephalitis virus B), was an important mosquito-borne zoonosis. Culex mosquitoes acted as the primary media of JE, the natural focus diseases. In development of JE, swine was considered the most important natural amplifying host and amplifier among several animals and also the transmitter mostly threatening the health of human beings. The infected patients showed encephalitis or suffered from neurological sequelae even fatal. JEV was the one of main pathogens caused reproductive disturbance in sows. Infected sows showed lower fatality rate, but may suffer from abortion, stillborn delivery or mummified fetuses. JEV also caused boar acute orchitis or infertility, which has been recognized as one of the most economically diseases in the swine industry worldwide.There were the most JE cases in China, but JEV could show mutation with the changes of location and time, which can make things difficult for monitoring and control of JE. A number of geographically diverse JEV strains have been isolated at different times from humans, mosquitoes and pigs. In recent years, lots of isolates of JEV strain were obtained from swine brain, stillborn or mosquito, and several strains have been cloned and fully sequenced. To understand the epidemic status of JE in Shaanxi province, epidemiological investigation was studied in part of areas in this paper. The new JEV strain was isolated from swine brain, and the full-length genome has been cloned and analyzed. The main antigenic protein was expressed in E. coli, and established the ELISA detection. Experimental results obtained as follows.1. The JEV antibody and nucleic acid in part of Shaanxi province were detected by LAT and one-step RT-nested-PCR. The results showed that the positive rate of antibody and nucleic acid in immunized farm were 94.80% and 0.56%, respectively, which may caused by the immunized failure. The positive rate of antibody and nucleic acid in non-immunized farm and house keeper were 11.72% and 10.73%, which can diagnose as JE. Culex tritaeniorhynchus and Culex pipiens pallens were the local predominant and may acted as the primary media of JE. 2. By nude mice inoculation and isolation and culture in BHK-21, the new JEV strain of specific pathogenicity and CPE was obtained from swine brain specimens. According to the results of CPE characteristic, hemagglutination test and indirect immunofluores- cence, the mew isolate was confirmed as JEV and named SXBJ07 strain. The results of amplification and sequencing the PrM gene and E gene by RT-PCR and homology analyzing the nucleotide and amino acid can further verified the SXBJ07 strain in molecular level.3. From RT-PCR and rapid-amplification of cDNA ends (RACE), the SXB07 strain genome full sequence of 10 965 bp length was obtained. Sequences analyze and homology comparison showed that there were many deletion and insertion in the 5’NTR and 3’NTR, and there were amino acid substitutions in 13 sites of the active donmains of E protein. Compared the full genome sequences of SXBJ07 with other reference strains, there were higher homology of nucleotide and amino acid with genotypeⅠ, and JEV/sw/Mie/40 has the nearest genetic relationship in all these strains. Identification of genotype based on PrM and E gene showed that SXBJ07 strain belong to genotypeⅠ. This was the first JEV genotypeⅠstrain isolated from Shaanxi.4. Inserting the JEV envelope protein gene into prokaryotic expression vector pET-32α, recombinant expression vector was constructed. The recombinant vector was translated to E. coli DH5αand successfully expressed the envelope protein. The results of SDS-PAGE analysis indicated that fusion protein was accord with target protein. Western blot analysis showed that the expressed envelope protein from prokaryotic expression can specifically combine with positive serum antibody. The purified JEV envelope protein acted as envelope antigen, this study established a specific and sensitive indirect ELISA detection through chessboard titration, determining the threshold quantity between negative and positive, specificity test, and within-run, run to run test. The clinical detection results showed that the ELISA detection has higher specificity and sensitivity.更多还原