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1.
检测猪乙型脑炎病毒抗体间接ELISA方法的建立与应用   总被引：3，自引：2，他引：1  
以乙型脑炎病毒(JEV)弱毒疫苗株作为诊断抗原,对ELISA反应条件进行优化,初步建立了检测猪乙型脑炎病毒血清抗体的间接ELISA方法。应用该法检测从江苏、安徽、山东、浙江4省部分地区收集到的1089份血样,对华东地区猪乙型脑炎血清流行病学进行初步调查。结果JEV抗体阳性率为68.2%,其中,种猪JEV抗体阳性率为82.1%,商品猪JEV抗体阳性率为62.6%。从中随机抽取135份血样与国产商品化试剂盒进行比较,间接ELISA方法的特异性和敏感性分别为92%和96.4%,2种方法的符合率为95.6%;与NS1蛋白包被建立的ELISA比较,两者的符合率为97.7%;同时,本法的阳性检出率显著高于临床普遍使用的乳胶凝集试验结果。由此表明,本试验建立的间接ELISA方法具有较高的敏感性和特异性,适于大规模猪乙型脑炎血清流行病学调查。  相似文献   

2.
Japanese encephalitis virus immunoglobulin M antibodies in porcine sera   总被引：1，自引：0，他引：1  
A solid-phase enzyme-linked immunosorbent assay (ELISA) was developed for detection of porcine immunoglobulin (Ig)M antibodies to Japanese encephalitis virus (JEV). Antibodies in sera were captured onto the solid phase of Microtiter plates sensitized with mouse monoclonal antibodies to porcine mu heavy chain. Virus antigen binding to the lawn of IgM was quantitated by subsequent binding of peroxidase-labeled human hyperimmune anti-JEV IgG, which in the final step, catalyzed a substrate color change. In sucrose density-gradient fractionated sera from recently infected pigs, the peak of ELISA JEV IgM activity corresponded to the peak of 18-S, 2-mercaptoethanol-sensitive hemagglutination-inhibiting (HAI) antibody activity. Within 2 to 3 days, JEV-infected sentinel pigs developed high JEV IgM activity; this activity decreased within 2 weeks. Among specimens collected from 99 random swine at abattoirs in Thailand during a period of low JEV transmission, none of 25 JEV HAI-negative sera had JEV IgM activity, 7 of 74 JEV HAI-positive sera did have JEV IgM activity, and the remaining 67 sera had readily detectable JEV HAI antibodies, but lacked JEV IgM. The JEV IgM solid-phase ELISA was useful for rapidly diagnosing active or recent JEV infections in swine.  相似文献   

3.
克隆表达乙脑病毒非结构蛋白NS1,并以其作为包被抗原,建立间接ELISA诊断方法。用此方法分别检测猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)及猪圆环病毒(PCV)阳性血清各3份,以及33份健康非免疫仔猪血清和205份乙型脑炎灭活疫苗免疫的猪血清,评价NS1-ELISA方法的特异性。取54份乙脑病毒感染的猪血清进行NS1-ELISA检测,评价该方法的敏感性。NS1-ELISA检测的特异性为95%,敏感性达90.7%。与商品化试剂盒比较,其符合率达到96.0%。在重复性试验中,NS1-ELISA检测方法重复性较好。本试验为进一步研究不同感染时期NS1抗体水平的差异奠定基础。  相似文献   

4.
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5.
Japanese encephalitis was recently reported from individuals in the mountain districts of Nepal without travel history to Japanese encephalitis virus (JEV) endemic areas. We performed a cross-sectional study to estimate the seroprevalence of JEV in pigs and subsequently conducted a survey of farmers to identify risk factors associated with seropositivity. In July and August, 2010, 454 pig serum samples were collected and tested by competitive ELISA. Data from a 35-question survey of 109 pig owners were analysed using multivariate logistic regression. Seventy-six (16.7, 95% CI 13.6-20.4) pigs tested positive for anti-JEV antibodies, none of which had been vaccinated against JEV or sourced from JEV endemic areas. Risk factors associated with JEV seropositivity were 'summer abortion', 'wells as a water source', 'urban location', 'reported presence of mosquitoes' and 'lower elevation'. Our results suggest that JEV is likely circulating in the mountain districts of Nepal, and that locally acquired JEV should be considered a risk for residents and travellers in these areas.  相似文献   

6.
The seroprevalence of Japanese encephalitis virus (JEV) among equines was evaluated from January 2006 to December 2009 in 13 different states of India by hemagglutination inhibition (HI) test and virus neutralization test (VNT). Antibodies against JEV were detected in 327 out of 3,286 (10%) equines with a maximum prevalence reported in the state of Manipur (91.7%) followed by Gujarat (18.5%), Madhya Pradesh (14.4%), and Uttar Pradesh (11.6%). Evidence of JEV infection was observed in equines in Indore (Madhya Pradesh) where a 4-fold or higher rise in antibody titer was observed in 21 out of 34 horses in November 2007 to October 2006. In March 2008, seven of these horses had a subsequent 4-fold rise in JEV antibody titers while this titer decreased in nine animals. JEV-positive horse sera had a JEV/WNV (West Nile virus) ratio over 2.0 according to the HI and/or VNT. These results indicated that JEV is endemic among equines in India.  相似文献   

7.
The attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) was cultured in BHK-21 cells. The viral supernatant was purified and concentrated with PEG (MW 20 000). A suitable concentration of JEV antigen was used to sensitize latex to prepare the latex antigen. The specificity, sensitivity and stability of the antigen were assessed. A latex agglutination test (LAT) was developed for rapidly detecting antibody against JEV infection. The LAT and haemagglutination inhibition (HI) assay were compared by simultaneously testing 35 porcine serum samples from five farms. Ninety per cent (20/23) of the samples were seropositive by both assays. No significant difference was found between the two methods (p > 0.05). Furthermore, when 1613 porcine sera from 120 farms were tested by LAT, the number of positive sera was 652, while that of negative sera was 961, ranging from 20% to 50% positive throughout the year. These results indicate that LAT is an appropriate candidate method for epidemiological surveys for and diagnosis of Japanese encephalitis.  相似文献   

8.
检测猪伪狂犬病病毒gE抗体红细胞凝集试验的建立及应用   总被引：2，自引：0，他引：2  
以纯化的抗人红细胞单链抗体(ScFv)—猪伪狂犬病病毒(PRV)gE蛋白双功能融合蛋白为抗原,建立了检测猪伪狂犬病毒gE抗体的红细胞凝集试验。利用方阵滴定试验筛选出最佳抗原工作浓度为55μg/mL,血清最佳稀释度为1∶20,作用时间15 min,与猪瘟(CSF)、猪细小病毒(PPV)、猪繁殖与呼吸综合征(PRRS)、猪乙型脑炎(JE)、猪布氏杆菌病(Brucellosis)阳性血清和PRV gE缺失疫苗接种的猪免疫血清均不出现红细胞凝集现象,与PRV标准阳性血清反应出现肉眼可见的凝集圈。与美国进口的PRV抗体检测gE-ELISA诊断试剂盒检测结果比较,50份猪血清的阴、阳性检出符合率均为100%。红细胞凝集试验检测方法具有操作简便、敏感性和特异性较高的特点,可用于PRV野毒感染的快速筛查。  相似文献   

9.
种公猪精液中与繁殖障碍有关的6种病毒的检测   总被引：2，自引：1，他引：1  
采用PCR和RT-PCR技术,于2006年4月～10月对上海及其周边地区的30个猪场和人工授精站的生产公猪精液样品355份进行了猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)、猪圆环病毒(PCV)、猪瘟病毒(CSFV)和日本脑炎病毒(JEV)等6种与猪繁殖障碍有关的病毒的检测,结果表明,日本脑炎病毒检测为阴性,检出PRRSV、PRV、CSFV、PPV和PCV阳性数和阳性率分别为6份(1.69%)、9份(2.54%)、5份(1.41%)、75份(21.1%)、6份(1.69%),有一个猪场的5份样品存在PRV和PPV混合感染.  相似文献   

10.
ABSTRACT: West Nile virus (WNV) is a positive-stranded RNA virus belonging to the Flaviviridae family, a large family with 3 main genera (flavivirus, hepacivirus and pestivirus). Among these viruses, there are several globally relevant human pathogens including the mosquito-borne dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), as well as tick-borne viruses such as tick-borne encephalitis virus (TBEV). Since the mid-1990s, outbreaks of WN fever and encephalitis have occurred throughout the world and WNV is now endemic in Africa, Asia, Australia, the Middle East, Europe and the Unites States. This review describes the molecular virology, epidemiology, pathogenesis, and highlights recent progress regarding diagnosis and vaccination against WNV infections.  相似文献   

11.
旨在建立一种可同时检测猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV-2)、猪细小病毒(PPV)和猪巨细胞病毒(PCMV)的多重PCR检测方法。参考相关文献及序列比对结果设计6对特异性引物,建立了可同时检测以上6种病毒的多重PCR方法并应用此方法对临床病例进行了检测。结果表明,所建立的多重PCR方法灵敏度高,对6种病毒的最低核酸检测量分别为12.8pg(PRRSV)、46pg(CSFV)、16pg(PRV)、23.5pg(PCV-2)、72pg(PPV)、8.6pg(PCMV),对猪流感病毒(SIV)、猪乙型脑炎病毒(JEV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪轮状病毒(PoRV)无特异性扩增。应用该方法对临床145份样品进行检测,总阳性率为83.45%,2种以上病毒混合感染阳性率为69.66%。说明所建立的多重PCR检测方法敏感,可用于猪群中上述6种猪病病原的单一感染或混合感染的鉴别诊断。  相似文献   

12.
In 2011, following severe flooding in Eastern Australia, an unprecedented epidemic of equine encephalitis occurred in South-Eastern Australia, caused by Murray Valley encephalitis virus (MVEV) and a new variant strain of Kunjin virus, a subtype of West Nile virus (WNVKUN). This prompted us to assess whether a delta inulin-adjuvanted, inactivated cell culture-derived Japanese encephalitis virus (JEV) vaccine (JE-ADVAX™) could be used in horses, including pregnant mares and foals, to not only induce immunity to JEV, but also elicit cross-protective antibodies against MVEV and WNVKUN. Foals, 74–152 days old, received two injections of JE-ADVAX™. The vaccine was safe and well-tolerated and induced a strong JEV-neutralizing antibody response in all foals. MVEV and WNVKUN antibody cross-reactivity was seen in 33% and 42% of the immunized foals, respectively. JE-ADVAX™ was also safe and well-tolerated in pregnant mares and induced high JEV-neutralizing titers. The neutralizing activity was passively transferred to their foals via colostrum. Foals that acquired passive immunity to JEV via maternal antibodies then were immunized with JE-ADVAX™ at 36–83 days of age, showed evidence of maternal antibody interference with low peak antibody titers post-immunization when compared to immunized foals of JEV-naïve dams. Nevertheless, when given a single JE-ADVAX™ booster immunization as yearlings, these animals developed a rapid and robust JEV-neutralizing antibody response, indicating that they were successfully primed to JEV when immunized as foals, despite the presence of maternal antibodies. Overall, JE-ADVAX™ appears safe and well-tolerated in pregnant mares and young foals and induces protective levels of JEV neutralizing antibodies with partial cross-neutralization of MVEV and WNVKUN.  相似文献   

13.
A competitive enzyme-linked immunosorbent assay (C-ELISA), based on a truncated E2 recombinant protein of the Alfort/187 strain of classical swine fever virus (CSFV) and a specific monoclonal antibody M1669, was evaluated using 2,000 sera from clinically healthy pigs in Canada (a CSFV-free country) and sera from experimentally infected pigs. The relative specificity and sensitivity of the C-ELISA were 100% and 86%, respectively, at a cutoff of 25% inhibition using negative and positive pig sera, as defined by the neutralizing peroxidase-linked assay (NPLA). A kappa value of 0.91 was obtained, indicating an excellent level of agreement between the NPLA and the C-ELISA. When sera from 120 infected pigs were used in the test at > or = 21 days postinfection, the sensitivity of the C-ELISA and the kappa value increased to 97% and 0.98, respectively. This C-ELISA will be useful when a large number of samples must be tested, as could occur during a disease outbreak or for surveillance or prevalence studies.  相似文献   

14.
为分析猪乙型脑炎病毒(JEV)E蛋白Ⅲ结构域的抗原性,本研究克隆了JEV疫苗株SA14-14-2的E蛋白结构域Ⅲ,并通过pET-28a载体进行融合表达和纯化。用纯化后蛋白作为免疫原,免疫8周龄BALB/c小鼠,通过SDS-PAGE、Western blotting、间接ELISA及间接免疫荧光方法(IFA)检测小鼠及猪抗体滴度,验证E蛋白结构域Ⅲ的抗原性。SDS-PAGE结果表明融合蛋白以包涵体形式表达;Western blotting、间接ELISA检测结果表明表达产物具有良好的抗原性;纯化的蛋白免疫BALB/c小鼠ELISA方法检测特异性抗体滴度可达1×105;猪JEV阳性血清ELISA抗体滴度可达5.1×104;IFA结果表明JEV E Ⅲ蛋白产生的抗血清能很好的识别乙型脑炎病毒抗原。以上结果表明,表达、纯化的重组JEV E Ⅲ蛋白具有良好的抗原性。本试验结果为建立以E蛋白结构域为抗原的诊断方法提供了重要依据。  相似文献   

15.
In order to analyze the antigenicity of porcine Japanese encephalitis virus (JEV) E protein domain Ⅲ, which was expressed by pET-28a vector with His-tag and purified through Ni-NTA, the BALB/c mice were immunized with the purified protein.We identified the antigenicity of domain Ⅲ of E protein and the anti-mice and anti-porcine JEV E Ⅲ protein specific antibody titers by SDS-PAGE, Western blotting, indirect ELISA and IFA.SDS-PAGE results showed the expressed target protein existed mainly in the form of inclusion body.Western blotting, ELISA test results showed that the protein had good reactivity with anti-serum.The mice immunized with the purified JEV E Ⅲ protein generated 1×105 anti-JEV E Ⅲ protein specific antibody titers by ELISA, and the porcine immunized with the porcine JEV generated 5.1×104 anti-JEV specific antibody titers.The IFA results showed that JEV E Ⅲ protein anti-serum could identify JEV antigen.The above results showed that the recombinant JEV E Ⅲ had good antigenicity.These results provided important basis for development of diagnostic antigen for JEV.  相似文献   

16.
为制备猪细小病毒VP2蛋白单克隆抗体(McAb),建立检测猪细小病毒的抗原捕捉ELISA方法 (AC-ELISA),本研究以原核表达的重组VP2蛋白作为免疫原,免疫6周龄BALB/c雌鼠,取其脾细胞与骨髓瘤细胞SP2/0进行融合,经间接ELISA方法筛选,成功获得了2株能稳定分泌抗猪细小病毒VP2蛋白的McAb,命名为3C4、5F8。以多克隆抗体作为捕获抗体、单克隆抗体5F8作为检测抗体,通过双抗夹心ELISA各个反应条件的优化,建立检测猪细小病毒抗原捕捉ELISA方法。该方法与日本乙脑病毒(JEV)、猪流行性腹泻病毒(PEDV)、伪狂犬病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)均不发生交叉反应;与RT-PCR相比较,符合率、敏感性和特异性分别为93.6%、90.9%、94.4%。本研究建立的猪细小病毒AC-ELISA有良好的重复性、敏感性和特异性,可应用于猪细小病毒感染的早期诊断。  相似文献   

17.
为了明确猪TREX1在JEV感染中的作用,本研究利用PCR扩增获得猪TREX1基因的ORF,并将该基因克隆至真核表达载体p3xFlag-CMV-14中,将构建的重组真核表达质粒命名为pFlag-pTREX1,利用lipofectamine 2000将构建成功的pFlag-pTREX1重组质粒瞬时转染至293T细胞中,通...  相似文献   

18.
本文报道了快速检测日本乙型脑炎（乙脑）抗体的被动血凝方法。用PEG提纯乙脑BHK抗原致敏醛化绵羊红细胞可用于人、猪血清中乙脑抗体的检测．该方法具有较高的敏感性和特异性，实验结果表明：该方法与血凝抑制试验具有同等的敏感性，两者符合率为85．12％，与ELISA符合率为91．70％。  相似文献   

19.
Antibody detection against selected potentially zoonotic vector‐borne alphaviruses and flaviviruses was conducted on sera from bats from all six parishes in Grenada, West Indies. Sera were tested for (i) antibodies to flaviviruses West Nile virus, St. Louis encephalitis virus, Ilhéus virus, Bussuquara virus (BSQV), Rio Bravo virus and all four serotypes of dengue virus (DENV) by plaque reduction neutralization test (PRNT); (ii) antibodies to alphaviruses western equine encephalitis virus, Venezuelan equine encephalitis virus and eastern equine encephalitis virus by epitope‐blocking enzyme‐linked immunosorbent assay (ELISA); and (iii) antibodies to the alphavirus chikungunya (CHIKV) by PRNT. Two species of fruit bats were sampled, Artibeus jamaicensis and Artibeus lituratus, all roosting in or within 1,000 m of human settlements. Fifteen (36%) of the 42 bats tested for neutralizing antibodies to CHIKV were positive. The CHIKV‐seropositive bats lived in localities spanning five of the six parishes. All 43 bats tested for epitope‐blocking ELISA antibody to the other alphaviruses were negative, except one positive for Venezuelan equine encephalitis virus. All 50 bats tested for neutralizing antibody to flaviviruses were negative, except one that had a BSQV PRNT80 titre of 20. The CHIKV serology results indicate that bats living close to and within human settlements were exposed to CHIKV in multiple locations. Importantly, bats for this study were trapped a year after the introduction and peak of the human CHIKV epidemic in Grenada. Thus, our data indicate that bats were exposed to CHIKV possibly during a time of marked decline in human cases.  相似文献   

20.
Japanese encephalitis virus (JEV) and West Nile virus (WNV) are two major mosquito borne flaviviruses belonging to same serocomplex. JEV is transmitted by Culex mosquitoes and the reservoir host for the virus is pigs and/or water birds. WNV is also transmitted by Culex mosquitoes and reservoir host in this case is birds. It can also be transmitted through contact with other infected animals, their blood, or other tissues. The envelope protein of these viruses is the major source of epitopes and provides protective immunity. Bioinformatics tools were used to identify conserved epitopes in the envelope protein of these viruses. A conserved peptide "TPVGRLVTVNPFV" present in both the viruses containing predicted T and B cell epitopes was found. The model of one of the predicted epitope was generated and upon docking it bound in the groove of HLA-A0201 Class I MHC molecule. Further, it was amenable to proteasomal cleavage enhancing its chances of processing by cytosolic pathway. The peptide was found to be non toxic, non allergenic and stable in mammalian cells based on database search. The population coverage was pan world and nearly 70% identity of the peptide was found in the Zika virus envelope protein. The peptide was located in the domain III of envelope protein which is the exposed domain therefore B cell receptors may recognize this peptide easily. The conserved peptide containing T and B cell epitopes can have future application for designing epitope based vaccines for both JEV and WNV.  相似文献   

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