Detection of African swine fever virus antigen and antibody by radioimmunoassay

Three groups of animals were infected with three different African swine fever virus isolates. Antibody responses were measured, using immuno electro-osmophoresis, reverse single radial immunodiffusion and radioimmunoassay (RIA). The RIA detected antibody at 3–4 days whereas the diffusion tests did not detect it until 10 days post inoculation. Virus survival in different transport media was compared, using haemadsorption assays and RIA. Glycerinated medium reduced infectivity titres, although it had no effect on the ability of the RIA to detect antigen.     

Swine fever: classical swine fever and African swine fever.

Because of the clinical and pathologic similarity to common endemic diseases, introduction of CSFV or ASFV strains of moderate to low virulence represents the greatest risk to North American swine herds. Producers, veterinarians, and diagnosticians should increase their awareness of these devastating diseases and request specific diagnostic testing whenever they are suspected. Production practices that improve biosecurity will reduce the risk of introduction of CSF and ASF and limit the spread if an incursion occurs. Additional resources. The following Web sites contain excellent color photographs that will assist producers and practitioners in identifying clinical signs and gross lesions associated with CSFV and ASFV: http://www.vet.uga.edu/vpp/gray_book/FAD and http://www.pighealth.com. The latter Web site and the OIE Web site (http://www.oie.int) offer updated information on current worldwide epizootics of ASF and CSF and other swine diseases. Details of biosecurity procedures can be found at http://www.agebb.missouri.edu; see publication G2340.     

非洲猪瘟病毒抗体检测间接ELISA方法的建立

以基因工程表达的非洲猪瘟病毒VP73蛋白作为包被抗原,建立了间接ELISA方法,用以检测猪血清中抗非洲猪瘟VP73蛋白的抗体。该方法对非洲猪瘟标准阳性血清的检测灵敏度可以达到1∶2 560,与同类进口ELISA试剂盒相当。此方法只特异性检出非洲猪瘟阳性血清,而对猪传染性胸膜肺炎等5种猪传染病阳性血清的检测结果均为阴性,表明其具有良好的特异性。批内和批间重复性试验结果发现,检测同一份血清的变异系数小于10%,表明其重复性较好。包被好的酶标板37℃放置5d后,对同一份血清的检测敏感性无明显变化,初步表明其稳定性较好。利用建立的间接ELISA方法和进口ELISA试剂盒分别对150份血清样品进行非洲猪瘟血清抗体检测,结果表明本方法的特异性和敏感性分别为99.1%和94.3%,2种方法检测结果的符合率为98%。以上试验表明,本试验建立的间接ELISA方法具有良好的特异性和敏感性、较好的重复性和稳定性,可以满足临床检测的需求。     

The role of antibody in protection against African swine fever virus

Intraperitoneal immunization of pigs with anti-African swine fever virus (ASFV) antibody protected them against the effects of challenge with ASFV. This protection, which was exemplified by a reduction in pyrexia and viraemia plus an increased survival time, appeared to be mediated through the effects of complement-dependent antibody-mediated cytotoxicity (CDAC) or antibody dependent cell mediated cytotoxicity (ADCC). Experiments suggested that the reduction in viraemia was associated with complement lysis whereas protection was conferred by ADCC.     

非洲猪瘟检测中的常见问题与分析

实验室检测是非洲猪瘟防控中的重要一环，但实验室检测不是仪器与试剂盒的简单叠加，是“人、机、料、法、环”多方面结合后的有效输出。在实验室广泛普及的同时，实验室的管理、质控、结果分析、报告解读及临床指导是重中之重。结果的正确解读及与临床信息的对接是其价值所在，临床及实验室的有效结合是综合诊断的基础。     

Neutralization of African swine fever virus by sera from African swine fever-resistant pigs

Sera from African swine fever-resistant pigs with infection-inhibitory activity decreased virus replication in infected porcine buffy coat cultures. This same effect was observed even after virus was adsorbed. The infection-inhibition was not reversed by removing the immune serum from the assay cultures. Reduction of African swine fever virus replication by immune sera was demonstrated by fluorescent focus assay on MS cell line cultures. Virus-neutralization tests showed a persistent fraction of non-neutralized virus, which was not demonstrable by infection-inhibition tests. One hypothesis for explaining this difference is proposed.     

African swine fever and classical swine fever: a review of the pathogenesis

This paper describes major pathogenetic mechanisms of African and Classical Swine Fever virus infections. The interactions between both viruses and the monocyte-macrophage-system result in the release of mediator molecules, which are important for the further progression of the diseases. The causes of the thrombocytopenia and the mechanisms of the haemorrhages, which are characteristic in both infections, are described. Apoptotic cell death is regarded as the predominant cause of lymphopenia in both virus infections.     

非洲猪瘟病毒无标签重组B602L抗原制备与鉴定

为了获得无标签重组抗原用于非洲猪瘟病毒(ASFV)抗体检测,本研究将B602L与类弹性蛋白多肽(ELP)基因进行融合表达,利用简单、经济的相变循环(ITC)纯化ELP-B602L融合蛋白,用烟草蚀纹病毒(TEV)蛋白酶活性包涵体切除ELP标签,再用ITC回收重组B602L蛋白;用抗体阳性猪血清对重组B602L进行鉴定;以重组B602L蛋白为包被抗原进行ELISA鉴定。结果显示重组大肠杆菌能正确表达ELP-B602L融合蛋白,纯化融合蛋白纯度大于85%;TEV蛋白酶活性包涵体切除ELP标签的效率大于90%,回收的重组B602L蛋白纯度大于90%,能与抗体阳性猪血清反应;以重组B602L蛋白为包被抗原建立的ELISA与抗体阳性血清反应为阳性,与抗体阴性血清反应为阴性,OD450值与血清稀释倍数具有线性相关性。     

Comparison of a radioimmunoprecipitation assay to immunoblotting and ELISA for detection of antibody to African swine fever virus

A radioimmunoprecipitation assay (RIPA) has been developed for detection of antibody to African swine fever virus (ASFV) and compared with the immunoblot assay with regard to sensitivity and specificity. Two hundred seven field sera, obtained from pigs in Spain from different geographic areas between 1975 and 1986, that were positive by ASFV enzyme-linked immunosorbent assay (ELISA) were also analysed by immunoblot assay and RIPA. By serum dilution experiments, the RIPA appeared at least as sensitive as the ELISA and immunoblotting tests, although ELISA and RIPA detected antibodies to ASFV earlier in natural infection than did the immunoblot assay, as disclosed by animal inoculation studies. The most antigenic ASFV-induced proteins in natural infection detected by RIPA were the viral proteins p243, p172, p73, p25.5, p15, and p12 and the infection proteins p30 and p23.5. In the immunoblot assay, the proteins that were most reactive with the same sera were the viral protein p25.5 and the infection proteins p30, p25, and p21.5. Only 1 serum, from an animal infected with ASFV, was negative by immunoblot assay but showed a positive result by RIPA. A modification of conventional RIPA was performed using a dot transference of immunoprecipitated proteins to a nitrocellulose filter. This modification simplified the conventional RIPA procedures by eliminating the electrophoresis of immunoprecipitated proteins without affecting sensitivity and specificity. The ease of use, specificity, and the sensitivity comparable to that of the immunoblot assay make the RIPA a useful confirmatory assay for sera that yield conflicting results in other ASFV antibody assays.     

Cytotoxic lymphocytes induced by African swine fever infection

The generation of lymphocytes cytotoxic to African swine fever virus infected testis cells during in vivo infection is described. Peripheral blood lymphocytes from 16 pigs developed cytotoxicity seven to eight days after infection but the lysis was not restricted to autologous cells.     

Diversity of African swine fever virus

An African swine fever virus is an heterogeneous population, consisting of clones having different biological characteristics in respect to hemadsorption, virulence, infectivity, plaque size, and antigenic determinants. The following observations were made: Nonhemadsorbing virus (NHV) have been segregated from field isolates from Haiti (HT-1) and a bone marrow- and buffy coat-passaged Portuguese isolate (L'60BM89BC1) and appear as a major, minor, or equal mixture with hemadsorbing viruses in the virus population. Biological characteristics of the virus inoculated into pigs often differed from viruses isolated later from the same pigs. Virulence and nonhemadsorbing characteristics of isolated clones were genetically stable. The lethal effect of 2 NHV clones of L'60BM89BC1 virus was dose-dependent; small doses of virus induced immunologic deaths or recoveries from the clinical disease in pigs, and large doses induced acute deaths. The NHV of Lisbon isolate of 1960 (L'60) and HT-1 isolate share the same antigenic determinants for inducing protection. Tengani isolate contained clones of distinctly different antigenic determinants, not shared by L'60 or HT-1 isolate that enabled it to overcome the protection induced by the other clones. Passaging of an African swine fever virus isolate in pigs or cell cultures may readily alter the proportions of the different clones in the population and thereby change its overall characteristics. A new virus population with atypical hemadsorption was found in HT-1 field isolate and L'60BM89BC1 virus.     

猪瘟病毒垂直感染的检测

猪瘟是一种急性、高度接触性传染的一种重要传染病。近年来猪瘟流行的方式发生了改变，典型猪瘟逐渐减少，非典型猪瘟逐渐增多，表现怀孕母猪流产，产死胎、木乃伊和病弱的仔猪等，尤其是新生仔猪猪瘟，临床症状不十分明显，但发病率、病死率较高，严重危害养猪业的发展。关于新生仔猪猪瘟的发生，多认为是母猪怀孕期间感染猪瘟病毒而通过垂直传播的方式引起仔猪感染，但到目前为止未见有关新生仔猪垂直感染猪瘟病毒的直接证据。     

非洲猪瘟病毒微滴数字PCR检测方法的建立

建立一种具有高灵敏度的检测非洲猪瘟病毒的微滴数字PCR方法,为非洲猪瘟的快速诊断和流行病学研究提供技术支持。在《OIE陆生动物诊断与疫苗手册》中提供的qPCR检测方法的基础上,建立了非洲猪瘟病毒微滴数字PCR方法,对该方法的敏感性、特异性和重复性进行了评估。结果显示,该方法的最低检测限可达0.8copies/μL,敏感性高于qPCR方法,不与猪常见的6种病毒发生交叉反应,而且重复性较好。采用建立的微滴数字PCR和OIE的qPCR方法分别对78份临床病料进行非洲猪瘟病毒的检测,结果显示2种方法的符合率为100%。本研究建立的微滴数字PCR方法具有理想的敏感性和特异性,适用于临床上非洲猪瘟病毒的检测。