Capripoxvirus is a genus of DNA viruses that includes sheep pox virus, goat pox virus and lumpy skin disease virus (LSDV). These viruses are considered high consequence viruses due to the severe clinical disease leading to economic losses to production and impact on trade. The recent spread of LSDV in Europe and Asia has demonstrated the transboundary nature of LSDV. The vaccines used to control Capripoxvirus infections currently do not have the ability to differentiate infected from vaccinated animals (DIVA) and the enzyme linked immunosorbent assays (ELISA) available for detection of antibodies to Capripoxvirus infections are not ideal. Previous research has been done to evaluate different Capripoxvirus antigens using E. coli expressed antigens; however, there has been no prior evaluation of baculovirus expressed Capripoxvirus antigens for use in ELISA. The objective of the current research was to produce 6 recombinant Capripoxvirus antigens using a baculovirus expression vector system (BEVS) and to evaluate these antigens in ELISAs to test bovine, sheep, and goat serum. The sequences used to produce the recombinant antigens were based on a consensus obtained using reference genomes consisting of all three viruses within the genus. All six antigens were produced and purified in-house using BEVS followed by benchtop or FPLC purification (Figure 1). To date, the antigens have been fully validated for performance in indirect ELISA using sheep sera (Figure 2) and partially using cattle sera, with goat sera validation to follow. The ELISAs were developed and optimized using known reference sera as controls. The performance of the ELISAs developed was assessed in comparison with the only commercially available Capripoxvirus serological test. The results demonstrate that some of the antigens used for the ELISAs completely outperformed the commercial test. Several antigens were found to be useful for ELISAs to detect responses in sheep and cattle sera. The sensitivities and specificities of the different ELISAs for sheep sera ranged from 99.54% to 53.57% sensitivity and 99.54 to 94.95% specificity with likelihood ratios above 10 using receiver operating characteristic curve analysis, while the commercial kit had a 42.85% sensitivity using the same positive sheep serum panel evaluated. The development of these serological assays allows for the simultaneous evaluation of immunogenicity of Capripoxvirus antigens through the testing of serum from experimentally infected animals. This study demonstrates the proof of principle, that sensitive and specific ELISAs can be developed for Capripoxviruses.

中文翻译：

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274 重组卡普波斯病毒抗原在间接酶联免疫吸附测定开发中的评价


卡普波克斯病毒是 DNA 病毒的一个属，包括绵羊痘病毒、山羊痘病毒和块状皮肤病病毒 （LSDV）。这些病毒被认为是高后果病毒，因为严重的临床疾病会导致生产经济损失和贸易影响。最近 LSDV 在欧洲和亚洲的传播证明了 LSDV 的跨界性质。用于控制卡普波病毒感染的疫苗目前不具备区分感染动物和接种疫苗的动物 （DIVA） 的能力，可用于检测卡普波病毒感染抗体的酶联免疫吸附测定 （ELISA） 并不理想。以前的研究已经使用大肠杆菌表达的抗原来评估不同的卡普波克斯病毒抗原;然而，之前尚未对杆状病毒表达的 Capripoxvirus 抗原用于 ELISA 进行评估。当前研究的目的是使用杆状病毒表达载体系统 （BEVS） 产生 6 种重组卡普波克斯病毒抗原，并在 ELISA 中评估这些抗原以检测牛、绵羊和山羊血清。用于产生重组抗原的序列基于使用由该属内所有三种病毒组成的参考基因组获得的共识。所有六种抗原均在内部使用 BEVS 生产和纯化，然后进行台式或 FPLC 纯化（图 1）。迄今为止，抗原在使用绵羊血清（图 2）和部分使用牛血清的间接 ELISA 中的性能已经过全面验证，随后将进行山羊血清验证。使用已知的参考血清作为对照开发和优化 ELISA。将开发的 ELISA 的性能与唯一市售的卡普波病毒血清学检测进行比较进行评估。 结果表明，用于 ELISA 的一些抗原的性能完全优于商业测试。发现几种抗原可用于 ELISA 检测绵羊和牛血清中的反应。使用受试者工作特征曲线分析，不同 ELISA 对绵羊血清的敏感性和特异性范围为 99.54% 至 53.57% 和 99.54% 至 94.95% 特异性，似然比高于 10，而商业试剂盒使用相同的阳性绵羊血清组的敏感性为 42.85%。这些血清学检测的开发允许通过检测实验感染动物的血清来同时评估 Capripoxvirus 抗原的免疫原性。本研究证明了原理证明，可以针对 Capripoxvirus 开发灵敏和特异性的 ELISA。