ICAR- National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Bengaluru, 560064, Karnataka, India.
Pox Virus Laboratory, Division of Virology, ICAR-Indian Veterinary Research Institute (IVRI), Mukteswar, 263138, Nainital (District), Uttarakhand, India.
J Virol Methods. 2018 Nov;261:112-120. doi: 10.1016/j.jviromet.2018.08.015. Epub 2018 Aug 24.
Orf or contagious ecthyma, is a highly contagious transboundary disease of sheep and goats. For sero-diagnosis of orf, recombinant antigen based assays are considered as alternatives to conventional approaches such as serum neutralization test (SNT) and counter-immuno-electrophoresis (CIE). A major envelope protein of orf virus (ORFV), F1L, is highly immunogenic and is a candidate for use in these assays. In this study, the F1L gene of the ORFV-59/05 strain encoding a recombinant mature F1L protein (M-D aa) with a C- terminal truncation, was produced as a fusion protein (∼50 kDa) in Escherichia coli. The immunogenic potential of purified rF1L was confirmed by detecting specific anti-F1L antibody responses in sera collected from immunized rabbits and guinea pigs using ELISA and SNT. An indirect-ELISA based on rF1L was developed and optimized. In comparison to SNT by ROC analysis in the detection of ORFV specific antibodies, this new assay exhibited a diagnostic specificity of 94.04% and 92.53% with sheep and goat sera, respectively, while the sensitivity was 89.22% and 94.25%, for sheep and goat sera. No cross reactivity was noted with sera collected from small ruminants infected with other transboundary diseases (goatpox, sheeppox, peste des petits ruminants, foot-and-mouth disease and bluetongue). Furthermore, the rF1L-ELISA applied to screen the vaccinated/challenged goat sera resulted in better detection (30%) than by SNT (28%) in spite of lower levels of antibodies which could be due to predominant cell mediated immune response in vaccinated animals. This study highlighted the potential utility of rF1L protein as a safe and novel diagnostic reagent in comparison to live virus antigen, in the development of sero-diagnostic assay for surveillance of ORFV infection in sheep and goats.
口疮或传染性接触性脓疱病，是一种高度传染性的绵羊和山羊跨物种疾病。为了对口疮进行血清学诊断，可以使用基于重组抗原的检测方法来替代血清中和试验（SNT）和对流免疫电泳（CIE）等传统方法。口疮病毒（ORFV）的一种主要包膜蛋白 F1L 具有高度的免疫原性，是这些检测方法的候选蛋白。在本研究中，ORFV-59/05 株的 F1L 基因被构建成编码一种带有 C 端截断的重组成熟 F1L 蛋白（M-D aa）的融合蛋白（约 50 kDa），并在大肠杆菌中表达。通过 ELISA 和 SNT 检测免疫兔和豚鼠血清中特异性抗 F1L 抗体反应，证实了纯化的 rF1L 的免疫原性。建立并优化了基于 rF1L 的间接 ELISA。与 ROC 分析比较 SNT 在检测 ORFV 特异性抗体中的诊断特异性，该新检测方法对绵羊和山羊血清的特异性分别为 94.04%和 92.53%，而敏感性分别为 89.22%和 94.25%。与感染其他跨物种疾病（山羊痘、绵羊痘、小反刍兽疫、口蹄疫和蓝舌病）的小反刍动物血清无交叉反应。此外，rF1L-ELISA 应用于筛选接种/攻毒的山羊血清，其检测结果（30%）优于 SNT（28%），尽管抗体水平较低，这可能是由于接种动物中主要存在细胞介导的免疫反应。本研究强调了 rF1L 蛋白作为一种安全且新型的诊断试剂的潜在应用价值，与活病毒抗原相比，在开发针对绵羊和山羊 ORFV 感染的血清学诊断检测方法方面具有优势。
