Nokhwal Alka, Kumar Ram, Chander Yogesh, Khandelwal Nitin, Verma Assim, Riyesh Thachamvally, Tripathi Bhupendra N, Kumar Naveen
National Centre for Veterinary Type Cultures, ICAR-National Research Centre on Equines, Hisar, India.
National Centre for Veterinary Type Cultures, ICAR-National Research Centre on Equines, Hisar, India; ICMR-National Institute of Virology, Pune, India.
J Virol Methods. 2025 Dec;338:115200. doi: 10.1016/j.jviromet.2025.115200. Epub 2025 Jun 8.
Mass vaccination to achieve disease-free status requires a serological test to differentiate infected and vaccinated animals (DIVA) towards the end of the control program. The lumpy skin disease (LSD) vaccines currently in use (licensed) are not DIVA-compatible. India recently approved a new live-attenuated LSD vaccine derived from the local LSD virus (LSDV) strain (Ranchi). Unlike field LSDV strains, the Ranchi strain has a distinct 801-nucleotide deletion in its inverted terminal repeat (ITR) region, affecting ORF003/ORF154. In this study, we successfully cloned and expressed LSDV ORF154 into pET28a and purified the His-tagged protein using Ni-NTA affinity chromatography. SDS-PAGE and Western blot analyses confirmed the presence of a ∼28 kDa protein, consistent with the predicted molecular weight. An optimized antigen concentration of 500 ng/well and serum dilution of 1:50, and at a positivity cut-off of 22.63 %, the assay showed high sensitivity (95.77 %) and specificity (95.42 %), effectively distinguishing infected from vaccinated cattle. These findings demonstrate the potential of an ORF154-based ELISA as a reliable serological diagnostic tool for LSDV surveillance and disease control programs, making the Ranchi strain-based LSDV vaccine the first DIVA-compatible vaccine.
大规模接种疫苗以实现无病状态需要在防控计划结束时进行血清学检测，以区分感染动物和接种疫苗的动物（DIVA）。目前使用的（已获许可的）牛结节性皮肤病（LSD）疫苗不具备DIVA兼容性。印度最近批准了一种源自当地LSD病毒（LSDV）毒株（兰契毒株）的新型减毒活LSD疫苗。与野外LSDV毒株不同，兰契毒株在其反向末端重复（ITR）区域有一个独特的801个核苷酸的缺失，影响ORF003/ORF154。在本研究中，我们成功地将LSDV ORF154克隆到pET28a中并进行表达，然后使用镍-氮三乙酸（Ni-NTA）亲和层析法纯化了带有His标签的蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹分析证实存在一种约28 kDa的蛋白，与预测的分子量一致。在优化的抗原浓度为500 ng/孔、血清稀释度为1:50且阳性判定值为22.63%的情况下，该检测方法显示出高灵敏度（95.77%）和特异性（95.42%），能有效区分感染牛和接种疫苗的牛。这些研究结果表明基于ORF154的酶联免疫吸附测定（ELISA）作为一种可靠的血清学诊断工具在LSDV监测和疾病防控计划中的潜力，使基于兰契毒株的LSDV疫苗成为第一种具备DIVA兼容性的疫苗。
