Evaluation of recombinant extracellular enveloped virion protein candidates for the detection of serological responses to lumpy skin disease virus in cattle

Lumpy skin disease virus (LSDV) is a significant threat to cattle, particularly in countries like Thailand, where outbreaks have necessitated the importation of diagnostic kits and vaccines. This study aimed to evaluate several recombinant extracellular enveloped virion (EEV) protein candidates, including F13L, A33R, A34R, and B5R, for their potential use in serological detection assays for LSDV specific antibodies in cattle. Given the challenges associated with LSDV research, such as its classification as a Class III biological agent in Thailand, gene synthesis was employed to produce these proteins. The recombinant proteins were expressed in a prokaryotic system and analyzed using SDS-PAGE and Western blotting. Among the candidates, F13L demonstrated the highest correlation with the results from a commercially available and validated ELISA, yielding 85.7%, and 75% positive for the infected and vaccinated groups, respectively, identifying it a promising candidate for serosurveillance activities during active LSDV outbreaks. Sequence analysis confirmed a 100% match between the F13L designed from the Neethling type strain 2490 and various Thai LSDV strains from the 2021 outbreaks, underscoring its potential as a conserved diagnostic marker. The availability of recombinant F13L and its reactivity with cattle sera from LSDV infected or vaccinated animals, demonstrated in this study, suggests it could also serve as a potential candidate for vaccine development. The study concludes that recombinant F13L shows great promise for the development of LSDV serological assays, though further optimization and validation are necessary to harness its diagnostic potential.

结节性皮肤病病毒（Lumpy skin disease virus, LSDV）对牛群构成重大威胁，尤以泰国等国家为甚——该国曾因疫情暴发不得不进口诊断试剂盒与疫苗。本研究旨在评估F13L、A33R、A34R及B5R等多种重组细胞外有包膜病毒颗粒（extracellular enveloped virion, EEV）蛋白候选物，用于检测牛血清中LSDV特异性抗体的血清学检测试剂盒开发潜力。鉴于LSDV研究面临诸多挑战（例如该病毒在泰国被归类为第三类生物制剂），本研究通过基因合成方式制备上述蛋白。重组蛋白在原核表达系统中完成表达，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）与蛋白质印迹法（Western blotting）进行分析。在所有候选蛋白中，F13L与市售且经过验证的酶联免疫吸附试验（ELISA）检测结果相关性最高：其对感染组与免疫组的阳性检出率分别为85.7%与75%，可作为LSDV活跃暴发期间血清监测工作的极具潜力的候选物。序列分析结果证实，基于尼瑟林型毒株2490设计的F13L基因序列，与2021年泰国暴发疫情中分离的多株LSDV毒株完全匹配，进一步凸显其作为保守性诊断标志物的潜力。本研究证实重组F13L可与LSDV感染或免疫牛的血清发生特异性反应，提示其亦可作为疫苗开发的潜在候选靶点。本研究最终得出结论：重组F13L在LSDV血清学检测试剂盒开发中极具应用前景，但仍需进一步优化与验证，以充分发挥其诊断潜能。