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Establishment of a pseudovirus neutralization assay for TGEV

文献类型： 外文期刊

作者： Wang, Haojie 1 ; Chen, Jianxing 1 ; Xue, Lihong 1 ; Sun, Yue 1 ; An, Tongqing 1 ; Wang, Yue 1 ; Chen, Hongyan 1 ; Yu, Changqing 2 ; Xia, Changyou 1 ; Zhang, He 1 ;

作者机构： 1.Chinese Acad Agr Sci, Harbin Vet Res Inst, State Key Lab Anim Dis Control & Prevent, Harbin, Peoples R China

2.Yibin Vocat & Tech Coll, Sch Adv Agr Sci, Yibin, Peoples R China

关键词： TGEV; pseudovirus; neutralizing antibody; pseudovirus neutralization test; ST cells

期刊名称：FRONTIERS IN IMMUNOLOGY （ 影响因子：5.9； 五年影响因子：6.8 ）

ISSN： 1664-3224

年卷期： 2025 年 16 卷

页码：

收录情况： SCI

摘要： Introduction Transmissible Gastroenteritis Virus (TGEV) is a major pathogen causing swine enteric diseases, necessitating effective control strategies. Vaccination plays a key role, but assessing vaccine efficacy remains challenging due to variations in immune response and existing detection limitations. Current antibody detection methods, such as neutralization assays and ELISA, are often subjective, labor-intensive, and time-consuming, highlighting the need for a more efficient evaluation approach.Methods and results The TGEV S gene was amplified and inserted into the eukaryotic vector PM2.G-Delta G-HA to construct the recombinant plasmid PM2.G-Delta G-TGEV-S-HA. Transfecting ST cells with this plasmid, followed by infection with G*VSV-GFP/LUC, successfully produced TGEV P0 pseudoviruses. Western blot and electron microscopy confirmed the presence of TGEV S and VSV N proteins and the distinct pseudovirus morphology. Optimization determined that 0.5 mu g/well of plasmid, 24 h transfection, and 24 h post-infection harvest yielded a viral titer of 106-107 TCID50/mL. The pseudoviruses exhibited strong ST cell tropism and were effectively neutralized by TGEV-positive sera. A pseudovirus-based neutralization test (pNT) was established, showing 100% sensitivity, 96.6% specificity, no cross-reactivity with PEDV, PPV, PDCoV, or PRoV, and a 94% concordance with the live virus neutralization test. The method effectively tracked antibody level changes post-TGEV vaccination.Discussion This study successfully developed a novel pseudovirus-based detection method, overcoming traditional assay limitations. The pNT method provides a scalable, efficient, and reliable tool for TGEV antibody evaluation, with broad potential applications in pathogen detection and vaccine assessment.

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