Establishment of a Pseudovirus neutralization assay for TGEV

Haojie Wang Haojie Wang ¹ Jianxing Chen ¹ Lihong Xue ¹ Yue Sun ¹ Tongqing An ¹ Yue Wang ¹ Hongyan Chen ¹ Changqing Yu ² Changyou Xia ¹ He Zhang ^1*

• ¹ Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang Province, China
• ² Yibin Vocational and Technical College, Yibin, Sichuan Province, China

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-ΔG-HA to construct the recombinant plasmid PM2.G-ΔG-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 μg/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.