Establishment of a pseudovirus neutralization assay for TGEV

Abstract

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.

Keywords: ST cells; TGEV; neutralizing antibody; pseudovirus; pseudovirus neutralization test.

Figure 1

Construction of the recombinant plasmid containing the TGEV S gene and validation of S protein expression. (A) PCR amplification of the TGEV S gene. M: 5000bp DNA marker. S1-S5: Partial fragments of TGEV S gene (883, 1235, 974, 1111, 1253 bp). Lane 7-8: Partial fragments of TGEV S gene linked by fusion PCR. S: The full length of TGEV S gene (4344 bp). (B) Double-enzyme digestion (EcoRI and XhoI) of the recombinant plasmid PM2.G-ΔG-TGEV-S-HA. M: 10000bp DNA marker. Lane 3 is the full-length recombinant plasmid PM2. G-ΔG-TGEV-S-HA. Lane 4 shows the double enzyme digestion result of recombinant plasmid PM2. G-ΔG-TGEV-S-HA. (C) Validation of TGEV S protein expression. M: Prestained Protein Marker 10-250 kDa. Lane 3-4 shows the results of TGEV positive serum detection after transfecting plasmids PM2. G-ΔG-TGEV-S-HA and PM2. G-ΔG-HA into cells, respectively.

Figure 2

Packaging and validation of TGEV pseudovirus. (A) Package the pseudovirus using ST cells. Transfection of recombinant plasmids PM2. G-ΔG-TGEV-S-HA and PM2. G-ΔG-HA using fluorescence inverted microscopy, and infection with G*VSV-GFP pseudovirus. (B) Western blot validation of TGEV pseudovirus. M: Prestained Protein Marker 10-250 kDa. Lane 3-4 shows the results of TGEV positive serum and VSV N protein antibody detection after transfecting plasmids PM2. G-ΔG-HA and PM2. G-ΔG-TGEV-S-HA into cells and infecting them with G virus, respectively. (C) Transmission electron microscopy images of TGEV pseudovirus.

Figure 3

Optimization of TGEV pseudovirus packaging conditions. (A) Selection of the cell line (ST, PK-15, 293T, BHK21), testing four types of cells for TGEV pseudovirus. (B) Optimization of the transfection dose (0.1, 0.5, 1.0, 1.5, 2.0) of recombinant plasmid PM2.G-ΔG-TGEV-S-HA. (C) Optimization of the transfection time (12, 24, 36, 48h) for recombinant plasmid PM2.G-ΔG-TGEV-S-HA; (D) Optimization of the collection time (12, 24, 36, 48h) for TGEV P0 pseudovirus.

Figure 4

Validation of TGEV pseudovirus neutralization activity and optimization of neutralization assay conditions. (A) Validation of TGEV pseudovirus neutralization activity. The test results for positive and negative serum ranged from 1:4 to 1:2048. (B) Determination of the optimal detection time (12, 24, 36, 48, 60, 72h) for the TGEV pNT method. (C) Determination of the optimal cell inoculation amount (1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000) using the TGEV pNT method. (D) Determination of the optimal TGEV pseudovirus infection level using the TGEV pNT method. The horizontal axis represents the TCID₅₀/50 μLof TGEV pseudovirus, and the vertical axis represents the relative light density (RLU) value. The linear equation is: Y=110x-8230.

Figure 5

Sensitivity, specificity, and repeatability validation of the TGEV S pseudovirus neutralization assay. (A) Results of the TGEV S pseudovirus neutralization assay testing 30 negative and 30 positive serum samples. (B) ROC curve analysis of the results from 60 serum samples. (C) Cross-reactivity with positive sera from PEDV (1:256), PPV (1:1024), PDCoV (1:512), and PRoV (1:512). (D) Intra-batch and inter-batch test results of the TGEV S pseudovirus neutralization assay.

Figure 6

Comparison with Blocking ELISA and TGEV Live Virus Neutralization Assay. (A) Simultaneous detection of 19 serum samples using the blocking ELISA and pNT methods. (B) Detection results of 50 serum samples using both the TGEV S pseudovirus neutralization assay and the TGEV live virus neutralization assay.

Figure 7

Detection results of serum samples at different time points after TGEV vaccination using the TGEV S pseudovirus neutralization assay.

Figure 8

Schematic diagram of the establishment and application of TGEV S pseudovirus packaging and neutralization assay.