{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T15:58:21Z","timestamp":1753891101567,"version":"3.41.2"},"reference-count":50,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,4,10]],"date-time":"2025-04-10T00:00:00Z","timestamp":1744243200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods and results<\/jats:title><jats:p>The TGEV S gene was amplified and inserted into the eukaryotic vector PM2.G-\u0394G-HA to construct the recombinant plasmid PM2.G-\u0394G-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 \u03bcg\/well of plasmid, 24\u00a0h transfection, and 24\u00a0h post-infection harvest yielded a viral titer of 10<jats:sup>6<\/jats:sup>-10<jats:sup>7<\/jats:sup> TCID<jats:sub>50<\/jats:sub>\/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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2025.1558604","type":"journal-article","created":{"date-parts":[[2025,4,10]],"date-time":"2025-04-10T05:11:28Z","timestamp":1744261888000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Establishment of a pseudovirus neutralization assay for TGEV"],"prefix":"10.3389","volume":"16","author":[{"given":"Haojie","family":"Wang","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jianxing","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lihong","family":"Xue","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yue","family":"Sun","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tongqing","family":"An","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yue","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hongyan","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Changqing","family":"Yu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Changyou","family":"Xia","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"He","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2025,4,10]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1093\/molbev\/msab364","article-title":"Phylogeography reveals association between swine trade and the spread of porcine epidemic diarrhea virus in China and across the world","volume":"39","author":"He","year":"2022","journal-title":"Mol Biol Evol"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1016\/j.heliyon.2024.e37128","article-title":"Surveillance strategies for SARS-CoV-2 infections through one health approach","volume":"10","author":"Huang","year":"2024","journal-title":"Heliyon"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1111\/tbed.13385","article-title":"Development of a multiplex RT-PCR for the detection of major diarrhoeal viruses in pig herds in China","volume":"67","author":"Ding","year":"2020","journal-title":"Transbound Emerg Dis"},{"key":"B4","doi-asserted-by":"publisher","first-page":"102","DOI":"10.1186\/s12985-018-1012-9","article-title":"Impact of TGEV infection on the pig small intestine","volume":"15","author":"Xia","year":"2018","journal-title":"Virol J"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1016\/j.rehab.2021.101627","article-title":"Functional status 1 year after severe childhood traumatic brain injury predicts 7-year outcome: Results of the TGE study","volume":"65","author":"Neumane","year":"2022","journal-title":"Ann Phys Rehabil Med"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.20506\/rst.34.1.2340","article-title":"International standards: the world organisation for animal health terrestrial animal health code","volume":"34","author":"Thiermann","year":"2015","journal-title":"Rev Sci Tech"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.3390\/v15020359","article-title":"Transmissible gastroenteritis virus: an update review and perspective","volume":"15","author":"Chen","year":"2023","journal-title":"Viruses"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1016\/j.micpath.2023.106320","article-title":"Prevalence of transmissible gastroenteritis among swine populations in China during 1983-2022: A systematic review and meta-analysis","volume":"183","author":"Chen","year":"2023","journal-title":"Microb Pathog"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1099\/jgv.0.001996","article-title":"Phloretin inhibits transmissible gastroenteritis virus proliferation via multiple mechanisms","volume":"105","author":"Duan","year":"2024","journal-title":"J Gen Virol"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.00461-24","article-title":"Transmissible gastroenteritis virus induces inflammatory responses via RIG-I\/NF-kappaB\/HIF-1alpha\/glycolysis axis in intestinal organoids and","volume":"98","author":"Zhang","year":"2024","journal-title":"vivo. J Virol"},{"key":"B11","doi-asserted-by":"publisher","first-page":"97","DOI":"10.1186\/s13567-024-01359-x","article-title":"In vitro and in vivo evaluation of thapsigargin as an antiviral agent against transmissible gastroenteritis virus","volume":"55","author":"Li","year":"2024","journal-title":"Vet Res"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.00128-23","article-title":"The TGEV membrane protein interacts with HSC70 to direct virus internalization through clathrin-mediated endocytosis","volume":"97","author":"Ji","year":"2023","journal-title":"J Virol"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1099\/jgv.0.001942","article-title":"Integrated metabolomics and transcriptomics analyses reveal metabolic responses to TGEV infection in porcine intestinal epithelial cells","volume":"104","author":"Zhang","year":"2023","journal-title":"J Gen Virol"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1016\/j.virusres.2016.04.009","article-title":"Porcine deltacoronavirus infection: Etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis","volume":"226","author":"Jung","year":"2016","journal-title":"Virus Res"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-3377-9_15","article-title":"Atomic force microscopy of viruses: stability, disassembly, and genome release","volume":"2694","author":"Cantero","year":"2024","journal-title":"Methods Mol Biol"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1111\/j.1439-0450.1989.tb00654.x","article-title":"Rapid diagnosis of TGEV-like coronavirus in fattened pigs by indirect immunofluorescence labelling in nasal cells","volume":"36","author":"Onno","year":"1989","journal-title":"Zentralbl Veterinarmed B"},{"key":"B17","doi-asserted-by":"publisher","first-page":"37","DOI":"10.1016\/j.jim.2004.10.012","article-title":"Evaluation of a safe and sensitive Spike protein-based immunofluorescence assay for the detection of antibody responses to SARS-CoV","volume":"296","author":"Manopo","year":"2005","journal-title":"J Immunol Methods"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2024.131842","article-title":"Development and application of a blocking ELISA based on a N protein monoclonal antibody for the antibody detection against porcine reproductive and respiratory syndrome virus 2","volume":"269","author":"Li","year":"2024","journal-title":"Int J Biol Macromol"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2024.1418959","article-title":"Development of a colloidal gold immunochromatographic strip for the simultaneous detection of porcine epidemic diarrhea virus and transmissible gastroenteritis virus","volume":"15","author":"Zhou","year":"2024","journal-title":"Front Microbiol"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1177\/104063879300500106","article-title":"Comparison of a monoclonal antibody capture ELISA (MACELISA) to indirect ELISA and virus neutralization test for the serodiagnosis of transmissible gastroenteritis virus","volume":"5","author":"Lanza","year":"1993","journal-title":"J Vet Diagn Invest"},{"key":"B21","doi-asserted-by":"publisher","first-page":"180","DOI":"10.1186\/s12917-015-0500-z","article-title":"Development of an indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of Porcine Epidemic Diarrhea Virus","volume":"11","author":"Okda","year":"2015","journal-title":"BMC Vet Res"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1016\/0042-6822(90)90521-r","article-title":"Mechanisms of transmissible gastroenteritis coronavirus neutralization","volume":"177","author":"Sune","year":"1990","journal-title":"Virology"},{"key":"B23","doi-asserted-by":"crossref","DOI":"10.2460\/ajvr.1988.49.03.300","article-title":"Neutralization of porcine transmissible gastroenteritis virus by complement-dependent monoclonal antibodies","volume":"49","author":"Woods","year":"1988","journal-title":"Am J Vet Res"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.3390\/vaccines12010011","article-title":"Prevention and control of swine enteric coronaviruses in China: A review of vaccine development and application","volume":"12","author":"Kong","year":"2023","journal-title":"Vaccines (Basel)"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.1080\/14760584.2023.2299380","article-title":"Pseudoviruses, a safer toolbox for vaccine development against enveloped viruses","volume":"23","author":"Thimmiraju","year":"2024","journal-title":"Expert Rev Vaccines"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.7150\/ijbs.59184","article-title":"Construction and applications of SARS-CoV-2 pseudoviruses: a mini review","volume":"17","author":"Chen","year":"2021","journal-title":"Int J Biol Sci"},{"key":"B27","doi-asserted-by":"publisher","first-page":"32","DOI":"10.1080\/22221751.2019.1702479","article-title":"HIV-1 pseudoviruses constructed in China regulatory laboratory","volume":"9","author":"Nie","year":"2020","journal-title":"Emerg Microbes Infect"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-2111-0_18","article-title":"Measuring neutralizing antibodies to SARS-coV-2 using lentiviral spike-pseudoviruses","volume":"2452","author":"Neerukonda","year":"2022","journal-title":"Methods Mol Biol"},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.7717\/peerj.16234","article-title":"The development and application of pseudoviruses: assessment of SARS-CoV-2 pseudoviruses","volume":"11","author":"Tan","year":"2023","journal-title":"PeerJ"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1007\/s00253-014-5521-1","article-title":"Bacteriophage lambda display systems: developments and applications","volume":"98","author":"Nicastro","year":"2014","journal-title":"Appl Microbiol Biotechnol"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1128\/ecosalplus.esp-0014-2023","article-title":"Research on phage lambda: a lucky choice","volume":"12","author":"Lewis","year":"2024","journal-title":"EcoSal Plus"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.1080\/22221751.2020.1743767","article-title":"Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2","volume":"9","author":"Nie","year":"2020","journal-title":"Emerg Microbes Infect"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-3961-0_20","article-title":"TCID (50) assay: A simple method to determine baculovirus titer","volume":"2829","author":"Zhang","year":"2024","journal-title":"Methods Mol Biol"},{"key":"B34","doi-asserted-by":"publisher","DOI":"10.1002\/mco2.517","article-title":"Development of a SARS-CoV-2 neutralization assay based on a pseudotyped virus using a HIV system","volume":"5","author":"Liang","year":"2024","journal-title":"MedComm (2020)"},{"key":"B35","doi-asserted-by":"publisher","first-page":"126585","DOI":"10.1016\/j.vaccine.2024.126585","article-title":"Pseudovirus nanoparticles targeting the receptor binding HA1 domains of influenza viruses elicited high HA1-specific antibody responses and protected mice against mortality caused by influenza virus challenges","volume":"46","author":"Xia","year":"2024","journal-title":"Vaccine"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.3402\/iee.v4.23011","article-title":"Detection of antibodies against H5 and H7 strains in birds: evaluation of influenza pseudovirus particle neutralization tests","volume":"4","author":"Wallerstrom","year":"2014","journal-title":"Infect Ecol Epidemiol"},{"key":"B37","doi-asserted-by":"publisher","first-page":"39","DOI":"10.1038\/s41429-023-00677-0","article-title":"Evaluation of A-ring hydroxymethylene-amino- triterpenoids as inhibitors of SARS-CoV-2 spike pseudovirus and influenza H1N1","volume":"77","author":"Kazakova","year":"2024","journal-title":"J Antibiot (Tokyo)"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2022.114540","article-title":"Comparison of plaque reduction and focus reduction neutralization tests for the measurement of neutralizing antibody titers against Japanese encephalitis virus","volume":"306","author":"Park","year":"2022","journal-title":"J Virol Methods"},{"key":"B39","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.antiviral.2015.11.003","article-title":"Teicoplanin inhibits Ebola pseudovirus infection in cell culture","volume":"125","author":"Wang","year":"2016","journal-title":"Antiviral Res"},{"key":"B40","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0023710","article-title":"Inhibition of SARS pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans","volume":"6","author":"Lang","year":"2011","journal-title":"PloS One"},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2022.927122","article-title":"A novel and secure pseudovirus reporter system based assay for neutralizing and enhancing antibody assay against marburg virus","volume":"13","author":"Bi","year":"2022","journal-title":"Front Microbiol"},{"key":"B42","doi-asserted-by":"publisher","DOI":"10.1038\/srep42769","article-title":"Development of in vitro and in vivo rabies virus neutralization assays based on a high-titer pseudovirus system","volume":"7","author":"Nie","year":"2017","journal-title":"Sci Rep"},{"key":"B43","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2021.826882","article-title":"Swine enteric coronaviruses (PEDV, TGEV, and PDCoV) induce divergent interferon-stimulated gene responses and antigen presentation in porcine intestinal enteroids","volume":"12","author":"Yin","year":"2021","journal-title":"Front Immunol"},{"key":"B44","doi-asserted-by":"publisher","DOI":"10.1016\/j.vetmic.2018.10.025","article-title":"p53 mediated IFN-beta signaling to affect viral replication upon TGEV infection","volume":"227","author":"Ding","year":"2018","journal-title":"Vet Microbiol"},{"key":"B45","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2015.06.057","article-title":"Efficacy and immunogenicity of recombinant swinepox virus expressing the A epitope of the TGEV S protein","volume":"33","author":"Yuan","year":"2015","journal-title":"Vaccine"},{"key":"B46","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1016\/j.virusres.2014.11.029","article-title":"Looking for a needle in a haystack: Cellular proteins that may interact with the tyrosine-based sorting signal of the TGEV S protein","volume":"202","author":"Trincone","year":"2015","journal-title":"Virus Res"},{"key":"B47","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2020.00444","article-title":"Comparison of VSV pseudovirus and focus reduction neutralization assays for measurement of anti-andes orthohantavirus neutralizing antibodies in patient samples","volume":"10","author":"Vial","year":"2020","journal-title":"Front Cell Infect Microbiol"},{"key":"B48","doi-asserted-by":"publisher","DOI":"10.1080\/22221751.2020.1815589","article-title":"Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells","volume":"9","author":"Xiong","year":"2020","journal-title":"Emerg Microbes Infect"},{"key":"B49","doi-asserted-by":"publisher","DOI":"10.1002\/pro.684","article-title":"Green fluorescent protein: a perspective","volume":"20","author":"Remington","year":"2011","journal-title":"Protein Sci"},{"key":"B50","doi-asserted-by":"publisher","first-page":"9","DOI":"10.1016\/0378-1135(89)90003-5","article-title":"A competitive inhibition ELISA for the differentiation of serum antibodies from pigs infected with transmissible gastroenteritis virus (TGEV) or with the TGEV-related porcine respiratory coronavirus","volume":"20","author":"Callebaut","year":"1989","journal-title":"Vet Microbiol"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1558604\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,4,10]],"date-time":"2025-04-10T05:11:29Z","timestamp":1744261889000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1558604\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,10]]},"references-count":50,"alternative-id":["10.3389\/fimmu.2025.1558604"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2025.1558604","relation":{},"ISSN":["1664-3224"],"issn-type":[{"type":"electronic","value":"1664-3224"}],"subject":[],"published":{"date-parts":[[2025,4,10]]},"article-number":"1558604"}}