{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,6]],"date-time":"2025-10-06T08:40:59Z","timestamp":1759740059597,"version":"build-2065373602"},"reference-count":47,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2025,10,5]],"date-time":"2025-10-05T00:00:00Z","timestamp":1759622400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32473044"],"award-info":[{"award-number":["32473044"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.mdpi.com"],"crossmark-restriction":true},"short-container-title":["Viruses"],"abstract":"<jats:p>Porcine circovirus type 3 (PCV3), initially identified in the United States in 2016, is associated with multisystemic inflammation, myocarditis, reproductive failure in sows, and growth retardation in piglets, posing a significant economic threat to the swine industry. In this study, prokaryotic-expressed recombinant PCV3 Cap protein was used to immunize mice and rabbits. A monoclonal antibody (mAb 4G1) was generated through hybridoma technology, targeting a novel linear epitope (37DYYDKK42) within the first \u03b2-sheet of the Cap structure. This epitope exhibits high conservation (99.35%, 1239\/1247) based on sequence alignment analysis, and residues 39 and 42 are critical residues affecting mAb binding. Subsequently, using rabbit polyclonal antibody (pAb) as the capture antibody and mAb 4G1 as the detection antibody, a double antibody sandwich ELISA (DAS-ELISA) method was developed. The assay demonstrates a cut-off value of 0.271, a detection limit for positive pig serum is 1:800, and shows no cross-reactivity with other swine pathogens. Intra- and inter-assay coefficients of variation were &lt;10%, with a linear detection range for Cap protein down to 3.4 ng\/mL. The coincidence rate between the DAS-ELISA and qPCR was 93.33% (70\/75) for PCV3 detection in serum, with a kappa value of 0.837. This study establishes a simple, sensitive, and operationally efficient DAS-ELISA and provides a reference for monitoring PCV3 infection in swine herds.<\/jats:p>","DOI":"10.3390\/v17101340","type":"journal-article","created":{"date-parts":[[2025,10,6]],"date-time":"2025-10-06T08:10:51Z","timestamp":1759738251000},"page":"1340","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of a Highly Specific Monoclonal Antibody-Based Sandwich ELISA for Rapid Detection of Porcine Circovirus Type 3"],"prefix":"10.3390","volume":"17","author":[{"given":"Zhen","family":"Li","sequence":"first","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiaying","family":"Zhao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8956-1300","authenticated-orcid":false,"given":"Ang","family":"Tian","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hao","family":"Wu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Huanchun","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture and Rural Affairs of China, Wuhan 430070, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yunfeng","family":"Song","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture and Rural Affairs of China, Wuhan 430070, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"da Silva, R.R., da Silva, D.F., da Silva, V.H., and de Castro, A. (2023). Porcine circovirus 3: A new challenge to explore. Front. Vet. Sci., 10.","DOI":"10.3389\/fvets.2023.1266499"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1007\/s00705-024-06107-2","article-title":"2024 taxonomy update for the family Circoviridae","volume":"169","author":"Varsani","year":"2024","journal-title":"Arch. Virol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1186\/s12985-016-0642-z","article-title":"Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation","volume":"13","author":"Phan","year":"2016","journal-title":"Virol. J."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Niu, G., Chen, S., Li, X., Zhang, L., and Ren, L. (2022). Advances in Crosstalk between Porcine Circoviruses and Host. Viruses, 14.","DOI":"10.3390\/v14071419"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"106715","DOI":"10.1016\/j.micpath.2024.106715","article-title":"Cytotoxicity effect and transcriptome analysis of PCV3-infected cells revealed potential viral pathogenic mechanisms","volume":"192","author":"Chang","year":"2024","journal-title":"Microb. Pathog."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1007\/s42770-021-00644-7","article-title":"A putative PCV3-associated disease in piglets from Southern Brazil","volume":"53","author":"Molossi","year":"2022","journal-title":"Braz. J. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e01879-16","DOI":"10.1128\/JVI.01879-16","article-title":"A Novel Porcine Circovirus Distantly Related to Known Circoviruses Is Associated with Porcine Dermatitis and Nephropathy Syndrome and Reproductive Failure","volume":"91","author":"Palinski","year":"2017","journal-title":"J. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Mora-Diaz, J., Pineyro, P., Shen, H., Schwartz, K., Vannucci, F., Li, G., Arruda, B., and Gim\u00e9nez-Lirola, L. (2020). Isolation of PCV3 from Perinatal and Reproductive Cases of PCV3-Associated Disease and In Vivo Characterization of PCV3 Replication in CD\/CD Growing Pigs. Viruses, 12.","DOI":"10.3390\/v12020219"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2936","DOI":"10.1111\/tbed.14204","article-title":"Porcine circovirus 3 (PCV-3) as a causal agent of disease in swine and a proposal of PCV-3 associated disease case definition","volume":"68","author":"Saporiti","year":"2021","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"jgv001502","DOI":"10.1099\/jgv.0.001502","article-title":"Pathogenicity and immune response against porcine circovirus type 3 infection in caesarean-derived, colostrum-deprived pigs","volume":"102","author":"Temeeyasen","year":"2021","journal-title":"J. Gen. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"198764","DOI":"10.1016\/j.virusres.2022.198764","article-title":"Five years of porcine circovirus 3: What have we learned about the clinical disease, immune pathogenesis, and diagnosis","volume":"314","author":"Kroeger","year":"2022","journal-title":"Virus Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e0034125","DOI":"10.1128\/jvi.00341-25","article-title":"Rescue of naive porcine circovirus type 3 and its pathogenesis in CD pigs","volume":"99","author":"Zhang","year":"2025","journal-title":"J. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2915","DOI":"10.1111\/tbed.14185","article-title":"What do we know about porcine circovirus 3 (PCV3) diagnosis so far?: A review","volume":"68","author":"Tan","year":"2021","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1004","DOI":"10.1111\/tbed.13125","article-title":"Molecular detection and phylogenetic analysis of porcine circovirus type 3 in 21 Provinces of China during 2015\u20132017","volume":"66","author":"Qi","year":"2019","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Ge, M., Ren, J., Xie, Y.L., Zhao, D., Fan, F.C., Song, X.Q., Li, M.X., and Xiao, C.T. (2021). Prevalence and Genetic Analysis of Porcine Circovirus 3 in China From 2019 to 2020. Front. Vet. Sci., 8.","DOI":"10.3389\/fvets.2021.773912"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Mai, J., Wang, D., Zou, Y., Zhang, S., Meng, C., Wang, A., and Wang, N. (2021). High Co-infection Status of Novel Porcine Parvovirus 7 With Porcine Circovirus 3 in Sows That Experienced Reproductive Failure. Front. Vet. Sci., 8.","DOI":"10.3389\/fvets.2021.695553"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Donneschi, A., Recchia, M., Romeo, C., Pozzi, P., Salogni, C., Maisano, A.M., Santucci, G., Scali, F., Faccini, S., and Boniotti, M.B. (2024). Infectious Agents Associated with Abortion Outbreaks in Italian Pig Farms from 2011 to 2021. Vet. Sci., 11.","DOI":"10.3390\/vetsci11100496"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1111\/tbed.12792","article-title":"Presence of Torque teno sus virus 1 and 2 in porcine circovirus 3-positive pigs","volume":"65","author":"Zheng","year":"2018","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2477","DOI":"10.14202\/vetworld.2024.2477-2487","article-title":"Coinfection of Mycoplasma suis and porcine circovirus type 3 is linked to reproductive failure in pig farms","volume":"17","author":"Ngo","year":"2024","journal-title":"Vet. World"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Zhang, P., Shen, H., Liu, X., Wang, S., Liu, Y., Xu, Z., and Song, C. (2020). Porcine Circovirus Type 3 Cap Inhibits Type I Interferon Induction Through Interaction with G3BP1. Front. Vet. Sci., 7.","DOI":"10.3389\/fvets.2020.594438"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"106997","DOI":"10.1016\/j.micpath.2024.106997","article-title":"Genotypic diversity and immunological implications of porcine circovirus: Inspiration from PCV1 to PCV4","volume":"196","author":"Yan","year":"2024","journal-title":"Microb. Pathog."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2439524","DOI":"10.1080\/21505594.2024.2439524","article-title":"Genomic composition and pathomechanisms of porcine circoviruses: A review","volume":"15","author":"Gao","year":"2024","journal-title":"Virulence"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1089\/vim.2020.0097","article-title":"Structural Perspectives of Beak and Feather Disease Virus and Porcine Circovirus Proteins","volume":"34","author":"Nath","year":"2021","journal-title":"Viral Immunol."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Qiu, H., Sun, M., Wang, N., Zhang, S., Deng, Z., Xu, H., Yang, H., Gu, H., Fang, W., and He, F. (2024). Efficacy comparison in cap VLPs of PCV2 and PCV3 as swine vaccine vehicle. Int. J. Biol. Macromol., 278.","DOI":"10.1016\/j.ijbiomac.2024.134955"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Maity, H.K., Samanta, K., Deb, R., and Gupta, V.K. (2023). Revisiting Porcine Circovirus Infection: Recent Insights and Its Significance in the Piggery Sector. Vaccines, 11.","DOI":"10.3390\/vaccines11081308"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1901004","DOI":"10.1002\/advs.201901004","article-title":"A Shift in Porcine Circovirus 3 (PCV-3) History Paradigm: Phylodynamic Analyses Reveal an Ancient Origin and Prolonged Undetected Circulation in the Worldwide Swine Population","volume":"6","author":"Franzo","year":"2019","journal-title":"Adv. Sci."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Cui, Y., Hou, L., Pan, Y., Feng, X., Zhou, J., Wang, D., Guo, J., Liu, C., Shi, Y., and Sun, T. (2022). Reconstruction of the Evolutionary Origin, Phylodynamics, and Phylogeography of the Porcine Circovirus Type 3. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.898212"},{"key":"ref_28","first-page":"1454","article-title":"Does porcine circovirus type 3 (PCV3) interfere with porcine circovirus type 2 (PCV2) vaccine efficacy?","volume":"66","author":"Wozniak","year":"2019","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"8858447","DOI":"10.1155\/2023\/8858447","article-title":"Porcine Circovirus Type 3: Diagnostics, Genotyping, and Challenges in Vaccine Development","volume":"2023","author":"Pranoto","year":"2023","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Pan, J., Zeng, M., Zhao, M., and Huang, L. (2023). Research Progress on the detection methods of porcine reproductive and respiratory syndrome virus. Front. Microbiol., 14.","DOI":"10.3389\/fmicb.2023.1097905"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1186\/s13568-019-0940-0","article-title":"Self-assembly into virus-like particles of the recombinant capsid protein of porcine circovirus type 3 and its application on antibodies detection","volume":"10","author":"Wang","year":"2020","journal-title":"AMB Express"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Wang, J., Lei, B., Zhang, W., Li, L., Ji, J., Liu, M., Zhao, K., and Yuan, W. (2024). Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3. Animals, 14.","DOI":"10.3390\/ani14020235"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/978-1-0716-2903-1_1","article-title":"Enzyme-Linked Immunosorbent Assay: Types and Applications","volume":"2612","author":"Hayrapetyan","year":"2023","journal-title":"Methods Mol. Biol."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Sun, M., Wang, S., Fang, Z., Zhao, M., Gao, Y., An, T., Tu, Y., Wang, H., and Cai, X. (2022). A Sandwich ELISA for Quality Control of PCV2 Virus-like Particles Vaccine. Vaccines, 10.","DOI":"10.3390\/vaccines10122175"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"474","DOI":"10.1002\/vms3.374","article-title":"Genotyping of PCV3 based on reassembled viral gene sequences","volume":"7","author":"Chung","year":"2021","journal-title":"Vet. Med. Sci."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Chen, S., Zhang, L., Li, X., Niu, G., and Ren, L. (2021). Recent Progress on Epidemiology and Pathobiology of Porcine Circovirus 3. Viruses, 13.","DOI":"10.3390\/v13101944"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1186\/s12985-024-02463-2","article-title":"Isolation of porcine circovirus 3 using primary porcine bone marrow-derived cells","volume":"21","author":"Hayashi","year":"2024","journal-title":"Virol. J."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Oh, T., and Chae, C. (2020). First isolation and genetic characterization of porcine circovirus type 3 using primary porcine kidney cells. Vet. Microbiol., 241.","DOI":"10.1016\/j.vetmic.2020.108576"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1016\/j.crimmu.2021.03.002","article-title":"Hybridoma technology: Is it still useful?","volume":"2","author":"Moraes","year":"2021","journal-title":"Curr. Res. Immunol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.jviromet.2018.07.014","article-title":"Production of a monoclonal antibody against Porcine circovirus type 3 cap protein","volume":"261","author":"Li","year":"2018","journal-title":"J. Virol. Methods."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.coi.2016.06.001","article-title":"Antibody-guided vaccine design: Identification of protective epitopes","volume":"41","author":"Lanzavecchia","year":"2016","journal-title":"Curr. Opin. Immunol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"6223","DOI":"10.1007\/s00253-020-10664-2","article-title":"Fine mapping of linear B cell epitopes on capsid protein of porcine circovirus 3","volume":"104","author":"Jiang","year":"2020","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1186\/s13568-023-01652-6","article-title":"Exploring the surface epitope and nuclear localization analysis of porcine circovirus type 3 capsid protein","volume":"13","author":"Chang","year":"2023","journal-title":"AMB Express"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"115080","DOI":"10.1016\/j.jviromet.2024.115080","article-title":"A novel linear B cell epitope of the porcine circovirus type 3 capsid protein identified by phage display technology","volume":"333","author":"Yang","year":"2025","journal-title":"J. Virol. Methods"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1007\/s00705-025-06351-0","article-title":"Multiplex quantitative PCR assay for porcine circoviruses 2 and 3 and its application for measuring infection rates at different stages of pig production","volume":"170","author":"Li","year":"2025","journal-title":"Arch. Virol."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Cobos, A., Sibila, M., and Segales, J. (Vet. Pathol., 2025). Review of porcine circovirus 3-associated lesions in swine: Challenges and advances in diagnostics, Vet. Pathol., ahead of print.","DOI":"10.1177\/03009858251347522"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1335","DOI":"10.1016\/j.jhep.2021.07.030","article-title":"Infection courses, virological features and IFN-alpha responses of HBV genotypes in cell culture and animal models","volume":"75","author":"Zhang","year":"2021","journal-title":"J. Hepatol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/10\/1340\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,6]],"date-time":"2025-10-06T08:13:35Z","timestamp":1759738415000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/10\/1340"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,5]]},"references-count":47,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2025,10]]}},"alternative-id":["v17101340"],"URL":"https:\/\/doi.org\/10.3390\/v17101340","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,5]]}}}