{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T09:24:39Z","timestamp":1787217879647,"version":"build-2736575974"},"reference-count":35,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T00:00:00Z","timestamp":1787184000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Japanese encephalitis virus (JEV) is a zoonotic pathogen transmitted primarily by Culex mosquitoes and causes severe neurological diseases in humans and animals. The main endemic areas are the Western Pacific and Southeast Asia, and its geographical distribution has expanded in recent years. The development of a diagnosis for orthoflavivirus infections is hampered by two main problems: the short duration of viremia, resulting in a narrow detection window, and severe cross-reactivity. NS1, a secreted nonstructural protein of orthoflavivirus, holds promise as a new target for overcoming these limitations. In this study, we established a highly specific and sensitive capture ELISA for the JEV NS1 protein. First, the JEV NS1 protein was successfully expressed in mammalian cells and purified using affinity chromatography. Seven mAbs recognizing JEV NS1 were generated, and the mAb 20B6 exhibited the strongest binding affinity. Based on 20B6, a capture ELISA was developed with an optimal coating concentration of 3 \u03bcg\/mL and an optimal detection antibody working concentration of 0.432 \u03bcg\/mL. No cross-reactivity was observed with other orthoflaviviruses (including WNV, KUNV, USUV, MVEV, SLEV, and ZIKV) or common porcine viruses. The method could effectively detect NS1 protein in cell culture medium, cell lysates, mouse tissues, and porcine serum samples from JEV-infected subjects. This study provides a solid foundation that may be further developed into an efficient and specific tool for epidemiological surveillance of Japanese encephalitis.<\/jats:p>","DOI":"10.3390\/ani16162601","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T08:10:20Z","timestamp":1787213420000},"page":"2601","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of JEV NS1 Specific Capture-ELISA Based on a Single Monoclonal Antibody"],"prefix":"10.3390","volume":"16","author":[{"given":"Shu-Jian","family":"Zhang","sequence":"first","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jian-Hui","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shi-Meng","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1920-9854","authenticated-orcid":false,"given":"Yu-Ting","family":"Huang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jin-Liang","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhi-Gao","family":"Bu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"},{"name":"Jiangsu Co-Innovation Centre for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7034-5766","authenticated-orcid":false,"given":"Rong-Hong","family":"Hua","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"968","DOI":"10.1080\/21505594.2021.1899674","article-title":"Pathogenicity and Virulence of Japanese Encephalitis Virus: Neuroinflammation and Neuronal Cell Damage","volume":"12","author":"Ashraf","year":"2021","journal-title":"Virulence"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3201\/eid1501.080311","article-title":"Past, Present, and Future of Japanese Encephalitis","volume":"15","author":"Erlanger","year":"2009","journal-title":"Emerg. Infect. Dis."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"766","DOI":"10.2471\/BLT.10.085233","article-title":"Estimated Global Incidence of Japanese Encephalitis: A Systematic Review","volume":"89","author":"Campbell","year":"2011","journal-title":"Bull. World Health Organ."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Xu, C., Zhang, W., Pan, Y., Wang, G., Yin, Q., Fu, S., Li, F., He, Y., Xu, S., and Wang, Z. (2022). A Bibliometric Analysis of Global Research on Japanese Encephalitis From 1934 to 2020. Front. Cell. Infect. Microbiol., 12.","DOI":"10.3389\/fcimb.2022.833701"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.vetmic.2017.01.014","article-title":"Japanese Encephalitis Virus Infection, Diagnosis and Control in Domestic Animals","volume":"201","author":"Mansfield","year":"2017","journal-title":"Vet. Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"9392","DOI":"10.1038\/s41598-022-13604-4","article-title":"Epidemiological Investigation of Swine Japanese Encephalitis Virus Based on RT-RAA Detection Method","volume":"12","author":"Nie","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Desingu, P.A., Ray, P.K., Patel, B.H.M., Singh, R., Singh, R.K., and Saikumar, G. (2016). Pathogenic and Genotypic Characterization of a Japanese Encephalitis Virus Isolate Associated with Reproductive Failure in an Indian Pig Herd. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0147611"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"eads7441","DOI":"10.1126\/sciadv.ads7441","article-title":"Sheep Serve as Amplifying Hosts of Japanese Encephalitis Virus, Increasing the Risk of Human Infection","volume":"11","author":"Zhang","year":"2025","journal-title":"Sci. Adv."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1126\/science.1247749","article-title":"Flavivirus NS1 Crystal Structures Reveal a Surface for Membrane Association and Regions of Interaction with the Immune System","volume":"343","author":"Akey","year":"2014","journal-title":"Science"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1016\/j.antiviral.2013.03.008","article-title":"The Flavivirus NS1 Protein: Molecular and Structural Biology, Immunology, Role in Pathogenesis and Application as a Diagnostic Biomarker","volume":"98","author":"Muller","year":"2013","journal-title":"Antivir. Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1186\/s12985-016-0590-7","article-title":"Flavivirus NS1: A Multifaceted Enigmatic Viral Protein","volume":"13","author":"Rastogi","year":"2016","journal-title":"Virol. J."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Zhang, S., He, Y., Wu, Z., Wang, M., Jia, R., Zhu, D., Liu, M., Zhao, X., Yang, Q., and Wu, Y. (2023). Secretory Pathways and Multiple Functions of Nonstructural Protein 1 in Flavivirus Infection. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1205002"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1400","DOI":"10.1002\/jmv.21814","article-title":"Clinical Evaluation of the NS1 Antigen-capture ELISA for Early Diagnosis of Dengue Virus Infection in Brazil","volume":"82","author":"Machado","year":"2010","journal-title":"J. Med. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"13924","DOI":"10.1128\/JVI.79.22.13924-13933.2005","article-title":"NS1 Protein Secretion during the Acute Phase of West Nile Virus Infection","volume":"79","author":"Macdonald","year":"2005","journal-title":"J. Virol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2544","DOI":"10.1038\/s41598-024-52123-2","article-title":"Development of a Quantitative NS1 Antigen Enzyme-Linked Immunosorbent Assay (ELISA) for Zika Virus Detection Using a Novel Virus-Specific mAb","volume":"14","author":"Morales","year":"2024","journal-title":"Sci. Rep."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1063","DOI":"10.1002\/jmv.22097","article-title":"Monoclonal Antibody-Based Antigen Capture Immunoassay for Detection of Circulating Non-Structural Protein NS1: Implications for Early Diagnosis of Japanese Encephalitis Virus Infection","volume":"83","author":"Kumar","year":"2011","journal-title":"J. Med. Virol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1016\/j.jviromet.2011.06.008","article-title":"A Specific and Sensitive Antigen Capture Assay for NS1 Protein Quantitation in Japanese Encephalitis Virus Infection","volume":"179","author":"Li","year":"2012","journal-title":"J. Virol. Methods"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"476","DOI":"10.1007\/s12250-020-00316-0","article-title":"Generation of A Stable GFP-Reporter Zika Virus System for High-Throughput Screening of Zika Virus Inhibitors","volume":"36","author":"Zhang","year":"2020","journal-title":"Virol. Sin."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Zhang, S.-J., Niu, B., Liu, S.-M., Zhu, Y.-M., Zhao, D.-M., Bu, Z.-G., and Hua, R.-H. (2024). Identification of Two Linear Epitopes on MGF_110-13L Protein of African Swine Fever Virus with Monoclonal Antibodies. Animals, 14.","DOI":"10.3390\/ani14131951"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/j.virusres.2014.03.001","article-title":"Comprehensive Mapping of a Novel NS1 Epitope Conserved in Flaviviruses within the Japanese Encephalitis Virus Serocomplex","volume":"185","author":"Hua","year":"2014","journal-title":"Virus Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1089\/hyb.2011.0027","article-title":"A Monoclonal Antibody Against PrM\/M Protein of Japanese Encephalitis Virus","volume":"30","author":"Hua","year":"2011","journal-title":"Hybridoma"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e00197-18","DOI":"10.1128\/JVI.00197-18","article-title":"Host Factor SPCS1 Regulates the Replication of Japanese Encephalitis Virus through Interactions with Transmembrane Domains of NS2B","volume":"92","author":"Ma","year":"2018","journal-title":"J. Virol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1038\/s44298-025-00114-z","article-title":"Flavivirus Infections and Diagnostic Challenges for Dengue, West Nile and Zika Viruses","volume":"3","author":"Madere","year":"2025","journal-title":"npj Viruses"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Chan, K.R., Ismail, A.A., Thergarajan, G., Raju, C.S., Yam, H.C., Rishya, M., and Sekaran, S.D. (2022). Serological Cross-Reactivity among Common Flaviviruses. Front. Cell. Infect. Microbiol., 12.","DOI":"10.3389\/fcimb.2022.975398"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"557","DOI":"10.1128\/JCM.02735-14","article-title":"Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans","volume":"53","author":"Chao","year":"2015","journal-title":"J. Clin. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"8003","DOI":"10.1073\/pnas.1017338108","article-title":"Secreted Dengue Virus Nonstructural Protein NS1 Is an Atypical Barrel-Shaped High-Density Lipoprotein","volume":"108","author":"Gutsche","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"370","DOI":"10.1016\/j.synbio.2025.10.008","article-title":"Engineering Genetic Elements for Microbial Protein Expression Systems: Advances, Challenges, Applications, and Prospects","volume":"11","author":"Li","year":"2026","journal-title":"Synth. Syst. Biotechnol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1023\/B:GLYC.0000046275.28315.87","article-title":"Comparing N-Glycan Processing in Mammalian Cell Lines to Native and Engineered Lepidopteran Insect Cell Lines","volume":"21","author":"Tomiya","year":"2004","journal-title":"Glycoconj. J."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/j.ymeth.2020.06.006","article-title":"Expression of Eukaryotic Membrane Proteins in Eukaryotic and Prokaryotic Hosts","volume":"180","author":"Kesidis","year":"2020","journal-title":"Methods"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"eadm8275","DOI":"10.1126\/sciadv.adm8275","article-title":"The Step-by-Step Assembly Mechanism of Secreted Flavivirus NS1 Tetramer and Hexamer Captured at Atomic Resolution","volume":"10","author":"Pan","year":"2024","journal-title":"Sci. Adv."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Khare, B., and Kuhn, R.J. (2022). The Japanese Encephalitis Antigenic Complex Viruses: From Structure to Immunity. Viruses, 14.","DOI":"10.3390\/v14102213"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/emi.2017.27","article-title":"West Nile Virus Infection in Suspected Febrile Typhoid Cases in Xinjiang, China: West Nile Virus and Febrile Tyhoid Co-Infection","volume":"6","author":"Cao","year":"2017","journal-title":"Emerg. Microbes Infect."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1089\/vbz.2012.0995","article-title":"West Nile Virus Infection in Xinjiang, China","volume":"13","author":"Li","year":"2013","journal-title":"Vector-Borne Zoonotic Dis."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Xiao, P., Li, C., Zhang, Y., Han, J., Guo, X., Xie, L., Tian, M., Li, Y., Wang, M., and Liu, H. (2018). Metagenomic Sequencing From Mosquitoes in China Reveals a Variety of Insect and Human Viruses. Front. Cell. Infect. Microbiol., 8.","DOI":"10.3389\/fcimb.2018.00364"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Mao, X., and Zhou, H. (2020). The Spatiotemporal Distribution of Japanese Encephalitis Cases in Yunnan Province, China, from 2007 to 2017. PLoS ONE, 15.","DOI":"10.1371\/journal.pone.0231661"}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/16\/2601\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T08:38:20Z","timestamp":1787215100000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/16\/2601"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,20]]},"references-count":35,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["ani16162601"],"URL":"https:\/\/doi.org\/10.3390\/ani16162601","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,20]]}}}