{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,20]],"date-time":"2026-02-20T06:02:24Z","timestamp":1771567344506,"version":"3.50.1"},"reference-count":40,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,2,18]],"date-time":"2026-02-18T00:00:00Z","timestamp":1771372800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100014103","name":"Key Research and Development Program of Shandong Province","doi-asserted-by":"crossref","award":["2022CXPT005-01-03"],"award-info":[{"award-number":["2022CXPT005-01-03"]}],"id":[{"id":"10.13039\/100014103","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["32172845"],"award-info":[{"award-number":["32172845"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"name":"National Key Research and Development Program for Young Scientists","award":["2022YFD1801900"],"award-info":[{"award-number":["2022YFD1801900"]}]},{"name":"China Agriculture Research System of MOF and MARA","award":["CARS-42-19"],"award-info":[{"award-number":["CARS-42-19"]}]},{"name":"Project Charter for the Shandong Provincial Key Research and Development Program in Agricultural Breeding Engineering","award":["2024LZGC021-04"],"award-info":[{"award-number":["2024LZGC021-04"]}]},{"name":"State-sponsored Postdoctoral Research Program","award":["GZC20231503"],"award-info":[{"award-number":["GZC20231503"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>To establish a rapid, sensitive, and reproducible method for evaluating the immunogenic performance of Tembusu virus (TMUV) vaccines, we developed and optimized a blocking enzyme-linked immunosorbent assay (bELISA) using the TMUV envelope (E) protein as the coating antigen. By systematically screening the coating antigen concentration, mAb dilution, serum dilution, and chromogenic reaction time, we determined the optimal reaction conditions for this assay. The results showed that bELISA exhibited high specificity, yielding positive reactions only with TMUV-positive sera and no cross-reactivity with sera against other common duck viruses; the cutoff value for positivity was 48.89%, and the lowest detectable serum dilution was 1:10. Neutralization assays confirmed that the TMUV E-specific mAb significantly inhibited viral replication, supporting the functional relevance and reliability of the established bELISA. In a comparative investigation, this assay was used to assess five TMUV vaccines, including both inactivated and attenuated variants, in Cherry Valley ducks. The DF2 inactivated vaccine was found to elicit the highest antibody levels and blocking rates. This was followed by the WF100 attenuated vaccine, which also demonstrated a strong immune response. The TC2B inactivated vaccine, although effective, showed a comparatively lower response, whereas the FX2010-180P strain and mosquito cell-derived WF100 attenuated vaccine showed weaker immunogenicity. Neutralization assays further confirmed that the TMUV E-specific mAb significantly inhibited viral replication, supporting the functional relevance and reliability of the established bELISA. In summary, the bELISA described here demonstrates high specificity, sensitivity, and reproducibility and is suitable for evaluating the immune efficacy of different TMUV vaccines, providing a reliable technical platform for vaccine immunology studies and optimization of immunization strategies.<\/jats:p>","DOI":"10.3390\/v18020256","type":"journal-article","created":{"date-parts":[[2026,2,18]],"date-time":"2026-02-18T09:22:46Z","timestamp":1771406566000},"page":"256","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Assessment of Functional Antibody Responses Induced by Tembusu Virus Vaccines Using a Blocking ELISA"],"prefix":"10.3390","volume":"18","author":[{"given":"Chengguang","family":"Lu","sequence":"first","affiliation":[{"name":"Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuanmingyuan West Road, Beijing 100091, China"},{"name":"College of Veterinary Medicine, Shandong Agricultural University, No. 7, Panhe Street, Tai\u2019an 271001, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Siming","family":"Zhu","sequence":"additional","affiliation":[{"name":"Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuanmingyuan West Road, Beijing 100091, China"},{"name":"College of Veterinary Medicine, Shandong Agricultural University, No. 7, Panhe Street, Tai\u2019an 271001, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wenjun","family":"Jiang","sequence":"additional","affiliation":[{"name":"Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuanmingyuan West Road, Beijing 100091, China"},{"name":"College of Veterinary Medicine, Shandong Agricultural University, No. 7, Panhe Street, Tai\u2019an 271001, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mingtian","family":"Mao","sequence":"additional","affiliation":[{"name":"Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuanmingyuan West Road, Beijing 100091, China"},{"name":"College of Veterinary Medicine, Shandong Agricultural University, No. 7, Panhe Street, Tai\u2019an 271001, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Huihui","family":"Li","sequence":"additional","affiliation":[{"name":"Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuanmingyuan West Road, Beijing 100091, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bing","family":"Li","sequence":"additional","affiliation":[{"name":"Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuanmingyuan West Road, Beijing 100091, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Meijuan","family":"Zhang","sequence":"additional","affiliation":[{"name":"Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuanmingyuan West Road, Beijing 100091, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mian","family":"Wu","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Shandong Agricultural University, No. 7, Panhe Street, Tai\u2019an 271001, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-5134-2441","authenticated-orcid":false,"given":"Zhuo","family":"Zhang","sequence":"additional","affiliation":[{"name":"Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuanmingyuan West Road, Beijing 100091, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dalin","family":"He","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Shandong Agricultural University, No. 7, Panhe Street, Tai\u2019an 271001, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Youxiang","family":"Diao","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Shandong Agricultural University, No. 7, Panhe Street, Tai\u2019an 271001, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yi","family":"Tang","sequence":"additional","affiliation":[{"name":"Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuanmingyuan West Road, Beijing 100091, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Hamel, R., Phanitchat, T., Wichit, S., Morales Vargas, R.E., Jaroenpool, J., Diagne, C.T., Pompon, J., and Miss\u00e9, D. (2021). New Insights into the Biology of the Emerging Tembusu Virus. Pathogens, 10.","DOI":"10.3390\/pathogens10081010"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Ti, J., Li, Z., Li, X., Lu, Y., Diao, Y., and Li, F. (2017). Identification of one B-cell epitope from NS1 protein of duck Tembusu virus with monoclonal antibodies. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0181177"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Truong, T.N., and Cheng, L.T. (2022). Development of a Subunit Vaccine against Duck Hepatitis A Virus Serotype 3. Vaccines, 10.","DOI":"10.3390\/vaccines10040523"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Taylor, S.C., Hurst, B., Charlton, C.L., Bailey, A., and Knight, V. (2021). A New SARS CoV-2 Dual Purpose Serology Test: Highly Accurate Infection Tracing and Neutralizing Antibody Response Detection. J. Clin. Microbiol., 59.","DOI":"10.1128\/JCM.02438-20"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Zhang, L., Li, Z., Jin, H., Hu, X., and Su, J. (2018). Development and application of a monoclonal antibody-based blocking ELISA for detection of antibodies to Tembusu virus in multiple poultry species. BMC Vet. Res., 14.","DOI":"10.1186\/s12917-018-1537-6"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"105080","DOI":"10.1016\/j.psj.2025.105080","article-title":"Development of a blocking ELISA employing a VP1-specific monoclonal antibody for the detection of DHAV3 antibodies","volume":"104","author":"Song","year":"2025","journal-title":"Poult. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"113160","DOI":"10.1016\/j.jim.2021.113160","article-title":"Qualification of ELISA and neutralization methodologies to measure SARS-CoV-2 humoral immunity using human clinical samples","volume":"499","author":"Larsen","year":"2021","journal-title":"J. Immunol. Methods"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1007\/s00253-025-13492-4","article-title":"A neutralizing nanobody-based liquid-phase blocking ELISA to assess the protective potency of Senecavirus A vaccine","volume":"109","author":"Mu","year":"2025","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"145622","DOI":"10.1016\/j.ijbiomac.2025.145622","article-title":"Development of a blocking ELISA to evaluate the neutralizing antibody level against canine distemper virus and identification of novel spatial conformation epitopes","volume":"319","author":"Wang","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Chen, H., Ou, Q., Tang, Y., Gao, X., Wu, L., Xue, C., Yu, C., Cui, J., and Diao, Y. (2014). Development and evaluation of a DAS-ELISA for rapid detection of Tembusu virus using monoclonal antibodies against the envelope protein. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0096366"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"114851","DOI":"10.1016\/j.jviromet.2023.114851","article-title":"Development and evaluation of a monoclonal antibody-based blocking ELISA to detect antibodies against the E2 protein of bovine viral diarrhea virus-1","volume":"323","author":"Liu","year":"2024","journal-title":"J. Virol. Methods"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"102326","DOI":"10.1016\/j.psj.2022.102326","article-title":"Development of a novel competitive ELISA based on nanobody-horseradish peroxidase fusion protein for rapid detection of antibodies against avian hepatitis E virus","volume":"102","author":"Chen","year":"2023","journal-title":"Poult. Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1016\/j.virs.2025.03.008","article-title":"Development and immunoprotection assessment of novel vaccines for avian infectious bronchitis virus","volume":"40","author":"Huang","year":"2025","journal-title":"Virol. Sin."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"198374","DOI":"10.1016\/j.virusres.2021.198374","article-title":"The spread of Tembusu virus in China from 2010 to 2019","volume":"300","author":"Qiu","year":"2021","journal-title":"Virus Res."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Xu, K., Li, J., Lu, X., Ge, X., Wang, K., Wang, J., Qiao, Z., Quan, Y., and Li, C. (2024). The Immunogenicity of CpG, MF59-like, and Alum Adjuvant Delta Strain Inactivated SARS-CoV-2 Vaccines in Mice. Vaccines, 12.","DOI":"10.3390\/vaccines12010060"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Lv, J., Liu, X., Cui, S., Yang, L., Qu, S., Meng, R., Yang, B., Feng, C., Wang, X., and Zhang, D. (2020). The Neutralizing Antibody Response Elicited by Tembusu Virus Is Affected Dramatically by a Single Mutation in the Stem Region of the Envelope Protein. Front. Microbiol., 11.","DOI":"10.3389\/fmicb.2020.585194"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e0045125","DOI":"10.1128\/jvi.00451-25","article-title":"Engineering a recombination-resistant live attenuated vaccine candidate with suppressed interferon antagonists for PEDV","volume":"99","author":"Liu","year":"2025","journal-title":"J. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1038\/s41541-022-00523-8","article-title":"TLR agonists induce sustained IgG to hemagglutinin stem and modulate T cells following newborn vaccination","volume":"7","author":"Clemens","year":"2022","journal-title":"NPJ Vaccines"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"22033","DOI":"10.1038\/s41598-025-05643-4","article-title":"Codon-deoptimized single-round infectious virus for therapeutic and vaccine applications","volume":"15","author":"Noguchi","year":"2025","journal-title":"Sci. Rep."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2209000","DOI":"10.1080\/21645515.2023.2209000","article-title":"Immunization technologies: Time to consider new preventative solutions for respiratory syncytial virus infections","volume":"19","author":"Salisbury","year":"2023","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1007\/978-1-4939-3389-1_1","article-title":"Challenges in Veterinary Vaccine Development and Immunization","volume":"1404","author":"Chambers","year":"2016","journal-title":"Methods Mol. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Li, X., Li, G., Teng, Q., Yu, L., Wu, X., and Li, Z. (2012). Development of a blocking ELISA for detection of serum neutralizing antibodies against newly emerged duck Tembusu virus. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0053026"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"114122","DOI":"10.1016\/j.jviromet.2021.114122","article-title":"Comparison of two commercial surrogate ELISAs to detect a neutralising antibody response to SARS-CoV-2","volume":"292","author":"Girl","year":"2021","journal-title":"J. Virol. Methods"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"684","DOI":"10.4049\/jimmunol.2200226","article-title":"An Antibody Neutralization Determinant on Domain III and the First \u03b1-Helical Domain in the Stem-Anchor Region of Tembusu Virus Envelope Protein","volume":"209","author":"Yang","year":"2022","journal-title":"J. Immunol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Qu, S., Wang, X., Yang, L., Lv, J., Meng, R., Dai, W., Li, Q., Liu, H., Zhang, B., and Zhang, D. (2020). Identification of a Neutralizing Monoclonal Antibody That Recognizes a Unique Epitope on Domain III of the Envelope Protein of Tembusu Virus. Viruses, 12.","DOI":"10.3390\/v12060647"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Yu, G., Lin, Y., Tang, Y., and Diao, Y. (2018). Evolution of Tembusu Virus in Ducks, Chickens, Geese, Sparrows, and Mosquitoes in Northern China. Viruses, 10.","DOI":"10.3390\/v10090485"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Wu, F., Xu, T., Lai, S.Y., Ai, Y.R., Zhou, Y.C., Ge, L.P., Sun, J., Liu, Z.H., Zeng, X., and Lang, L.Q. (2024). Prevalence and genetic evolution analysis of porcine epidemic diarrhea virus and porcine circovirus type 2 in Sichuan Province, China, from 2023 to 2024. Front. Vet. Sci., 11.","DOI":"10.3389\/fvets.2024.1475347"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Nambala, P., and Su, W.C. (2018). Role of Zika Virus prM Protein in Viral Pathogenicity and Use in Vaccine Development. Front. Microbiol., 9.","DOI":"10.3389\/fmicb.2018.01797"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Jia, Z., Ma, C., Yang, X., Pan, X., Li, G., and Ma, D. (2021). Oral Immunization of Recombinant Lactococcus lactis and Enterococcus faecalis Expressing Dendritic Cell Targeting Peptide and Hexon Protein of Fowl Adenovirus 4 Induces Protective Immunity Against Homologous Infection. Front. Vet. Sci., 8.","DOI":"10.3389\/fvets.2021.632218"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Huang, A.T., Salje, H., Escoto, A.C., Chowdhury, N., Ch\u00e1vez, C., Garcia-Carreras, B., Rutvisuttinunt, W., Maljkovic Berry, I., Gromowski, G.D., and Wang, L. (2022). Beneath the surface: Amino acid variation underlying two decades of dengue virus antigenic dynamics in Bangkok, Thailand. PLoS Pathog., 18.","DOI":"10.1371\/journal.ppat.1010500"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Rohaim, M.A., Naggar, R.F.E., AbdelSabour, M.A., Ahmed, B.A., Hamoud, M.M., Ahmed, K.A., Zahran, O.K., and Munir, M. (2021). Insights into the Genetic Evolution of Duck Hepatitis A Virus in Egypt. Animals, 11.","DOI":"10.3390\/ani11092741"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Alpert, M.D., Harvey, J.D., Lauer, W.A., Reeves, R.K., Piatak, M., Carville, A., Mansfield, K.G., Lifson, J.D., Li, W., and Desrosiers, R.C. (2012). ADCC develops over time during persistent infection with live-attenuated SIV and is associated with complete protection against SIV(mac)251 challenge. PLoS Pathog., 8.","DOI":"10.1371\/journal.ppat.1002890"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e39","DOI":"10.4110\/in.2021.21.e39","article-title":"SARS-CoV-2 Antibody Neutralization Assay Platforms Based on Epitopes Sources: Live Virus, Pseudovirus, and Recombinant S Glycoprotein RBD","volume":"21","author":"Septisetyani","year":"2021","journal-title":"Immune Netw."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"752","DOI":"10.1080\/10717544.2017.1321060","article-title":"Cell-penetrating peptide conjugates to enhance the antitumor effect of paclitaxel on drug-resistant lung cancer","volume":"24","author":"Duan","year":"2017","journal-title":"Drug Deliv."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Singanallur, N.B., Dekker, A., Ebl\u00e9, P.L., Van Hemert-Kluitenberg, F., Weerdmeester, K., Horsington, J., and Vosloo, W.W. (2020). Emergency Foot-and-Mouth Disease Vaccines A Malaysia 97 and A(22) Iraq 64 Offer Good Protection against Heterologous Challenge with A Variant Serotype A ASIA\/G-IX\/SEA-97 Lineage Virus. Vaccines, 8.","DOI":"10.3390\/vaccines8010080"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Tang, M., Shi, X., Xiong, Y., Yuan, C., Zhu, Q., Huang, M., Xu, B., Liu, Q., Pan, X., and Zhang, Z. (2025). Development of a Blocking ELISA for Detection of Serum Neutralizing Antibodies Against Duck Adenovirus Type 3. Microorganisms, 13.","DOI":"10.3390\/microorganisms13112607"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e1130","DOI":"10.1111\/tbed.14402","article-title":"Tembusu virus infection in laying chickens: Evidence for a distinct genetic cluster with significant antigenic variation","volume":"69","author":"Yu","year":"2022","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"103469","DOI":"10.1016\/j.psj.2024.103469","article-title":"NS1: A promising novel target antigen with strong immunogenicity and protective efficacy for avian flavivirus vaccine development","volume":"103","author":"Huang","year":"2024","journal-title":"Poult. Sci."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Sun, M., Zhang, L., Cao, Y., Wang, J., Yu, Z., Sun, X., Liu, F., Li, Z., Liu, P., and Su, J. (2020). Basic Amino Acid Substitution at Residue 367 of the Envelope Protein of Tembusu Virus Plays a Critical Role in Pathogenesis. J. Virol., 94.","DOI":"10.1128\/JVI.02011-19"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Kanse, S., Khandelwal, M., Pandey, R.K., Khokhar, M., Desai, N., and Kumbhar, B.V. (2023). Designing a Multi-Epitope Subunit Vaccine against VP1 Major Coat Protein of JC Polyomavirus. Vaccines, 11.","DOI":"10.20944\/preprints202304.1067.v1"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/2\/256\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,20]],"date-time":"2026-02-20T05:10:57Z","timestamp":1771564257000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/2\/256"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,18]]},"references-count":40,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["v18020256"],"URL":"https:\/\/doi.org\/10.3390\/v18020256","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,18]]}}}