Development and application of a NP-cELISA for the detection of nucleoprotein antibodies of equine influenza virus.
Abstract: Equine influenza (EI), caused by the equine influenza virus (EIV), is an acute respiratory disease that has become enzootic worldwide, resulting in frequent outbreaks and substantial economic losses within the equine industry. In this study, we developed a competitive enzyme-linked immunosorbent assay (NP-cELISA) for the detection of antibodies against the EIV nucleoprotein (NP). The assay was designed by coating plates with purified monoclonal antibodies (mAbs) against the NP protein, followed by simultaneous incubation of the test serum samples and HRP-NP antigen in a competitive binding reaction. Receiver operating characteristic (ROC) curve analysis demonstrated that the assay achieved 100% sensitivity and specificity. To assess the diagnostic performance of the NP-cELISA, we evaluated 119 clinical samples in parallel using the NP-cELISA, a commercially available competitive ELISA (ID.vet-cELISA), and the hemagglutination inhibition (HI) assay as the reference standard. The results indicated that the NP-cELISA showed an 87.4% concordance rate with the HI assay, outperforming the 78.2% concordance rate observed between the ID.vet-cELISA and the HI test. Additionally, in a serological surveillance study conducted using the developed NP-cELISA in China from 2021 to 2023, equine serum samples showed an average annual seroprevalence of 37.96% for EIV antibodies. In conclusion, the NP-cELISA developed in this study demonstrates significant potential as a reliable and efficient diagnostic tool for the serological detection of EI, with broad applicability in various settings. Objective: Equine influenza (EI) is a highly contagious respiratory disease that poses significant economic and health challenges to the global equine industry. Current diagnostic methods, such as hemagglutination inhibition (HI), are accurate but complex and impractical for widespread use, especially in regions like China where commercial kits are unavailable. This study developed a competitive ELISA (cELISA) for detecting EI virus antibodies, offering a simpler, faster, and more cost-effective alternative. The assay demonstrated higher concordance with HI than existing commercial kits and effectively monitored antibody responses in vaccinated horses. Additionally, it enabled the first large-scale serological survey of EI in China, providing critical insights into the virus's prevalence. This advancement supports timely disease detection and control, benefiting veterinary practices and the equine industry worldwide.
Publication Date: 2025-08-25 PubMed ID: 40853243PubMed Central: PMC12502703DOI: 10.1128/spectrum.00939-25Google Scholar: Lookup
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- Journal Article
Summary
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Overview
- Researchers developed a new competitive ELISA test (NP-cELISA) to detect antibodies against equine influenza virus (EIV) nucleoprotein in horse serum.
- This test offers a simpler, faster, and highly accurate alternative to traditional methods, enabling improved diagnosis and surveillance of EI infections.
Background and Significance
- Equine influenza (EI) is a contagious respiratory disease in horses caused by the equine influenza virus (EIV), leading to frequent outbreaks and economic losses worldwide.
- Accurate detection of antibodies against EIV is essential for disease diagnosis, vaccine evaluation, and epidemiological surveillance.
- Existing diagnostic methods such as hemagglutination inhibition (HI) assay are highly accurate but complex, time-consuming, and not easily implemented on a wide scale, especially in regions lacking commercial kits like China.
Development of the NP-cELISA
- The study developed a competitive enzyme-linked immunosorbent assay targeting the nucleoprotein (NP) antibody of EIV.
- The assay protocol involved coating microtiter plates with purified monoclonal antibodies (mAbs) specific to the NP protein of EIV.
- Test serum samples were incubated simultaneously with horseradish peroxidase (HRP)-conjugated NP antigen, allowing competition for binding sites between serum antibodies and the labeled antigen.
- This competitive binding format ensures only serum antibodies specific to NP can inhibit the binding of the HRP-NP antigen, enabling detection via a measurable enzymatic reaction.
Performance Evaluation
- Receiver operating characteristic (ROC) curve analysis showed the assay achieved 100% sensitivity and 100% specificity, indicating no false positives or negatives in the test population.
- To validate diagnostic utility, 119 clinical serum samples were tested simultaneously with:
- The newly developed NP-cELISA
- A commercial competitive ELISA (ID.vet-cELISA)
- The HI assay as a reference standard
- Results demonstrated:
- NP-cELISA had an 87.4% concordance rate with the HI assay, indicating high agreement.
- ID.vet-cELISA showed a lower concordance rate (78.2%) with the HI assay, suggesting the NP-cELISA outperformed the commercial kit.
Application in Serological Surveillance
- The NP-cELISA was applied in a large-scale serological survey of equine populations in China from 2021 to 2023.
- The study found an average annual seroprevalence of 37.96% for EIV antibodies, providing valuable data on the extent of viral exposure in horses within China.
- This represents the first extensive use of such a diagnostic tool in this region, filling a gap due to previously unavailable commercial kits.
Advantages and Implications
- The NP-cELISA is simple, rapid, and cost-effective compared to traditional HI assays and commercial ELISA kits.
- Its high sensitivity and specificity make it a reliable diagnostic tool for serological detection of EI.
- It facilitates:
- Timely diagnosis of EI outbreaks
- Monitoring of antibody responses post-vaccination
- Large-scale epidemiological surveillance in regions lacking commercial assays
- Wider application of this assay can improve disease control strategies globally, benefiting veterinary medicine and the equine industry by minimizing economic losses and improving animal health.
Cite This Article
APA
Yang Y, Guo K, Xu L, Guo W, Dong M, Liu W, Li S, Zhang Z, Chu X, Wang Y, Zhang Z, Hu Z, Wang X.
(2025).
Development and application of a NP-cELISA for the detection of nucleoprotein antibodies of equine influenza virus.
Microbiol Spectr, 13(10), e0093925.
https://doi.org/10.1128/spectrum.00939-25 Publication
Researcher Affiliations
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
- Harbin Guosheng Biotechnology Co. Ltd., Harbin, China.
- Harbin Guosheng Biotechnology Co. Ltd., Harbin, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
- University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, USA.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang, China.
- Institute of Western Agriculture, the Chinese Academy of Agricultural Sciences, Changji, Xinjiang, China.
MeSH Terms
- Horses
- Animals
- Antibodies, Viral / blood
- Antibodies, Viral / immunology
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horse Diseases / immunology
- Enzyme-Linked Immunosorbent Assay / methods
- Enzyme-Linked Immunosorbent Assay / veterinary
- Orthomyxoviridae Infections / veterinary
- Orthomyxoviridae Infections / diagnosis
- Orthomyxoviridae Infections / virology
- Orthomyxoviridae Infections / immunology
- Sensitivity and Specificity
- Influenza A Virus, H3N8 Subtype / immunology
- Influenza A Virus, H3N8 Subtype / isolation & purification
- Nucleoproteins / immunology
- Antibodies, Monoclonal / immunology
- Hemagglutination Inhibition Tests
- ROC Curve
Grant Funding
- IWA2023 / Tianchi Talent Introduction Plan
- No. 2021YFD1800500 / The National Key Research and Development Program of China
- ZZYD2023010 / Xinjiang Talent Development Fund
- TD2022C006 / The Nature Science Foundation of Heilongjiang Province
Conflict of Interest Statement
The authors declare no conflict of interest.
References
This article includes 37 references
- Parrish CR, Murcia PR, Holmes EC. Influenza virus reservoirs and intermediate hosts: dogs, horses, and new possibilities for influenza virus exposure of humans.. J Virol 89:2990–2994.
- Kirkland PD, Finlaison DS, Crispe E, Hurt AC. Influenza virus transmission from horses to dogs, Australia.. Emerg Infect Dis 16:699–702.
- Tong S, Li Y, Rivailler P, Conrardy C, Castillo DAA, Chen L-M, Recuenco S, Ellison JA, Davis CT, York IA. A distinct lineage of influenza A virus from bats.. Proc Natl Acad Sci USA 109:4269–4274.
- Te Velthuis AJW, Fodor E. Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis.. Nat Rev Microbiol 14:479–493.
- Borland S, Gracieux P, Jones M, Mallet F, Yugueros-Marcos J. Influenza A virus infection in cats and dogs: a literature review in the light of the “one health” concept.. Front Public Health 8:83.
- Zhang M, Liu M, Bai S, Zhao C, Li Z, Xu J, Zhang X. Influenza a virus–host specificity: an ongoing cross-talk between viral and host factors.. Front Microbiol 12.
- Palese P, Schulman JL. Mapping of the influenza virus genome: identification of the hemagglutinin and the neuraminidase genes.. Proc Natl Acad Sci USA 73:2142–2146.
- Shao W, Li X, Goraya MU, Wang S, Chen J-L. Evolution of influenza A virus by mutation and re-assortment.. Int J Mol Sci 18:1650.
- Murcia PR, Wood JLN, Holmes EC. Genome-scale evolution and phylodynamics of equine H3N8 influenza A virus.. J Virol 85:5312–5322.
- Cullinane A, Elton D, Mumford J. Equine influenza – surveillance and control.. Influenza Other Respir Viruses 4:339–344.
- Paillot R, El-Hage CM. The use of a recombinant canarypox-based equine influenza vaccine during the 2007 Australian outbreak: a systematic review and summary.. Pathogens 5:42.
- Gildea S, Fitzpatrick DA, Cullinane A. Epidemiological and virological investigations of equine influenza outbreaks in Ireland (2010–2012).. Influenza Other Respir Viruses 7:61–72.
- Fougerolle S, Fortier C, Legrand L, Jourdan M, Marcillaud-Pitel C, Pronost S, Paillot R. Success and limitation of equine influenza vaccination: the first incursion in a decade of a Florida clade 1 equine influenza virus that shakes protection despite high vaccine coverage.. Vaccines (Basel) 7:174.
- Shittu I, Meseko CA, Sulaiman LP, Inuwa B, Mustapha M, Zakariya PS, Muhammad AA, Muhammad U, Atuman YJ, Barde IJ, Zecchin B, Quaranta EG, Shamaki D, Alabi O, Monne I, Fusaro A, Joannis TM. Fatal multiple outbreaks of equine influenza H3N8 in Nigeria, 2019: the first introduction of Florida clade 1 to West Africa.. Vet Microbiol 248:108820.
- Diallo AA, Souley MM, Issa Ibrahim A, Alassane A, Issa R, Gagara H, Yaou B, Issiakou A, Diop M, Ba Diouf RO, Lo FT, Lo MM, Bakhoum T, Sylla M, Seck MT, Meseko C, Shittu I, Cullinane A, Settypalli TBK, Lamien CE, Dundon WG, Cattoli G. Transboundary spread of equine influenza viruses (H3N8) in West and Central Africa: molecular characterization of identified viruses during outbreaks in Niger and Senegal, in 2019.. Transbound Emerg Dis 68:1253–1262.
- Laabassi F, Lecouturier F, Amelot G, Gaudaire D, Mamache B, Laugier C, Legrand L, Zientara S, Hans A. Epidemiology and genetic characterization of H3N8 equine influenza virus responsible for clinical disease in Algeria in 2011. Transbound Emerg Dis 62:623–631.
- Sack A, Daramragchaa U, Chuluunbaatar M, Gonchigoo B, Bazartseren B, Tsogbadrakh N, Gray GC. Low prevalence of enzootic equine influenza virus among horses in Mongolia. Pathogens 6:61.
- Gahan J, Garvey M, Asmah Abd Samad R, Cullinane A. Whole genome sequencing of the first H3N8 equine influenza virus identified in Malaysia. Pathogens 8:62.
- Favaro PF, Fernandes WR, Reischak D, Brandão PE, Silva S de S, Richtzenhain LJ. Evolution of equine influenza viruses (H3N8) during a Brazilian outbreak, 2015. Braz J Microbiol 49:336–346.
- Perglione CO, Gildea S, Rimondi A, Miño S, Vissani A, Carossino M, Cullinane A, Barrandeguy M. Epidemiological and virological findings during multiple outbreaks of equine influenza in South America in 2012. Influenza Other Respir Viruses 10:37–46.
- Alves Beuttemmüller E, Woodward A, Rash A, Dos Santos Ferraz LE, Fernandes Alfieri A, Alfieri AA, Elton D. Characterisation of the epidemic strain of H3N8 equine influenza virus responsible for outbreaks in South America in 2012. Virol J 13:45.
- Mena J, Brito B, Moreira R, Tadich T, González I, Cruces J, Ortega R, van Bakel H, Rathnasinghe R, Pizarro-Lucero J, Medina R, Neira V. Reemergence of H3N8 equine influenza a virus in Chile, 2018. Transbound Emerg Dis 65:1408–1415.
- Olguin-Perglione C, Vissani MA, Alamos F, Tordoya MS, Barrandeguy M. Multifocal outbreak of equine influenza in vaccinated horses in Argentina in 2018: epidemiological aspects and molecular characterisation of the involved virus strains. Equine Vet J 52:420–427.
- Bibby DC, Savanovic M, Zhang J, Torelli A, Jeeninga RE, Gagnon L, Harris SL. Interlaboratory reproducibility of standardized hemagglutination inhibition assays. mSphere 7:e0095321.
- Hu Y, Sneyd H, Dekant R, Wang J. Influenza A virus nucleoprotein: a highly conserved multi-functional viral protein as a hot antiviral drug target. Curr Top Med Chem 17.
- Grant E, Wu C, Chan KF, Eckle S, Bharadwaj M, Zou QM, Kedzierska K, Chen W. Nucleoprotein of influenza A virus is a major target of immunodominant CD8 T-cell responses. Immunol Cell Biol 91:184–194.
- Guo K, Zhang Z, Yang Y, Zhang W, Wang J, Li S, Chu X, Guo W, Liu D, Wang Y, Hu Z, Wang X. Development and application of an iELISA for the detection of antibody against Salmonella Abortusequi. Transbound Emerg Dis 2023:1403180.
- Dou D, Revol R, Östbye H, Wang H, Daniels R. Influenza a virus cell entry, replication, virion assembly and movement. Front Immunol 9:1581.
- Narkhede YB, Gonzalez KJ, Strauch E-M. Targeting viral surface proteins through structure-based design. Viruses 13:1320.
- Zhang Y, Xu Z, Cao Y. Host–virus interaction: how host cells defend against influenza A virus infection. Viruses 12:376.
- Rak A, Isakova-Sivak I, Rudenko L. Nucleoprotein as a promising antigen for broadly protective influenza vaccines. Vaccines (Basel) 11:1747.
- Myers ML, Gallagher JR, Kim AJ, Payne WH, Maldonado-Puga S, Assimakopoulos H, Bock KW, Torian U, Moore IN, Harris AK. Commercial influenza vaccines vary in HA-complex structure and in induction of cross-reactive HA antibodies. Nat Commun 14:1763.
- Guo K, Guo W, Liu D, Zhang W, Yang Y, Zhang Z, Li S, Wang J, Chu X, Wang Y, Hu Z, Wang X. Development and application of a competitive ELISA for the detection of antibodies against Salmonella Abortusequi in equids. J Clin Microbiol 61:e0027323.
- Schardt JS, Pornnoppadol G, Desai AA, Park KS, Zupancic JM, Makowski EK, Smith MD, Chen H, Garcia de Mattos Barbosa M, Cascalho M, Lanigan TM, Moon JJ, Tessier PM. Discovery and characterization of high-affinity, potent SARS-CoV-2 neutralizing antibodies via single B cell screening. Sci Rep 11:20738.
- Plata-Hipólito CB, Cedillo-Rosales S, Obregón-Macías N, Hernández-Luna CE, Rodríguez-Padilla C, Tamez-Guerra RS, Contreras-Cordero JF. Genetic and serologic surveillance of canine (CIV) and equine (EIV) influenza virus in Nuevo León State, México. PeerJ 7:e8239.
- Bahadoran A, Lee SH, Wang SM, Manikam R, Rajarajeswaran J, Raju CS, Sekaran SD. Immune responses to influenza virus and its correlation to age and inherited factors. Front Microbiol 7:1841.
- McFarlane D. Immune dysfunction in aged horses. Vet Clin North Am Equine Pract 32:333–341.
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