{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T19:26:44Z","timestamp":1780514804062,"version":"3.54.1"},"reference-count":24,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2025,2,28]],"date-time":"2025-02-28T00:00:00Z","timestamp":1740700800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Hendra virus (HeV) is a bat-borne zoonotic agent which can cause a severe and highly fatal disease and can be transferred from animals to humans. It has caused over 100 deaths in horses since it was discovered in 1994. Four out of seven infected humans have died. Since the release of the HeV vaccine (Equivac\u00ae HeV Hendra Virus Vaccine for Horses, Zoetis Australia Pty Ltd., Rhodes, NSW 2138) in Australia, there has been an urgent requirement for a serological test for differentiating infected from vaccinated animals (DIVA). All first-line diagnostic serological assays at the Australian Centre for Disease Preparedness (ACDP) incorporate recombinant HeV soluble G glycoprotein (sG) as the antigen, which is also the only immunogen present in the Equivac\u00ae HeV vaccine. Problems therefore arose in that antibody testing results were unable to distinguish between prior vaccination or infection with HeV. This study describes the development of a HeV DIVA ELISA strategy using recombinant sG and HeV nucleoprotein (N), paired with specific monoclonal antibodies in a competition ELISA format. The validation of this assay strategy was performed using a positive cohort of 19 serum samples representing post-infection sera, a negative cohort of 1138 serum samples representing horse sera collected pre-vaccine release and a vaccination cohort of 502 serum samples from horses previously vaccinated with Equivac\u00ae HeV vaccine. For the sG glycoprotein, the diagnostic sensitivity (DSe) was 100.0% (95% CI: 99.3\u2013100.0%) and diagnostic specificity (DSp) 99.91% (95% CI: 99.5\u2013100.0%), using a percentage inhibition cut-off value of &gt;36, whereas for the N protein, DSe was 100.0% (95% CI: 82.4\u2013100.0%) and DSp 100.0% (95% CI: 99.7\u2013100.0%), using a percentage inhibition cut-off value of &gt;49. Taken together, these results demonstrate that the HeV DIVA ELISA strategy developed here is now an essential and critical component of the testing algorithm for HeV serology testing in Australia.<\/jats:p>","DOI":"10.3390\/v17030354","type":"journal-article","created":{"date-parts":[[2025,2,28]],"date-time":"2025-02-28T10:12:33Z","timestamp":1740737553000},"page":"354","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Development and Validation of a Differentiating Infected from Vaccinated Animals (DIVA) Enzyme-Linked Immunosorbent Assay (ELISA) Strategy for Distinguishing Between Hendra-Infected and Vaccinated Horses"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0935-5495","authenticated-orcid":false,"given":"Leanne","family":"McNabb","sequence":"first","affiliation":[{"name":"CSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amy","family":"McMahon","sequence":"additional","affiliation":[{"name":"CSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ezana Getachew","family":"Woube","sequence":"additional","affiliation":[{"name":"Private Consultant, Melbourne, VIC 3000, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9934-306X","authenticated-orcid":false,"given":"Kalpana","family":"Agnihotri","sequence":"additional","affiliation":[{"name":"CSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Axel","family":"Colling","sequence":"additional","affiliation":[{"name":"CSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5382-0510","authenticated-orcid":false,"given":"Christopher C.","family":"Broder","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Uniformed Services University, Bethesda, MD 20814, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1761-0089","authenticated-orcid":false,"given":"Indre","family":"Kucinskaite-Kodze","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Centre, Vilnius University, LT-01513 Vilnius, Lithuania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5676-2993","authenticated-orcid":false,"given":"Rasa","family":"Petraityte-Burneikiene","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Centre, Vilnius University, LT-01513 Vilnius, Lithuania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Timothy R.","family":"Bowden","sequence":"additional","affiliation":[{"name":"CSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1279-5010","authenticated-orcid":false,"given":"Kim","family":"Halpin","sequence":"additional","affiliation":[{"name":"CSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,2,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1038\/nrmicro1323","article-title":"Hendra and Nipah viruses: Different and dangerous","volume":"4","author":"Eaton","year":"2006","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1927","DOI":"10.1099\/0022-1317-81-8-1927","article-title":"Isolation of Hendra virus from pteropid bats: A natural reservoir of Hendra virus","volume":"81","author":"Halpin","year":"2000","journal-title":"J. Gen. Virol."},{"key":"ref_3","unstructured":"(2024, December 16). Queensland Business, Available online: https:\/\/www.business.qld.gov.au\/industries\/service-industries-professionals\/service-industries\/veterinary-surgeons\/guidelines-hendra\/incident-summary."},{"key":"ref_4","unstructured":"OIE (2018). Nipah and Hendra virus Diseases. OIE Terrestrial Manual 2018, OIE. Chapter 3.1.14."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/S0166-0934(01)00377-9","article-title":"A rapid immune plaque assay for the detection of Hendra and Nipah viruses and anti-virus antibodies","volume":"99","author":"Crameri","year":"2002","journal-title":"J. Virol. Methods"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Amaya, M., Yin, R., Yan, L., Borisevich, V., Adhikari, B.N., Bennett, A., Malagon, F., Cer, R.Z., Bishop-Lilly, K.A., and Dimitrov, A.S. (2023). A Recombinant Chimeric Cedar Virus-Based Surrogate Neutralization Assay Platform for Pathogenic Henipaviruses. Viruses, 15.","DOI":"10.3390\/v15051077"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"6690","DOI":"10.1128\/JVI.79.11.6690-6702.2005","article-title":"Receptor binding, fusion inhibition, and induction of cross-reactive neutralizing antibodies by a soluble G glycoprotein of Hendra virus","volume":"79","author":"Bossart","year":"2005","journal-title":"J. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"362","DOI":"10.1177\/1040638718760102","article-title":"A network approach for provisional assay recognition of a Hendra virus antibody ELISA: Test validation with low sample numbers from infected horses","volume":"30","author":"Colling","year":"2018","journal-title":"J. Vet. Diagn. Investig."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1016\/j.jviromet.2014.01.010","article-title":"Henipavirus microsphere immuno-assays for detection of antibodies against Hendra virus","volume":"200","author":"McNabb","year":"2014","journal-title":"J. Virol. Methods"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2839","DOI":"10.1099\/vir.0.81218-0","article-title":"Location of, immunogenicity of and relationships between neutralization epitopes on the attachment protein (G) of Hendra virus","volume":"86","author":"White","year":"2005","journal-title":"J. Gen. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"113731","DOI":"10.1016\/j.jviromet.2019.113731","article-title":"Optimization and diagnostic evaluation of monoclonal antibody-based blocking ELISA formats for detection of neutralizing antibodies to Hendra virus in mammalian sera","volume":"274","author":"McNabb","year":"2019","journal-title":"J. Virol. Methods"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1111\/avj.12694","article-title":"Virus-neutralising antibody responses in horses following vaccination with Equivac(R) HeV: A field study","volume":"96","author":"Tan","year":"2018","journal-title":"Aust. Vet. J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1016\/j.cveq.2022.11.009","article-title":"Hendra Virus: An Update on Diagnosis, Vaccination, and Biosecurity Protocols for Horses","volume":"39","author":"Wang","year":"2023","journal-title":"Vet. Clin. N. Am. Equine Pract."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Halpin, K., Graham, K., and Durr, P.A. (2021). Sero-Monitoring of Horses Demonstrates the Equivac((R)) HeV Hendra Virus Vaccine to Be Highly Effective in Inducing Neutralising Antibody Titres. Vaccines, 9.","DOI":"10.3390\/vaccines9070731"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/j.virusres.2006.10.008","article-title":"Generation of henipavirus nucleocapsid proteins in yeast Saccharomyces cerevisiae","volume":"124","author":"Juozapaitis","year":"2007","journal-title":"Virus Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"114296","DOI":"10.1016\/j.jviromet.2021.114296","article-title":"Development and validation of an IgM antibody capture ELISA for early detection of Hendra virus","volume":"298","author":"McNabb","year":"2021","journal-title":"J. Virol. Methods"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1006\/viro.2000.0882","article-title":"Tioman virus, a novel paramyxovirus isolated from fruit bats in Malaysia","volume":"283","author":"Chua","year":"2001","journal-title":"Virology"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1079\/AHR200479","article-title":"Application of DIVA vaccines and their companion diagnostic tests to foreign animal disease eradication","volume":"5","author":"Pasick","year":"2004","journal-title":"Anim. Health Res. Rev."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"933","DOI":"10.1177\/1040638720962070","article-title":"Inter-laboratory comparison of 2 ELISA kits used for foot-and-mouth disease virus nonstructural protein serology","volume":"32","author":"Browning","year":"2020","journal-title":"J. Vet. Diagn. Investig."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1461","DOI":"10.1007\/s007050050390","article-title":"Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus","volume":"143","author":"Sorensen","year":"1998","journal-title":"Arch. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Carey, K.J., Smith, I., Barr, J., Caruso, S., Au, G.G., Hartley, C.A., Bailey, K.E., Perriam, W., Broder, C.C., and Gilkerson, J.R. (2024). Foals of mares vaccinated for Hendra virus have a suboptimal response to HeV vaccination. Vet. Microbiol., 295.","DOI":"10.1016\/j.vetmic.2024.110167"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Zhu, W., Pickering, B., Smith, G., Pinette, M., Truong, T., Babiuk, S., Kobasa, D., Banadyga, L., and Yang, M. (2023). Development and laboratory evaluation of a competitive ELISA for serodiagnosis of Nipah and Hendra virus infection using recombinant Nipah glycoproteins and a monoclonal antibody. Front. Vet. Sci., 10.","DOI":"10.3389\/fvets.2023.1120367"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1016\/S1286-4579(01)01382-X","article-title":"Laboratory diagnosis of Nipah and Hendra virus infections","volume":"3","author":"Daniels","year":"2001","journal-title":"Microbes Infect."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Balkema-Buschmann, A., Fischer, K., McNabb, L., Diederich, S., Singanallur, N.B., Ziegler, U., Keil, G.M., Kirkland, P.D., Penning, M., and Sadeghi, B. (2022). Serological Hendra Virus Diagnostics Using an Indirect ELISA-Based DIVA Approach with Recombinant Hendra G and N Proteins. Microorganisms, 10.","DOI":"10.3390\/microorganisms10061095"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/3\/354\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T16:44:47Z","timestamp":1760028287000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/3\/354"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2,28]]},"references-count":24,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2025,3]]}},"alternative-id":["v17030354"],"URL":"https:\/\/doi.org\/10.3390\/v17030354","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,2,28]]}}}