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
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...
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2025-02-01
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| author | Leanne McNabb Amy McMahon Ezana Getachew Woube Kalpana Agnihotri Axel Colling Christopher C. Broder Indre Kucinskaite-Kodze Rasa Petraityte-Burneikiene Timothy R. Bowden Kim Halpin |
| author_facet | Leanne McNabb Amy McMahon Ezana Getachew Woube Kalpana Agnihotri Axel Colling Christopher C. Broder Indre Kucinskaite-Kodze Rasa Petraityte-Burneikiene Timothy R. Bowden Kim Halpin |
| author_sort | Leanne McNabb |
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| description | 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<sup>®</sup> 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<sup>®</sup> 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<sup>®</sup> HeV vaccine. For the sG glycoprotein, the diagnostic sensitivity (DSe) was 100.0% (95% CI: 99.3–100.0%) and diagnostic specificity (DSp) 99.91% (95% CI: 99.5–100.0%), using a percentage inhibition cut-off value of >36, whereas for the N protein, DSe was 100.0% (95% CI: 82.4–100.0%) and DSp 100.0% (95% CI: 99.7–100.0%), using a percentage inhibition cut-off value of >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. |
| format | Article |
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| institution | Kabale University |
| issn | 1999-4915 |
| language | English |
| publishDate | 2025-02-01 |
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| spelling | doaj-art-c2e31b1d722248b88d1ddd94de3be2f72025-08-20T03:44:03ZengMDPI AGViruses1999-49152025-02-0117335410.3390/v17030354Development and Validation of a Differentiating Infected from Vaccinated Animals (DIVA) Enzyme-Linked Immunosorbent Assay (ELISA) Strategy for Distinguishing Between Hendra-Infected and Vaccinated HorsesLeanne McNabb0Amy McMahon1Ezana Getachew Woube2Kalpana Agnihotri3Axel Colling4Christopher C. Broder5Indre Kucinskaite-Kodze6Rasa Petraityte-Burneikiene7Timothy R. Bowden8Kim Halpin9CSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, AustraliaCSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, AustraliaPrivate Consultant, Melbourne, VIC 3000, AustraliaCSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, AustraliaCSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, AustraliaDepartment of Microbiology and Immunology, Uniformed Services University, Bethesda, MD 20814, USAInstitute of Biotechnology, Life Sciences Centre, Vilnius University, LT-01513 Vilnius, LithuaniaInstitute of Biotechnology, Life Sciences Centre, Vilnius University, LT-01513 Vilnius, LithuaniaCSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, AustraliaCSIRO Australian Centre for Disease Preparedness (ACDP), 5 Portarlington Road, Geelong, VIC 3220, AustraliaHendra 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<sup>®</sup> 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<sup>®</sup> 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<sup>®</sup> HeV vaccine. For the sG glycoprotein, the diagnostic sensitivity (DSe) was 100.0% (95% CI: 99.3–100.0%) and diagnostic specificity (DSp) 99.91% (95% CI: 99.5–100.0%), using a percentage inhibition cut-off value of >36, whereas for the N protein, DSe was 100.0% (95% CI: 82.4–100.0%) and DSp 100.0% (95% CI: 99.7–100.0%), using a percentage inhibition cut-off value of >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.https://www.mdpi.com/1999-4915/17/3/354Hendra virusHenipavirusserologyDIVAELISA |
| spellingShingle | Leanne McNabb Amy McMahon Ezana Getachew Woube Kalpana Agnihotri Axel Colling Christopher C. Broder Indre Kucinskaite-Kodze Rasa Petraityte-Burneikiene Timothy R. Bowden Kim Halpin 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 Viruses Hendra virus Henipavirus serology DIVA ELISA |
| 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | Hendra virus Henipavirus serology DIVA ELISA |
| url | https://www.mdpi.com/1999-4915/17/3/354 |
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