Evaluation of an N1 NA antibody-specific enzyme-linked lectin assay for detection of H5N1 highly pathogenic avian influenza virus infection in vaccinated birds

Authors: IBRAHIM, SHERIF - Orise Fellow; Spackman, Erica; Suarez, David; Goraichuk, Iryna; Lee, Chang

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/14/2025
Publication Date: 2/15/2025
Citation: Ibrahim, S., Spackman, E., Suarez, D.L., Goraichuk, I., Lee, C.W. 2025. Evaluation of an N1 NA antibody-specific enzyme-linked lectin assay for detection of H5N1 highly pathogenic avian influenza virus infection in vaccinated birds. Journal of Virological Methods. Journal of Virological Methods 334:115127. 2025. https://doi.org/10.1016/j.jviromet.2025.115127.
DOI: https://doi.org/10.1016/j.jviromet.2025.115127

Interpretive Summary: Vaccination for highly pathogenic avian influenza (HPAI) has remained as an important non-tariff trade barrier that has prevented most international trade in vaccinated poultry and poultry products. However, trade can potentially be allowed if a robust surveillance system allows reasonable assurance that vaccinated poultry have not been infected during the production cycle and that the risk of transmission through vaccinated poultry or poultry products is manageably small. Thus, in addition to developing effective vaccines, it is imperative to establish surveillance tools that can identify whether vaccinated animals have ever been infected (DIVA) to quickly detect and depopulate infected birds and ensure that the country specific region of the country, or poultry compartment is free from HPAI virus infection. In this study, we applied enzyme-linked lectin assay (ELLA) to sensitively and specifically detect N1 neuraminidase (NA) antibody in avian sera. NA inhibition (NI)-ELLA showed relatively high sensitivity with great potential for DIVA application with the widest range of vaccine usage including subunit vaccines and heterologous neuraminidase strategies. Compared to traditional NI assay, ELLA does not require toxic reagents and uses similar reagents and workflow as indirect ELISA, so could be easily implemented by veterinary diagnostic labs. ELLA can be a viable cost-effective option for large-scale serological surveillance of detecting infection of vaccinated poultry with influenza virus.

Technical Abstract: Unprecedented H5N1 highly pathogenic avian influenza (HPAI) outbreaks are occurring around the world and there is growing interest in the use of vaccines in affected regions. Vaccination when properly applied can contribute to HPAI control by significantly reducing virus shedding and breaking the transmission chain, but it requires robust surveillance to ensure that international trade is not affected. Thus, it is imperative to establish a test to differentiate vaccinated only animals from vaccinated and then infected animals (DIVA). In this study, we applied enzyme-linked lectin assay (ELLA) to specifically detect N1 neuraminidase (NA) antibody by inhibition of NA activity and provide a proof-of-concept bench validation using reference and experimental serum samples. We used a wild-type low pathogenic H7N1 virus of North American lineage as the ELLA antigen. The NA inhibition ELLA (NI-ELLA) was evaluated for its specificity and sensitivity using reference and experimental samples. The results demonstrated that the NI-ELLA was highly specific with low background NI activity against influenza-negative sera from different species although varying level of cross-reactivity was observed against sera of different NA subtypes. Using a conservative positive cut-off threshold of 50% NI activity, NI-ELLA provides 100% specificity with reference sera. The relative sensitivity of NI-ELLA was evaluated in detecting H5N1 infection in vaccinated and then challenged birds that showed higher sensitivity of the NI-ELLA compared with commercial NP ELISAs and real-time RT-PCR. Overall, the NI-ELLA shows high specificity and sensitivity and has the potential for application in DIVA surveillance with further validation.