Development and Qualification of a Pseudotyped Virus‐Based Microneutralisation Assay for Influenza D Virus

BACKGROUND: Epidemiological surveillance of influenza D virus (IDV) has gained increased priority following recent serological findings indicating its potential zoonosis in humans. In this context, it is crucial to develop strong, reproducible, reliable and scalable immunological assays that can be quickly implemented in the surveillance of new emerging threats. Serology is a powerful tool for immune monitoring prior to infection and conducting epidemiological surveillance. However, the traditional microneutralisation (MN) assay requires wild-type viruses, considerably limiting its accessibility for some laboratories. Pseudotyped viruses (PVs) allow for expanded usage since they are safer and more flexible for adaptation to specific strains and enable application in laboratories without implementation in high biosecurity containment. METHODS: In this study, we conducted the qualification of a PV-based MN (pMN) assay with IDV-PVs that express the HEF glycoprotein of the D/Swine/Italy/199724-3/2015 strain. The assay functionality was examined using 14 bovine serum samples, assessing key analytical parameters including accuracy, specificity, precision, linearity and robustness. RESULTS: The findings demonstrate the IDV pMN assay to be an effective method for the detection of neutralising antibodies. CONCLUSIONS: Therefore, the assay can be a valuable tool to facilitate large-scale surveillance and provide data to inform immunisation strategy development.