Using pseudotyped virus-based systems and structural approaches to explore the potential cross-reactivity between influenza C and D viruses

Influenza C (ICV) and influenza D (IDV) viruses are less studied members of the Orthomyxoviridae family, characterised by the hemagglutinin-esterase fusion glycoprotein (HEF), which mediates both receptor binding and new virion release. HEFs from ICV and IDV share about 53% homology at the genetic level. Although ICV and IDV are not currently classified as major threats to human health, it is essential to understand their biology and infection mechanisms to prevent and mitigate potential outbreaks. Pseudotyped viruses (PVs) offer a flexible alternative to wild-type viruses, as they are safe to handle, easily adaptable to specific strains, suitable for laboratories without high-containment facilities, and usable even when viral isolates are unavailable. In this thesis, lentiviral PVs expressing the HEF glycoprotein of ICV (C/Minnesota/33/2015 strain) and IDV (D/Swine/Italy/199724-3/2015, D/Bovine/France/5920/2014, and D/Bovine/Ibaraki/7768/2016 strains) were generated and optimised. The results demonstrated that PVs can effectively mimic ICV and IDV entry. The swine testis cell line was the most permissive, and the human airway trypsin-like protease was the most effective in promoting HEF maturation. Using these PVs, a pseudotyped virus-based microneutralisation (pMN) assay to measure neutralising antibody responses was established, and an esterase activity assay to evaluate HEF enzymatic activity was adapted. Specific antisera neutralised PVs but failed to inhibit esterase activity. A qualification of the IDV pMN assay was conducted assessing key analytical parameters. The findings proved the IDV pMN assay to be an effective method for the detection of neutralising antibodies, facilitating large-scale surveillance. The observation of strong neutralising activity of cow serum samples against both ICV and IDV prompted an investigation into its molecular basis. Structural modelling, epitope prediction, and receptor docking analyses identified five potential epitopes. Targeted amino acid substitutions were introduced into HEFs to generate ICV- and IDV-mutant PVs. Although only IDV mutants yielded functional PVs, the ICV antiserum showed high neutralising activity against one construct, suggesting the presence of an ICV-like antigenic site within the IDV framework. Overall, this study provides novel methodological tools for the characterisation of ICV and IDV, qualifies a safe and standardisable serological assay, and identifies potential shared antigenic determinants that may inform the development of cross-protective influenza vaccines and improve interspecies influenza surveillance.