Influenza A viruses (IAVs) of the H1N1 and H3N2 serotypes are the major cause of seasonal influenza epidemics. IAVs are labeled based on the antigenic properties of the two surface glycoproteins and main antigens: hemagglutinin (HA/H) and neuraminidase (NA/N). HA and NA have opposing roles, with HA binding and NA enzymatically removing terminal acid residues from glycoconjugates. As HA is more abundant in the viral envelope and easily quantified, current vaccines are standardized only to HA amount. However, the seasonal vaccines have low efficacy, in part because they mainly elicit an immune response to the HA protein. Suggestions to actively include and standardize to NA amount as well has been made. Unfortunately, the active and most immunogenic form of NA is an unstable tetramer that easily disassociate during vaccine production. Knowledge of the stabilizing properties of NA is therefore needed. Additionally, due to the influenza error-prone polymerase and the many avian serotypes that could potentially make zoonotic jumps in the future, identifying residues and regions of the surface proteins that are conserved across HA and NA subtypes could help provide a more universal response to IAVs.

This thesis presents several studies aimed to increase the knowledge about influenza and the NA protein. First, a method is presented where labeled oligonucleotides (padlock probes) hybridize with the viral genome, allowing for identification of genomic segments during an infection. We show that using a handful of probes against highly conserved regions, the vast majority of IAV serotypes can be identified. Second, we show that the conserved central calcium site of NA is vital for enzymatic function and that the oligomeric structure allows for the rescue of inactive monomers by formation of enzymatically active heterotetramers. The final studies take aim at the conserved N-linked glycosylation sites and cysteines of NA ectodomains. We show that glycosylation of the head domain influences NA virion incorporation and provides stability to the protein. We also identify conserved NA residues, including a surface accessible tryptophan that is entirely conserved across a majority of IAV NA subtypes.

Together, these results provide a better knowledge of influenza neuraminidase and present several targets for next-generation vaccines.