Investigating the impact of influenza neuraminidase on viral replication
AuthorAllen, Robert John
AffiliationMicrobiology & Immunology
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2021-06-05.
© 2019 Dr. Robert John Allen
Infection with influenza A virus remains an important issue for healthcare systems worldwide due to the direct impact of seasonal epidemics on human morbidity and mortality and also the risk of emergent pandemics. Currently, preventive measures and treatments are not fully effective in controlling influenza virus infection. The mainstay of disease prevention is vaccination, with vaccines being updated annually and most commonly produced in embryonated chicken eggs. Influenza A viruses are classified by the surface glycoproteins haemagglutinin (HA) and neuraminidase (NA) that they express. The established role of influenza NA is the enzymatic cleavage of sialic acid from newly formed virions and from viral receptors on the surface of infected host cells at the site of virus budding. This is necessary to prevent virus aggregation and reattachment to the infected cell by the sialic acid-binding function of the HA, which would reduce the effectiveness of virus release. In this body of research, attention has been given to the potential effect of influenza NA on infectious virus yields in a variety of in vitro and in vivo systems. This project sought to determine whether an NA with intrinsically low enzyme activity for sialic acid would confer lower viral fitness and, if so, which stages of the infection cycle of influenza A virus are most affected. Specifically, this project has investigated the importance of sialidase activity of different H3N2 influenza viruses. Reverse engineered viruses differing only in the expression of different N2 NAs were constructed and tested for their yields in different host systems and for their ability to selectively remove sialic acids expressed in discrete linkage configurations (SA alpha2-3Gal and SA alpha2-6Gal) on host glycoproteins. The impact of altering the expression levels of sialic acid on host cell surfaces on the replication of viruses with strong and weak NA activity was also explored. It was shown that reverse engineered viruses expressing an NA with low cleavage activity for removal of sialic acid from a complex substrate yielded low infectious virus titres in embryonated chicken eggs and mouse lung, but not MDCK or A549 cells. This was despite the fact that wild-type virus from which the NA was selected did not show this deficit. In addition, viral fitness could not be restored in engineered viruses when NA was paired with the matched HA suggesting a gene other than HA was compensating for the weak NA activity in the wild-type virus. Mutation of the NA with lower activity to change amino acids near the active site to corresponding residues in a highly active NA was shown to increase the overall enzyme activity, but did not rescue high viral yields in chicken eggs. The reduced replication in eggs and mouse lung of viruses with weaker NA correlated with a pronounced decrease in the ability to cleave SA alpha2-3Gal. Importantly, low NA activity of reverse engineered viruses and associated reduction of viral yields in eggs was found to be due to a significant reduction in the amount of NA present on the virion. The low yield in eggs was also observed in viruses in which the weakly active NA and its matched HA were engineered onto the backbone of the virus strain used in reassortment to produce vaccine seeds for seasonal vaccine production. Acknowledging the present limitations of influenza disease prevention, this research indicates that NA may be inadequately represented in current seasonal vaccines, which may reduce their effectiveness. The study provides impetus to further understand mechanisms of virulence and optimise controls that limit influenza virus infection.
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