Inhibiting influenza virus replication using novel and established antiviral compounds

Abstract

Circulating influenza viruses have the potential to cause pandemics, as seen in 2009 with the emergence of a novel H1N1 virus that rapidly disseminated worldwide. In such an event, huge reliance is placed upon antiviral drugs for the prophylaxis and treatment of influenza infections. To date, only two different classes of antiviral drug are licensed for the treatment of influenza and only one of these, oseltamivir, has been stockpiled by major world governments in preparation for an influenza pandemic. However, widespread resistance to this drug has been documented in seasonal circulating strains, which emerged in the absence of intense drug use and predominated over drug-sensitive phenotypes. Furthermore, examples of oseltamivir resistant viruses have been found sporadically in pandemic A/H1N1 2009 influenza strains. The need for novel antivirals is therefore essential for effective infection control of influenza viruses in future years. A potential source of novel therapies are licensed compound libraries. We begin with the screening of two compound libraries, resulting in the identification of several compounds which inhibited influenza virus replication in vitro. After completing a traditional library screen, a novel approach to high-throughput compound screening using reporter plasmids expressed in a stable cell line was attempted. Although these cell-lines did not prove maintainable in the long-term, this work resulted in the generation of a reporter plasmid that is directly initiated by influenza infection in vitro and is thus a useful tool for assaying polymerase fitness of different influenza strains. The investigation then focused on one of the newly discovered hits and attempted to identify the spectrum of activity of this compound, a glycosylation inhibiting molecule, which was shown to be efficacious against influenza A strains only. The drug was shown to have low toxicity and proved active against the recently emerged pandemic influenza virus. The thesis then documents the effects of a common oseltamivir-resistant mutation, H275Y, on the neuraminidase protein of a representative 2009 pandemic influenza virus. This mutation was well tolerated in vitro by the virus and did not handicap replication in a human airway epithelial model. However, subtle impediments to the neuraminidase activity of the mutant were observed biochemically which may suggest why this mutation has not emerged more readily in the field