Neuraminidase inhibitor resistance in influenza viruses

The neuraminidase inhibitors are a relatively new class of antiviral drugs designed for the treatment or prevention of influenza A and B infections, and are an essential tool in the control ofboth human and animal influenza strains. Currently, two neuraminidase inhibitors are available on the market, oseltamivir and zanamivir, although more are undergoing clinical trials. These drugs bind to the neuraminidase ofthe influenza virus and prevent the effective release ofprogeny virions that are produced following replication. Viral resistance is typically associated with mutations in the neuraminidase protein and can rapidly develop, rendering these drugs ineffective for the treatment or prophylaxis of influenza. Clinical trials conducted prior to the release ofthe drugs (pre 200 l), demonstrated a low incidence of oseltamivir resistance in individuals under treatment (1-6%) and very low levels of resistance in community isolates from untreated patients « 1%). This thesis presents eight studies that involved both human and avian influenza viruses and investigated the frequency of neuraminidase inhibitor resistance, the role of key neuraminidase mutations in conferring resistance and the impact that these mutations had on viral infectivity, growth and transmissibility. Specifically, the studies show that the frequency of oseltamivir resistance in seasonal A(Hl Nl) viruses was found to increase dramatically in South Africa, South East Asia and Oceania during 2008, from less than I % to over 80% in some countries. Zanamivir resistance remained rare in community isolates, although a small number of seasonal A(H 1 N I) isolates were found to contain a previously undescribed mutation that conferred zanamivir resistance, but appeared to not compromise the in vitro or in vivo fitness of the virus. The fitness of two oseltamivir resistant viruses was also investigated, using a novel 'competitive mixtures' method that involved the co-infection of ferrets with a mixture of two influenza viruses and the changes in the relative proportion of those viruses was monitored over the course of the infection and upon transmission. The susceptibility of a number of highly pathogenic A(H5N 1) avian influenza viruses was also determined, with two previously undescribed neuraminidase mutations found to confer a mildly reduced sensitivity to oseltamivir or zanamivir. Two drug sensitive A(H5Nl) strains were also investigated for their potential to develop resistance under oseltamivir and zanamivir pressure in vitro and in vivo. A dual neuraminidase mutation following serial passage of one A(H5Nl) virus in increasing concentrations of oseltamivir caused a >8000-fold reduction in oseltamivir susceptibility, while other novel oseltamivir and zanamivir resistance mutations were also detected. Treatment of A(H5Nl) infected ferrets with a range of oseltamivir and zanamivir concentrations did not generate any oseltamivir resistance, but did result in the selection ofa zanamivir resistant strain with a novel neuraminidase mutation. The studies presented here have significantly added to the overall understanding of neuraminidase inhibitor resistance, by detecting a number of previously undescribed mutations, characterising these viruses to assess their impact on drug susceptibility and viral fitness and by investigating novel methods that may better assist researchers in the future to determine the relative fitness of influenza viruses.