There are two specific anti-influenza drugs which are effective against all strains of influenza, zanamivir and oseltamivir. Since they bind to highly conserved residues in the active site of the virus neuraminidase (NA) they are effective against all strains of influenza. With the spread of the highly pathogenic H5N1 avian influenza in Asia and now across into Europe, the possibility of a pandemic strain emerging is more likely. Since it will take several months to produce a vaccine the NA inhibitors, especially oseltamivir, are being stockpiled as the first line of defense. While resistance to zanamivir has not yet been seen in immunocompetent patient, there are 3 common mutations in conserved residues in the NA active site which confer oseltamivir resistance. However, these mutations do not confer resistance in all NA subtypes. Resistance rates of up to 18% have now been seen after oseltamivir treatment, and there are already reports of H5N1 oseltamivir resistant viruses, hence there are increasing concerns about drug resistance. Through the Neuraminidase Inhibitor Susceptibility Network (NISN) global surveillance program we have also identified several other viruses which have mutations in non-conserved residues outside of the active site. There must therefore be secondary structural effects outside the active site which can impact on drug binding. Hence our central hypothesis is that """"""""Additional secondary structural characteristics in the NA play a critical role in determining drug sensitivity and whether a mutation confers drug resistance"""""""". In addition to the NA mutations, we also have HA mutations in one of the isolates which confer resistance in vitro. Hence we also have a minor hypothesis that """"""""HA mutations in clinical isolates can play a role in resistance to the NA inhibitors in vivo"""""""". The long term aim of this project is to use structural and biological studies to understand the mechanisms of influenza resistance which contribute to differences in N1/N2 drug sensitivity, N1/N2 subtype specific resistance, how mutations in non-conserved residues outside the active site affect inhibitor binding, as well as the role of HA mutations in resistance in vivo.
We aim to determine the structures of the wild type and mutant NAs from either egg-grown or baculovirus expressed protein. We will assess the impacts of the NA and HA mutations on growth and drug sensitivity in vitro and in vivo as an indication of the """"""""fitness"""""""" of the viruses , to understand the likelihood of them being transmissible and drug resistant in the clinical situation. Relevance Understanding the mechanisms of drug resistance, either in naturally occurring or drug selected variants, is critical for appropriate management of epidemic and pandemic outbreaks when resistant virus is circulating, and for the potential long term development of alternative inhibitors.
