Defining functional neuraminidase inhibitor drug resistance motifs in avian influenza viruses and the consequential impact on virus fitness in chicken cells.
Neuraminidase inhibitors (NAIs) are antiviral agents recommended worldwide to treat or prevent influenza virus infections in humans. Mapping of functional resistance to currently licensed NAIs has been limited to human influenza viruses with only sporadic reports investigating avian influenza viruses (AIV). However past pandemics as well as the increasing number of humans infected with AIV have shown the importance of having information about avian NAs that could cross the species barrier. In this study we introduced four NAI resistance-associated mutations previously found in human strains into the NA of six prevalent AIV subtypes that threaten the poultry industry and human health: H7N9, H6N1, H4N6 and highly pathogenic H5N8, H5N6 and H5N2. Using the established MUNANA assay we show that R292K substitution significantly impaired NA activity in all strains, whereas E119V, H274Y and N294S have more variable effects on NA activity. The impact of these mutations on NAI susceptibility was drug- and strain-specific. We have shown that despite compromised NA activity drug-resistant H5N6 and H6N1 viruses replicated to comparable or significantly higher titres in primary chicken cells as compared to wild type. The replicative fitness of NAI-resistant H5N6 was also confirmed in ovo. Two drug resistant H5N6 viruses had single amino acid substitutions in their haemagglutinin (HA) which reduced receptor binding properties. Our results demonstrate that there are no universal NAI resistance determinants for all strains and although some are clearly deleterious for the virus, others can be rapidly compensated by acquiring concurrent changes in other gene segments.\n\nIMPORTANCEThe number of human infections caused by avian influenza viruses (AIV) keeps increasing. This together with the rapid emergence of influenza strains resistant to neuraminidase inhibitor drugs (NAIs) observed in the past raises a significant concern to public health. We studied the NAI resistance-associated molecular changes, previously reported in neuraminidase (NA) of human influenza, in AIV background. We found that single amino acid substitution can confer a multidrug resistance, or lead to a single-drug resistance across multiple virus subtypes. We also found that the drug-resistant viruses retained or showed enhanced fitness properties as compared to the corresponding wild-type, and this could be achieved by quick acquisition of concurrent mutations in haemagglutinin. Our study highlights the need for constant monitoring of NAI-resistance in AIV and understanding the molecular basis of antiviral resistance, as such information would be invaluable for pandemic preparedness and may facilitate the development of novel therapeutics.