Mutations in influenza A virus N8 and N9 glycoproteins that confer resistance to neuraminidase inhibitors

Abstract

Neuraminidase inhibitors (NAI) remain the mainstay therapeutic option against influenza infections. Drug resistant viruses usually acquire mutations at conserved residues that directly interact with the compound (catalytic sites) or at residues that maintain the structure for the NA (framework sites). Previous studies have identified mutations in the NA proteins of seasonal H1N1 and H3N2 viruses that may confer resistance to NAI after clinical use. Nevertheless, such information is meager for N9 and N8 subtypes that recently caused human zoonotic infections. This project aims to evaluate the mutations that may confer resistance to NAI in H7N9 and H10N8 viruses with zoonotic potential. Random mutagenesis was first applied to introduce mutations in the N8 and N9 glycoproteins followed by generation of recombinant A/Puerto Rico/8/34 (H1N8 and H1N9) viruses containing random mutations in the NA head region. Recombinant viruses (7+1) were serially passaged in MDCK-SIAT1 cells under increasing concentrations of zanamivir and oseltamivir to select resistant variants. In addition, targeted mutagenesis was applied to introduce NA mutations known to confer resistance in seasonal influenza viruses into N8 and N9 glycoproteins. Recombinant H7N9 and H10N8 viruses with different NA mutations were generated and were assessed for their drug resistance phenotype to oseltamivir, zanamivir and peramivir by NA inhibition assays and plaque reduction assays. A266V and T87A, T87S and T247A mutations were enriched after serial passage of the recombinant H1N8 and H1N9 viruses under increasing concentration of zanamivir respectively. However, after introducing these mutations into the respective H10N8 and H7N9 viruses, we observed no reduction in susceptibility to NAI. Among the targeted mutations introduced into H10N8 virus, H274Y and N294S conferred highly reduced sensitivity (>100 fold change in IC50) to oseltamivir and E119V as well as Q136K conferred highly reduced inhibition to zanamivir. The mutations H274Y, E119Vand Q136K conferred highly reduced sensitivity to peramivir as well. I222R conferred reduced sensitivity (10-100 fold change in IC50) to oseltamivir. Among the targeted mutations introduced into H7N9 viruses R292K and E119V conferred highly reduced sensitivity (>100 fold change in IC50) to oseltamivir and Q136K to zanamivir. Other mutations such as N294S conferred reduced sensitivity (10-100 fold change in IC50) to oseltamivir and zanamivir and I222R conferred reduced sensitivity to all three NAI in N9 background. Impact of these mutations on the fitness varied according to specific subtype. The resistant mutations generated in N8 background impaired the replicative potential of the viruses in vitro to a certain extent whereas the viruses harboring the drug resistant mutations in N9 background remained apparently fit in comparison to wild type (WT). Since novel viruses are emerging very rapidly and considering the limited options of anti-influenza drugs available, our results highlights that continuous monitoring of antiviral drug resistance is of utmost priority.