Drug Susceptibility Evaluation of an Influenza A(H7N9) Virus by Analyzing Recombinant Neuraminidase Proteins

J Infect Dis. 2017 Sep 15;216(suppl_4):S566-S574. doi: 10.1093/infdis/jiw625.

Abstract

Background: Neuraminidase (NA) inhibitors are the recommended antiviral medications for influenza treatment. However, their therapeutic efficacy can be compromised by NA changes that emerge naturally and/or following antiviral treatment. Knowledge of which molecular changes confer drug resistance of influenza A(H7N9) viruses (group 2NA) remains sparse.

Methods: Fourteen amino acid substitutions were introduced into the NA of A/Shanghai/2/2013(H7N9). Recombinant N9 (recN9) proteins were expressed in a baculovirus system in insect cells and tested using the Centers for Disease Control and Prevention standardized NA inhibition (NI) assay with oseltamivir, zanamivir, peramivir, and laninamivir. The wild-type N9 crystal structure was determined in complex with oseltamivir, zanamivir, or sialic acid, and structural analysis was performed.

Results: All substitutions conferred either reduced or highly reduced inhibition by at least 1 NA inhibitor; half of them caused reduced inhibition or highly reduced inhibition by all NA inhibitors. R292K conferred the highest increase in oseltamivir half-maximal inhibitory concentration (IC50), and E119D conferred the highest zanamivir IC50. Unlike N2 (another group 2NA), H274Y conferred highly reduced inhibition by oseltamivir. Additionally, R152K, a naturally occurring variation at the NA catalytic residue of A(H7N9) viruses, conferred reduced inhibition by laninamivir.

Conclusions: The recNA method is a valuable tool for assessing the effect of NA changes on drug susceptibility of emerging influenza viruses.

Keywords: A(H7N9); Neuraminidase; bird flu; drug resistance; recombinant protein.

MeSH terms

  • Acids, Carbocyclic
  • Antiviral Agents / pharmacology*
  • Cyclopentanes / pharmacology
  • Databases, Genetic
  • Drug Resistance, Multiple, Viral / genetics*
  • Enzyme Inhibitors / pharmacology
  • Guanidines / pharmacology
  • Humans
  • Influenza A Virus, H7N9 Subtype / drug effects*
  • Influenza A Virus, H7N9 Subtype / genetics
  • Influenza, Human / drug therapy
  • Inhibitory Concentration 50
  • Neuraminidase / antagonists & inhibitors*
  • Neuraminidase / genetics
  • Oseltamivir / pharmacology
  • Protein Conformation
  • Pyrans
  • Recombinant Proteins / genetics
  • Sialic Acids
  • Viral Proteins / antagonists & inhibitors*
  • Viral Proteins / genetics
  • Zanamivir / analogs & derivatives
  • Zanamivir / pharmacology

Substances

  • Acids, Carbocyclic
  • Antiviral Agents
  • Cyclopentanes
  • Enzyme Inhibitors
  • Guanidines
  • Pyrans
  • Recombinant Proteins
  • Sialic Acids
  • Viral Proteins
  • Oseltamivir
  • laninamivir
  • Neuraminidase
  • Zanamivir
  • peramivir