Selection of avian influenza A (H9N2) virus with reduced susceptibility to neuraminidase inhibitors oseltamivir and zanamivir

•Avian influenza H9N2 susceptibility to oseltamivir & zanamivir was studied in eggs.•NA substitution R292 K was selected in Passage 1 with oseltamivir.•NA substitution E119D was selected in Passage 2 with zanamivir.•NAI assays showed highly reduced susceptibility of mutant viruses to respective drugs.•Mutant viruses reverted to wild type without drug pressure in P2 and P3. Identification of amino-acid substitutions in the neuraminidase (NA) of low-pathogenic avian influenza (AI) H9N2 viruses is important to study the susceptibility to NA inhibitors (NAI). To identify mutations under NAI selective pressure, the virus was serially passaged with increasing levels of either oseltamivir or zanamivir in ovo, and the growth of the viruses in the presence and absence of NAI’s compared. Mutations R292 K in the presence of oseltamivir and E119D in presence of zanamivir were observed within passage one and two respectively. The R292 K mutation reduced oseltamivir susceptibility significantly (2,523-fold) and moderately reduced susceptibility to zanamivir. The E119D mutation significantly reduced susceptibility to zanamivir (415-fold) and remained susceptible to oseltamivir. Genetic stability of the mutations assessed by serial passages of the mutant viruses in eggs without drug pressure resulted in the loss of these mutations, making the virus susceptible to both the drugs. Molecular modeling and dynamics simulations revealed that the R292 K mutation disrupted oseltamivir binding similar to other group 2 NAs, while a different mechanism was noted for zanamivir binding for both R292 K and E119D mutations. The study highlights the need for regular susceptibility screening of circulating AI viruses.