Generation and Characterization of Recombinant Influenza A(H1N1) Viruses Resistant to Neuraminidase Inhibitors

OBJECTIVES: To examine the effect of neuraminidase (NA) mutations on the NA inhibitor (NAI) resistance phenotype, the recombinant influenza A/Chungbuk/4448/2008(H1N1) virus isolated in South Korea during the 2008–2009 season was generated by reverse genetics. METHODS: Site-directed mutagenesis was introduced on the NA gene of A/Chungbuk/4448/2008(H1N1) virus, and a total of 23 single, double, and triple mutants were generated. Resistance phenotype of these recombinant viruses was determined by NA-inhibition (NAI) assays based on a fluorometric method using two NAIs (oseltamivir and zanamivir). RESULTS: NA-inhibition assays showed that all the single and double mutants containing the Y275 except the single Y275-E119V mutant conferred important levels of resistance to oseltamivir, whereas all the single, double, and triple mutants containing the E119V mutation were associated with the resistance to zanamivir. CONCLUSION: Considering the effect of mutations in NA gene on the resistance to NAIs, it is important to monitor the possible emergence and dissemination of multidrug-resistant variants in the human population due to amino acid changes at NA gene as well as to develop novel NAIs.