Competitive Fitness of Influenza B Viruses with Neuraminidase Inhibitor-Resistant Substitutions in a Coinfection Model of the Human Airway Epithelium

Abstract

Influenza A and B viruses are human pathogens that are regarded to cause almost equally significant disease burdens. Neuraminidase (NA) inhibitors (NAIs) are the only class of drugs available to treat influenza A and B virus infections, so the development of NAI-resistant viruses with superior fitness is a public health concern. The fitness of NAI-resistant influenza B viruses has not been widely studied. Here we examined the replicative capacity and relative fitness in normal human bronchial epithelial (NHBE) cells of recombinant influenza B/Yamanashi/166/1998 viruses containing a single amino acid substitution in NA generated by reverse genetics (rg) that is associated with NAI resistance. The replication in NHBE cells of viruses with reduced inhibition by oseltamivir (recombinant virus with the E119A mutation generated by reverse genetics [rg-E119A], rg-D198E, rg-I222T, rg-H274Y, rg-N294S, and rg-R371K, N2 numbering) or zanamivir (rg-E119A and rg-R371K) failed to be inhibited by the presence of the respective NAI. In a fluorescence-based assay, detection of rg-E119A was easily masked by the presence of NAI-susceptible virus. We coinfected NHBE cells with NAI-susceptible and -resistant viruses and used next-generation deep sequencing to reveal the order of relative fitness compared to that of recombinant wild-type (WT) virus generated by reverse genetics (rg-WT): rg-H274Y > rg-WT > rg-I222T > rg-N294S > rg-D198E > rg-E119A ≫ rg-R371K. Based on the lack of attenuated replication of rg-E119A in NHBE cells in the presence of oseltamivir or zanamivir and the fitness advantage of rg-H274Y over rg-WT, we emphasize the importance of these substitutions in the NA glycoprotein. Human infections with influenza B viruses carrying the E119A or H274Y substitution could limit the therapeutic options for those infected; the emergence of such viruses should be closely monitored. IMPORTANCE Influenza B viruses are important human respiratory pathogens contributing to a significant portion of seasonal influenza virus infections worldwide. The development of resistance to a single class of available antivirals, the neuraminidase (NA) inhibitors (NAIs), is a public health concern. Amino acid substitutions in the NA glycoprotein of influenza B virus not only can confer antiviral resistance but also can alter viral fitness. Here we used normal human bronchial epithelial (NHBE) cells, a model of the human upper respiratory tract, to examine the replicative capacities and fitness of NAI-resistant influenza B viruses. We show that virus with an E119A NA substitution can replicate efficiently in NHBE cells in the presence of oseltamivir or zanamivir and that virus with the H274Y NA substitution has a relative fitness greater than that of the wild-type NAI-susceptible virus. This study is the first to use NHBE cells to determine the fitness of NAI-resistant influenza B viruses.