Competitive Fitness of Influenza B Viruses Possessing E119A and H274Y Neuraminidase Inhibitor Resistance–Associated Substitutions in Ferrets

Abstract

Neuraminidase (NA) inhibitors (NAIs) are the only antiviral drugs recommended for influenza treatment and prophylaxis. Although NAI-resistant influenza B viruses that could pose a threat to public health have been reported in the field, their fitness is poorly understood. We evaluated in ferrets the pathogenicity and relative fitness of reverse genetics (rg)–generated influenza B/Yamanashi/166/1998-like viruses containing E119A or H274Y NA substitutions (N2 numbering). Ferrets inoculated with NAI-susceptible rg–wild-type (rg-WT) or NAI-resistant (rg-E119A or rg-H274Y) viruses developed mild infections. Growth of rg-E119A virus in the nasal cavities was delayed, but the high titers at 3 days post-inoculation (dpi) were comparable to those of the rg-WT and rg-H274Y viruses (3.6–4.1 log10TCID50/mL). No virus persisted beyond 5 dpi and replication did not extend to the trachea or lungs. Positive virus antigen-staining of the nasal turbinate epithelium was intermittent with the rg-WT and rg-H274Y viruses; whereas antigen-staining for the rg-E119A virus was more diffuse. Virus populations in ferrets coinoculated with NAI-susceptible and -resistant viruses (1:1 mixture) remained heterogeneous at 5 dpi but were predominantly rg-WT (>70%). Although the E119A substitution was associated with delayed replication in ferrets, the H274Y substitution did not measurably affect viral growth properties. These data suggest that rg-H274Y has undiminished fitness in single virus inoculations, but neither rg-E119A nor rg-H274Y gained a fitness advantage over rg-WT in direct competition experiments without antiviral drug pressure. Taken together, our data suggest the following order of relative fitness in a ferret animal model: rg-WT > rg-H274Y > rg-E119A.

Chimeric Hemagglutinin Constructs Induce Broad Protection against Influenza B Virus Challenge in the Mouse Model

“…Furthermore, a recent study found that the proportion of deaths in children hospitalized with influenza attributable to influenza B virus is 1.1%, compared to 0.4% for influenza A virus (14). Finally, oseltamivir treatment seems to be less effective against influenza B viruses than against influenza A viruses (15)(16)(17), and oseltamivir-resistant mutants emerge (18)(19)(20), sometimes without a loss of fitness (21,22). Current influenza virus vaccines are an effective prophylactic countermeasure against influenza B virus infections.…”

Characterization of influenza B virus variants with reduced neuraminidase inhibitor susceptibility

“…We show that the MUT-Y273 virus had equivalent NA activity and expression to the WT-H273 virus, but delayed growth in cell culture. Previous experiments using a rg-H273Y virus showed that relative to the rg-WT, the rg-H273Y virus had significantly higher NA activity and surface expression, and superior replication kinetics in a competitive mixtures experiment in normal human bronchial epithelial cells (14, 22, 23). The discrepancy in these fitness outcomes between our 2015 H273Y virus and a 1998 rg-H273Y virus may be due to differences in viral background (16 amino acid differences).…”

“…A number of different NA substitutions at conserved amino acid positions (e.g. E117, D197, I221 and H273) have previously been described to confer reduced inhibition by the NAIs in vitro (8, 12-21), but the impact of these substitutions on enzyme function, virus replication or transmissibility, has only been assessed in a limited number of studies (14, 22, 23). The fitness of influenza B viruses with either the H273Y or D197N NA substitution is of particular interest as a number of viruses with either substitution have been recently found in patients in community settings who, unlike hospitalized or immunocompromised patients, do not typically receive NAI treatment (8, 9, 17, 18, 24).…”

Selection of Multi-Drug Resistant Influenza A and B Viruses under Zanamivir Pressure and their Replication Fitness in Ferrets

“…In contrast, Abed et al [12] reported attenuated replication fitness and instability of the E119D mutation variants both in vitro (MDCK and ST6GalI-MDCK) and in vivo (mice and guinea pigs). For other subtypes, mutations at the 119 NA residue, such as E119V of influenza A(H3N2), and E117A of influenza B virus (B numbering is used throughout for influenza B NA residues) have been reported to confer resistance to multiple NAIs and reduce replication fitness in vitro [14][15][16].…”