Characterization of Influenza B Virus Variants with Reduced Neuraminidase Inhibitor Susceptibility

Abstract

Treatment options for influenza B virus infections are limited to neuraminidase inhibitors (NAIs), which block the neuraminidase (NA) glycoprotein on the virion surface. The development of NAI resistance would therefore result in a loss of antiviral treatment options for influenza B virus infections. This study characterized two contemporary influenza B viruses with known resistance-conferring NA amino acid substitutions, D197N and H273Y, detected during routine surveillance. The D197N and H273Y variants were characterized in vitro by assessing NA enzyme activity and affinity, as well as replication in cell culture compared to those of NAI-sensitive wild-type viruses. In vivo studies were also performed in ferrets to assess the replication and transmissibility of each variant. Mathematical models were used to analyze within-host and between-host fitness of variants relative to wild-type viruses. The data revealed that the H273Y variant had NA enzyme function similar to that of its wild type but had slightly reduced replication and transmission efficiency in vivo The D197N variant had impaired NA enzyme function, but there was no evidence of reduction in replication or transmission efficiency in ferrets. Our data suggest that the influenza B virus variant with the H273Y NA substitution had a more notable reduction in fitness compared to wild-type viruses than the influenza B variant with the D197N NA substitution. Although a D197N variant is yet to become widespread, it is the most commonly detected NAI-resistant influenza B virus in surveillance studies. Our results highlight the need to carefully monitor circulating viruses for the spread of influenza B viruses with the D197N NA substitution.

Keywords: influenza B; neuraminidase inhibitor; resistance; viral fitness.

FIG 1

The mean NA surface expression and activity of influenza B variants relative to the corresponding WT. HEK-293T cells were transfected with plasmids containing the NA gene encoding WT and variant proteins. At 20 h posttransfection, cells were analyzed for NA activity using a MUNANA-based assay and for NA expression using a fluorescence-activated cell sorter (FACS)-based assay. The activity and expression data of the variants were normalized relative to their corresponding WT and expressed as the mean ± standard error of the mean (SEM). Data are derived from two independent experiments, each containing triplicate samples, and were compared between relevant groups using the nonparametric Mann-Whitney U test.

FIG 2

Multicycle replication of influenza B WT and MUT viruses in MDCK and SIAT cells. Cells were infected at an MOI of 0.01 and supernatants collected and assayed for infectious virus at 2, 24, 36, and 48 h postinfection. Data show the mean (± SEM) of triplicate samples for MUT-Y273 versus WT-H273 (a and b) and MUT-N197 versus WT-D197 (c and d). A two-way ANOVA with Bonferroni's posttest analysis was done to compare mean viral titers at each time point between WT and MUT viruses. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

Viral shedding in nasal wash samples from donor and recipient ferrets infected with WT or MUT influenza B viruses. Donor ferrets (n = 4 per group) were experimentally infected with 10⁵ TCID₅₀ of pure WT or MUT virus. Naive recipients were cohoused with each donor 1 day after experimental infection of the donors. Daily nasal wash samples were collected from both donors and recipients over a period of 11 days, and titers of infectious virus in nasal wash samples were determined using a TCID₅₀ assay. (A to D) Summary of TCID₅₀ results, with the bar graphs showing the mean (± SEM) titers at each day for each group. At each time point, mean viral titers were compared between donors infected with either WT or MUT virus by two-way ANOVA with Bonferroni's posttest analysis. Similarly, analysis was done to compare titers between recipients infected with either WT or MUT virus. Significant differences in mean viral titers were observed between WT-H273- and MUT-Y273-infected recipients on day 3 and 4 postexposure and between WT-D197- and MUT-N197-infected donors on day 1 postinoculation. The AUCs of the TCID₅₀ graphs were also calculated and compared by Mann-Whitney's U test; no significant differences were detected between donor animals infected with WT and corresponding MUT viruses. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

Summary of changes in mutant virus proportions in nasal washes within-host and during transmission in competitive mixture ferret experiments. To assess within-host and transmission fitness of mutant viruses relative to their corresponding wild types, ferrets were experimentally coinfected with mixtures of WT and MUT viruses at different ratios (50:50, 80:20, and 20:80) and cohoused with naive recipients. The proportion of WT versus MUT was determined in the collected nasal wash samples by pyrosequencing, and data were analyzed only from animals where the proportion of WT and MUT viruses could be accurately determined. (a and b) Individual points that correspond to the change in the proportion of mutant virus in one ferret between the first day of infection and last day of infection (y axis). This allows for a visual representation of within-host fitness. (c and d) Individual points that correspond to a transmission event between two ferrets. In these plots, the abscissa is the proportion of MUT virus in donor ferrets the day preceding confirmed transmission, and the ordinate shows the proportion of MUT virus in recipient ferrets within the first 24 h of confirmed infection. The solid gray line is the representative curve of the shape parameter, calculated by fitting our data to a model described in more detail in Materials and Methods and the supplemental material. The 95% confidence interval is represented by the shaded region. If our fitted model lies above the symmetry line (y = x solid line), the mutant virus is fitter than the wild-type virus, whereas if the fitted model lies below the symmetry line, the converse is true.