Evaluation of Recombinant 2009 Pandemic Influenza A (H1N1) Viruses Harboring Zanamivir Resistance Mutations in Mice and Ferrets

Recombinant influenza A(H1N1) pdm09 wild-type (WT) and zanamivir-resistant E119G and Q136K neuraminidase mutants were generated to determine their enzymatic and replicative properties in vitro, as well as their infectivity and transmissibility in mice and ferrets. Viral titers of recombinant E119G and Q136K mutants were significantly lower than those of the WT in the first 36 h postinoculation (p.i.) in vitro. The E119G and Q136K mutations were both associated with a significant reduction of total neuraminidase (NA) activity at the cell surface of 293T cells, with relative total NA activities of 14% (P < 0.01) and 20% (P< 0.01), respectively, compared to the WT. The E119G mutation significantly reduced the affinity (8-fold increase in Km) but not the V-max. The Q136K mutation increased the affinity (5-fold decrease in Km) with a reduction in Vmax (8% Vmax ratio versus the WT). In mice, infection with the E119G and Q136K mutants resulted in lung viral titers that were significantly lower than those of the WT on days 3 p.i. (3.4 x 106 x 0.8 x 10(6) and 2.1 x 10(7) x 0.4 x 10(7) PFU/ml, respectively, versus 8.8 x 10(7) x 1.1 x 10(7); P< 0.05) and 6 p.i. (3.0 x 105 +/- 0.5 x 105 and 8.6 x 10(5) +/- 1.4 x 105 PFU/ml, respectively, versus 5.8 x 107 x 0.3 x 107; P< 0.01). In experimentally infected ferrets, the E119G mutation rapidly reverted to the WT in donor and contact animals. The Q136K mutation was maintained in ferrets, although nasal wash viral titers from the Q136K contact group were significantly lower than those of the WT on days 3 to 5 p.i. Our results demonstrate that zanamivir-resistant E119G and Q136K mutations compromise viral fitness and transmissibility in A(H1N1) pdm09 viruses.