Impact of R152K and R368K neuraminidase catalytic substitutions on in vitro properties and virulence of recombinant A(H1N1)pdm09 viruses

Abstract Neuraminidase (NA) mutations conferring resistance to NA inhibitors (NAIs) are expected to occur at framework or catalytic residues of the NA enzyme. Numerous clinical and in vitro reports already described NAI-resistant A(H1N1)pdm09 variants harboring various framework NA substitutions. By contrast, variants with NA catalytic changes remain poorly documented. Herein, we investigated the effect of R152K and R368K NA catalytic mutations on the NA enzyme properties, in vitro replicative capacity and virulence of A(H1N1)pdm09 recombinant viruses. In NA inhibition assays, the R152K and R368K substitutions resulted in reduced inhibition [10- to 100-fold increases in IC 50 vs the wild-type (WT)] or highly reduced inhibition (>100-fold increases in IC 50 ) to at least 3 approved NAIs (oseltamivir, zanamivir, peramivir and laninamivir). Such resistance phenotype correlated with a significant reduction of affinity observed for the mutants in enzyme kinetics experiments [increased Km from 20 ± 1.77 for the WT to 200.8 ± 10.54 and 565.2 ± 135 μM (P  in vitro passages as well as in lungs of infected mice. Due to the multi-drug resistance phenotypes and conserved fitness, the emergence of NA catalytic mutations accompanied with potential compensatory HA changes should be carefully monitored in A(H1N1)pdm09 viruses.