Characterization of the R292K mutation that confers resistance to the neuraminidase inhibitors in a novel H7N9 human isolate

Abstract

We characterized the A/Shanghai/1/2013 virus isolated from the first confirmed human case of A/H7N9 disease in China. The A/Shanghai/1/2013 isolate contained a mixed population of R (65%; 15/23 clones)/K (35%; 8/23 clones) at the neuraminidase (NA) residue 292, as determined by clonal sequencing. A/Shanghai/1/2013 with mixed R/K at residue 292 exhibited a sensitive phenotype to zanamivir and oseltamivir carboxylate by the enzyme based NA inhibition assay and to zanamivir in vitro. The plaque purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones) showed decreased sensitivity to zanamivir by 30-fold and to oseltamivir carboxylate by >100-fold compared to its plaque purified wild-type counterpart possessing dominant R292 (93%, 14/15 clones). In MDCK cells, the plaque purified A/Shanghai/1/2013-NAK292 virus exhibited no reduction in viral titer under increasing concentrations of oseltamivir carboxylate (range 0-1000 μM), whereas the replication of the plaque purified A/Shanghai/1/2013-NAR292 and the A/Shanghai/2/2013 viruses were completely inhibited at 250 μM and 31.25 μM of oseltamivir carboxylate, respectively. Although the plaque purified A/Shanghai/1/2013-NAK292 virus exhibited lower NA enzyme activity and a higher Km for 2’-(4-methylumbelliferryl)-α-D-N-acetylneuraminic acid than that of the wild-type A/Shanghai/1/2013-NAR292 virus, the A/Shanghai/1/2013-NAK292 virus formed large plaques and replicated efficiently in vitro. Our results confirmed that the NA R292K mutation confers resistance to oseltamivir and zanamivir in the novel human H7N9 viruses. Importantly, detection of the resistance phenotype may be masked in the clinical samples containing a mixed population of R/K at NA residue 292 while applying the in vitro based assay or the fluorescence based NA inhibition assay.