Resistance to neuraminidase inhibitors conferred by an R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population

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) and K (35%; 8/23 clones) at neuraminidase (NA) residue 292, as determined by clonal sequencing. A/Shanghai/1/2013 with mixed R/K at residue 292 exhibited a phenotype that is sensitive to zanamivir and oseltamivir carboxylate by the enzyme-based NA inhibition assay. The plaque-purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones) showed sensitivity to zanamivir that had decreased by >30-fold and to oseltamivir carboxylate that had decreased by >100-fold compared to its plaque-purified wild-type counterpart possessing dominant R292 (93%, 14/15 clones). In Madin-Darby canine kidney (MDCK) cells, the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited no reduction in viral titer under conditions of increasing concentrations of oseltamivir carboxylate (range, 0 to 1,000 µM) whereas the replication of the plaque-purified A/Shanghai/1/2013-NAR292 and the A/Shanghai/2/2013 viruses was 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 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, peramivir, 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 in the enzyme-based NA inhibition assay.

Importance: The neuraminidase (NA) inhibitors oseltamivir and zanamivir are currently the front-line therapeutic options against the novel H7N9 influenza viruses, which possess an S31N mutation that confers resistance to the M2 ion channel blockers. It is therefore important to evaluate the sensitivity of the clinical isolates to NA inhibitors and to monitor for the emergence of resistant variants. We characterized the A/Shanghai/1/2013 (H7N9) isolate which contained a mixed population of R/K at NA residue 292. While the clinical isolate exhibited a phenotype of sensitivity to NA inhibitors using the enzyme-based NA inhibition assay, the plaque-purified A/Shanghai/1/2013 virus with dominant K292 was resistant to zanamivir, peramivir, and oseltamivir. Resistance to NA inhibitors conferred by the R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population, and this should be taken into consideration while monitoring antiviral resistance in patients with H7N9 infection.

FIG 1

Human H7N9 influenza virus sensitivity to zanamivir, oseltamivir, and favipiravir (T-705) in vitro. MDCK cells were infected with A/Shanghai/1/13 (mixed R/K at NA residue 292) or A/Shanghai/2/13 virus at MOI = 0.001 TCID₅₀/cell. Supernatants were collected at 48 h postinfection and were titrated in MDCK cells (log₁₀ TCID₅₀/ml).

FIG 2

Morphology of the plaque-purified A/Shanghai/1/2013 viruses. (A) A/Shanghai/1/2013 was grown in the absence or presence of 10 µM oseltamivir carboxylate for plaque purification. The arrows indicated the hole left after plaque picking. (B) Morphology of the plaque-purified A/Shanghai/1/2013 NA^R292 (WT no. 6) and NA^K292 (MUT no. 6) viruses.

FIG 3

Dose-response curve of the plaque-purified A/Shanghai/1/2013 and the A(H1N1) pdm09 viruses in the fluorescence-based neuraminidase inhibition assay. A/Shanghai/1/13 NA^R292 (WT no. 6), A/Shanghai/1/13 NA^K292 (MUT no. 6), RG-A/CA/04/09, and RG-A/CA/04/09 NA^H274Y viruses (A) and A/Shanghai/1/13 NA^R292 (WT no. 1 and WT no. 6) and A/Shanghai/1/13 NA^K292 (MUT no. 2, MUT no. 3, MUT no. 5, and MUT no. 6) viruses (B) were preincubated with zanamivir, peramivir, or oseltamivir carboxylate for 45 min at 37°C before incubation with the fluorogenic substrate MUNANA at a final concentration of 167 µM at 37°C for 30 min. The neuraminidase-cleaved product (4-methylumbelliferone) was detected using an FLUOstar OPTIMA microplate reader (BMG Labtech). EX, excitation; EM, emission (in nm).

FIG 4

Sensitivity of the plaque-purified A/Shangai/1/2013 viruses to zanamivir and oseltamivir carboxylate in vitro. MDCK cells were preincubated with NA inhibitors for 2 h prior to infection with A/Shanghai/1/13 NA^R292 (WT no. 6), A/Shanghai/1/13 NA^K292 (MUT no. 6), or A/Shanghai/2/13 viruses at MOI = 0.001 TCID₅₀/cell for 1 h and overlaid with media containing oseltamivir carboxylate (0 to 1,000 µM for the A/Shanghai/1/13 NA^K292 MUT no. 6 virus and 0 to 500 µM for the A/Shanghai/1/13 NA^R292 WT no. 6 and the A/Shanghai/2/2013 viruses) (A) or zanamivir (0 to 500 µM for the A/Shanghai/1/13 NA^K292 MUT no. 6 virus and 0 to 125 µM for the A/Shanghai/1/13 NA^R292 WT no. 6 and the A/Shanghai/2/2013 viruses) (B). Supernatants were collected at 48 h postinfection and were titrated in MDCK cells (log₁₀ TCID₅₀/ml).

FIG 5

Replication kinetics of the plaque-purified A/Shanghai/1/2013 NA^R292 and NA^K292 viruses in MDCK-SIAT cells. Cells were infected with an MOI = 0.001 TCID₅₀/cell. The supernatants were collected at the time points indicated and titrated in MDCK cells (log₁₀ PFU/ml).