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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.

Yen HL, McKimm-Breschkin JL, Choy KT, Wong DD, Cheung PP, Zhou J, Ng IH, Zhu H, Webby RJ, Guan Y, Webster RG, Peiris JS - MBio (2013)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Centre of Influenza Research, School of Public Health, LKS Faculty of Medicine, the University of Hong Kong, Hong Kong SAR.

ABSTRACT

Unlabelled: 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.

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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 NAR292 (WT no. 6) and NAK292 (MUT no. 6) viruses.
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fig2: 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 NAR292 (WT no. 6) and NAK292 (MUT no. 6) viruses.

Mentions: Since the clinical H7N9 isolate A/Shanghai/1/2013 contained a mixed R/K population at NA residue 292, we performed plaque purification in the presence and absence of 10 µM of oseltamivir carboxylate. Plaque purification (Fig. 2A) yielded three clones of wild-type populations containing R292 in the NA protein and four mutant populations containing K292 in the NA protein as confirmed by Sanger sequencing. To monitor if any compensatory mutation might have arisen together with the NA R292K mutation, full-length hemagglutinin (HA) and NA sequences of these plaque-purified viruses were verified by Sanger sequencing. HA mutations were found in 3 of 4 plaque-purified mutant viruses and 1 of 3 of the wild-type viruses at different residues (Table 2). Overall, we observed that the wild-type viruses bearing the R292 in NA protein replicated to approximately 10-fold-lower peak titers (range, 7.07 to 7.57 log10 50% tissue culture infective doses [TCID50]/ml) than the mutant viruses carrying the K292 in the NA protein (range, 8.21 to 8.96 log10 TCID50/ml) (Table 2). Plaque-purified WT variant no. 6 and MUT variant no. 6, which differed only by the NA R292K mutation in their HA and NA genes, were selected for further analysis. In addition to Sanger sequencing, we verified the ratio of R/K at residue 292 for both WT no. 6 and MUT no. 6 using clonal sequencing. It was observed that the WT no. 6 strain was dominated by R292 (93%, 14/15), with detection of one clone of G292 (1/15). The MUT no. 6 strain was dominated by K (94%, 15/16) with one clone of R292 (1/16) after two rounds of expansion under conditions of exposure to 10 µM oseltamivir carboxylate. Both the plaque-purified WT no. 6 and MUT no. 6 strains formed large plaques in MDCK cells (Fig. 2B); however, it was noted that the MUT no. 6 strain formed a more diffused plaque morphology than the WT no. 6 strain.


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.

Yen HL, McKimm-Breschkin JL, Choy KT, Wong DD, Cheung PP, Zhou J, Ng IH, Zhu H, Webby RJ, Guan Y, Webster RG, Peiris JS - MBio (2013)

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 NAR292 (WT no. 6) and NAK292 (MUT no. 6) viruses.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3735122&req=5

fig2: 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 NAR292 (WT no. 6) and NAK292 (MUT no. 6) viruses.
Mentions: Since the clinical H7N9 isolate A/Shanghai/1/2013 contained a mixed R/K population at NA residue 292, we performed plaque purification in the presence and absence of 10 µM of oseltamivir carboxylate. Plaque purification (Fig. 2A) yielded three clones of wild-type populations containing R292 in the NA protein and four mutant populations containing K292 in the NA protein as confirmed by Sanger sequencing. To monitor if any compensatory mutation might have arisen together with the NA R292K mutation, full-length hemagglutinin (HA) and NA sequences of these plaque-purified viruses were verified by Sanger sequencing. HA mutations were found in 3 of 4 plaque-purified mutant viruses and 1 of 3 of the wild-type viruses at different residues (Table 2). Overall, we observed that the wild-type viruses bearing the R292 in NA protein replicated to approximately 10-fold-lower peak titers (range, 7.07 to 7.57 log10 50% tissue culture infective doses [TCID50]/ml) than the mutant viruses carrying the K292 in the NA protein (range, 8.21 to 8.96 log10 TCID50/ml) (Table 2). Plaque-purified WT variant no. 6 and MUT variant no. 6, which differed only by the NA R292K mutation in their HA and NA genes, were selected for further analysis. In addition to Sanger sequencing, we verified the ratio of R/K at residue 292 for both WT no. 6 and MUT no. 6 using clonal sequencing. It was observed that the WT no. 6 strain was dominated by R292 (93%, 14/15), with detection of one clone of G292 (1/15). The MUT no. 6 strain was dominated by K (94%, 15/16) with one clone of R292 (1/16) after two rounds of expansion under conditions of exposure to 10 µM oseltamivir carboxylate. Both the plaque-purified WT no. 6 and MUT no. 6 strains formed large plaques in MDCK cells (Fig. 2B); however, it was noted that the MUT no. 6 strain formed a more diffused plaque morphology than the WT no. 6 strain.

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Centre of Influenza Research, School of Public Health, LKS Faculty of Medicine, the University of Hong Kong, Hong Kong SAR.

ABSTRACT

Unlabelled: 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.

Show MeSH
Related in: MedlinePlus