The effect of the MDCK cell selected neuraminidase D151G mutation on the drug susceptibility assessment of influenza A(H3N2) viruses.
Propagation of influenza A(H3N2) viruses in MDCK cells has been associated with the emergence of neuraminidase (NA) variants carrying a change at residue 151. In this study, the pyrosequencing assay revealed that ∼90% of A(H3N2) virus isolates analyzed (n=150) contained more than one amino acid variant (D/G/N) at position 151. Susceptibilities of the virus isolates to zanamivir and oseltamivir were assessed using the chemiluminescent and fluorescent NA inhibition (NI) assays. In the chemiluminescent assay, which utilizes NA-Star® substrate, up to 13-fold increase in zanamivir-IC50 was detected for isolates containing a high proportion (>50%) of the G151 NA variant. However, an increase in zanamivir-IC50s was not seen in the fluorescent assay, which uses MUNANA as substrate. To investigate this discrepancy, recombinant NAs (rNAs) were prepared and tested in both NI assays. Regardless of the assay used, the zanamivir-IC50 for the rNA G151 was much greater (>1500-fold) than that for rNA D151 wild-type. However, zanamivir resistance conferred by the G151 substitution was masked in preparations containing the D151 NA which had much greater activity, especially against MUNANA. In conclusion, the presence of NA D151G variants in cell culture-grown viruses interferes with drug susceptibility assessment and therefore measures need to be implemented to prevent their emergence.
Virus Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA.
Vasiliy P
VP
Mishin
Virus Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA.
Katrina
K
Sleeman
Virus Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA; Battelle Memorial Institute, Atlanta, GA 30333, USA.
Marnie
M
Levine
Virus Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA.
Paul J
PJ
Carney
Virus Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA.
James
J
Stevens
Virus Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA. Electronic address: LGubareva@cdc.gov.
Larisa V
LV
Gubareva
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D004285:Dogs; D024882:Drug Resistance, Viral; D005470:Fluorometry; D006801:Humans; D053122:Influenza A Virus, H3N2 Subtype / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D020128:Inhibitory Concentration 50; D008163:Luminescent Measurements; D061985:Madin Darby Canine Kidney Cells; D008826:Microbial Sensitivity Tests; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D053139:Oseltamivir / Q000494:pharmacology; D012641:Selection, Genetic; D014776:Virus Cultivation; D053243:Zanamivir / Q000494:pharmacology
D000998:Antiviral Agents; D050505:Mutant Proteins; D053139:Oseltamivir; D009439:Neuraminidase; D053243:Zanamivir
NA-Fluor™ kit; NA-Star® kit; Neuraminidase inhibition assay; Pyrosequencing; Recombinant protein