Host cell selection of influenza neuraminidase variants: Implications for drug resistance monitoring in A(H1N1) viruses

The neuraminidase inhibitors (NAIs), oseltamivir and zanamivir, are essential for treatment and prevention of influenza A and B infections. Oseltamivir resistance among influenza A (H1N1) viruses rapidly emerged and spread globally during the 2007–2008 and 2008–2009 influenza seasons. Approximately 20% and 90% of viruses tested for NAI susceptibility at CDC during these seasons, respectively, were resistant to oseltamivir (IC50 ∼100–3000 time > those of sensitive viruses), based on the chemiluminescent NA inhibition assay. Pyrosequencing analysis confirmed H274Y mutation (H275Y in N1 numbering) in the neuraminidase (NA) gene of oseltamivir-resistant viruses. Full NA sequence analysis of a subset of oseltamivir-resistant and sensitive virus isolates from both seasons (n = 725) showed that 53 (7.3%) had mutations at residue D151 (D → E/G/N), while 9 (1.2%) had mutations at Q136 (Q → K) and 2 (0.3%) had mutations at both residues. Viruses with very high IC50 for oseltamivir and peramivir, and elevated IC50 for zanamivir, had H274Y in addition to mutations at D151 and/or Q136, residues which can potentially confer NAI resistance based on recent N1 NA crystal structure data. Mutations at D151 without H274Y, did not elevate IC50 for any tested NAI, however, Q136K alone significantly reduced susceptibility to zanamivir (36-fold), peramivir (80-fold) and A-315675 (114-fold) but not oseltamivir. Mutations at D151 and Q136 were present only in MDCK grown viruses but not in matching original clinical specimens (n = 33) which were available for testing, suggesting that these variants were the result of cell culture selection or they were present in very low proportions. Our findings provide evidence that propagation of influenza virus outside its natural host may lead to selection of virus variants with mutations in the NA that affect sensitivity to NAIs and thus poses implications for drug resistance monitoring and diagnostics.