Generation and characterization of recombinant pandemic influenza A(H1N1) viruses resistant to neuraminidase inhibitors

Abstract

Background: Neuraminidase inhibitors (NAIs) play a key role in the management of influenza epidemics and pandemics. Given the novel pandemic influenza A(H1N1) (pH1N1) virus and the restricted number of approved anti-influenza drugs, evaluation of potential drug-resistant variants is of high priority.

Methods: Recombinant pH1N1 viruses were generated by reverse genetics, expressing either the wild-type or any of 9 mutant neuraminidase (NA) proteins (N2 numbering: E119G, E119V, D198G, I222V, H274Y, N294S, S334N, I222V-H274Y, and H274Y-S334N). We evaluated these recombinant viruses for their resistance phenotype to 4 NAIs (oseltamivir, zanamivir, peramivir, and A-315675), NA enzymatic activity, and replicative capacity.

Results: The E119G and E119V mutations conferred a multidrug resistance phenotype to many NAIs but severely compromised viral fitness. The oseltamivir- and peramivir-resistance phenotype was confirmed for the H274Y and N294S mutants, although both viruses remained susceptible to zanamivir. Remarkably, the I222V mutation had a synergistic effect on the oseltamivir- and peramivir-resistance phenotype of H274Y and compensated for reduced viral fitness, raising concerns about the potential emergence and dissemination of this double-mutant virus.

Conclusions: This study highlights the importance of continuous monitoring of antiviral drug resistance in clinical samples as well as the need to develop new agents and combination strategies.

Figure 1.

Replication kinetics of recombinant A/Québec/144147/09 pH1N1 viruses in vitro. Confluent MDCK α2,6 cells were infected with a multiplicity of infection (MOI) of .001 plaque-forming units (PFU) per cell. Supernatants were harvested at the indicated time points and titrated by standard plaque assays. A, E119G, E119V, D198G, H274Y, and N294S mutants. B, I222V, S334N, I222V-H274Y, and H274Y-S334N mutants. The mean values for 3 experiments with standard deviations (SDs) are presented. *P < .05; **P < .01 (for differences in viral titers when compared with the wild-type virus using the t test). The time points with significant differences relative to the wild-type virus are as follows: E119G (24, 36, 48, and 60 h), E119V (24, 36, 48, 60, and 72 h), D198G (72 h), I222V (24, 48, and 60 h), H274Y (36, 60, and 72 h), N294S (24 h), S334N (72 h), and I222V-H274Y (24 h).