Zanamivir-resistant influenza viruses with a novel neuraminidase mutation

Abstract

The neuraminidase inhibitors zanamivir and oseltamivir are marketed for the treatment and prophylaxis of influenza and have been stockpiled by many countries for use in a pandemic. Although recent surveillance has identified a striking increase in the frequency of oseltamivir-resistant seasonal influenza A (H1N1) viruses in Europe, the United States, Oceania, and South Africa, to date there have been no reports of significant zanamivir resistance among influenza A (H1N1) viruses or any other human influenza viruses. We investigated the frequency of oseltamivir and zanamivir resistance in circulating seasonal influenza A (H1N1) viruses in Australasia and Southeast Asia. Analysis of 391 influenza A (H1N1) viruses isolated between 2006 and early 2008 from Australasia and Southeast Asia revealed nine viruses (2.3%) that demonstrated markedly reduced zanamivir susceptibility and contained a previously undescribed Gln136Lys (Q136K) neuraminidase mutation. The mutation had no effect on oseltamivir susceptibility but caused approximately a 300-fold and a 70-fold reduction in zanamivir and peramivir susceptibility, respectively. The role of the Q136K mutation in conferring zanamivir resistance was confirmed using reverse genetics. Interestingly, the mutation was not detected in the primary clinical specimens from which these mutant isolates were grown, suggesting that the resistant viruses either occurred in very low proportions in the primary clinical specimens or arose during MDCK cell culture passage. Compared to susceptible influenza A (H1N1) viruses, the Q136K mutant strains displayed greater viral fitness than the wild-type virus in MDCK cells but equivalent infectivity and transmissibility in a ferret model.

FIG. 1.

Proportion of Q136K mutant in viral isolates following serial passage in MDCK cells. Means and standard deviations of the proportion of Q136K mutant virus in populations were determined following pyrosequencing analysis of amplified PCR product from three separate RT-PCR assays performed on separate occasions. Viruses A/Christchurch/62/2007 (•) from the original specimen (OS) to MDCK11 and A/Macau/229/2008 (○) from MDCK2 to MDCK11 were assayed (no result was obtained for the A/Macau/229/2008 original specimen, and the A/Macau/229/2008 MDCK1 isolate was not available for testing). Arrows indicate MDCK isolates that were obtained in the Christchurch or Macau laboratories. All subsequent isolates were obtained in the WHO Collaborating Centre for Reference and Research on Influenza, Melbourne.

FIG. 2.

Virus growth curves of plaque-purified viruses and recombinant viruses in MDCK cells. (A) Growth curves of A/Philippines/1279/2006 plaque-purified viruses PP-1 (Q136 wild-type) (•) and PP-2 (K136 mutant) (○) in MDCK cells over 72 h. (B) Growth curves of recombinant viruses RG-1 (Q136 wild-type) (•) and RG-2 (K136 mutant) (○) in MDCK cells over 72 h.

FIG. 3.

Modeling of resistance mutations based on the crystal structure of the H5N1-zanamivir complex. (A) Stick representation of wild-type H5N1 (white sticks) with zanamivir (orange sticks). (B) Mutation Q136K (green sticks) results in a loss of the hydrogen bond network. Figures were produced with the modeling software package PyMol (W. L. DeLano, DeLano Scientific, Paolo Alto, CA) using the sequence of the Protein Data Bank code 2HTQ protein (27) downloaded from the RCS Protein Data Bank.