Effect of the H275Y neuraminidase mutation on viral fitness of oseltamivir-resistant pandemic 2009 and seasonal H1N1 influenza Aviruses

Abstract

Neuraminidase (NA) inhibitors are one of two classes of antiviral compounds available for the control of influenza infections. The H275Y NA mutation confers resistance to the NA inhibitor oseltamivir carboxylate among the N1 influenza subtype and has been identified from resistant variants with distinct epidemiological outcomes in human. Specifically, dominance of oseltamivir-resistant variant of the A/Brisbane/59/2007-like viruses was reported during the 2008 to 2009 influenza season, until it was replaced by the 2009 pandemic H1N1 virus [A(H1N1)pdm09]. Since the emergence of the 2009 pandemic, the fitness and transmission potential of oseltamivir-resistant A(H1N1)pdm09 variants carrying the H275Y mutation has been a concern. This project aims to systematically evaluate the fitness of viruses carrying the H275Y mutation for A(H1N1)pdm09 and seasonal H1N1 viruses. A panel of recombinant viruses with their NA gene derived from the A(H1N1)pdm09 A/California/04/09 (CA04), seasonal H1N1 A/New Caledonia/20/1999 (NewCal) or A/Brisbane/59/2007 (Brisbane) was generated in the genetic background of CA04. The H275Y mutation in all three viruses led to a reduced affinity for 3’-sialylactose (3’SL) or 6’-sialylactose (6’SL). Similarly, lowered enzyme activity was observed across H275Y-carrying viruses in 3’SL and 6’SL, with the exception of RG-CA04NA-H275Y at catalyzing 3’SL. Differential 3’SL and 6’SL substrate usage was observed between the NA of seasonal H1N1 and A(H1N1)pdm09 viruses. Reduced infectivity was also observed for recombinant CA04 viruses carrying the H275Y mutation with decreased infectivity in mucin-secreting primary human airway epithelial cells when compared to their oseltamivir-sensitive counterparts. In the ferret model, the pathogenicity of RG-CA04NA-H275Y and RG-CA04BrisbaneNA-H275Y viruses was attenuated albeit the transmissibility was minimally affected when compared to RG-CA04 wild-type virus. In parallel, recombinant seasonal H1N1 viruses encoding the surface glycoproteins of NewCal and Brisbane were tested in ferrets. Results indicated that NewCal and Brisbane viruses carrying the H275Y mutation displayed comparable transmission efficiencies to the wild-type NewCal virus via direct-contact and respiratory-droplet settings. These results suggest that the H275Y NA mutation only leads to a minor reduction in viral fitness, with its transmission potential being minimally affected in the naïve ferret model.