Role of permissive neuraminidase mutations in influenza A/Brisbane/59/2007-like (H1N1) viruses

Neuraminidase (na) mutations conferring resistance to na inhibitors were believed to compromise influenza virus fitness. unexpectedly,an oseltamivir-resistant a/brisbane/59/2007 (bris07)-like h1n1 h275y na variant emerged in 2007 and completely replaced the wild-type (wt) strain in 2008-2009. the na of such variant contained additional na changes (r222q,v234m and d344n) that potentially counteracted the detrimental effect of the h275y mutation on viral fitness. here,we rescued a recombinant bris07-like wt virus and 4 na mutants/revertants (h275y,h275y/q222r,h275y/m234v and h275y/n344d) and characterized them in vitro and in ferrets. a fluorometric-based na assay was used to determine vmax and km values. replicative capacities were evaluated by yield assays in st6gal1-mdck cells. recombinant na proteins were expressed in 293t cells and surface na activity was determined. infectivity and contact transmission experiments were evaluated for the wt,h275y and h275y/q222r recombinants in ferrets. the h275y mutation did not significantly alter km and vmax values compared to wt. the h275y/n344d mutant had a reduced affinity (km of 50 vs 12 μm) whereas the h275y/m234v mutant had a reduced activity (22 vs 28 u/sec). in contrast,the h275y/q222r mutant showed a significant decrease of both affinity (40 μm) and activity (7 u/sec). the wt,h275y,h275y/m234v and h275y/n344d recombinants had comparable replicative capacities contrasting with h275y/q222r mutant whose viral titers were significantly reduced. all studied mutations reduced the cell surface na activity compared to wt with the maximum reduction being obtained for the h275y/q222r mutant. comparable infectivity and transmissibility were seen between the wt and the h275y mutant in ferrets whereas the h275y/q222r mutant was associated with significantly lower lung viral titers. in conclusion,the q222r reversion mutation compromised bris07-like h1n1 virus in vitro and in vivo. thus,the r222q na mutation present in the wt virus may have facilitated the emergence of nai-resistant bris07 variants. © 2011 abed et al.