title: Role of permissive neuraminidase mutations in influenza A/Brisbane/59/2007-like (H1N1) viruses.
abstract: 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.
authors.affiliation: Research Center in Infectious Diseases of the CHUQ-CHUL and Laval University, Québec City, Québec, Canada.
authors.forename: Yacine
authors.initials: Y
authors.lastname: Abed
authors.forename: Andrés
authors.initials: A
authors.lastname: Pizzorno
authors.forename: Xavier
authors.initials: X
authors.lastname: Bouhy
authors.forename: Guy
authors.initials: G
authors.lastname: Boivin
mesh_terms: D000818:Animals; D024882:Drug Resistance, Viral / Q000235:genetics*; D005289:Ferrets; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics* / Q000472:pathogenicity*; D009154:Mutation; D009439:Neuraminidase / Q000235:genetics* / Q000378:metabolism; D009976:Orthomyxoviridae Infections / Q000235:genetics* / Q000635:transmission; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014779:Virus Replication / Q000235:genetics
chemical_list: D009439:Neuraminidase