Neuraminidase (NA) inhibitors (NIs) are the first line of defense against influenza virus. Reverse genetics experiments allow the study of resistance mechanisms by anticipating the impacts of mutations to the virus. To look at the possibility of an increased effect on the resistance phenotype of a combination of framework mutations, known to confer resistance to oseltamivir or zanamivir, with limited effect on virus fitness, we constructed 4 viruses by reverse genetics in the A/Moscow/10/99 H3N2 background containing double mutations in their neuraminidase genes: E119D+I222L, E119V+I222L, D198N+I222L, and H274Y+I222L (N2 numbering). Among the viruses produced, the E119D+I222L mutant virus was not able to grow without bacterial NA complementation and the D198N+I222L mutant and H274Y+I222L mutant were not stable after passages in MDCK cells. The E119V+I222L mutant was stable after five passages in MDCK cells. This E119V-and-I222L combination had a combinatorial effect on oseltamivir resistance. The total NA activity of the E119V+I222L mutant was low (5% compared to that of the wild-type virus). This drop in NA activity resulted from a decreased NA quantity in the virion in comparison to that of the wild-type virus (1.4% of that of the wild type). In MDCK-SIAT1 cells, the E119V+I222L mutant virus did not present a replicative advantage over the wild-type virus, even in the presence of oseltamivir. Double mutations combining two framework mutations in the NA gene still have to be monitored, as they could induce a high level of resistance to NIs, without impairing the NA affinity. Our study allows a better understanding of the diversity of the mechanisms of resistance to NIs.