Introduction.?Influenza B viruses with a novel I221 L substitution in neuraminidase (NA) conferring high level resistance to oseltamivir were isolated from an immunocompromised patient after prolonged oseltamivir treatment.Methods.?Enzymatic characterization of the NAs (Km, Ki) and the in vitro fitness of viruses carrying wild-type (wt) or mutated (I221 L) NA genes were evaluated. Proportions of wt and mutated NA genes were directly quantified in the patient samples. Structural characterizations by X-ray crystallography of a wt and I221 L variant NA were performed.Results.?The Km and Ki revealed that the I221 L variant NA had approximately 84 and 51 times lower affinity for oseltamivir carboxylate and zanamivir respectively compared to wt NA.Viruses with a wt or I221 L variant NA had similar growth kinetics in MDCK cells and five passages in MDCK cells revealed no reversion of the I221 L substitution. The crystal structure of the I221 L NA and oseltamivir complex showed that the leucine side-chain protrudes into the hydrophobic pocket of the active site that accommodates the pentyloxy substituent of oseltamivir.Conclusions.?Enzyme kinetic and NA structural analyses provide an explanation for the high resistance to oseltamivir, while retaining good virus fitness of viruses carrying I221 L variant NA.