Comparison of 60 min IC50 values for enzyme inhibition assays with and without preincubation with inhibitor for wild type and mutant viruses.

a(−)Virus, inhibitor and MUNANA substrate were added simultaneously with no preincubation. (+) virus and inhibitor were preincubated for 30 min, then MUNANA was added. Both reactions were followed for 60 min. Values are the means of duplicate reactions.bSlow binding is demonstrated by a higher IC50 without preincubation compared to with preincubation; ratio of (−)/(+) >2.0.c(−)/(+) ratio ∼1 shows changed kinetics, which can be due to fast binding and fast dissociation, as seen for all the I222 mutants with oseltamivir, or slow binding, but fast dissociation as seen for the P154S mutant with zanamivir and oseltamivir, as shown in Fig. 2.