Given that a large percentage of seasonal influenza circulating viruses are resistant to oseltamivir, we next assessed the activity and synergy of the TCAD regimen against oseltamivir-resistant viruses to evaluate the spectrum of activity of the TCAD regimen, and to determine whether oseltamivir contributed to the activity of the TCAD regimen against oseltamivir-resistant viruses. Two H1N1 oseltamivir-resistant viruses were used, both of which bear the H274Y substitution in NA which has been demonstrated to confer resistance to oseltamivir [20] : A/Mississippi/3/01 (MS H274Y) and A/Hawaii/21/07 (HI H274Y). As a single agent, oseltamivir carboxylate had no antiviral activity against either virus below 1 mg/mL (data not shown). The synergy volumes of double and triple combinations of amantadine, ribavirin, and oseltamivir were determined against both viruses, and the data are presented in Figure 3 . As double combinations, amantadine/ oseltamivir carboxylate, amantadine/ribavirin, and ribavirin/ oseltamivir carboxylate were all additive ( Figure 3A) . As was seen with the 2009 H1N1 viruses, the TCAD regimen was synergistic, and the synergy volume was greater than the synergy volume of any double combination, with each drug making a contribution to the synergy of the TCAD regimen against the oseltamivir-resistant viruses ( Figure 3B-D) . Importantly, oseltamivir carboxylate contributed to the synergy of the TCAD regimen starting at 0.1 mg/mL against MS H274Y (P,0.05) and at 0.32 mg/mL against HI H274Y (P,0.01). At these concentrations, which are achievable clinically, oseltamivir carboxylate is not active as a single agent against these resistant strains. Synergy plots for the TCAD regimen against MS H274Y and HI H274Y are provided in Figure S3 , and reveal increasing synergy with increasing concentrations of oseltamivir carboxylate. At the highest concentration of oseltamivir carboxylate tested (3.2 mg/mL), synergy occurred over wide concentrations of ribavirin and amantadine. At lower concentrations of oseltamivir carboxylate, synergy occurred at higher concentrations of amantadine (0.1 mg/mL or higher), and at wide concentrations of ribavirin. No significant antagonism was detected at any concentrations of the three drugs. We also determined the EC 50 of oseltamivir carboxylate as a single agent and in double and triple combinations against both oseltamivir-resistant viruses. As summarized in Table 3 , the EC 50 of oseltamivir carboxylate as a single agent was 74 mg/mL against MS H274Y and 15 mg/mL against HI H274Y. These represent 1480-and 300-fold reductions in susceptibility compared to the published values for a wild-type virus [11] . The EC 50 of oseltamivir carboxylate was not reduced in double combination with 1mg/mL ribavirin or 0.032 mg/mL amantadine against MS H274Y, and was reduced by only 1.7-and 1.4-fold, respectively, against HI H274Y. However, in triple combination with ribavirin and amantadine at the same concentrations used in double combination, the EC 50 of oseltamivir carboxylate was reduced by 21-fold against MS H274Y and by 5.8-fold against HI H274Y.