Summary of Phenotype and Genotype of Supernatants after Serial Passage under Escalating Drug Concentrations.

Wild type influenza A/Hawaii/31/07 (H1N1) virus was passaged in the presence of escalating concentrations of each drug regimen for a total of ≥25 cumulative days in culture, or until the drug concentration reached the 50% of the cytotoxic concentration (TC50) of the drug as a single agent. Each drug regimen was tested starting at 1/8th the EC50 of each drug as a single agent. The drug concentration was increased 1X, 2X, or 4X at each passage starting at passage 2, depending on whether virus-induced cytopathic effect (CPE) was apparent during the passage. Phenotypic testing using neutral red assay in MDCK cells was performed on the supernatants from each regimen to determine the susceptibility to each drug as a single agent after serial passage. In addition, the supernatant from selected passages from each regimen was sequenced by the Sanger method at the M2, hemagglutinin (HA), and/or neuraminidase (NA) genes to detect the presence of mutations.
aEC50 values are the mean of 6 replicate wells from two plates with standard deviations.
bAmino acid positions in HA are designated using universal H3 numbering [42].