Virus Breakthrough and the Presence of Resistance-Associated Substitutions as a Function of the Concentration of the Third Drug in TCAD.

MDCK cells in 96-well plates were infected with influenza A/Hawaii/31/2007 (H1N1) virus in the presence of a combination of two drugs at fixed concentrations with varying concentrations of the third drug, using 12 replicates for each condition. The fixed concentrations of the double combinations were 0.30 µg/mL OSC and 0.60 µg/mL RBV, 0.6 µg/mL AMT and 0.6 µg/mL RBV, or 0.6 µg/mL AMT and 0.3 µg/mL OSC. Following 5 serial passages, the number of wells for each condition having virus breakthrough, defined as >50% cytopathic effect (CPE), was determined by neutral red uptake.
aStatistical analysis of the number of wells with virus breakthrough at each concentration of third drug compared to no third drug was performed using the Fisher's exact test.
bThe supernatants from wells with virus breakthrough were analyzed by Sanger sequencing at the M2, HA, and NA genes to determine the presence of resistance-associated mutations.
cSubtitutions are listed if they were detected in any well by Sanger sequencing. Amino acid positions for NA and HA are presented using N2 and H3 numbering, respectively.