FIG 8 .
Passage of CHIKV in the presence of digoxin enriches for drug-resistant viruses. U-2 OS cells were incubated with DMSO, 10 µM 5-NT, or increasing concentrations of digoxin for 1 h prior to adsorption with CHIKV stocks that had been passaged 14 times in the presence of either DMSO (SL15649DMSO) or digoxin (SL15649Digoxin) at an MOI of 5 PFU/cell. After 1 h, virus was removed, and cells were incubated with medium containing DMSO or inhibitor for 5 h. Cells were stained with CHIKV-specific antiserum and DAPI to detect nuclei and imaged by fluorescence microscopy. Results are presented as percentages of infected cells normalized to DMSO-treated cells. Error bars indicate standard errors of the means. (B) U-2 OS cells were incubated with DMSO or 500 nM digoxin for 1 h prior to adsorption at an MOI of 5 PFU/cell with virus clones that were plaque purified from either the SL15649DMSO or SL15649Digoxin stock. After 1 h, virus was removed, and cells were incubated with medium containing DMSO or digoxin for 5 h. Cells were stained with CHIKV-specific antiserum and DAPI to detect nuclei and imaged by fluorescence microscopy. Results are presented as percentages of infected cells normalized to DMSO-treated cells for duplicate experiments. Error bars indicate standard errors of the means. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, in comparison to DMSO-treated or DMSO-passaged virus-infected cells as determined by ANOVA followed by Tukey’s post hoc test.