Figure 7. Both ECTV and VACV are sensitive to the anti-poxvirus drug IBT.

(A) BS-C-1 cells were infected (MOI=10) with either ECTV or VACV for 24 hrs. prior to staining for dsRNA (red) and cell nuclei/virus factories (DAPI; blue). IBT (45 µM) was added at the time of infection. Due to space constraints, only the merged images are shown, which were prepared using ImageJ software. All images are at 400× total magnification and representative of three independent trials. (B) BS-C-1 cells were infected (MOI=10) with either ECTV or VACV for 24 hrs. prior to staining for dsRNA. IBT (45 µM) was added at the time of infection. The flow cytometric data are depicted in histogram format and representative of three independent trials. Percentages represent the fraction of cells within the positive gate. (C) RNase L activation assays are shown for BS-C-1 cells infected (MOI=5) with ECTV or VACV either in the presence of absence of 45 µM IBT. Total RNA was isolated at 18 hrs. post-infection and run on a 1% bleach TBE gel. The data are representative of two independent trials. (D) Plaque reduction assays were performed to determine the effect of IBT on virus replication. Confluent monolayers of BS-C-1 cells in six-well plates were infected with approximately 50 plaque forming units of either ECTV or VACV. IBT was added to the cells at the indicated concentrations. Plaques were visualized using a 0.5% crystal violet solution after two days of incubation for VACV and five days for ECTV. The indicated percentages are relative to the no drug control wells. Symbols depict the average values and each error bar represents the standard deviation of the mean. The data are an assembly of eight independent assays. Statistical analysis [performed using GraphPad Prism software (version 7.0a)] was carried out using the Mann-Whitney test. * denotes a p value <0.05.