Fig. 8.

MPXV sensitivity to IBT. (A) MPXV sensitivity to IBT: Plaque Reduction. BSC-40 cells were infected with ~100 pfu of virus and treated with two-fold increasing concentrations of IBT. Viruses used were VACV, VACV-A24R-R1, VACV-E3LΔ37N, two West African strains of MPXV (7-61 and US-2003), and MPXV Zaire (Central African strain). Plaques were stained with crystal violet and counted. Results appear as the percentage of the number of plaques present in the untreated cells from three replicates. (B) MPXV sensitivity to IBT: Multi-cycle Growth. Multi-cycle growth kinetics assays were performed in BSC-40 cells. The cells were infected with VACV, MPXV, and VACV-A24R-R1 at an MOI of 0.01 in the presence of absence of 60 µM IBT. Viruses were harvested at 0 and 72 hpi and titered by plaque assay in RK-E3L cells. Data presented are means with standard error of multiple experiments. (C) MPXV sensitivity to IBT: Accumulation of Free dsRNA. HeLa cells were mock infected or infected with either wt VACV, VACV A24R-R1 or MPXV. Infected cells were either untreated or treated with 45 µM IBT. After 6 hpi, cells were trypsinized and fixed. Cells were stained with antibodies against total VACV and dsRNA and were subjected to flow cytometry the next day. Total cells were gated to exclude non-viable cells. (D) Quantitation of replicate results from Panel C. Statistical difference was determined by t test using Holm-Sidak method with alpha=5%. (E) MPXV sensitivity to IBT: PKR Phosphorylation. Hela cells were infected with VACV, MPXV, or VACV-A24R-R1 at an MOI of 5 and treated one hour post infection with two-fold increasing concentrations of IBT. Western blot analyses were performed with antibodies directed against the phosphorylated form of PKR and with antibodies to the viral protein E3/F3. Western blot band intensities were measured with Image Quant, normalized to GAPDH levels, and shown as a percentage of the band intensity at the highest (15 µM) level of IBT treatment.