The F13L gene from the final plaque isolates were amplified by PCR and sequenced to confirm the presence of the D217N amino acid change. Data presented in this work were expressed as mean ± SD (standard deviation). selleck inhibitor The results of one test group were compared to another
group and analyzed statistically with unpaired Student’s t-test. The results of more than two sets of measurements in one experiment were analyzed statistically by one-way analysis of variance (ANOVA) followed by Dunnett’s and Tukey’s Multiple Comparison Tests. A p value <0.05 was considered statistically significant. All analyses were performed using Prism v 5.01 (GraphPad Software, Inc.). Previous results from our group indicated that CTGV has an overall lower dissemination rate and yield production in cell culture when compared to other VACV strains (Damaso et al., 2000 and Jesus et al., 2009a). Therefore, we first evaluated the growth rates of CTGV and two other VACV strains in two different cell lines before further testing the antiviral effect of ST-246 in these cell types. We observed that in RK-13, BSC-40 and BHK-21, CTGV produced
less infectious particles than VACV-WR at 24 and 48 h post-infection (p < 0.01) ( Fig. 1A–C). VACV-IOC showed similar growth kinetics as CTGV in all cell lines tested (p > 0.05). Despite the lower rates of replication, both CTGV and VACV-IOC were able to develop their replicative Antiinfection Compound Library solubility dmso cycle and produce virus particles over the course of infection in these cells. All subsequent experiments were done in BSC-40 cells. ST-246 has been previously evaluated for toxicity to BSC-40 cells (Yang et al., 2005). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-based assays confirmed that the drug was not toxic to the monolayers revealing that 97.3 ± 13.94% of the cells were viable after 48 h in the presence of 100 μM ST-246 (p > 0.05; Student’s t-test) (data not
shown). To evaluate the antiviral effect of ST-246 on the replication of CTGV, we analyzed the formation of virus plaques in the presence of the drug for 48 h. As shown in Fig. 2A, ST-246 inhibited CTGV plaque formation at 48 h post-infection and this effect Y-27632 2HCl appeared to be more dramatic than that observed for VACV strains IOC and WR. Similar effects on plaque formation were observed at 96 h post-infection (p < 0.001; one-way Anova followed by Tukey’s tests) or when RK-13 or BHK-21 cells were infected with these viruses (p < 0.01; one-way Anova followed by Tukey’s tests) (data not shown). We extended the concentration range of ST-246 and included other orthopoxviruses in the assay ( Fig. 2B). The antiviral effect of ST-246 was dose-dependent for all viruses tested, but CTGV was significantly more susceptible to the effect of ST-246 than other orthopoxviruses. At 0.02 μM ST-246, a 95.