Figure 6. Effects of C003 and C052 on BTV-induced caspase-3/7 activation. BSR cells were mock infected or infected with BTV at MOI of 0.01, and treated with C003 or C052 at their EC90 concentrations (A) or EC50 concentrations (B). At 24, 48 and 72 h.p.i, caspase-3/-7 activities were determined using the Caspase-Glo-3/7 reagent, and presented as relative fluorescent unit (RLU). Each data points represented mean values and SD from triplicates experiments. doi:10.1371/journal.pone.0043341.g006 similar concentrations (1.8 mM and 10 mM), including 2.0- and 2.3-fold decreases of caspase-3/7 activity at 48 h.p.i. (Fig. 6A and B). The above results indicated that C003 and/or C052 could inhibit BTV-induced caspase-3/7 activation, which further prevented BTV-induced apoptosis, consequently CPE and cell lysis. The Effect of C003 and C052 on Virus Progeny Production To understand whether C003 and C052 could disrupt the productive BTV viral life-cycle, we analyzed their effect on BTV progeny productions. BSR were infected with BTV at MOI of 0.01 and cells were treated with C003 or C052 at 10 mM or 2.5 mM, respectively. At different h.p.i, cells and supernatants were collected, and BTV titers in these samples were determined by the conventional plaque assay. At 24 h.p.i., there were one log differences of virus progeny production in cells treated with C003 Virostatic Agents against Bluetongue Virus or C052 at 10.0 mM and 2.5 mM, respectively, when compared with that in samples collected from BTV-infected only cells. There were three- or two-log reductions of virus progeny production in samples collected at 48 h.p.i. and 72 h.p.i. after treated with C003 or C052, respectively (Fig. 7A). Similarly, the reduction of progeny virus production was also noticeable in the supernatant samples after treated with C003 or C052, with three- or two-log reductions of virus progeny titer, respectively, comparing with that in samples collected from BTV-infected supernatant without virostatic compound treatment (Fig. 7B). The inhibition of C003 and C052 to BTV virus progeny production was further confirmed using the plaque reduction assay. C003 or C052 at different concentration were mixed with the agrose overlay and applied to BTV infected cells (MOI of 0.01) directly. The inhibition of BTV plaque formation was then examined at 72 h.p.i. The effects of C003 and C052 on the BTV plaque reduction were in a dose-dependent fashion. At 72 h.p.i., a one-log reduction of plaque formation was observed, when C003 at 2.5 mM and C052 at 1.25 mM were applied to the BTV infected cells, respectively. When applied at higher concentrations, i.e. 20.0 mM of C003 or C052, to the infected cells, more than two-log of plaque reductions were observed (Fig. 7C). There was no difference in terms of the size or formation of the plaques (results not shown). This result showed that C003 and C052 could reduce the viral plaque formation directly. Time-of-addition Assay Since C003/C052 showed no overt toxicity in vitro, yet prevented BTV induced CPE/apoptosis/cell lysis and reduced the virus progeny production, we initiated the mechanism of action studies with the time-of-addition assay to determine the possible stage(s) of viral life-cycle that could be affected by C003 and C052. In order to observed both the enhancement and reduction of virostatic efficacy at different time of addition, we used two concentrations of virostatic compound for both C003 and C052, i.e., the C003 at 1.76 mM and 20 mM, and C052 at 0.27 mM and 2.5 mM, respectively. We first observed that adding virostatic compound C003 or C052 at 1 or 2 hours prior to BTV infection, i.e. 21 and 22 h.p.i., did not change their virostatic efficacies. This result indicated that both C003 and C052 might not act on the early stage of viral life-cycle, such as virus entry (Fig. 8A and B). Interestingly, when the compounds were added to BTV infected cells as later as 24 h.p.i., both C003 and C052 showed similar protection in term of the percentage of viable cells. When added at 32 h.p.i., the percentage of viable cells decreased in both C003 and C052 treatment samples, comparing with their protection in the initial 24 h.p.i., indicating that both C003 and C052 were less protective post certain stage(s) of viral life-cycle. When C003 and C052 were added at 48 h.p.i., both compounds showed no protection to BSR cells from BTV-induced CPE (Fig. 8A and B). Since the first cycle of BTV viral replication usually completed in infected cells within 24 h.p.i., our results suggested that C003 and C052 might not act at the early stage of BTV viral life-cycle, including virus entry and uncoating, but rather at the late stage of BTV viral life-cycle, such as virus replication, packaging, maturation and egress. Meanwhile, it is also possible that these compounds may act on host cellular machineries that were involved during late viral life-cycle. Effect of C003 and C052 on BTV Viral Genomic RNA Replication To determine whether C003/C052 acts on BTV viral life-cycle directly, we examined the effect of C003/C052 on viral genomic RNA replication in infected cells. BTV viral genomic RNA copies PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e43341