Virostatic Agents against Bluetongue Virus Figure 3. The de novo synthesis of C052 (2a) and C055 (3a) were illustrated. Each number here represents each intermediate compound (ic) as described in the text. doi:10.1371/journal.pone.0043341.g003 Both C003 and C052 Prevented BTV Induced CPE The CellTiter-Glo cell viability assay determines the number of viable cells in culture based on quantitation of cellular ATP presented, which signals the presence of metabolically active living cells [34,35,36]. To further confirm that C003 or C052 could Figure 4. The virostatic efficacy and cytotoxicity of C052 were showed as cells were infected with BTV at MOI of 0.01 in the presence of various concentrations of C052. At 72 h.p.i, cell viability (triangles) or cytotoxicity (circles) was determined using CellTiter-Glo reagent. Each data point represents means and SD from five replicates. doi:10.1371/journal.pone.0043341.g004 prevent BTV-induced CPE, cell morphology changes at different h.p.i. were examined post BTV infection at MOI of 0.01. Without C003 or C052 treatment, CPE was observed as early as 48 h.p.i. in infected cells. At 72 h.p.i., the majority of infected cells were dislodged from the plate indicating CPE and cell lysis (Fig. 5B). When C003 at 10 mM or C052 at 2.5 mM was added to BTV- infected cells at 0 h.p.i., CPE was not observed up till 72 h.p.i. There was no sign of cell dislodging and lysis (Fig. 5D and F). Similar to previous observation, uninfected cells treated with C003 at 10 mM or C052 at 2.5 mM (Fig. 5C and E) showed no overt toxicity, with regard to cell division, morphology changes, with comparable cell viability to that of the control cells (Fig. 5A). This result confirmed that BTV-induced CPE was prevented after cells were treated with C003 or C052. C003 and C052 Prevented BTV-induced Apoptosis Previous studies have showed that BTV infection in mammalian cells triggers apoptosis leading to CPE/cell lysis in vitro and the virus-induced pathogenesis in vivo [1,37]. In order to further confirmed that C003 and/or C052 prevented BTV-induced CPE via blocking apoptosis, we examined the activation of caspase-3/7 in infected cells treated with the virostatic compounds at different h.p.i. Our result showed that BTV-induced caspase-3/7 activa- tion, the main characteristics of apoptosis, was inhibited in C003/ C052 treated cells. When BTV-infected cells were treated with PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e43341