Virostatic Agents against Bluetongue Virus Figure 8. The time-of-addition assay for C003 (A) and C052 (B). C003 at 20 and 1.76 mM, and C052 at 2.5 mM and 0.27 mM, respectively, were added to BTV infected cells at different h.p.i. as indicated, and the protection of C003 and C052 against BTV induced CPE, or cell viability, was measured using CellTiter-Glo reagent at 72 h.p.i. Each data points represented the average values and SD from of eight independent replicates. doi:10.1371/journal.pone.0043341.g008 were determined in both cell and supernatant samples using the real time qRT-PCR (Fig. 9A and B). The correspondence of viral genomic RNA replication and virus progeny production in these samples were confirmed by evaluating the amount of infectious virus using standard plaque assay (Fig. 9C and D). Samples were collected post BTV infection and divided into cells and super- natants portion at different time in the initial 24 h.p.i., with and without treatments of C003 (10 mM) and C052 (2.5 mM), re- spectively. When infected at low MOI (0.01), the viral genomic RNA copies and progeny virus titers obtained from in cell samples represent the events occurred inside the cells for the initial viral-life cycle, including virus entry, replication and maturation within the cells. The viral genomic RNA copies and virus titers in the supernatant samples denotes the events post virus life-cycle inside the cells in the initial 24 h.p.i., including the amount of viruses exited from the cells. At early time points including 3 and 6 h.p.i., there were no differences in viral genomic RNA copies in samples collected from both supernatant and cells, whether treated with C003/C052 or not, indicating that early viral life cycle, such as the entry of BTV into the cells, were not interfered (Fig. 9 A and B). Meanwhile, there is no detectable progeny virus production in all Figure 7. The effects of C003 and C052 on BTV progeny production. BTV infected cells were treated with C003 (10 mM) or C052 (2.5 mM). At different time post infection as indicated in figures, cell (A) and supernatant (B) samples were collected separately. BTV progeny productions were evaluated using the standard plaque assay. In a separate experiment, the plaque reduction assay (C) was carried by mixing C003 or C052 with the overlay and applied directly to BTV- infected cells. At 72 h.p.i, plaques were counted and analyzed. Each data points represented the average values and SD from of three independent replicates. doi:10.1371/journal.pone.0043341.g007 PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43341