samples (Fig. 9C and D). At 24 h.p.i., there were pronounced reductions, around two-log, of virus genomic RNA copies and virus titers in supernatant samples with C003/C052 treatment, when compared with these in the supernatant samples without C003/C052 treatment. In supernatant samples collected at 24 h.p.i from BTV infected but without C003/C052 treatment, there were 6.176106 copies of BTV viral genomic RNA, while there were only 4.816104 or 4.226104 copies of BTV viral genomic RNA in samples treated with C003 or C052, respectively (Fig. 9B). This result indicated a pronounced effect of these virostatic agents at the late stage of viral life-cycle. However, C003/C052 might also inhibit virus replication directly, since there was a one-log difference in the viral genomic RNA copies and virus titers when comparing samples from infected cells with and without C003/ C052 treatments (Fig. 9A). Effect of C003/C052 on VP6 Expression To further confirm the effect of C003/C052 on BTV viral life- cycle, we also analyzed viral protein synthesis in the above cell samples infected at MOIs of 0.01, with and without C003 or C052 treatments. VP6 of BTV possesses nucleoside triphosphatase, RNA binding, and helicase activities [9,11,38]. For a successful BTV life-cycle, VP6 is an integral part of a transcription complex essential for primary replication [14]. At MOI of 0.01, VP6 was not detectable at 24 h.p.i. in all samples, but was detected at 48 Virostatic Agents against Bluetongue Virus h.p.i. in BTV-infected cells without C003/C052 treatment (Fig. 10). When BTV-infected cells were treated with C003 at10 mM and C052 at 2.5 mM, respectively, there were either very low or no detectable VP6 expression in the cells at 48 h.p.i. (Fig. 10). This result supported our previous observations that C003/C052 reduced viral protein expression when infected at low MOI of 0.01. Effect of C003 and C052 on BTV-induced Autophagy Based on the data presented above, we hypothesized that C003/C052 might act on host machineries/pathways involved in the viral life-cycle, including the autophagic pathway. Autophagy is one of the major pathways for the degradation and turnover of long-lived proteins and organelles in cells during cell starvation or upon various stimuli, such as virus infection [39,40,41,42]. Growing evidences suggest that autophagy actively participates in various pathogenic infections, including the formation of autophagosome for the replication of Influenza A virus [43,44,45], hepatitis C virus [46,47] and Dengue virus [48]. The turnover of microtubule-associated protein light chain 3-I (LC3-I) to LC3-II, or the ratio of LC3-I and LC3-II, is the only biomarker that is reliably associated with the formation of autophagosome. This is also related to virus replication and the leading indicator of virus-induced apoptosis. Since C003 and C052 prevented BTV- induced apoptosis, we examined the activation and formation of Figure 9. The effects of C003 and C052 on BTV viral genomic RNA synthesized and progeny virus production at MOI of 0.01. BTV infected cells were treated with C003 (10 mM) or C052 (2.5 mM), at different h.p.i. as indicated in figures. The viral genomic RNA was extracted and the copy number was determined using real-time qRT-PCR in cell (A) and supernatant (B) samples. The progeny virus titer in cells (C) and supernatants (D) were also analyzed using standard plaque assay. doi:10.1371/journal.pone.0043341.g009 PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e43341