swirling or inverting the contents until the substrate is thoroughly dissolved to form the Caspase-GloH 3/7 reagent. The premixed Caspase-Glo 3/7 reagents (Promega) were added at 1:1 ratio (reagent : media) to the treated cells at 48 h.p.i., or otherwise noted in the text. After 40 minutes incubation in the dark at room temperature, luminescent signals were read with the Synergy-II multimode microplate reader (BioTek). Real-time Quantitative Reverse Transcriptase PCR (qRT- PCR) Total RNA was extracted from cells and supernatants as described for TRIzol LS Reagent (Invitrogen; Carlsbad, CA) according to the manufacturer’s instructions. Dried RNA pellets were re-suspended in 50 ml DEPC treated-water and stored at 280uC before RT- PCR amplification. Following the manufac- tures’ instruction, the real-time qRT-PCR was carried out using the ‘‘one step SRBY Ex Taq qRT-PCR’’ kit (Takara, Japan). The real-time qRT-PCR was performed in a single tube and amplified products were monitored in real-time using the 7500 Fast Real- Time PCR System (AB applied biosystems). Two BTV-specific primers were designated and used for the real-time qRT-PCR: Forward primer: 59-GATTGATGTTTACAGGGATGAGGT- 39 Reverse primer: 59-TCTTCCTCTGCTTGCGTCCT- 39 SDS-PAGE and Western Blot Analysis Samples for SDS-PAGE and western blot analysis were prepared after boiled in protein dissociation buffer [10% (v/v) b- mercaptoethanol, 10% (wt/vol) SDS, 25% (v/v) glycerol, 10 mM Tris-HCl, pH 6.8), and 0.02% (wt/vol) bromophenol blue] for 10 min. Protein samples were loaded in 16 sample buffer and electrophoresis carried out on 12.5% SDS-polyacrylamide gels. Proteins were electroblotted on to Immobilon-P poly-vinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) at 100 mA for 2 h by using a semidry electrotransfer apparatus. The PVDF membranes were immediately placed into 5% skimmed milk in TBS20.1% Tween-20 (TBST) and incubated overnight. This step was followed by 1 h incubation in primary antibody diluted with 3% BSA in TBS. Mouse monoclonal anti-VP6 [75], mouse monoclonal anti-human LC3-II (Abgent, Inc., San Diego, CA) were used at the concentrations recommended by the manufacturers. Blots were washed three times for 10 min each in TBST and then incubated for 1 h in horseradish peroxidase- conjugated secondary antibody diluted in 3% BSA–TBST. Goat anti-mouse or anti-rabbit secondary antibodies conjugated to horseradish peroxidase (BD Biosciences, San Jose, CA) were used References 1. Li Q, Li H, Blitvich BJ, Zhang J (2007) The Aedes albopictus inhibitor of apoptosis 1 gene protects vertebrate cells from bluetongue virus-induced apoptosis. Insect Mol Biol 16: 93–105. 2. Schwartz-Cornil I, Mertens PP, Contreras V, Hemati B, Pascale F, et al. (2008) Bluetongue virus: virology, pathogenesis and immunity. Vet Res 39: 46. 3. Forzan M, Marsh M, Roy P (2007) Bluetongue virus entry into cells. J Virol 81: 4819–4827. 4. Mellor PS, Wittmann EJ (2002) Bluetongue virus in the Mediterranean Basin 1998–2001. Vet J 164: 20–37. 5. Paweska JT, Venter GJ, Mellor PS (2002) Vector competence of South African Culicoides species for bluetongue virus serotype 1 (BTV-1) with special reference to the effect of temperature on the rate of virus replication in C. imicola and C. bolitinos. Med Vet Entomol 16: 10–21. 6. Mellor PS, Carpenter S, Harrup L, Baylis M, Mertens PP (2008) Bluetongue in Europe and the Mediterranean Basin: history of occurrence prior to 2006. Prev Vet Med 87: 4–20. 7. Mertens PP, Diprose J, Maan S, Singh KP, Attoui H, et al. (2004) Bluetongue virus replication, molecular and structural biology. Vet Ital 40: 426–437. Virostatic Agents against Bluetongue Virus at 1:5,000. Bound antibodies were detected by an enhanced chemiluminescence kit (Amersham, Piscataway, NJ, USA). Plaque Reduction Assay The plaque reduction assay was carried out in the 24-well plates by including compounds in the overlay agarose, which was applied directly to the BTV infected cell culture as described previously [76,77]. Cells were initially inoculated with BTV at ten-fold series dilution with known initial BTV titer. After absorbing for 2 h at 37uC, 5% CO2 with 80–95% humidity, the inoculation was removed, washed with media and replaced by 1% agarose mixed with compound at the indicated concentration, ranging from 20.0 mM to 0.16 mM. Each concentration was triplicated. At 72 h.p.i., the number of plaques formed at different compound concentration was counted and the final virus titer was determined accordingly. De novo Compound Synthesis The structure and modification of C003 were illustrated in Figure 2 and 3. The preparation of intermediate compound 4 (IC4) followed an adaptation of the Gewald reaction [78] beginning with commercially available compound 4-methylaceto- phenone. IC7 and IC8 were achieved by modification of the Horner-Wadsworth-Emmons procedure [79] on commercially available 2-thiophenecarboxaldehyde and 2-pyridinecarboxalde- hyde followed by a standard acid chloride formation with thionyl chloride. Combining IC4 with IC7 and IC8 was pursued as described in the literature [79,80] for related systems. Standard saponification of the resultant IC9 and IC10 produced IC2b and IC3b that became the final products C052 (IC2a) and C055 (IC3a) upon routine acidification (Fig. 3). All compounds were characterized by high field NMR (Bruker AM 400) and high resolution mass spectral (Waters Q Premier API Quadrupole TOF Tandem mass spectrometer) analyses with purity confirmed by micro analytical elemental determinations. Acknowledgments We thank the technical assistances from Ms. Pulin Che and Mr. Hairui Su as assistant students during the course of the work. Author Contributions Conceived and designed the experiments: QL. Performed the experiments: LG VM ZB AQ SWS QL. Analyzed the data: LG VM ZB AQ SWS QL. Contributed reagents/materials/analysis tools: LG VM ZB AQ SWS QL. Wrote the paper: LG VM ZB SWS QL. 8. MacLachlan NJ, Conley AJ, Kennedy PC (2000) Bluetongue and equine viral arteritis viruses as models of virus-induced fetal injury and abortion. Anim Reprod Sci 60–61: 643–651. 9. Roy P (2005) Bluetongue virus proteins and particles and their role in virus entry, assembly, and release. Adv Virus Res 64: 69–123. 10. Roy P (2008) Functional mapping of bluetongue virus proteins and their interactions with host proteins during virus replication. Cell Biochem Biophys 50: 143–157. 11. Roy P (2008) Bluetongue virus: dissection of the polymerase complex. J Gen Virol 89: 1789–1804. 12. Boyce M, Celma CC, Roy P (2008) Development of reverse genetics systems for bluetongue virus: recovery of infectious virus from synthetic RNA transcripts. J Virol 82: 8339–8348. 13. Boyce M, Roy P (2007) Recovery of infectious bluetongue virus from RNA. J Virol 81: 2179–2186. 14. Matsuo E, Roy P (2009) Bluetongue virus VP6 acts early in the replication cycle and can form the basis of chimeric virus formation. J Virol 83: 8842–8848. 15. Maclachlan NJ, Drew CP, Darpel KE, Worwa G (2009) The pathology and pathogenesis of bluetongue. J Comp Pathol 141: 1–16. PLOS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e43341