less attentions [70]. This is mainly due to the lack of information on the mechanism of egress/dissemination. Interestingly, further studies showed that autophagy may assist poliovirus egress. During Poliovirus and Coxsackievirus B3 infection, the co-localization of non-structural proteins with autophagy markers LC3-II, together with gene silencing and pharmacological experiments, indicate that autophagy proteins enhance viral replication and progeny virus yields. In BTV, the viral NS3 may act like the membrane protein of enveloped viruses and is responsible for intracellular trafficking and budding of virus particles [71]. Virostatics targeting virus egress would be predicted to prevent or slow down virus release and spread in an infected host, thus allowing more time for the individual’s immune system to respond and control the infection [72]. Further studies on the role of autophagy during BTV progeny virus egress/dissemination may help to understand the exact mechanism of action for C003/C052. Our results extend our previous findings that showed cluster of lead compounds that prevent BTV induced CPE [34]. While further studies might be needed to define the exact mechanism of action for the virostatic agents C003/C052, our studies showed that these compounds may not interact with host apoptotic machinery, rather posses virostatic activity which block various signals to induce host apoptosis. The finding that host autophagy was involved in BTV life-cycle and C003/C052 might act on host autophagic machinery may open a new avenue to identify and develop novel small molecule antivirals that specifically interact with host autophagy machinery. Materials and Methods Cells, Virus Stock and Use of Compounds BSR cells [73], a cloned derivative of baby hamster kidney (BHK) cells, were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA), supplemen- ted with 5% fetal calf serum (FCS), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen). For all assays, unless otherwise noted, cells were plated in DMEM with optimized conditions as previously determined, including 1% FCS [34]. All cells were incubated and grown at 37uC, 5% CO2. The type 10 BTV (BTV-10) was plaque purified and propagated as described previously [1]. BTV-10 was titrated by standard plaque assay, stocked at 2.56107 PFU/ml and further used for infection with the indicated multiplicity of infection (MOI). For virus replication, cell monolayers were washed with FCS-free growth medium and then incubated with viruses at the indicated MOI. Virus adsorptions were carried out for 2 h at 37uC, 5% CO2, followed by incubation in growth medium supplemented with 1% FCS. At various hours post infection (h.p.i.), cells and supernatants were collected. Cells were sonicated for 40 s and cell debris were removed by low-speed centrifugation (2,500 g). The virus titers in the resulting super- natants were determined on BSR cells at 37uC, 5% CO2, using a standard plaque assay as described previously [74]. CPE-based Cell Viability Assay The CPE-based cell viability assays, using the CellTiter-GloH Luminescent Cell Viability kit (Promega Inc., Madison, WI), were carried out as described previously [34]. Cell viability was determined at indicated time points using CellTiter-Glo reagents following manufacturer’s instruction (Promega). Briefly, CellTiter- Glo buffer and the lyophilized CellTiter-Glo substrate (Promega) were thawed and equilibrated to room temperature prior to use. The homogeneous CellTiter-Glo reagent solution was reconsti- tuted after mixing the lyophilized enzyme/substrate and the buffer Virostatic Agents against Bluetongue Virus reagent according to the manufacturer’s instructions. Meanwhile, the assay plates were also equilibrated to room temperature for 15 min. An equal volume (25 ml) of CellTiter-Glo reagents was added to each well by a MicroFlo select dispenser (BioTek, Winooski, VT). After incubated for 15 minutes at room temper- ature, luminescence signals were measured using Synergy-II multi- mode microplate reader (BioTek) with an integration time of 0.1 s. The Ten-concentration Dose Response Assay and Cytotoxicity Assays The virostatic efficacy assay for each compound was carried out under the optimized conditions as described previously [34]. Briefly, BSR cells were seeded into a 384-well black plate (Corning) using the MicroFlo select dispenser (BioTek). After 2 hours incubation at the 37uC, 5% CO2 and 80–95% humidity, ten different concentrations of each compound were serially diluted and added to each well. Cells were then infected with BTV at MOI of 0.01 via the Bio-Tek dispenser. Each compound treatment group, with BTV infection at denoted MOIs, includes eight replicates. Mock infection (cell only) and virus infection only controls were also included as positive and negative controls, respectively. Compound cytotoxicity assay were carried out in the same fashion, except without BTV infection. After incubated at 37uC, 5% CO2 and 80–95% humidity for 72 h, cell viability was determined using CellTiter-Glo reagents as described previously. Time-of-Addition Assay The Time-of-Addition assay was performed to further un- derstand the virostatic activity of the previously identified compounds [34]. Briefly, BSR cells were seeded and incubated at 37uC, 5% CO2 and 80–95% humidity. Cells were then inoculated with BTV at MOI of 0.01, and incubated at the optimized conditions as described previously [34]. Selected virostatic compounds were diluted to the indicated concentrations and added to each well at different time pre- or post- BTV infection. The denoted 22 and 21 h.p.i. indicate that BSR cells were incubated with the selected virostatic compounds prior to BTV infection. For 0 h.p.i., the compound and BTV were added to the culture simultaneously. Treated cells were incubated at 37uC, 5% CO2 with 80–95% humidity. In parallel, cells without compounds and virus treatments were included as the mock infection control. Cells with compound treatment only were served as the control to monitor compound cytotoxicity. Cells with BTV infection but without compound treatment were also included as the virus infection only control. Each BTV and compound treatment group includes eight replicates. After incubated for 72 h, cell viability was determined using CellTiter-Glo reagent as described above. The ability of virostatic compound in inhibiting BTV-induced CPE were normalized using controls designated in the assay, where cell viability in the mock infected cells was designated as 100%, and cell viability in BTV infected cells as 0%. The Caspase-GloH 3/7 Apoptosis Assay The Caspase-GloH 3/7 apoptosis assay was accomplished using the Caspase-GloH 3/7 assay kit (Promega) following the manufacturer’s instruction. Cell plating and incubation conditions, as well as the plate layout, were similar to that used in the CPE- based assay. BTV infection and compound addition were also in the similar fashion as described in the CPE-based virostatic efficacy assay. Caspase-GloH 3/7 buffer and lyophilized Caspase- GloH 3/7 substrate were equilibrated to room temperature before use. The Caspase-GloH 3/7 buffer was transferred into the amber bottle containing Caspase-GloH 3/7 substrate, and then mix by PLOS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e43341