Inhibition of novel ß coronavirus replication by a combination of interferon-a2b and ribavirin

Falzarano, Darryl; de Wit, Emmie; Martellaro, Cynthia; Callison, Julie; Munster, Vincent J.; Feldmann, Heinz

Virus and cells. Human betacoronavirus EMC (hCoV-EMC/2012) was kindly provided by Erasmus Medical Center (Rotterdam, Netherlands). Vero (African green monkey kidney) and LLC-MK2 (rhesus monkey kidney) were maintained at 37uC in 5% CO 2 in Dulbecco's modified Eagle's media (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin and 50 mg/ml of streptomycin. HCoV-EMC was subsequently propagated on Vero cells using DMEM as above with 2% FBS (complete DMEM). Antiviral assays. Confluent Vero and LLC-MK2 cells in 24-well culture plates (Costar, Corning, NY ) were infected in triplicate with hCoV-EMC/2012 diluted in complete DMEM at an MOI 5 0.001. Following 1 h adsorption at 37uC, the inoculum was removed and the cells were washed 3 times with DMEM. Subsequently, complete DMEM containing IFN-a2b (0-5000 U/ml) (PBL Interferon Source, Piscataway, NJ) and/or ribavirin (0-2000 mg/ml) (MP Biomedicals, Solon, OH) was added to the cells. Cells were incubated for 24 h at 37uC, 5% CO 2 in a humidified environment and the supernatant was removed, an aliquot was inactivated with AVL (Qiagen, Germantown, MD) for viral load quantification and the remainder was stored at 280uC for subsequent virus titration. The supernatant was replaced with fresh complete DMEM containing IFN-a2b and/or ribavirin. Supernatant was also collected at 72 h and 120 h. Five days post-infection representative wells were photographed to document cytopathic effect (CPE) and cells were subsequently collected for protein analysis in 43 SDS-PAGE loading buffer.