Betacoronavirus HCoV-OC43 Viral Stocks

The original Betacoronavirus 1 HCoV-OC43 VR-759 and the HCoV-OC43 rOC/US183-2 double S protein mutant (H183R & Y241H) derived from VR-759 (herein described as VR-759 dm; gifts from Dr. Talbot, INRS, QC, Canada) were passaged twice on HRT-18 cells and harvested as previously described (Favreau et al., 2009 (link)). The HCoV-OC43 variant VR-1558 (ATCC, Manassas, VA, USA) was passaged twice on MRC-5 cells to obtain a working P3 stock. P3 viral stocks were independently prepared from the supernatant and the cell-associated fractions. Hence, cells were scraped and centrifuged at low speed (500×g, 10 min, 4 °C) to separate extracellular and cell-associated viruses. Extracellular virions were next concentrated by ultracentrifugation (60,000×g, 1 h, 4 °C) and resuspended in a minimal volume of DMEM while the intracellular viruses were released by two rounds of freeze-thawing. Both fractions were sonicated 15 times for 1s at a power of 8 of a Sonic Dismembrator Model 100 using a cup-horn setting (Fischer Scientific, Hampton, NH, USA), flash frozen in liquid nitrogen and stored at −80 °C. These stocks were then used to infect cells as described below.