SARS-CoV-2 Variant Cell-Based Assays

Vero E6, Vero-ACE2-TMPRSS2, Huh-7, Vero 81, and 293T cells were maintained in Dulbecco’s minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics [chlortetracycline (25 µg/mL), penicillin (250 U/mL), and streptomycin (250 µg/mL)]. Calu-3 2B4 cells were grown in DMEM supplemented with 20% FBS and antibiotics. MA-SARS-CoV-2 (SARS2-N501Y_MA30) (56 (link)) and MA-MERS-CoV (57 (link)) were propagated in Calu-3 2B4 and Huh-7, respectively. SARS-CoV-2 XBB.1.16 omicron variant (isolate hCoV-19/USA/CA-Stanford-139_S23/2023) was obtained from BEI. Lentivirus-based pseudoviruses expressing SARS-CoV-2 Wuhan-Hu-1 strain were generated with the second-generation lentiviral packaging plasmid psPAX2 (Addgene, Watertown, MA), a reporter plasmid pUCGFP-Luc (Addgene), and pAbVec-SARS2-S (42 (link)). Both plasmids expressing hACE2 or TMPRSS2 were obtained from Origen (Rockville, MD). MDL28170 (calpain and cathepsin B inhibitor) and Nafamostat (TMPRSS2 inhibitor) were purchased from Sigma-Aldrich (St Louis, MO). SARS-CoV-2 3CLpro inhibitors, 5c/d and 11c/d were recently reported from our labs (41 (link)). Polybrene was purchased from Santa Cruze Biotechnology (Santa Cruz, CA).