Ma104 cells

SARS-CoV-2 (strain 2019-nCoV/USA-WA1/2020) was obtained from the US Centers for Disease Control (CDC) and propagated on MA-104 monkey kidney cells, and the virus titer was determined by focus forming and plaque assays. The virus stock was sequenced by next-generation sequencing, and the spike protein sequence was identical to the original WA1 isolate. However, approximately 50% of the sequences, contained a 30 nucleotide deletion at the furin-cleavage of the spike protein. Vero E6 (CRL-1586, American Type Culture Collection (ATCC), were cultured at 37°C in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, and 100 U/ml of penicillin–streptomycin. MA-104 cells (CRL-2378, ATCC) were cultured in Medium 199, supplemented with 5% FBS, 100 U/ml of penicillin–streptomycin, and 1 m L Amphotericin B. All work with infectious SARS-CoV-2 was performed in Institutional Biosafety Committee approved BSL3 and A-BSL3 facilities at Washington University School of Medicine using appropriate positive pressure air respirators and protective equipment.

MA104 cells (ATCC) were cultured in M199 media (Invitrogen) supplemented with 25 mM HEPES and 10% HI-FBS (Invitrogen). BSC-1 cells (ATCC) were cultured in DMEM (Invitrogen) supplemented with 10% HI-FBS (Invitrogen). For VP4 and VP7 expression, full-length genomic sequences from rhesus rotavirus (G3 serotype, NCBI:txid444185) were amplified by PCR and cloned into pFastbac (Thermo Fisher Scientific) expression vector. Mutations were introduced by quick-change mutagenesis in DH10α cells (Thermo Fisher Scientific). Purified plasmid constructs were transfected into DH10-Bac cells (Thermo Fisher Scientific). Purified bacmids were transfected into SF9 cells (ATCC) grown in SF900 II SFM media supplemented with 1% pen-strep.