Cells and virus

Vero E6 cells (ATCC^® CRL-1586™, American Type Culture Collection, Manassas, VA, USA) were used for virus propagation and titration. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Corning, New York, NY, USA), supplemented with 5% fetal bovine serum (FBS, R&D Systems, Minneapolis, MN, USA) and antibiotics/antimycotics (ThermoFisher Scientific, Waltham, MA, USA), and maintained at 37 °C under a 5% CO₂ atmosphere. The SARS-CoV-2 USA-WA1/2020 strain was acquired from BEI Resources (Manassas, VA, USA) and passaged three times in Vero E6 cells to establish a stock virus (1 × 10⁶ TCID₅₀/mL) for inoculation of animals. This stock virus was sequenced by next generation sequencing (NGS) using the Illumina MiSeq and its consensus sequence was found to be homologous to the original USA-WA1/2020 strain (GenBank accession: MN985325.1). To determine infectious virus titer, 10-fold serial dilutions were performed on Vero E6 cells. The presence of cytopathic effects (CPE) after 96 hours incubation was used to calculate the 50% tissue culture infective dose (TCID₅₀)/mL using the Spearman-Karber method.