Sars cov 2 strain wa1

As established viral target cells infected by HCoV-NL63 and SARS-CoV-2, Calu-3 human metastatic lung epithelial adenocarcinoma (HTB-55), Caco-2 human colorectal epithelial adenocarcinoma (HTB-37) and Vero normal epithelial monkey kidney (CCL‐81) cells (all from ATCC, Manassas, VA, USA) were maintained according to published standard procedures [19 (link)–22 (link)]. In brief, all cells (Calu-3, Caco-2 and Vero) were cultured in Eagle’s Minimum Essential Medium (MEM) medium (Corning, Manassas, VA) supplemented with 10% bovine calf serum (BCS, HyClone™ Laboratories, Logan, UT). Coronavirus HCoV-NL63 (NR-470) and its genomic RNA (NR-44105) were obtained from BEI Resources (NIAID, NIH). SARS-CoV-2 strain WA1 (NR-52281; BEI Resources) was propagated in Vero cells unless specified otherwise [6 (link)]. For viral stocks, cells were infected at a multiplicity of infection (MOI) of 0.01 and cultured for 48 h. At that point, cells were harvested, homogenized, subjected to a single freeze-thaw cycle, and then combined with the culture supernatant followed by centrifugation (3000 rpm, 10 min). The viral titers of the final supernatant (after serial dilution) was determined by plaque forming assay. All work with SARS-CoV-2 was performed under BSL3 conditions in a facility with negative pressure and PPE that included Tyvek suits and N95 masks for respiratory protection.