Quantitative RT-PCR Analysis of SARS-CoV Infection

Cells were incubated with SARS-CoV-1 or SARS-CoV-2 at a multiplicity of infections (MOI) of 1 in the presence of the cathepsin inhibitor, 25 μM E-64d (Abcam, Cambridge, MA, USA) or DMSO as control. After 1 h absorption, cells were washed with PBS and cultured in maintenance medium. At 4 h post-infection (hpi), total RNAs were extracted from inoculated cells using ISOGEN (Nippon Gene, Tokyo, Japan) and a Direct-zol RNA MiniPrep Kit (Zymo Research, Orange, CA, USA). Extracted RNAs were subjected to qRT-PCR analysis with the THUNDERBIRD Probe One-step qRT-PCR Kit (TOYOBO, Osaka, Japan). The sequences of primers and probes targeting the N gene of SARS-CoV-1 and SARS-CoV-2 have been described previously [33 (link),34 (link)]. Human ACTB (Beta Actin) Endogenous Control (Applied Biosystems, Foster City, CZ, USA) and nonhuman primate β-actin were employed as endogenous controls [35 (link)].