Antiviral activity assay

A clinical isolate of SARS-CoV-2 (nCoV-2019BetaCoV/Wuhan/WIV04/2019) was propagated in Vero E6 cells, and viral titer was determined by 50% tissue culture infective dose (TCID₅₀) using immunofluorescence assay. Briefly, Vero E6 cells were fixed with 4% paraformaldehyde and permeabilised with 0.5% Triton X-100 before blocked with 5% BSA for 2 h at 25 °C. The blocked cells were incubated with the primary antibody of polyclonal antibody against viral nucleocapsid protein of a bat SARS-CoV²⁶ and followed by the second antibody of Alexa 488-labeled goat anti-rabbit (Abcam). The nuclei were stained with Hoechst 33258 dye (Beyotime) before visualised by fluorescence microscopy. For the antiviral assay, pre-seeded Vero E6 cells (5 × 10⁴ cells/well) were pre-treated with the different concentration of compound for 1 h and the virus was subsequently added (MOI of 0.01) to allow infection for 1 h. At 24-h post infection, the cell supernatant was collected and extracted viral RNA using MiniBEST Viral RNA/DNA Extraction Kit (Tanaka, #RR047A). Reverse transcription was conducted using PrimeScript RT Reagent Kit with gDNA eraser (Tanaka, #RR047A) to prepare cDNA template. qRT-PCR analysis was carried out on StepOne Plus Real-time PCR (Applied Biosystem) with TB Green Premix Ex Taq II (Tanaka, #RR820A). Receptor binding domain (RBD) of spike gene was amplified by PCR from the cDNA template with primers: RBD-F: 5′-GCTCCATGGCCTAATATTACAAACTTGTGCC3′; RBD-R: 5′-TGCTCTAGACTCAAGTGTCTGTGGATCAC-3′, cloned into pMT/BiP/V5-His vector (Invitrogen) and used as the standard plasmid. A standard curve was generated by the determination of copy numbers from serially dilutions (10³–10⁹ copies) of the standard plasmid. The primers used for quantitative PCR were RBD-qF1: 5′-CAATGGTTTAACAGGCACAGG-3′ and RBD-qR1: 5′-CTCAAGTGTCTGTGGATCACG-3′²⁶. PCR amplification was performed as follows: 95 °C for 5 min followed by 40 cycles consisting of 95 °C for 15 s, 54 °C for 15 s, 72 °C for 30 s. For cytotoxicity assays, pre-seeded Vero E6 cells were treated with appropriate concentrations of compound. After 24 h, the relative numbers of surviving cells were measured by the CCK8 (Beyotime) assay in accordance with the manufacturer’s instructions. All experiments were performed in triplicate, and all the infection experiments were performed at biosafety level-3.