WGS of SARS-CoV-2 in Vero E6 cells
Independent triplicate cultures were infected with SARS-CoV-2 (WA1 strain) in the presence of different concentrations of NHC, MRK-A, or culture medium alone at a final volume of 1.5 ml/well. SARS-CoV-2 was inoculated into each well at 50% tissue culture infectious dose (TCID50) of 5,000 units for the initial culture. NHC or MRK-A (dissolved in 100 mM DMSO) were added to the cell culture across a range of concentrations in a 3-fold dilution series based on the IC50 of each drug. After addition of virus, cells were incubated at 37C and 5% CO2 for 72 hours. The culture supernatant (100 L) from wells with the highest drug concentration exhibiting >10% cytopathic effect were used to seed the next passage. Viral RNA from culture supernatants was collected for each replicate culture, at each passage, and purified using the PureLink Pro 96 Viral RNA/DNA Purification Kit (ThermoFisher, Massachusetts, USA). Whole genome SARS-CoV-2 sequencing libraries were prepared via the QIAseq DIRECT SARS-CoV-2 kit (Qiagen, Hilden, Germany). Sequencing was performed at the DNA Sequencing & Genotyping Center, University of Delaware, Ammon-Pinizzotto Biopharmaceutical Innovation Center (Newark, Delaware, USA) on the Illumina platform, yielding 2  151 nucleotide paired-end reads.