Serial passaging and mutation sequencing of SARS-CoV-2 BA.2.3 in the presence of ISM3312 and Nirmatrelvir

Passaging SARS-CoV-2-BA.2.3 in VeroE6 cells was conducted with escalating concentrations of ISM3312 and Nirmatrelvir with three replicates, along with the efflux inhibitor Elacridar. For each passage, multiple new cultures were initiated with the same concentration, as well as lower and higher concentrations. The passage exhibiting the maximum sustainable compound concentration was chosen for subsequent passaging. Viral RNA was extracted for Next-Generation Sequencing (NGS) at passages 7 and 18.