2.15 Comparison of the infectivity of WT and D614G mutant of pSARS-CoV-2
To generate pSARS-CoV-2 harboring D614G mutation on spike protein, a single nucleotide A-to-G substitution was introduced by site-directed mutagenesis into SARS-CoV-2 spike plasmid using the primer sequence 5′-GTG GCC GTG CTG TAC CAG GGC GTG AAT TGC ACC GAG GTG -3′. WT and D614G pSARS-CoV2 viruses were produced as described above. The culture supernatants containing viral particles were filtered on 0.45 µm pore filter and concentrated by ultracentrifugation at 28,000 rpm for 2 h at 4°C in a Beckman SW28 rotor and an Optima XE-100 Ultracentrifuge (Beckman Coulter). The virus pellets were resuspended in PBS buffer and then viral titers were determined by qRT-PCR method using Lentivirus Titration Kit (LV900, ABM). The equivalent amount of WT and D614G pSARS-CoV2 viruses were used to infect host cells for pSARS-CoV-2 entry assay.