P2 virus stock was propagated in Vero-E6 cells cultured in a Nunc cell factory system (Thermo Fisher Scientific, Waltham, MA, USA) at multiplicity of infection (MOI) 0.005, then cells were microscopically investigated daily. The virus-infected culture supernatant was clarified by centrifugation at 4000 rpm for 15 min at 4 °C twice. The harvested virus was titrated by plaque titration assay [5 (link)]. Harvested SARS-CoV-2 was inactivated with 0.1% β-Propiolactone (Invitrogen), then the treated virus was incubated at 4 °C for two days in a cooling shaking incubator. The β-Propiolactone treated virus was tested for loss of its infectivity by inoculating it in Vero-E6 cells for seven days. No cytopathic effect (CPE) was observed on cells infected with inactivated virus. Virus harvest (1000 mL) was concentrated by ultra-filtration system (Amersham Bioscience, Amersham, UK) hollow fiber cartridge of 50 KDa pore size and a circulation rate of 150 rpm. The concentrated virus was further concentrated by ultracentrifugation (80,000× g, 4 °C, 1 h with 20% sucrose as a cushion). Total protein content was measured by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).
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