Vero Cell Virus Production and Purification

Twenty-four hours before transfection, 1.2x10⁶ low-passage Vero cells were seeded into a 12.5 cm² flask in 5 mL of DMEM media supplemented with 10% FBS and 1x PSG solution. Next day, Vero cells were transfected with 5 μg of plasmid DNA using Lipofectamine 2000 reagent (Invitrogen) according to manufacturer’s instructions. Cells were maintained in DMEM at 37°C, 5% CO₂ for 5 days. Cell culture supernatant was harvested, and virus was titrated on Vero cell monolayers and was purified by one-step terminal dilution as described earlier [42 (link)]. Experimental virus stocks were prepared by two consecutive passages in Vero cells (passages 143–149) maintained in Opti-Pro (Invitrogen), followed by titration of viruses in Vero cells. Complete genomes of biologically-cloned viruses were sequenced to ensure genetic integrity.