Effect of tomatidine on the inactivation, attachment, entry, replication, and release of PEDV. A Inactivated assay. Four groups: A. PEDV (10 μM) + tomatidine, B. PEDV (0.01 MOI) + DMSO, C. tomatidine (10 μM), and D. DMSO, were prepared and incubated at 37 °C for 3 h. Group A was then mixed with group D, and group B was mixed with group C, and the mixtures were added into Vero cells seeded in 24-well plates. After incubation at 37 °C for another 1 h, culture supernatants were replaced with fresh DMEM and incubated for an additional 12 h. The cells were washed with PBS, and the mRNA levels of PEDV N and GAPDH in the cells were measured using qRT-PCR. B Virus attachment assay. C Virus internalization assay. D Virus replication assay. E Virus release assay. The results are from one of three independent experiments. Error bars represent the SD. The asterisks in the figures indicate significant differences (*P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant).