*2.5. Antiviral Activity Assay*

To analyze the effect of Rg1 on PRRSV infection, Marc-145 cells and PAMs were grown to 70%–80% confluence respectively in six-well plates. PRRSV strains (0.1 MOI) diluted in DMEM or RPMI 1640 were incubated with Marc-145 cells or PAMs at 37 ◦C for 1 h. The supernatants were removed, and the cells were washed twice with PBS. Then, fresh DMEM containing different concentrations of each compound was added and incubated at 37 ◦C in 5% CO2. After treatment, the cells and supernatants were collected at the indicated time points of post-infection. The supernatants were used to titrate the production of progeny virus, and the viral titers were defined and calculated as TCID50/mL [22]. The cell plates were washed with PBS and harvested for immunofluorescence assay (IFA), qRT-PCR, and western blotting analysis. The 50% effective concentrations (EC50) value (the concentration of Rg1 required to protect 50% cells from infection) of Rg1 against different type 2 PRRSV strains was determined as previously described [14], and calculated with the GraphPad Prism 7.0 software.
