*3.1. Ginsenoside Rg1 Treatment Supressed PRRSV Replication in Marc-145 Cells and PAMs*

The cytotoxicity of ginsenoside Rg1 (Rg1) on Marc-145 cells and PAMs was analyzed by WST-1 assay. Rg1 does not impair Marc-145 and PAM cell viability at a concentration as high as 400 μM (Figure 1B,C). In the course of the experiment, we notice that Marc-145 cells cultured with Rg1 exhibit better cellular morphology under the serum deprivation. Therefore, whether Rg1 affects cell proliferation was analyzed in Marc-145 cells. The results indicate that Rg1 does not influence Marc-145 cell proliferation significantly at doses from 5 μM to 400 μM, and cell proliferation increases in 800 μM and 1600 μM (Figure 1D). These results suggest that Rg1 has minimal cytotoxicity on Marc-145 cells and PAMs within the tested doses.

To determine the anti-PRRSV activity of Rg1, Marc-145 cells and PAMs were infected with PRRSV XH-GD (0.1 MOI) for 1 h and then treated with indicated concentrations of Rg1 for 24 h. As shown in Figure 1E, treatment with Rg1 results in a significant dose-dependent reduction in PRRSV Nsp9 mRNA levels both in Marc-145 cells and PAMs. The expression level of N protein, evaluated by western blotting, decreases in proportion to the amount of Rg1 used in the treatment (Figure 1F). This result indicates that Rg1 treatment inhibits PRRSV replication, and 10 μM Rg1 could inhibit Nsp9 mRNA and N protein expression. Therefore, PRRSV inhibition kinetics by Rg1 in Marc-145 cells and PAMs are further analyzed at 10 μM and 50 μM. Nsp9 mRNA expression, representing the PRRSV replication rate in the treated groups, was compared to that in the DMSO-treated control (Figure 1G). The results reveal that the decrease in Nsp9 mRNA is more pronounced from 24 h.p.i. (hours post infection) to 48 h.p.i. in PAMs, and from 36 h.p.i. to 72 h.p.i. in Marc-145 cells. Collectively, these results suggest that Rg1 treatment suppresses PRRSV infection.

**Figure 1.** Cytotoxicity and anti-PRRSV activity of Rg1 in Marc-145 cells and PAMs. (**A**) The chemical structures of ginsenoside Rg1 (Rg1). (**B**,**C**) Cytotoxicity of Rg1 in Marc-145 cells (**B**) and PAMs (**C**) were analyzed by using the WST-1 assay. Results are showed as the relative cell viability of PAMs or Marc-145 cells cultured without Rg1 (set as 100%). (**D**) Rg1 affects the proliferation of Marc-145 cells. Cells were seeded and cultured without FBS for 12 h and the medium was replaced with DMEM contains 0, 5, 10, 50, 100, 200 and 400 μM Rg1 respectively. (**E**,**F**) PRRSV XH-GD (0.1 MOI) infected Marc-145 cells or PAMs for 1 h at 37 ◦C, and then cells were cultured in DMEM or RPMI 1640 supplemented with 2% FBS and indicated concentrations of Rg1. The samples were collected at 48 hpi to analyze PRRSV Nsp9 mRNA levels in different groups by RT-PCR (**E**). N protein expression levels in cells treated with different concentrations of Rg1 were detected by western blot (**F**). (**G**) Marc-145 cells and PAMs infected with PRRSV XH-GD (0.1 MOI) for 1 h at 37 ◦C and then cultured in fresh medium supplemented with 10 or 50 μM Rg1. The expression levels of PRRSV Nsp9 in Marc-145 cells and PAMs were detected by RT-PCR analysis at the indicated time points. Each data represents results of three independent experiments (means ± SD). Significant differences compared with the control group are denoted by \* (*p* < 0.05), \*\* (*p* < 0.01), \*\*\* (*p* < 0.001) and \*\*\*\* (*p* < 0.0001).
