*3.2. Rg1 Treatment A*ff*ect PRRSV Attachment, Replication and Release in Marc-145 Cells*

To explore the effects of Rg1 upon the PRRSV life cycle, virus entry was analyzed in cell attachment (from 0–2 h.p.i.) and internalization assays (from 2–5 h.p.i.), virus replication was analyzed between 6–10 h.p.i. and virus assembly or release was determined after 12 h.p.i., based on the ratio of Nsp9 mRNA in infected cells and supernatant. The experimental strategy was as previously described [14]. The pre-treatment of Marc-145 with Rg1 does not reduce viral RNA levels, suggesting that Rg1 does not affect the susceptibility of Marc-145 cell to PRRSV significantly. For virus attachment, the experiment was designed to allow virus binding, but not cellular internalization, and the results show that the Nsp9 mRNA levels in cell lysates are reduced both in the 10 μM and 50 μM treatment groups (Figure 2A). Virus internalization was analyzed from 2–5 h.p.i. in the presence of Rg1, and the Nsp9 expression levels only decrease in the 50 μM group (Figure 2A). The inhibitory effect on PRRSV replication is significant in both the 10 μM and 50 μM groups (Figure 2A). As described previously, PRRSV progeny viruses are released 8 h.p.i. [13,28]. In addition, our results of PRRSV inhibition kinetics by Rg1 in Marc-145 cells indicate that viral mRNA significantly reduces from 12 h.p.i. to 72 h.p.i. (Figure 1G). Thus, we evaluated the virus release rate with Rg1 treatment at 12 h.p.i. Marc-145 cells were infected for 12 h at 37 ◦C, and the cells were then cultured in DMEM containing 10 μM or 50 μM Rg1 for another 2 h. The Nsp9 mRNA levels in infected cells and culture supernatants were quantified by RT-PCR, and the ratio of cell/supernatant Nsp9 corresponds to the virus progeny release rate. The results indicate that this PRRSV virus released from Marc-145 cells is remarkably suppressed by Rg1 treatment (Figure 2B).

**Figure 2.** Inhibitory effects on the virus lifecycle of Rg1 in Marc-145 cells. In the Pre-treatment assay, Marc-145 cells were pretreated with DMEM supplemented with 10 or 50 μM Rg1 for 2 h, then cells were washed twice with PBS before being infected with type 2 PRRSV XH-GD (0.1 MOI), and then samples were collected at 48 h.p.i. For the attachment and internalization assay, Marc-145 cells were pre-cultivated at 4 ◦C for 1 h and then infected with virus (0.1 MOI) at 4 ◦C for 2 h. During virus attachment upon PRRSV infection, cells were cultured with DMEM or DMEM containing 10 or 50 μM Rg1 to analyze PRRSV Nsp9 mRNA level. Marc-145 cells were infected with XH-GD at 4 ◦C for 2 h and then cultured with or without Rg1 for 3 h at 37 ◦C. To avoid interference of other steps of viral lifecycle on replication assay, Marc-145 cells were infected with XH-GD for 6 h and then incubated with DMEM with or without 10 or 50 μM Rg1 at 37 ◦C, and samples were collected at 4 h.p.i. In all of the trials, GAPDH was used as a housekeeping gene for normalization, and cells treated with 0.4% DMSO was used as a reference control. (**A**) The effect of Rg1 on viral attachment, internalization, replication, and Rg1 pretreatment was analyzed by evaluating Nsp9 mRNA expression levels. (**B**) The effect of Rg1 on PRRSV release was detected by the ratio of Nsp9 RNA copy numbers in the supernatant and the cell lysate detected by qPCR. The analysis above was performed in triplicate. Statistical significance is denoted by \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001.
