*2.8. Rg1 Treatment on PRRSV Life Cycle Assay*

Marc-145 cells were grown to 70%–80% confluence in six-well plate at 37 ◦C in 5% CO2. Cells were infected with PRRSV. Four steps in PRRSV life cycle including attachment, internalization, replication and release were analyzed at different time-points after post-infection and the pre-treatment of Rg1 on PRRSV infection was also analyzed as previously described [14].

#### *2.9. Animal Experiment*

Thirty-two four-week old piglets, which were free of African swine fever virus (ASFV), PRRSV, antibody against PRRSV, pseudorabies virus (PRV) and swine influenza virus (SIV), were randomly divided into four groups with eight piglets in each group. For each group, five piglets were randomly selected and raised together as a sub-group used for collecting samples and recording clinical performance, morbidity and mortality, while the other three piglets were raised in another pigsty and euthanized at seven days post-challenge for pathological detection. The groups PRRSV and PRRSV+Rg1 were challenged intranasally (1 mL) and intramuscularly (1 mL) with the HP-PRRSV JXA1 strain (5 <sup>×</sup> 105 TCID50), and the Mock group was inoculated in the same way with the same volume of DMEM and continued administration for 10 days, and then used as a negative control. At 12 h post infection, the group PRRSV+Rg1 was injected intramuscularly with Rg1 (10 mg/kg/day) and continued administration for 10 days. The PRRSV group was injected with 1 mL DMEM at 12 h post-infection and continued administration for 10 days, and then used as a challenge control group. The group Rg1 was injected with Rg1 (10 mg/kg/day) and continued administration for 10 days, being used as the drug control group. All of the piglets were planned to be monitored for 14 days after the challenge. Clinical signs and rectal temperatures of all of the groups were evaluated and scored daily. Blood samples of each piglet were obtained by venipuncture at 0, 2, 4, 7, 10 and 14 days post-infection (dpi) for the detection of the PRRSV viral load and anti-PRRSV antibody (Idexx PRRSV Elisa Kit, Idexx, US). The tissue sample of lung, thymus and lymph node were collected at 7 and 15 dpi. Tissue (1g) and serum (100 μL) samples were used to extract total RNA. RNA extraction and cDNA synthesis were performed as previous report [23]. Real-time quantification PCR was carried out on a CFX96TM real-time system (Bio-Rad, USA). Standard, serially-diluted PRRSV strain JXA1 (100–107 TCID50/mL) was used to generate a standard curve (slope = <sup>−</sup>3.717; R2 = 0.995) [24,25]. All pigs were euthanized for pathological detection at 15 dpi.
