3.6.1. Clinical Signs and Mortality

After being challenged with virulent HP-PRRSV JXA1, all piglets in the PRRSV group (piglets challenged with JXA1) displayed high rectal temperature (Figure 6A) and exhibited typical severe clinical signs, including in-appetence, lethargy, dyspnea, periocular and eyelid edema and hyperspasmia. Piglets treated with Rg1 after JXA1 challenge (PRRSV+Rg1 group) showed moderate anorexia and depression. Animals in Rg1 group (piglets only treated with Rg1) and mock group (piglets only treated with DMEM) behaved normally during the course of the experiment. The scores of the evaluation of clinical signs in the PRRSV+Rg1 group were significantly lower than PRRSV group at 7 d.p.i. (*p* < 0.05) (Table 3), a time-point of peak period of morbidity and mortality. All of the piglets displayed increased rectal temperature higher than 40 ◦C after challenge with HP-PRRSV JXA1, while piglets treated with Rg1 showed gradual descending since 8 dpi (Figure 6A). The survival rate of piglets in PRRSV group is 0% to 11 dpi, however, 40% animals in the PRRSV+Rg1 group (piglets treated with Rg1 after JXA1 challenge) survived to 14 dpi (Figure 6B). Since piglets showed different loss of appetite during infection course, the body weight of PRRSV and PRRSV+Rg1 displayed obviously drop after challenge, while the body weight of piglets in PRRSV+Rg1 displayed slower drop trend and begin to rise at 10 dpi (Figure 6C).

**Table 3.** The scores of clinical signs and lung lesions of the different groups at 7 d.p.i.a.


<sup>a</sup> Values followed by letters 1 represents significant difference (*p* < 0.05) between PRRSV and PRRSV+Rg1; letter 2 represents significant difference (*p* < 0.05) between Mock/Rg1 and PRRSV/PRRSV+Rg1. <sup>b</sup> The clinical sign score was calculated by sum of behavior performance, appetite, respiration according to the extent of severity. <sup>c</sup> Analyzing the percentage of the macroscopic lesion features of pneumonia in entire lung.

### 3.6.2. Pathological Examination

As previously described, piglets infected with JXA1 began to die within 6–8 d.p.i. [9], and animals in the PRRSV group start to die at 6 d.p.i. in our study. Therefore, three living pigs of each group were euthanized for pathological examination at 7 d.p.i. to evaluate whether Rg1 treatment relieved lung injury.

The lungs of pigs in PRRSV+Rg1 group showed fewer pathological lesions and got significantly lower scores of macroscopic injury of lungs than those in the PRRSV group as shown in Table 3 (*p* < 0.05). Further histological examination of lung tissue showed that PRRSV+Rg1 group exhibited moderate interstitial pneumonia, while the PRRSV group were characterized by thickened alveolar walls, interstitial fibro-plastic proliferation and intensive mononuclear cell infiltration, which revealed the severe viral pneumonia (Figure 6D). At 15 dpi, all of the living pigs were euthanized for pathological examination. Due to no piglet survived at 14 dpi in the PRRSV group, the deceased pigs at 10 dpi of it were collected to perform pathological examination. The results displayed the same trend as 7 dpi. In the PRRSV+Rg1 group, the survived piglets at 15 dpi exhibited mild pneumonia compared with that of the mock group and Rg1 control group (Figure 6D).

#### 3.6.3. Viremia and Tissue Viral Load

Blood samples of the piglets were collected at 2, 4, 7, 10, 14 dpi to determine viral load and N protein antibody of PRRSV. And, the viral load in tissues, include lung, lymph node and thymus were determined by qRT-PCR as described previously [31]. Since all of the animals in the PRRSV group died at 11 d.p.i., there was no viremia and tissue viral load analysis on it at 14 or 15 h.p.i. The data revealed that piglets treated with Rg1 showed significantly lower viremia, indicated by antibody against PRRSV and viral load in serum, in the blood than those in PRRSV group at 7 (*p* < 0.05) and 10 dpi (*p* < 0.01) (Figure 6E,F). Meanwhile, PRRSV+Rg1 group displayed significantly reduced viral load in the lung, lymph node and thymus at 7 dpi (*p* < 0.01) (Figure 6G).

**Figure 6.** Rg1 exhibits anti-PRRSV activity in 4-week-piglet. (**A**) Daily rectal temperature of the pigs in the PRRSV, PRRSV+Rg1, Rg1 and mock groups. Rectal temperature reach or beyond 40 ◦C was defined as fever. (**B**) The mortality of each group was recorded daily and calculated as survival rate until 14 dpi. (**C**) The body weight gain of different groups during the experiment. (**D**) Severe lung lesions in the PRRSV group characterized by swelling, congestion, fibrosis, and inflammatory cell aggregates; however, in the PRRSV+Rg1 group, these index were moderate. Due to no piglet survived at 14 dpi in the challenge control group (PRRSV), the lung of the deceased pigs at 10 dpi was used to perform pathological analysis. (**E**) The anti-PRRSV antibody levels in serum at different time-points. The value of S/*p* ratio ≥ 0.4 was considered antibody positive. (**F**) The level of PRRSV mRNA in the serum was measured by real-time PCR. (**G**) The expression level of PRRSV mRNA level in lungs, lymph node and thymus was measured by qRT-PCR. Each tissue sample was measured three times, and the error bars represent the standard deviations of samples.

#### **4. Discussion**

PRRSV was identified in Europe in 1991 and emerged in United States in 1992 [32,33]. This virus is classified into European genotype (type 1) and North American genotype (type 2) [3], and the widespread outbreaks of PPRS in China are associated with constant evolution of viruses through high frequency of recombination and immune suppression events in recent decades. The protective immunity to PRRS elicited by current vaccines is effective only against homologous infections and exhibits partial protection from heterologous PRRSV. New antiviral therapeutic strategies became an urgent need. In the present study, we demonstrate that Rg1 suppressed broad lineages of type 2 PRRSV infection, including HP-PRRSV, classical strain and NADC30-like strains, both in Marc-145 cells and PAMs. It suggests that Rg1, as a natural herbal molecular, could be applied in broad-spectrum anti-PRRSV medicament.

PRRSV infection triggers the up-regulated release of IL-1β, IL-6, IL-8 and TNF-α [6,34,35]. These pro-inflammatory factors contribute to the stimulation of protective immune responses, while it also leads to the development of excessive systemic inflammatory reactions and causing inflammatory lesions [36]. Extensive studies have illustrated the potent anti-inflammatory effect of Rg1 treatment in various diseases by regulating inflammatory cytokine expression [37–39]. However, the anti-viral activity of Rg1 associated with modulating inflammatory response was poorly described. In our study, the results reveal that the expression level of several pro-inflammatory factors, including IL-1β, IL-6, IL-8 and TNF-α, is significantly reduced by Rg1 treatment in Marc-145 cell and PAMs upon PRRSV infection. It suggested that Rg1 moderate the PRRSV-induced inflammatory responses by decreasing mRNA expression of pro-inflammatory cytokines in vitro.

PRRSV infects host via membrane receptor mediated endocytosis process including virus attachment and binding, membrane fusion, and followed internalization [40,41]. Once the viral genome, single strand positive-sense RNA, is released into the cytoplasm, it substantially process translation to generate replication and transcription complex. Here, we performed analysis to determine whether Rg1 affect PRRSV lifecycle. The results indicated that pre-treatment of Rg1 could not affect virus host-cell tropism. Marc-145 cells treated with Rg1 during different period of infection process were analyzed respectively, the results showed virus attachment, internalization, and release were impaired (Figure 2). Meanwhile, the inhibitory effect or Rg1 on PRRSV replication stage was more obvious.

NF-κB, key transcription factor that regulates the activation of inflammatory cytokines, can be activated by virus infection, viral gene expression or by LPS stimulation [29], and it could be exploited by influenza viruses or type 1 HIV to sustain a high viral replication [42,43]. In view of the role of Rg1 played in suppressing pro-inflammatory factor expression triggered by PRRSV (Figure 4) and PRRSV-induced NF-κB activation facilitated its replication was demonstrated in previous report [30]. Therefore, we wondered Rg1 inhibited virus replication were associated with cellular process indirectly. In the present study, we identified that Rg1 alleviated PRRSV infection induced IκB degradation, phosphorylation level of p65 and p65 nuclear aggregation, important factors contribute to NF-κB activation, in Marc-145 cells (Figure 5). Moreover, our data indicated that Rg1 treatment suppressed NF-κB activity in LPS-treated Marc-145 cells (Figure 5), which was consistent with a previous study in mouse RAW 264.7 cells and macrophages [44]. This result suggested that the decreased NF-κB activation in Marc-145 cells, which treated with Rg1 upon PRRSV infection, was not due to a reduction in virus infection, it involved in the interaction between Rg1 and cellular process. Overall, the inhibitory effect on both IκB degradation and NF-κB nuclear translocation signaling contributes to the anti-PRRSV replication activity of Rg1. Possibly, it can be speculated that Rg1 mediated cellular process contributes to its anti-viral activity and it might be widely applied in other viral infection.

Recently, multiple Chinese traditional medicines were shown to possess anti-PRRSV ability in vitro [12–14]. However, the antiviral activities of these identified natural herbal extracts, active ingredients, compound or drugs have not been further evaluated in vivo. In present study, piglets challenged with HP-PRRSV JXA1 under Rg1 treatment showed increased survival rate, moderate pneumonia and lower serum and tissue viral loads compared to those in the PRRSV group (challenge

control) (Figure 6). It suggests Rg1 might be a natural anti-PRRSV agent that could be considered as an adjuvant therapy in the pig herd production. Furthermore, it has been confirmed that ginseng extract inhibited virus infection including influenza virus and hepatitis C virus, and Rg1, which purified from the roots or stems of Panax notoginseng (PN) and Panax ginseng (PG), could also be used as an immunoadjuvant to improve immune responses [45,46]. In the current status, there are several Modified live Virus (MLV), which induced immune response did confer protection to vaccinated animals against homologous PRRSV challenge, licensed in various countries and extensively applied in pig farms [47,48]. However, existing evidence suggests that these MLV vaccines of type 1 or type 2 both stimulates limited humoral and cellular immunity and fail to against heterogeneous strain [49,50]. Therefore, it would be possible that ginseng extract used as a natural supplement in feeding management for disease prevention. Besides, it might be used in a combination with some direct-acting antivirals to achieve widely effective disease prevention or as immune-adjuvant of vaccination to reach immune enhancement. For the proper application of it, the pharmacokinetic data of ginseng extract in swine model needs further systematic study. Meanwhile, the optimum dosage and administration mode should be defined reasonably to avoid drug residues in meat production. Considering the economic benefit and most of the nutrient component were similar between PN and PG, PN might possess more potential value of application in the scaled raising of pigs.

In summary, our study demonstrated that ginsenoside Rg1 with low cytotoxicity and possess anti-PRRSV activity both in vitro and in piglets. And, it suppressed different lineages of type 2 PRRSV infections. These findings not only provide new insights into the molecular mechanism of Rg1 against PRRSV infection but also suggest a potential immune-modulatory and anti-viral agent in the control of the PRRS.

**Author Contributions:** Conceptualization, G.-h.Z. and Z.-q.Y.; Methodology, Z.-q.Y. and Y.C.; Software, H.-y.Y.; Validation, Q.L. and R.-t.Z.; Formal Analysis, M.-k.C.; Investigation, Z.-q.Y., H.-y.Y., J.M., C.-x.Q., G.L., Q.D., Y.-j.C., and X.-l.H.; Resources, G.-h.Z., H.W. and K.T.; Data Curation, Y.-f.W.; Writing—Original Draft Preparation, Z.-q.Y.; Writing—Review & Editing, G.-h.Z.; Visualization, Z.-q.Y.; Supervision, H.W.; Project Administration, G.-h.Z. and Z.-q.Y.

**Funding:** This project was supported by the National Natural Science Foundation of China (Grant No. 31872489), the Modern Agro-industry Technology Research System (Grant number CARS-35) and the Natural Science Foundation of Guangdong province (Grant No. 2018A030313712).

**Conflicts of Interest:** The authors declare no conflict of interest.
