**1. Introduction**

Porcine reproductive and respiratory syndrome (PRRS), characterized by respiratory distress, reproductive failure in pregnant sows and high mortality in piglets [1], is one of the most epidemic porcine infectious diseases that cause huge economic losses in the worldwide pig industry. Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-sense, single-stranded RNA virus,

and it belongs to the Arteriviridae family [2]. PRRSV is divided into two genotypes, including European strains (type 1) and North American strains (type 2) [3]. Type 2 PRRSV is dominant in China for decades, and it is further classified into nine lineages based on the nucleotide sequence of the ORF5 gene [4,5].

Marc-145 cells, purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA) and saved in our lab, were cultured in Dulbecco's Minimum Essential Medium (DMEM, Biological Industries, Kibbutz Beit Haemek, Israel), supplemented with 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit Haemek, Israel) at 37 ◦C with 5% CO2. Porcine alveolar macrophages (PAMs) were collected from the fresh lungs of 4-week-old Large-White piglets, which were free of the PRRSV and anti-PRRSV antibody, and prepared as previously described [6]. PAMs were grown in RPMI 1640 (Gibco, UT, USA) which contains 10% FBS and 100 IU/mL penicillin and 100 μg/mL streptomycin.

In recent decades, the emergence of a novel PRRSV strain and worldwide transmission attracted increasing attention [7–11]. Current major preventive strategies focus on the vaccine application. However, the poorly studied immunosuppression of pigs to PRRSV infection, virus evolution, multiple-recombination event between wild-type strain and Modified Live Virus (MLV), and currently licensed vaccines, fail to offer effective protection against the challenge of a heterogeneous strain, posing a great challenge to vaccine development. Thus, a new strategy for controlling this infectious disease is urgently needed.

Many natural compounds and herbal components have been confirmed to possess antiviral activities in Traditional Chinese Medicine (TCM). Natural herbal extracts contain many bioactive compounds featuring anti-inflammatory, antiviral and immune-regulatory activities, especially flavaspidic acid AB [12], glycyrrhizin [13] and platycodin D [14], which have been demonstrated to suppress PRRSV infection in vitro. The development of novel drugs might become an effective means to fight the global PRRS epidemic. However, the in vivo study of confirming TCM as a potential natural and effective anti-PPRSV agent was poorly described. Therefore, further studies in swine are necessary.

Ginsenosides are biologically-active components of Panax ginseng and Panax notoginseng saponins that were widely used as a traditional herbal tonic in China for a thousand years, and ginsenoside Rg1 is a major bioactive component therein [15] (Figure 1A). A previous study demonstrates that Rg1 treatment enhances the immune responses induced by recombinant Toxoplasma gondii SAG1 antigen [16], and Rg1 could be used as an adjuvant to promote both T helper (Th) 1 and Th2 responses [17,18]. Treatment with Rg1 is found to significantly relieve the cellular inflammatory response in neurons [19] and reduce the expression of TNF-α, IL-1β and IL-6 in vivo [20]. However, the antiviral activity of Rg1 is poorly described. PRRSV infection results in the release of IL-1β, IL-6, IL-8 and TNF-α, and these pro-inflammatory cytokines contribute to the development of excessive systemic inflammatory reactions and pathological injury [6]. Thus, we set out to determine whether Rg1 has anti-PRRSV effects and possesses protective effects against viruses-induced injury.

Here we demonstrate that Rg1 exhibits an antiviral effect against a broad range of type 2 PRRSV in Marc-145 cells and PAMs, and Rg1 treatment reduces the mRNA levels of several pro-inflammatory cytokines triggered by PRRSV infection, and also inhibits the activation of the NF-κB signaling pathway. More importantly, piglets treated with Rg1 display decreased viremia, alleviated lung injury and increased survival rate after challenging with HP-PRRSV JXA1. Together, these data suggested that Rg1 might be a potential natural compound that could be applied in a PRRSV control strategy.
