Exopolysaccharides from Enterococcus faecium WH strain inhibit porcine epidemic diarrhea virus infection in vitro

Porcine epidemic diarrhea virus (PEDV) primarily triggers acute enteric infection in neonatal piglets, leading to high mortality and severe economic losses in the swine industry. Probiotics and their extracellular products have shown antiviral potential, but the active components and mechanisms remain unclear. In this study, both Enterococcus faecium WH strain and its cell-free culture supernatant (CFS) significantly inhibited PEDV infection in vitro. Exopolysaccharides (EPSs) isolated from the CFS suppressed PEDV infection in a dose-dependent manner, as demonstrated by quantitative real-time PCR (RT-qPCR), Western blotting, 50 % tissue culture infective dose (TCID₅₀), and immunofluorescence assay (IFA). Further purification by DEAE-650M chromatography yielded neutral (EPS-1) and acidic (EPS-2) fractions, with EPS-1 identified as the major bioactive component. EPS-1 significantly reduced PEDV N protein expression and viral titers, exhibiting stronger inhibition than EPS-2. Structural analysis revealed that EPS-1 is a heteropolysaccharide with a molecular weight (Mw=2.54 × 10⁴ Da) composed of glucose, mannose, galactose, and glucosamine (60:37:2:1) with FT-IR confirming α- and β-configurations in pyranose rings. Functional assays demonstrated that EPS-1 primarily interfered with the replication stage of PEDV lifecycle and suppressed PEDV-induced activation of the MAPK pathway (p38 and JNK), reduced intracellular reactive oxygen species (ROS) accumulation, and modulated inflammatory cytokine expression by downregulating IL-8 and TNF-α while enhancing TGF-β. Collectively, these findings indicate that EPS from E. faecium WH contributes to the inhibition of PEDV infection and provide a scientific basis for the development of probiotic-derived polysaccharides as antiviral agents in swine production.