Magnolol inhibits porcine epidemic diarrhea virus infection by suppressing cathepsin L expression <i>in vitro</i> and <i>in vivo</i>

ABSTRACT Porcine epidemic diarrhea (PED), caused by the porcine epidemic diarrhea virus (PEDV), is an acute and highly contagious intestinal disease in pigs. The high variability of the PEDV S gene has rendered current commercial vaccines ineffective, indicating the need for safe, effective, and cost-effective antiviral agents to control PEDV. Here, we aimed to screen and identify compounds with anti-PEDV activity and elucidate the antiviral mechanisms of magnolol. A compound library was screened using an in-cell Western blotting assay to identify potential antiviral agents. The in vitro antiviral activity of magnolol was evaluated using viral titer assays, time-of-addition experiments, and Western blotting. The in vivo efficacy of magnolol against PEDV was assessed by monitoring body weight, rectal temperature, histopathological changes, and diarrhea scores. Cathepsin L (CTSL) expression and distribution were analyzed in vitro and in vivo using RT-qPCR, Western blotting, and confocal immunofluorescence. A total of 25 potential antiviral candidates were identified, among which magnolol exhibited significant anti-PEDV activity. Magnolol effectively inhibited the entry of PEDV, porcine deltacoronavirus, and porcine enteric alphacoronavirus by suppressing CTSL expression. In vivo experiments showed that magnolol treatment alleviated PED-associated pathological damage and altered CTSL distribution in the intestinal epithelium of piglets, thereby reducing PEDV infection. Mechanistically, magnolol inhibited PEDV infection by downregulating CTSL expression and altering its distribution in intestinal epithelial cells. This study provides valuable experimental evidence and suggests potential therapeutic strategies for the clinical treatment of porcine enteric coronavirus infections. IMPORTANCE Porcine epidemic diarrhea (PED) is an acute and highly contagious intestinal disease in pigs that causes severe intestinal damage and death, especially in piglets. Currently available treatments and vaccines for the disease have been unable to provide complete protection due to the high mutation/variability of the causative virus, porcine epidemic diarrhea virus (PEDV). In this study, we demonstrated for the first time that magnolol, an extract from the roots and bark of Magnolia officinalis , inhibits PEDV infection by downregulating cathepsin L expression and altering its distribution in intestinal epithelial cells, thereby alleviating PED-associated pathological damage in the intestines of piglets. Our findings suggest that magnolol has potential applications in the treatment of PED.