In Vitro Assessment of Antiviral Activity of Saikosaponin B2 Against Porcine Epidemic Diarrheal Virus

Porcine epidemic diarrhea virus (PEDV) variant strains lead to vaccine failures and significant economic losses in swine farming, highlighting the need for new natural small-molecule antiviral candidates. In this study, we screened a library of 45 small molecules using a cytopathic effect (CPE)-based cytoprotective assay. Saikosaponin B2 (SSB2), a triterpenoid saponin derived from Bupleuri Radix, emerged as the most promising candidate, boasting a selectivity index of 19.77, which surpasses that of most natural compounds. Dose–response tests confirmed SSB2’s concentration-dependent anti-PEDV activity, with an inhibitory plateau reached at concentrations above 60 μmol/L (μM). The 50 half-maximal effective concentration (EC50) values differed between CPE reduction and viral titer readouts, a discrepancy commonly due to varying detection sensitivities. Mechanistic assays revealed that SSB2 does not possess direct virucidal properties; instead, its antiviral efficacy depends on sustained treatment to inhibit viral adsorption and internalization. Time-of-addition experiments further demonstrated that SSB2 effectively suppresses both early viral entry and mid-stage intracellular genome replication, without affecting viral release. Notably, post-infection treatment resulted in stronger inhibition compared to continuous administration, possibly due to disrupted cellular proliferation homeostasis from prolonged exposure. Additionally, SSB2 maintained strong inhibitory effects against prevalent G2a (JL-06) and G2b (JL-08) field variants, though its efficacy was reduced for both strains and slightly weaker against JL-08 than JL-06. Overall, this study systematically characterizes SSB2’s dual-stage anti-PEDV profile and its broad-spectrum activity against spike-mutant strains, supporting SSB2 as a promising herbal lead for developing veterinary antivirals targeting variant PEDV.