Hesperetin inhibits <i>Siniperca chuatsi</i> rhabdovirus replication by reducing mitochondrial apoptosis in EPC cells

Hesperetin is one of the natural dihydroflavonoid widely extracted from various plants, and a large number of studies have demonstrated that hesperetin has a wide range of biological activities. In this study, we explored its antiviral function against Siniperca chuatsi rhabdovirus (SCRV), one strain of important fish viral viruses, resulting in multiple transmissions and devastating damage in aquaculture. Experiments were performed to confirm that hesperetin could effectively delay the occurrence of cytopathic effects (CPEs) in epithelioma papulosum cyprinid cells (EPC), and inhibit SCRV replication by microscopy observation and viral titer assay, respectively. Time-of-addition assay showed that the best inhibitory effect of hesperetin was achieved by blocking SCRV invasion into cells. In addition, qRT-PCR and western blot analysis confirmed that hesperetin could significantly inhibit the expression of SCRV nucleoprotein. Flow cytometry analysis showed that hesperetin could reduce SCRV-inducing apoptosis of EPC cells after annexin V-FITC/PI double staining and immunofluorence microscopy observation after JC-1 incubation. Down-regulation of caspase-3 and caspase-9 enzymatic activity after hesperetin treatment indicated that it might protect cells from SCRV infection by decreasing mitochondrial intrinsic apoptosis in infected cells. After hesperetin treatment, a decrease in ROS and an increase in mitochondrial membrane potential (MMP) in SCRV-infected EPC cells further confirmed the inhibition of the apoptotic pathway mediated by hesperetin. Thus, our present study indicated that hesperetin possessed antiviral ability against SCRV, suggesting its candidate role for potential effective control of fish rhabdovirus spreading.