Action mechanism of Semen Ziziphi Spinosae alcohol extracts and betulinic acid against nervous necrosis virus

【Objective】 To investigate the action mechanism of Semen Ziziphi Spinosae methanol extracts（SZSE）and its active ingredient betulinic acid（BA） in resistance of fish nervous necrosis virus（NNV） infection, which could provide theoretical basis for the development of green, efficient and pollution-free anti-NNV fishery drugs. 【Method】 The safe working concentration of SZSE and its active ingredient BA was determined by optical microscope observation, CCK-8 assay, and immunofluorescence. Then, the optical microscope observation, crystal violet staining observation and real-time fluorescence quantitative PCR were used to analyze the antiviral effects of SZSE and its active ingredient BA against NNV. The anti-NNV mechanisms of the two were further explored via real-time fluorescence quantitative PCR. 【Result】 The safe working concentrations of SZSE and its active ingredient BA were SZSE≤50 μg/mL and BA≤6.25 μg/mL respectively. Furthermore, 50 μg/mL SZSE or 6.25 μg/mL BA showed no cytotoxic effects on Trachinotus ovatus splenic fibroblasts cells（TOSF）. NNV co-incubated with TOSF cells at concentrations of 50, 25, 12.5 μg/mL of SZSZ or 6.25, 3.125, 1.5625 μg/mL of BA respectively. After 48 h, the cytopathic effects（CPEs） in drug-treated TOSF cells were found to be greatly lower than in the NNV group, the number of viable TOSF cells in the drug-treated group was greatly higher than in the NNV group after crystal violet staining, the real-time fluorescence quantitative PCR results showed that the relative expression of NNV CP gene in drug-treated TOSF cells was extremely significantly lower than in the NNV group（P＜0.01）, and in a dose-dependent manner. SZSE and its active ingredient BA showed antiviral effects when both were used to treat TOSF cells 2 h before and after NNV infection,indicating that they could not only prevent viral infection but also treat it. In addition, SZSE and its active ingredient BA exerted antiviral effects by interacting with NNV particles and disrupting the attachment, invasion and replication stages of NNV in host cells,as well as regulating the expression of immune-related genes such as CAT, GSH-Px, HO-1, TGF, Keap1 and Nrf2. 【Conclusion】 SZSE and its active ingredient BA have the function of inhibiting NNV infection. They can exert antiviral effects not only by interfering with the adsorption, invasion and replication stages during the process of NNV infecting host cells, but also by regulating the expression of immune-related genes. It can be seen that SZSE and its active ingredient BA have the potential to be developed into green and highly effective anti-NNV fishery drugs.