桑叶水提物及异槲皮苷成分抗石斑鱼虹彩病毒作用机制研究

【目的】探究桑叶提取物成分对石斑鱼虹彩病毒（SGIV）的抑制作用，并对其抗病毒机制进行研究，为利用桑叶提取物成分研发抗SGIV药物提供理论依据，也为开发高效安全的新型抗病毒渔用药物提供新思路。【方法】利用石斑鱼脾脏组织细胞系（GS），通过光学显微镜观察及CCK-8检测分析桑叶水提物（Morus alba L.water extracts，MAE）及其活性成分异槲皮苷（Isoquercetin，IQ）的安全工作浓度，然后使用实时荧光定量PCR及核酸适配体荧光分子探针技术（Q2-AFMP）分析MAE和IQ对SGIV的抗病毒效果；通过分析SGIV感染GS细胞中MCP基因和VP19基因的表达水平，分析IQ对SGIV的粒子结构及其与宿主细胞表面结合、侵入宿主细胞和在宿主细胞中复制的影响，探究IQ体外抗SGIV的作用机制。【结果】桑叶源化合物成分（MAE和IQ）对GS细胞的最高安全工作浓度为:MAE≤10.0 mg/mL，IQ≤1000.0μg/mL。与接入SGIV的GS细胞相比，在以安全工作浓度的MAE （10.0 mg/mL）和IQ （1000.0μg/mL）分别接入SGIV孵育感染的GS细胞后细胞病变（CPEs）明显减少，MCP基因和VP19基因的相对表达量均呈极显著下降趋势（P<0.01，下同），GS细胞荧光值也极显著降低，表明桑叶源化合物成分能有效抑制SGIV感染。以IQ处理SGIV孵育感染的GS细胞，其细胞内MCP基因和VP19基因的相对表达量均呈极显著下降趋势，提示IQ能破坏SGIV的粒子结构，并干扰SGIV对宿主细胞的吸附、侵入和复制。【结论】MAE和IQ对SGIV具有良好的抗病毒效果，其中IQ能在病毒吸附、侵入和复制阶段发挥抗病毒作用，即桑叶源化合物成分在防治SGIV感染方面具有潜在的应用价值，可作为研发抗SGIV感染的有效药用成分。

The inhibitory activities and antiviral mechanism of Morus alba L. water extracts and isoquercitrin components against grouper iridovirus infection

【Objective】This study aimed to explore the antiviral effects and mechanisms of Morus alba L. extracts on grouper iridovirus(SGIV), and provide data support and theoretical basis for developing anti-SGIV drugs with Morus alba extracts and offer new ideas for developing efficient and safe antiviral fishing drugs.【Method】The safe working concentrations of M. alba water extracts(MAE) and its active component isoquercetin(IQ) were determined on grouper spleen tissue cell line(GS) by light microscope observation and CCK-8 cell viability assay. Then, real-time fluorescence quantitative PCR(RT-qPCR) and aptamer Q2-based fluorescent molecular probe assay(Q2-AFMP) were used to analyze the antiviral effects of MAE and IQ against SGIV in vitro. RT-qPCR was further applied to detect the expression level of MCP gene and VP19 gene in SGIV infected cells, and explore the antiviral mechanism of IQ, including the effects of IQ onSGIV particles, SGIV bound to host cell surface, SGIV invasion and replication in host cells.【Result】The safe working concentrations of M. alba extracts on GS cells were that, MAE ≤ 10.0 mg/mL, IQ ≤ 1000.0 μg/mL. The research results of MAE and IQ against SGIV showed that, compared to GS cells only incubated with SGIV, the cytopathic effects(CPEs) of GS cells incubated with both SGIV and M. alba extracts(10.0 mg/mL MAE and 1000.0 μg/mL IQ) in the experimental groups were greatly reduced. MCP gene and VP19 gene in experimental group cells decreased extremely significantly(P<0.01, the same below), and the fluorescence intensity of GS cells decreased significantly. The above results indicated that MAE and IQ could effectively combat SGIV infection. GS cells incubated with SGIV treated with IQ showed extremely significant decrease in the relative expression of both the intracellular MCP gene and the VP19 gene, it showed that, IQ could destroy the SGIV particles structure and interfere with the adsorption, invasion and replication of SGIV to host cells.【Conclusion】MAE and IQ have great antiviral effects on SGIV, and IQ plays an antiviral role in the stages of virus adsorption, invasion and replication.M. alba extracts have potentials in the treatments of SGIV infection, and can be used in the development of fishery drugs for inhibiting SGIV infection.