Studies on betanodavirus induces oxidative stress and this stress modulated by viral protein B1 in GF-1 cells

• Main Authors: Chih-wei Chang, 張芝瑋
• Other Authors: Jiann-Ruey Hong
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/66192862884001725430

碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 97 === Betanodavirus is the causative agent of a highly infectious disease in larva of grouper fish and in other economic fishes and results in severe mortality and significant economic losses. In previous studies, RGNNV can induce necrotic cell death via mitochondrial membrane permeability (MMP) loss in fish cells. In this present, we want to know whether MMP loss connects to produce the reactive oxygen species (ROS) within oxidative stress. First, to investigate that RGNNV could induce intracellular ROS production in grouper cells (GF-1), we used the fluorescent probe carboxy-H2DCFDA staining assay and Amplex Red to measure concentration of hydrogen peroxide. Our data shows that RGNNV infection leads to accumulation of the ROS at 24 p.i. RGNNV infection also induce antioxidant enzyme response, such as Cu/Zn SOD and catalase in GF-1. Further, either treatment with DPI (diphenyliodonuim), an inhibitor of NADPH oxidase activity, or NAC (N-acetylcysteine), a ROS scavenger, also can effectively block ROS production and reduce MMP loss in early-middle stages of replication cycle. Second, we want to remove hydrogen peroxide by overexpression of catalase for reducing cell damage-induced by oxidative stress. Our data shows that overexpression of zebrafish catalase in GF-1 can prevent RGNNV-induced ROS production and delay cell death. These data suggest that RGNNV induces oxidative stress and mitochondria-mediated cell death may via ROS for triggering MMP loss in grouper cells. On the other hand, the non-structure proteins B1 which play a role on anti-necrotic death function. To understand how protein B1 effect between cell death and oxidative stress are examined. In the present, knockdown of B1 expression by antisense RNA promoted ROS production and decreased Cu/Zn SOD and catalase expression. Moreover, we found that protein B1 can modulate viral replication by northern blot analysis, which reduce viral replication at 12 and 24 p.i. Our results suggest that RGNNV can induce the oxidative stress response and viral protein B1 can against oxidative injury. These results provide insights into RGNNV infection pathogenesis that provide a firm theoretical basis for the investigation of antioxidant therapies in this important disease in future.