| Summary: | 碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 98 === White spot syndrome virus (WSSV) is the causative pathogen of white spot disease (WSD), a disease with great impact on the cultured shrimp industry. Pathogenesis of WSSV, however, is poorly understood. It has been postulated that mitochondria may play an important role during WSSV pathogenesis, and that mitochondrial dysfunction may be involved in cell death. Materials move in and out of the mitochondria via the mitochondrial permeability transition pore (MPTP). The outermost area of the pore is critically important in permeability and is composed of a single protein, the voltage dependent anion channel (VDAC) protein. Previous studies found that host VDAC is upregulated after WSSV infection, suggesting a mitochondrial role in pathogenesis. Preliminary microarray analysis showed that hexokinase was down-regulated after WSSV infection. Hexokinase mediates the opening of MPTP, and is also involved in a key metabolic pathway, the glycolytic pathway. There are 3 parts to this study. Because imbalanced VDAC-hexokinase interaction may result in mitochondrial membrane permeabilization (MMP), the study is the first investigation about the impact of WSSV on mitochondrial activity. Three factors (mitochondrial membrane potential, energy production, and oxidative stress) were assessed over a pre- and post-infection time period (0, 12, 24, 36, 48, 60 and 72 hours post-infection). The data showed that beginning at 24h post-WSSV challenge, and increasing over time, host hemocytes showed loss of mitochondrial membrane potential. Also beginning 24 h post-infection, energy production was disrupted (shown in increased ADP/ATP ratio). No oxidative stress was detected. This clearly shows that mitochondrial function was disrupted. Because hexokinase is also involved in glycolysis, fatty acid metabolism and the pentose phosphate pathway (PPP), the second part of this study analyzed their post-infection changes. Amounts of glucose and lactate (for glycolysis), triglycerides (for fatty acid metabolism) and the activity of G6PDH, the key enzyme in the pentose phosphate pathway (PPP) were measured at the same post-infection time points. The data show that at 24 h post-infection glucose concentration was higher than in control group. This indicates that glycolysis was down-regulated. Beginning at 12 h, triglyceride concentration in the infected group was significantly lower than in the control group. G6PDH activity was higher than the control group at 12 and 36 h. As the infection progressed, there was a temporal flux in metabolites. In order to clarify the viral life cycle, and explain the metabolic flux, we further quantified WSSV replication efficiency and found that replication efficiency was highest at 24 h. It appears that the replication cycle and the changed metabolic flux are parallel. Overall the results of this study support the view that viral replication and release are clearly related to metabolic changes in the cell as well as changes in mitochondrial activity.
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