傳染性胰臟壞死病毒(Infectious Pancreatic Necrosis Virus, IPNV)為一種分佈廣泛的水生生物病毒，可以感染多種經濟性魚貝類。於先前的研究中證實此病毒感染大眼鮭魚胚胎細胞株(CHSE-214)後會誘發細胞凋亡(Apoptosis)。然而，病毒個別基因是否參與誘發宿主細胞凋亡仍不清楚。本論文嘗試證實IPNV病毒外殼蛋白VP3能誘發魚類細胞株凋亡並探討之。此論文的研究發現將VP3蛋白基因接合到綠色螢光蛋白(EGFP)基因後，經轉染至斑馬魚肝臟上皮細胞株(ZLE)中大量表現此VP3蛋白，可觀察到VP3蛋白主要分佈在細胞質中，於轉染第48小時後，會導致約七成的細胞發生凋亡。凋亡特徵除了細胞形態產生變化，還有細胞膜 phosphatidylserine (PS)外翻及細胞核內染色體DNA斷裂。於凋亡分子機制方面，可測得粒線體膜電位之改變，促凋亡蛋白(Bad)被誘導表現上升之情形，以及caspase-8、caspase-9及caspase-3有活化的現象。最後於共轉染Bcl-2家族蛋白zfBcl-xL或zfMcl-1a與VP3後，發現此抗凋亡因子可降低發生凋亡細胞的比例與caspase-3活化的情形。綜合上述結果，證明VP3不僅扮演結構蛋白的角色，亦具有誘發細胞走向凋亡的功能，且分子機制是透過粒線體調控(mitochondria-mediated)與caspase活化(caspase activation)的途徑。希望此論文之研究能助於魚類疾病之預防和治療。

Infectious Pancreatic Necrosis Virus (IPNV) is one of the widespread fish pathogens and infects many economic finfish and shellfish. In the previous studies, IPNV infection could induce apoptosis in chinook salmon embryo (CHSE-214) cell line. However, it was still unclear whether viral protein could involve in triggering host cell death. In the present study, firstly VP3 was fused with EGFP for tracing their proteins localization, which found it mostly distributed in cytoplasm. Then, overexpression of IPNV capsid protein VP3 could induce almost 70% apoptosis in zebrafish liver epithelium (ZLE) cells at 48 h post-transfection (p.t.) time. The VP3 overexpressing ZLE cells exhibited the following apoptotic features: cell morphological change, phosphatidylserine (PS) exposure and chromosome fragmentation. In molecular death mechanism studies, VP3 can (1) disrupt the mitochondrial membrane potential, (2) induce the Bad expression and (3) activate the caspases that include initiator capsase-8 and -9 and effector caspase-3 from 24 h to 48 h p.t. Finally, when Bcl-2 member zfBcl-xL or zfMcl-1a was cotransfected with VP3 in ZLE cells, the VP3-mediated apoptotic cell death could be prevented. Our results suggest that the VP3-induced apoptotic cell death may via up-regulating the pro-apoptotic gene bad expression and go through the mitochondria-mediated pathway for activating the caspases and triggering the apoptotic cell death. These results may provide new insight on prevention of IPNV infection.

官柏志 傳染性胰臟壞死病毒感染大眼鮭魚胚胎細胞株引發轉錄因子
NF-κB 之活化以造成細胞凋亡。台灣大學漁業科學研究所碩士論文。
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周宜黎 傳染性胰臟壞死病毒凋亡基因vp3所誘發細胞凋亡分子機制
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