傳染性胰臟壞死病毒(Infectious pancreatic necrosis virus, IPNV)屬於典型的雙股RNA病毒，是感染經濟性魚貝類的重要水生病毒，因此對於其致病機轉的瞭解相當重要。根據先前研究與文獻證實傳染性胰臟壞死病毒感染後會引發宿主細胞造成細胞凋亡。然而內質網在許多細胞中扮演著重要的功能，包括蛋白質的合成或摺疊、細胞內鈣離子的平衡，及與細胞凋亡有關。在病毒感染宿主細胞的過程中，大量病毒蛋白在被感染的細胞內合成，未摺疊或錯誤摺疊的蛋白質會引發內質網壓力。為了抵抗內質網壓力，細胞會啟動一連串反應來紓解內質網壓力，稱作不完全摺疊蛋白反應(unfolded protein response)。本實驗室初步發現隨著傳染性胰臟壞死病毒感染時間增加，內質網壓力的指標GRP78和caspase-12分別表現增加及活化。因此本論文更進一步想要探討以傳染性胰臟壞死病毒感染大眼鮭魚胚胎(CHSE-214)細胞引發內質網壓力的下游訊息傳遞是如何影響粒線體失衡來造成細胞壞死。實驗結果發現在病毒感染下會引發內質網壓力的IRE1及ATF6路徑，增加XBP1活化態蛋白及ATF6蛋白受到切割。另外也會造成PERK蛋白磷酸化並且磷酸化下游eIF2α蛋白，進而促使CHOP轉錄因子的活化來抑制Bcl-2家族抗凋亡蛋白的表現。使用專一性GRP78抑制劑(vomitoxin)時，發現在病毒感染下不會影響PERK路徑中最上游PERK蛋白磷酸化的表現量，但卻能使下游eIF2α蛋白磷酸化減少、進而調控CHOP來增加Bcl-2家族抗凋亡蛋白的表現。此外處理GRP78抑制劑後也發現能抑制病毒的複製，並且利用PKR抑制劑來佐證，病毒會透過PKR路徑來影響eIF2α蛋白磷酸化。更進一步發現處理GRP78抑制劑會影響下游粒線體cytochrome c蛋白的釋出及caspase-3的活化。另外發現在病毒感染下，細胞內鈣離子表現量會增加。使用鈣離子螯合劑(BAPTA)能在病毒感染下螯合細胞內的鈣離子，並且增加細胞存活率、減少cytochrome c蛋白的釋出及粒線體膜電位的改變來影響細胞凋亡。本論文釐清在細胞凋亡時產生內質網壓力訊息與導致粒線體失衡而引發細胞凋亡之間的相互關係。透過此研究能提供我們對於傳染性胰臟壞死病毒的致病機轉能有所貢獻。

Infectious Pancreatic Necrosis Virus (IPNV) is one of the widespread fish pathogen and infects many economically important finfish and shellfish. It is a double-stranded, bi-segmented RNA genome virus (designated segments A and B) and within a medium-sized, non-enveloped single-shelled icosahedral particle. In previous study, IPNV-infected in CHSE-214 cells could induce cell death through apoptosis and post-apoptotic necrosis. The endoplasmic reticulum (ER) plays a vital role in a variety of cellular functions, including protein synthesis, folding, homeostasis of intracellular calcium, and apoptosis. ER stress is triggered when unfolded proteins accumulate in the ER due to increased input of proteins, such as virus infection, a large amount of viral proteins are synthesized in infected cells, which unfolded or misfolded proteins activate the ER stress responses. To cope with the stress, cells activate intracellular signaling pathway-the unfolded protein response (UPR) to provide adaptive responses for survival. In preliminary results, the ER stress markers either GRP78 or caspase-12 was up-regulated and activated with IPNV infection. Thus, we want to investigate IPNV-induced ER stress and this stress signaling how to correlate to induce mitochondrial disfunction in fish necrotic cells. We first found that IPNV infection can activate two components inositol-requiring 1 (IRE1) and activating transcription factor 6 (ATF6). We further observed that it also can activate phosphorylation of protein kinase-like ER kinase (PERK) and eukaryotic translation initiation factor 2α (eIF2α), then induce the expression of transcription factor C/EBP homologous protein (CHOP) leading to down-regulate anti-apoptotic Bcl-2 family proteins such as Bcl-2, Mcl-1 and Bcl-xL. We further treated specific GRP78 inhibitor vomitoxin, which can’t affect PERK phosphorylation, but inhibit eIF2α phosphorylation to block the expression of CHOP and then increase anti-apoptotic Bcl-2 family proteins. Moreover, GRP78 inhibitor also can inhibit IPNV viral replication, which actives double-stranded RNA- dependent protein kinase (PKR) leading to increased eIF2α phosphorylation, and affect mitochondrial cytochrome c release. Furthermore, IPNV infection can introduce calcium releasing from ER, which calcium release blocked by its specific chelator BATPA that effectively rescues cell apoptosis, inhibits cytochrome c release and mitochondria membrane potential loss. Taken together, these results indicate that IPNV-induced ER stress signaling pathway connects with mitochondrial disfunction via down-regulation anti-apoptotic Bcl-2 family proteins in CHSE-214 cell, which may provide insight into IPNV pathogenesis.

邱謙禮，傳染性胰臟壞死病毒外殼蛋白VP3造成斑馬魚肝臟細胞株凋亡分子機制之研究。
成功大學生物科技研究所碩士論文，台南，2006。
陳栢均，傳染性胰臟壞死病毒造成宿主細胞大眼鮭魚胚胎細胞株因粒線體膜電位消失而凋亡之研究。
成功大學生物科技研究所碩士論文，台南，2007。
劉嫣婷，傳染性胰臟壞死病毒與其促凋亡基因VP3誘發凋亡功能之探討。
成功大學生物科技研究所碩士論文，台南，2008。
Ashkenazi, A., and Dixit, V. M. (1998). Death receptors: signaling and modulation. Science. 281(5381): 1305-1308.
Baltzis, D., Qu, L. K., Papadopoulou, S., Blais, J. D., Bell, J. C., Sonenberg, N., and Koromilas, A. E. (2004). Resistance to vesicular stomatitis virus infection requires a functional cross talk between the eukaryotic translation initiation factor 2alpha kinases PERK and PKR. J Virol. 78(23): 12747-12761.
Bitko, V., and Barik, S. (2001). An endoplasmic reticulum-specific stress- activated caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells by respiratory syncytial virus. J Cell Biochem. 80(3): 441-454.
Boya, P., Cohen, I., Zamzami, N., Vieira, H. L., and Kroemer, G. (2002). Endoplasmic reticulum stress-induced cell death requires mitochondrial membrane permeabilization. Cell Death Differ. 9(4): 465-467.
Braakman, I., Hoover-Litty, H., Wagner, K. R. and Helenius, A. (1991). Folding of influenza hemagglutinin in the endoplasmic reticulum. J Cell Biol. 114:401-411.
Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72: 248-254.
Brown, D. G., Sun, X. M., and Cohen, G. M. (1993). Dexamethasone-induced apoptosis involves cleavage of DNA to large fragments prior to internucleosomal fragmentation. J Biol Chem. 268(5): 3037-3039.
Chen, S. N., Chi, S. C., Guu, J. J., Chen, J. C. and Kou, G. H. (1984). Pathogenicity of birnavirus isolated from loach CoA Fisheries series No. 10. Fish Disease Res. 6: 6-11.
Christie, K. E., Havarstein, L. S., Djupvik, H. O., Ness, S., and Endresen, C. (1988). Characterization of a new serotype of infectious pancreatic necrosis virus isolated from Atlantic salmon. Arch Virol. 103(3-4): 167-177.
Cortez-San Martin, M., Villanueva, R. A., Jashes, M., and Sandino, A. M. (2009). Molecular characterization of IPNV RNA replication intermediates during the viral infective cycle. Virus Res.
Cregan, S. P., Dawson, V. L., and Slack, R. S. (2004). Role of AIF in caspase- dependent and caspase-independent cell death. Oncogene. 23(16): 2785- 2796.
Dobos, P. and Roberts, T. E. (1983). The molecular biology of infectious pancreatic necrosis virus: a review. Can J Microbiol. 29: 377-384.
Dobos, P. (1976). Size and structure of the genome of infectious pancreatic necrosis virus. Nucleic Acids Res. 3(8): 1903-1924.
Dobos, P., and Rowe, D. (1977). Peptide map comparison of infectious pancreatic necrosis virus-specific polypeptides. J Virol. 24(3): 805-820.
Duncan, R., Nagy, E., Krell, P. J., and Dobos, P. (1987). Synthesis of the infectious pancreatic necrosis virus polyprotein, detection of a virus-encoded protease, and fine structure mapping of genome segment A coding regions. J Virol. 61(12): 3655-3664.
Erwig, L. P., and Henson, P. M. (2007). Immunological consequences of apoptotic cell phagocytosis. Am J Pathol. 171(1): 2-8.
Fadeel, B., and Orrenius, S. (2005). Apoptosis:a basic biological phenomenon with wide-ranging implications in human disease. J Intern Med. 258(6): 479-517.
Fridholm, H., Eliasson, L., and Everitt, E. (2007). Immunogenicity properties of authentic and heterologously synthesized structural protein VP2 of infectious pancreatic necrosis virus. Viral Immunol. 20(4): 635-648.
Gale, M., Jr., and Katze, M. G. (1998). Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon- induced protein kinase. Pharmacol Ther. 78(1): 29-46.
Garner, J. N., Joshi, B., and Jagus, R. (2003). Characterization of rainbow trout and zebrafish eukaryotic initiation factor 2alpha and its response to endoplasmic reticulum stress and IPNV infection. Dev Comp Immunol. 27(3): 217-231.
Green, D. R., and Reed, J. C. (1998). Mitochondria and apoptosis. Science. 281(5381): 1309-1312.
Green, D. R., Knight, R. A., Melino, G., Finazzi-Agro, A., and Orrenius, S. (2004). Ten years of publication in cell death. Cell Death Differ. 11(1): 2-3.
Harrison, S. M., and Bers, D. M. (1987). The effect of temperature and ionic strength on the apparent Ca-affinity of EGTA and the analogous Ca-chelators BAPTA and dibromo-BAPTA. Biochim Biophys Acta. 925(2): 133-143.
He, B. (2006). Viruses, endoplasmic reticulum stress, and interferon responses. Cell Death Differ. 13(3): 393-403.
Hedrick, R. P., Fryer, J. L., Chen, S. N. and Kou, G. H. (1983). Characteristics of four birnavirus isolated from fish in Taiwan. Fish Pathol. 18: 91-97.
Hill, B. J. and Way, K. (1983). Serological classification of fish and shellfish birnaviruses. In abstract of Fish Interactional Conference of the European Association of Fish pathologists. pp.10.
Hong, J. R., Lin, T. L., Hsu, Y. L., and Wu, J. L. (1998 a). Induction of apoptosis and secondary necrosis by infectious pancreatic necrosis virus in fish embryonic cells. J Fish Diseases. 21: 463-467.
Hong, J. R., Lin, T. L., Hsu, Y. L., and Wu, J. L. (1998 b). Apoptosis precedes necrosis of fish cell line with infectious pancreatic necrosis virus infection. Virology. 250(1): 76-84.
Hong, J. R., Lin, T. L., Yang, J. Y., Hsu, Y. L., and Wu, J. L. (1999 a). Dynamics of nontypical apoptotic morphological changes visualized by green fluorescent protein in living cells with infectious pancreatic necrosis virus infection. J Virol. 73(6): 5056-5063.
Hong, J. R., Hsu, Y. L., and Wu, J. L. (1999 b). Infectious pancreatic necrosis virus induces apoptosis due to down-regulation of survival factor MCL-1 protein expression in a fish cell line. Virus Res. 63(1-2): 75-83.
Hong, J. R., Gong, H. Y., and Wu, J. L. (2002 a). IPNV VP5, a novel anti-apoptosis gene of the Bcl-2 family, regulates Mcl-1 and viral protein expression. Virology. 295(2): 217-229.
Hong, J. R., and Wu, J. L. (2002 b). Induction of apoptotic death in cells via Bad gene expression by infectious pancreatic necrosis virus infection. Cell Death Differ. 9(2): 113-124.
Hong, J. R., Guan, B. J., Her, G. M., Evensen, O., Santi, N., and Wu, J. L. (2008). Aquatic birnavirus infection activates the transcription factor NF-kappaB via tyrosine kinase signalling leading to cell death. J Fish Diseases. 31(6): 451-460.
Hoyer-Hansen, M., and Jaattela, M. (2007). Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium. Cell Death Differ. 14(9): 1576-1582.
Hsu, Y. L., Chen, B. S., and Wu, J. L. (1989). Comparison of RNAs and polypeptides of infectious pancreatic necrosis virus isolates from eel and rainbow trout. J Gen Virol. 70: 2233-2239.
Hu, Y., and Conway, T. W. (1993). 2-Aminopurine inhibits the double- stranded RNA-dependent protein kinase both in vitro and in vivo. J Interferon Res. 13(5): 323-328.
Imajoh, M., Hirayama, T., and Oshima, S. (2005). Frequent occurrence of apoptosis is not associated with pathogenic infectious pancreatic necrosis virus (IPNV) during persistent infection. Fish Shellfish Immunol. 18(2): 163-177.
Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 26(4): 239-257.
Kim, R., Emi, M., Tanabe, K., Murakami, S., Uchida, Y., and Arihiro, K. (2006). Regulation and interplay of apoptotic and non-apoptotic cell death. J Pathol. 208(3): 319-326.
Kochan, G., Gonzalez, D., and Rodriguez, J. F. (2003). Characterization of the RNA-binding activity of VP3, a major structural protein of Infectious bursal disease virus. Arch Virol. 148(4): 723-744.
Kroemer, G. (1997). The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat Med. 3(6): 614-620.
Kroemer, G., Dallaporta, B., and Resche-Rigon, M. (1998). The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol. 60: 619-642.
Kuang, E., Wan, Q., Li, X., Xu, H., Liu, Q., and Qi, Y. (2005). ER Ca2+ depletion triggers apoptotic signals for endoplasmic reticulum (ER) overload response induced by overexpressed reticulon 3 (RTN3/HAP). J Cell Physiol. 204(2): 549-559.
Le Bras, M., Rouy, I., and Brenner, C. (2006). The modulation of inter- organelle cross-talk to control apoptosis. Med Chem. 2(1): 1-12.
Lo, C. F., Hong, Y. W., Huang, S. Y., and Wang, C. H. (1988). The characteristics of the virus isolated from the gill of clam, Meretrix lusoria. Fish Pathol. 23: 147-154.
Lo, E. G. (1987). Biochemical and immunological Microterus salmmoids (Lacpede). Master Thesis.
Macdonald, R. D., and Gower, D. A. (1981). Genomic and phenotypic divergence among three serotypes of aquatic birnaviruses (infectious pancreatic necrosis virus). Virology. 114: 187-195.
Magnelli, L., Cinelli, M., Turchetti, A., and Chiarugi, V. P. (1994). Bcl-2 overexpression abolishes early calcium waving preceding apoptosis in NIH-3T3 murine fibroblasts. Biochem Biophys Res Commun. 204(1): 84-90.
Majno, G., and Joris, I. (1995). Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol. 146(1): 3-15.
McCullough, K. D., Martindale, J. L., Klotz, L. O., Aw, T. Y., and Holbrook, N. J. (2001). Gadd153 sensitizes cells to endoplasmic reticulum stress by down- regulating Bcl2 and perturbing the cellular redox state. Mol Cell Biol. 21(4): 1249-1259.
Meyer, M., Caselmann, W. H., Schluter, V., Schreck, R., Hofschneider, P. H., and Baeuerle, P. A. (1992). Hepatitis B virus transactivator MHBst: activation of NF-kappa B, selective inhibition by antioxidants and integral membrane localization. EMBO J. 11(8): 2991-3001.
Momoi, T. (2004). Caspases involved in ER stress-mediated cell death. J Chem Neuroanat. 28(1-2): 101-105.
Nakagawa, T., and Yuan, J. (2000). Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. J Cell Biol. 150(4): 887-894.
Nishitoh, H., Matsuzawa, A., Tobiume, K., Saegusa, K., Takeda, K., Inoue, K., Hori, S., Kakizuka, A., and Ichijo, H. (2002). ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev. 16(11): 1345-1355.
Ohoka, N., Yoshii, S., Hattori, T., Onozaki, K., and Hayashi, H. (2005). TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death. EMBO J. 24: 1243-1255.
Packard, B. Z., and Komoriya, A. (2008). Intracellular protease activation in apoptosis and cell-mediated cytotoxicity characterized by cell-permeable fluorogenic protease substrates. Cell Res. 18(2): 238-247.
Pahl, H. L., and Baeuerle, P. A. (1996). Activation of NF-kappa B by ER stress requires both Ca2+ and reactive oxygen intermediates as messengers. FEBS Lett. 392(2): 129-136.
Pahl, H. L., Sester, M., Burgert, H. G. and Baeuerle, P. A. (1996). Activation of transcription factor NF-kappaB by the adenovirus E3/19K protein requires its ER retention. J Cell Biol. 132: 511-522.
Pedersen, T., Skjesol, A., and Jorgensen, J. B. (2007). VP3, a structural protein of infectious pancreatic necrosis virus, interacts with RNA- dependent RNA polymerase VP1 and with double-stranded RNA. J Virol. 81(12): 6652- 6663.
Petit, S., Lejal, N., Huet, J. C., and Delmas, B. (2000). Active residues and viral substrate cleavage sites of the protease of the birnavirus infectious pancreatic necrosis virus. J Virol. 74(5): 2057-2066.
Rao, R. V., Castro-Obregon, S., Frankowski, H., Schuler, M., Stoka, V., del Rio, G., Bredesen, D. E., and Ellerby, H. M. (2002). Coupling endoplasmic reticulum stress to the cell death program. An Apaf-1-independent intrinsic pathway. J Biol Chem. 277(24): 21836-21842.
Rao, R. V., Ellerby, H. M., and Bredesen, D. E. (2004). Coupling endoplasmic reticulum stress to the cell death program. Cell Death Differ. 11(4): 372-380.
Reed, J. C., and Kroemer, G. (2000). Mechanisms of mitochondrial membrane permeabilization. Cell Death Differ. 7(12): 1145.
Rock, K. L., and Kono, H. (2008). The inflammatory response to cell death. Annu Rev Pathol. 3: 99-126.
Ron, D., and Walter, P. (2007). Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol. 8: 519-529.
Rong, Y., and Distelhorst, C. W. (2008). Bcl-2 protein family members: versatile regulators of calcium signaling in cell survival and apoptosis. Annu Rev Physiol. 70: 73-91.
Rutkowski, D. T., and Kaufman, R. J. (2004). A trip to the ER: coping with stress. Trends Cell Biol. 14(1): 20-28.
Samali, A., Gorman, A. M., and Cotter, T. G. (1996). Apoptosis -- the story so far. Experientia. 52(10-11): 933-941.
Schroder, M., and Kaufman, R. J. (2005). The mammalian unfolded protein response. Annu Rev Biochem. 74: 739-789.
Shi, Y. (2002). Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell. 9(3): 459-470.
Shwed, P. S., Dobos, P., Cameron, L. A., Vakharia, V. N., and Duncan, R. (2002). Birnavirus VP1 proteins form a distinct subgroup of RNA- dependent RNA polymerases lacking a GDD motif. Virology. 296(2): 241-250.
Snieszko, S. F., Wolf, K., Camper, J. E., and Pettijohn, L. L. (1959). Infectious nature of pancreatic necrosis. Trans Am Fish Soc. 88: 289-293.
Su, H. L., Liao, C. L., and Lin, Y. L. (2002). Japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response. J Virol. 76(9): 4162-4171.
Tardif, K. D., Mori, K., and Siddiqui, A. (2002). Hepatitis C virus subgenomic replicons induce endoplasmic reticulum stress activating an intracellular signaling pathway. J Virol. 76(15): 7453-7459.
Underwood, B. O., Smale, C. J., Brown, F., and Hill, B. J. (1977). Relationship of a virus from Tellina tenuis to infectious pancreatic necrosis virus. J Gen Virol. 36(1): 93-109.
Wang, K. K. (2000). Calpain and caspase: can you tell the difference? Trends Neurosci. 23(1): 20-26.
Widlak, P., and Garrard, W. T. (2005). Discovery, regulation, and action of the major apoptotic nucleases DFF40/CAD and endonuclease G. J Cell Biochem. 94(6): 1078-1087.
Wolf, K., and Mann, J. A. (1980). Pokilother vertebrate cell line and virus: a current listing for fish. Virology. 16: 168-179.
Wolf, K., Snieskol, S. F., Dumbar, C. E., and Pyle, E. (1960). Virus nature of infectious pancreatic necrosis in trout. Proc Soc Exp Biol Med. 104: 105-108.
Wood, D. E., Thomas, A., Devi, L. A., Berman, Y., Beavis, R. C., Reed, J. C., and Newcomb, E. W. (1998). Bax cleavage is mediated by calpain during drug-induced apoptosis. Oncogene. 17(9): 1069-1078.
Wood, E. M., Snieszko, S. F., and Yasutake, W. T. (1955). Infectious Pancreatic Necrosis in Brook Trout. AMA Arch Pathol. 60: 26-28.
Wu, J. L., Chang, C. Y., and Hsu, Y. L. (1987). Characteristics of an infectious pancreatic necrosis like virus isolated from Japanese eel (Amguolla japonica). Bulletin of the Institute of Zoology. Academia Sinica. 26: 210-214.
Yang, G. H., Li, S., and Pestka, J. J. (2000). Down-regulation of the endoplasmic reticulum chaperone GRP78/BiP by vomitoxin (Deoxynivalenol). Toxicol Appl Pharmacol. 162(3): 207-217.
Yoneda, T., Imaizumi, K., Oono, K., Yui, D., Gomi, F., Katayama, T., and Tohyama, M. (2001). Activation of caspase-12, an endoplastic reticulum (ER) resident caspase, through tumor necrosis factor receptor-associated factor 2-dependent mechanism in response to the ER stress. J Biol Chem. 276(17): 13935-13940.
Zecchin, K. G., Seidinger, A. L., Chiaratti, M. R., Degasperi, G. R., Meirelles, F. V., Castilho, R. F., and Vercesi, A. E. (2007). High Bcl-2/Bax ratio in Walker tumor cells protects mitochondria but does not prevent H2O2-induced apoptosis via calcineurin pathways. J Bioenerg Biomembr. 39(2): 186-194.
Zong, W. X., Li, C., Hatzivassiliou, G., Lindsten, T., Yu, Q. C., Yuan, J., and Thompson, C. B. (2003). Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis. J Cell Biol. 162(1): 59-69.