神經壞死病毒是屬於結病毒屬，會感染許多高經濟魚種，造成魚類的高死亡率，而使得漁業經歷重大經濟損失衝擊。在這項實驗中，我們研究神經壞死病毒在石斑魚細胞所引起的細胞死亡現象，經由兩個部分來探討，第一個部分為神經壞死病毒感染細胞後所產生死亡的外型變化，另一部份則是神經壞死病毒誘發石斑魚細胞死亡的分子機制。在型態方面，(一)我們建立帶有綠螢光蛋白的穩定表現之細胞株來觀察其形態方面的改變，例如細胞圓化，從培養盤分離、最後造成細胞死亡。(二)以TUNEL assay偵測細胞內的核酸片段化現象。(三)以annexin V標定早期凋亡細胞，由這些方法顯示細胞受病毒感染後早期會發生細胞凋亡而晚期以annexin V/PI方法推測以細胞壞死方式死亡。

　　除了確定神經壞死病毒感染石斑魚細胞引起的細胞凋亡現象之外，我們將研究神經壞死病毒在石斑魚細胞所引起凋亡相關的基因和訊息路徑。根據之前研究指出神經壞死病毒在海鱸魚細胞中，可能經由Caspase路徑引起細胞凋亡，在本論文中我們證實神經壞死病毒誘發細胞死亡和cytochrome c的釋放相關，而cytochrome c的釋放和粒線體膜電位去極化有相關。而在細胞凋亡扮演重要角色且有高度保留性的Bcl-2 family，經由在細胞中大量表現，則具有保護神經壞死病毒誘發細胞凋亡的能力。

　　希望藉由探討石斑魚細胞受到神經壞死病毒感染後細胞外型變化與凋亡分子機制之探討對致病機轉有更進一步的了解，進而能對疾病之控制有所幫助。

　Nervous necrosis virus (NNV), a member of the virus family Nodaviridae, causes an acute, contagious disease in a number of economically important fish species and resulted in severe mortality and significant economic losses to the aquaculture industry. Apoptosis is the process whereby an individual cell of multicellular organisms undergoes systematic self-destruction in response to a wide variety of stimuli, such as virus infection. In this study, we investigated that NNV induced host grouper liver cell (GL-av) death from morphological changes to death molecular mechanism. In the morphological changes of NNV-infected cell studies: (1) We establish the EGFP stable cell, which could observe a series of the morphological changes of NNV-infected cells that are include cellular detachment, rounding up, and cellular proteins aggregation around the nucleus; (2) In TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling) positive assay, we found that NNV infection can induces DNA fragment gradually at different time points as from 0 h to 48 h post-infection (p.i.).

　On the other hand, in the molecular mechanism studies, in our results, we found: (1) NNV-induced apoptosis in GL-av cells is need newly protein synthesis, because CHX (0.33 ug/ml) treated with GL-av cells could prevented cell death during NNV infection; (2) NNV-induced apoptosis in GL-av cells may mediate to alter the mitochondrial permeability changed for cytochrome c releasing from intermembrane; (3) The related genes of Bcl-2 family such as zebrafish Bcl-XL and Mcl-1a that all could block GL-av apoptotic cell death induced by NNV.

　In summary, the NNV induces the GL-av apoptotic cell death that may be via modulated the cytochrome c releasing for triggered the cell death, which some potent drugs may be useful for rational design of strategies for the prevention and/or treatment of NNV induces diseases in the future.

羅秉真，南台灣養殖石斑魚病毒性神經壞死症之研究。國立台灣大學
動物學研究所碩士論文，台北，1997
Adams J. M., and Cory S. The Bcl-2 protein family: arbiters
of ell survival. Science 281(5381): 1322-1326. (1998).
Anne R., Richard C. M., and Philip E. B. Viruses and apoptosis. Annu. Rev. Microbiol. 53: 577-628. (1999).
Ashkenazi A., and Dixit V. M. Death receptors: signaling and
modulation. Science 281(5381): 1305-1308. (1998).
Bingle C. D., Craig R. W., Swales B.M., Singleton V., Zhou P., and Whyte M.K. Exon Skipping in Mcl-1 Results in a Bcl-2 Homology Domain 3 (BH3)-only Gene Product that Promotes Cell Death.J. Biol. Chem.275(29): 22136-46. (2000).
Bae J., Leo C., Hsu S.,and Hsueh A. Mcl-1S, a splicing variant of the antiapoptotic Bcl-2 family member Mcl-1, encodes a roapoptotic protein possessing only the BH3-domain.
J. Biol.Chem. 275(33): 25255-261. (2000).
Boonyaratpalin S., Supamattaya K., Kasornchandra J.,and Hoffmann R. W. Picona - like virus associated with mortality and a spongious encephalopathy in grouper Epinephelus malabaricus. Dis. Aquat. Org. 26:75-80. (1996)
Brown D. G., Sun X. M., and Cohen G. M. Dexamethasone-induced
apoptosis involves cleavage of DNA to large fragments prior
internucleosomal fragmentation. J. Biol. Chem. 268: 3037-3039.
(1993).
Chao D. T., and Korsmeyer S. J. BCL-2 family: regulators of cell death. Annu. Rev. Immunol. 16: 395-419. (1998).
Chou J. J., Li H., Salvesen G. S., Yuan J., and Wagner G.
Solution structure of BID, an intracellular amplifier of
apoptotic signaling. Cell 96(5): 615-624. (1999).
Desagher S., and Martinou J. C. Mitochondria as the central
control point of apoptosis. Trends Cell Biol. 10(9): 369-377.
(2000).
Eick D., and Hermeking H. Viruses as pacemakers in the evolution of defence mechanisms against cancer. Trends Genet. 12: 4-6.(1996).
Eleouet J.F., Druesne N., Chilmonczyk S., Monge D., Dorson M., and Delmas B. Comparative study of in-situ cell death induced by the viruses of viral haemorrhagic septicaemia (VHS) and infectious pancreatic necrosis (IPN) in rainbow trout. J. Comp. Pathol.124(4): 300-7. (2001).
Essbauer S., and Ahne W. The epizootic haematopoietic necrosis virus(Iridoviridae) induces apoptosis in vitro. J.Vet.Med. B Infect.Dis.Vet.Public Health 49(1): 25-30. (2002).
Franceschi C. Cell proliferation, cell death and aging. Aging
1(1): 3-15. (1989).
Green D. R., and Reed J. C. Mitochondria and apoptosis. Science 281(5381): 1309-1312. (1998).
Guido K., Bruno D., and Michele R. R. The mitochondrial
death/life regulator in apoptosis and necrosis. Annu. Rev.
Physiol. 60: 619-42. (1998).
Guo Y.X., Wei T,, Dallmann K., and Kwang J. Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein alpha as an apoptosis inducer. Virology 308(1): 74-82.(2003).
Harrington E. A., Bennett M. R., Fandini A., and Evan G. I.
Modulation of myc–induced apoptosis by specific cytokines.
EMBO J.13: 3286-3295. (1994).
Hong J. R., and Wu J. L. VP5, a novel anti-apoptosis gene of the Bcl-2 family, regulates Mcl-1 and viral protein expression. Virology 295(2): 217-29. (2002).
Hong J. R., and Wu J. L. Induction of apoptosis death in cell via Bad gene expression by infectious pancreatic necrosis virus
infection. Cell Death Differ. 9: 113-124. (2002).
Hong J. R., and Wu J. L. Molecular regulation of cellular apoptosis by fish infectious pancreatic necrosis virus
infection.Current Topics in Virology (Review)2: 151-160. (2002).
Imajoh M., Sugiura H., and Oshima S. Morphological changes contribute to apoptotic cell death and are affected by caspase-3 and caspase-8 inhibitors during red sea bream iridovirus permissive replication. Virology 322(2): 220-30. (2004).
Kelekar A., and Thompson C. B. Bcl-2-family proteins: the role of the BH3 domain in apoptosis. Trends Cell Biol. 8: 324-329. (1998).
Kerr J. F. R., Wyllie A. H., and Currie A. R. Apoptosis : a basic biological phenomenon with wide ranging implications in tissue kinetics. Br.J. cancer 26: 1790-1794. (1972).
Kerr J. F. R. Neglected apportunities in apoptosis research.
Trends Cell Biol. 5: 55-57. (1995).
Kroemer G. The proto-oncogene Bcl-2 and its role in regulation apoptosis. Nature Med. 3: 614-620. (1997).
Kumar S., and Harvey N. L. Role of multiple cellular protease in the execution of programmed cell death. FEBS Lett. 375: 169-173.(1995).
Ball L.A. Nodavirus RNA recombination. Seminar in virology. 8:95-100.(1997).
McDonnell J. M., Fushman D., Milliman C. L., Korsmeyer S. J.,and Cowburn D. Solution structure of the proapoptotic
molecule BID: a structural basis for apoptotic agonists and
antagonists. Cell 96(5): 625-634. (1999).
Monti D., Troiano L., Grassilli E., Agnesini C., Tropea F., Barbieri D., Capri M., Cristofalo E. A., Salvioli S., and
Ronchetti I. Cell proliferation and cell death in immunosenescence. Ann. N. Y. Acad.Sci. 21: 663:250-61. (1992).
Mori K. I., Nakai T., Muroga K., Arimoto M., Mushiake K., and Furusaea I. Properties of a new virus belonging to nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis. Virology. 187:368-371. (1992)
Mori K I., Nakai T., Nagahara M., Muroga K., Mekuchi T., and Kanno T. A viral disease in hatchery - reared larvae and juveniles of redspotted grouper.Fish Pathol. 26: 209-210. (1991)
Munday B. L., Nakai T., and Nguyen H. D. Antigenic relationship of the picorna -like virus of larval barramundi, lates calcarifer Bloch to the nodavirus of larval striped jacj, pseudocaranx dentex. Aust Vet J. 71: 384-385. (1994)
Muroga K. Viral and bacterial disease in larval and juvenile marine fish and shellfish: a review. Fish Pathol. 30: 71-85. (1995)
Oldstone M. B. A. How viruses escape from cytotoxic T lymphocytes :molecular parameters and players. Virology 234: 179-185.(1997).
Reynold J. E., Yang T., Qian L., Jenkinson D., Zhou P., Estanam A., and Craig R. W. Mcl-1 , a member of the Bcl-2 family, delays apoptosis induced by c-Myc overexpression in Chinese hamster ovary cells. Cancer Res. 4: 6348-6352. (1994)
Rechsteiner M., and Rogers S. W. PEST sequences and regulationby proteolysis.. Trend. Biochem. Sci.21: 267-271. (1996).
Saelens X., Festjens N., Vande Walle L., van Gurp M., van Loo G., and Vandenabeele P. Toxic proteins released from mitochondria in cell death. Oncogene. 23(16):2861-74.(2004).
Samali A., Gorman A. M., and Cotter G. Apoptosis—the story so far… Experientia. 52(10-11): 933-941. (1996).
Sattler M., Liang H., Nettesheim D., Meadows R. P., Harlan
J. E., Eberstadt M., Yoon H. S., Shuker S. B., Chang B.
S., Minn A. J., Thompson C. B., and Fesik S. W. Structure
of Bcl-xL-Bak peptide complex: recognition between regulators
of apoptosis. Science 275(5302): 983-986. (1997).
Shimizu S., Ide T., Yanagida T., and Tsujimoto Y.
Electrophysiological study of a novel large pore formed by Bax
and the voltage-dependent anion channel that is permeable to
cytochrome c. J. Biol. Chem. 275(16): 12321-12325. (2000).
Shimizu S., Konishi A., Kodama T., and Tsujimoto Y. BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death. Proc. Natl. Acad. Sci. USA 97(7): 3100-3105. (2000).
Shimizu S., Narita M., and Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature 399(6735): 483-487.
(1999).
Shoshan-Barmatz V., and Gincel D. The voltage-dependent anion channel: characterization, modulation, and role in mitochondrial function in cell life and death. Cell Biochem Biophys. 39(3):279-92. (2003)
Steller H. Mechanisms and genes of cellular suicide. Science 267:144-9. (1995).
Suzuki M., Youle R. J., and Tjandra N. Structure of Bax:
coregulation of dimer formation and intracellular localization. Cell 103(4): 645-654. (2000).
Tanaka S., Mori K., Arimoto M., Iwamoto T., and Nakai T. Protective immunity of sevenband grouper, Epinephelus septemfasciatus Thunberg, against experimental viral nervous necrosis. J.of Fish Dise. 24: 15-22.(2001)
Vaux D.L., and Strasser A. The molecular biology of apoptosis.Pro. Nat. Acad. Sci. USA 933: 2239-2244. (1996).
Von A. O., Renken C., Perkins G., Kluck R. M., Bossy-Wetzel E., and Newmeyer D. D. Preservation of mitochondrial structure and function after Bid- or Bax-mediated cytochrome c release. J. Cell. Biol. 150(5): 1027-1036. (2000).
Wyllie A. H. Apoptosis : an overview. British. med. Bull. 53 (3):451-465. (1997).
Yang T., Kozopas K., and Craig R.W. The intracellular
distribution and pattem of expression of Mcl-1 overlap with,
but not identical to, this of Bcl-2 . J. Cell. Biol. 128:
1173-1184 (1995).
Yoshikoshi K., and Inoue K. Virus nervous necrosis in hatchery-reared larve and juveniles of Japanase parrotfish, Oplegnathus fasciatus . Journal of fish disease. 13: 69-77.
Zhou P., Qian L., Kozopas K. M., and Craig R. W. Mcl-1, a Bcl-2 family member, delays the death of hematopoietic cells under a variety of apoptosis-inducing conditions. Blood 89: 630-643.(1997).