神經壞死病毒(Nervous Necrosis Virus, NNV)是一種可感染多種高經濟性魚種如石斑魚、金目鱸等，且致死率極高並對水產養殖造成大量的經濟損失。本實驗室先前研究證實神經壞死病毒感染石斑魚鰭細胞株(GF-1)會產生由活性氧分子(O2-, H2O2)所介導的自噬作用。因此本研究想要接著探討病毒的表現、複製與細胞自噬作用誘導之關聯性如何。分別利用UV光、RNA1 siRNA與蛋白質合成抑制劑Cycloheximide來抑制病毒基因與蛋白質產物之表現與合成。結果發現，病毒複製下降時其所誘導產生的活性氧分子也跟著減少，進而使得自噬作用上游分子發現AMPK與Akt活性下降及mTOR活性上升，同時發現mTOR的活化也減緩了自噬作用進行。進而分析自噬作用參與蛋白Bcl-2與Beclin 1之互動關係。結果發現病毒複製下降時，會使得自噬作用參與蛋白Bcl-2活性下降，Beclin 1活性上升。當進一步分析其上游調控分子時，發現c-Jun NH2-terminal kinase (JNK) 表現量上升，得知病毒感染時能活化JNK路徑來誘導細胞自噬之啟動。尤其以Cycloheximide處理效果最為顯著。

As we known, many fish species are infected by betanodavirus such as grouper, which has resulted in severe mortality and significant economic losses on the aquaculture industry worldwide.Autophagy is a cellular metabolic process to degrade and recycle the older or damaged organelles. Autophagic is also induced under stress and virus infection. In previous study, we know GF-1cell induced autophagy by reactive oxygen species (ROS) (O2-,H2O2) after virus infection. Here, we focused on the role of virus replication on the autophagy induction.In this study, we confirmed GF-1 induced autophagy after virus infection 48 hr by electron microscopy. Antioxidant drug DPI was demonstrated can reduced autophagy. Then, we inactivated virus by UV light、RNA1 siRNA、Cycloheximide. We found that inactived virus produced less reactive oxygen species (ROS). Less ROS caused mTOR activated by AMPK expression level decrease and Akt expression level decrease, as mTOR activated reduce autophagy after inactivated virus. Autophagy related protein Bcl-2 and Beclin 1. Bcl-2 expression level decrease and Beclin 1 expression level increase after inactivated virus. Furtermore we found the upstream factor c-Jun NH2-terminal kinase (JNK) expression level increase after virus infection. Taken together our results suggest that RGNNV induced ROS signals can induced the autophagic process by Bcl-2 phosphorylation through JNK activated. Our finding provided insight into RNA virus molecular mechanism.

呂明偉、韓宛娟，石斑魚的病毒性疾病，科學發展472，66-71，2012。

沈士新、鄭安倉、劉秉忠、林正輝、冉繁華，台灣石斑魚產業現況與趨勢，水產養殖生技38，1-7，2014。

李昂融，抗凋亡家族蛋白Bcl-2與Bcl-xL對野田病毒所誘導魚類宿主細胞死亡與自噬作用之研究，成功大學生物科技研究所碩士論文，2014。

林彥宏、余祁暐、孫智麗，石斑魚產業概況及趨勢，農業生技產業季刊40，12-19，2014。

張芝瑋，神經壞死病毒誘發石斑魚細胞氧化壓力與病毒蛋白 B1 影響氧
化壓力之探討，成功大學生物科技研究所碩士論文，2009。

陳俐伽，神經壞死病毒非結構蛋白B1基因選殖及其功能之探討，成功大
學生物科技研究所碩士論文，2007。

陳博榕，神經壞死病毒所誘導之活性氧化分子對於自噬作用的調節，成功大學生物科技研究所碩士論文，2014。

黃淑敏，石斑魚虹彩病毒疫苗研發，行政院農業委員會家畜衛生試驗所，獸醫專訊3，31-34，2011。

楊玉婷、陳葦芋、陳政忻，石斑魚產業概況及趨勢，農業生技產業季刊19，24-29，2009。

楊惠郎、林翰佑、林青丘，石斑養殖科技，科學發展462，66-71，2011。

廖浤鈞，神經壞死病毒所誘導細胞內氧化壓力能影響細胞自噬體吞噬作用及病毒複製之研究，成功大學生物科技研究所碩士論文，2012。

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，1997。

Arsham, A. M., and Neufeld, T. P. Thinking globally and acting locally with TOR. Current Opinion in Cell Biology 18, 589-597, 2006.
Axe, E. L., Walker, S. A., Manifava, M., Chandra, P., Roderick, H. L., Habermann, A., Griffiths, G., and Ktistakis, N. T. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. The Journal of Cell Biology 182, 685-701, 2008.
Azad, M. B., Chen, Y., and Gibson, S. B. Regulation of autophagy by reactive oxygen species (ROS): implications for cancer progression and treatment. Antioxidants and Redox Signaling 11, 777-790, 2009.
Baehrecke, E. H. Autophagy: dual roles in life and death? Nature Reviews Molecular Cell Biology 6, 505-510, 2005.
Balaban, R. S., Nemoto, S., and Finkel, T. Mitochondria, oxidants, and aging. Cell 120, 483-495, 2005.
Bannister, J. V., Bannister, W. H., and Rotilio, G. Aspects of the structure, function, and applications of superoxide dismutase. Critical Reviews in Biochemistry 22, 111-180, 1987.
Bossy-Wetzel, E., Newmeyer, D. D., and Green, D. R. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. The European Molecular Biology Organization Journal 17, 37-49, 1998.
Bursch, W., Ellinger, A., Kienzl, H., Torok, L., Pandey, S., Sikorska, M., Walker, R., and Hermann, R. S. Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy. Carcinogenesis 17, 1595-1607, 1996.
Carpenter, J. E., Jackson, W., Benetti, L., and Grose, C. Autophagosome formation during varicella-zoster virus infection following endoplasmic reticulum stress and the unfolded protein response. Journal of Virology 85, 9414-9424, 2011.
Chang, C. W., Su, Y. C., Her, G. M., Ken, C. F., and Hong, J. R. Betanodavirus induces oxidative stress-mediated cell death that prevented by anti-oxidants and zfcatalase in fish cells. PLoS One 6, e25853, 2011.
Chen, L. J., Su, Y. C., and Hong, J. R. Betanodavirus non-structural protein B1: A novel anti-necrotic death factor that modulates cell death in early replication cycle in fish cells. Virology 385, 444-454, 2009.
Chen, S. P., Wu, J. L., Su, Y. C., and Hong, J. R. Anti-Bcl-2 family members, zfBcl-x(L) and zfMcl-1a, prevent cytochrome c release from cells undergoing betanodavirus-induced secondary necrotic cell death. Apoptosis : An International Journal on Programmed Cell Death 12, 1043-1060, 2007.
Chen, S. P., Yang, H. L., Her, G. M., Lin, H. Y., Jeng, M. F., Wu, J. L., and Hong, J. R. Betanodavirus induces phosphatidylserine exposure and loss of mitochondrial membrane potential in secondary necrotic cells, both of which are blocked by bongkrekic acid. Virology 347, 379-391, 2006.
Chen, Y., and Klionsky, D. J. The regulation of autophagy - unanswered questions. Journal of Cell Science 124, 161-170, 2011.
Chi, S. C., Shieh, J. R., and Lin, S. J. Genetic and antigenic analysis of betanodaviruses isolated from aquatic organisms in Taiwan. Disease of Aquatic Organisms 55, 221-228, 2003.
Chiramel, A. I., Brady, N. R., and Bartenschlager, R. Divergent roles of autophagy in virus infection. Cells 2, 83-104, 2013.
Danial, N. N., and Korsmeyer, S. J. Cell death: critical control points. Cell 116, 205-219, 2004.
Datan, E., Roy, S. G., Germain, G., Zali, N., McLean, J. E., Golshan, G., Harbajan, S., Lockshin, R. A., and Zakeri, Z. Dengue-induced autophagy, virus replication and protection from cell death require ER stress (PERK) pathway activation. Cell Death and Disease 7, e2127, 2016.
Delgado, M., Singh, S., De Haro, S., Master, S., Ponpuak, M., Dinkins, C., Ornatowski, W., Vergne, I., and Deretic, V. Autophagy and pattern recognition receptors in innate immunity. Immunological Reviews 227, 189-202, 2009.
Deter, R. L., and De Duve, C. Influence of glucagon, an inducer of cellular autophagy, on some physical properties of rat liver lysosomes. The Journal of Cell Biology 33, 437-449, 1967.
Dreux, M., Gastaminza, P., Wieland, S. F., and Chisari, F. V. The autophagy machinery is required to initiate hepatitis C virus replication. Proceedings of the National Academy of Sciences of the United States of America 106, 14046-14051, 2009.
Edinger, A. L., and Thompson, C. B. Death by design: apoptosis, necrosis and autophagy. Current Opinion in Cell Biology 16, 663-669, 2004.
Esclatine, A., Chaumorcel, M., and Codogno, P. Macroautophagy signaling and regulation. Current Topics in Microbiology and Immunology 335, 33-70, 2009.
Gagne, N., Johnson, S. C., Cook-Versloot, M., MacKinnon, A. M., and Olivier, G. Molecular detection and characterization of nodavirus in several marine fish species from the northeastern Atlantic. Disease of Aquatic Organisms 62, 181-189, 2004.
Gallagher, L. E., Williamson, L. E., and Chan, E. Y. Advances in Autophagy Regulatory Mechanisms. Cells 5, 2016.
Gaudernak, E., Seipelt, J., Triendl, A., Grassauer, A., and Kuechler, E. Antiviral effects of pyrrolidine dithiocarbamate on human rhinoviruses. Journal of Virology 76, 6004-6015, 2002.
Gerba, C. P., Gramos, D. M., and Nwachuku, N. Comparative inactivation of enteroviruses and adenovirus 2 by UV light. Applied and Environmental Microbiology 68, 5167-5169, 2002.
Granato, M., Santarelli, R., Farina, A., Gonnella, R., Lotti, L. V., Faggioni, A., and Cirone, M. Epstein-barr virus blocks the autophagic flux and appropriates the autophagic machinery to enhance viral replication. Journal of Virology 88, 12715-12726, 2014.
Hahn-Windgassen, A., Nogueira, V., Chen, C. C., Skeen, J. E., Sonenberg, N., and Hay, N. Akt activates the mammalian target of rapamycin by regulating cellular ATP level and AMPK activity. Journal Biological Chemistry 280, 32081-32089, 2005.
Itakura, E., Kishi, C., Inoue, K., and Mizushima, N. Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG. Molecular Biology of the Cell 19, 5360-5372, 2008.
Jackson, C. A., Messinger, J., Peduzzi, J. D., Ansardi, D. C., and Morrow, C. D. Enhanced functional recovery from spinal cord injury following intrathecal or intramuscular administration of poliovirus replicons encoding IL-10. Virology 336, 173-183, 2005.
Jackson, W. T. Viruses and the autophagy pathway. Virology 479-480, 450-456, 2015.
Jheng, J. R., Ho, J. Y., and Horng, J. T. ER stress, autophagy, and RNA viruses. Frontiers in Microbiology 5, 388, 2014.
Jordan, T. X., and Randall, G. Manipulation or capitulation: virus interactions with autophagy. Microbes and Infection / Institut Pasteur 14, 126-139, 2012.
Joubert, P. E., Meiffren, G., Gregoire, I. P., Pontini, G., Richetta, C., Flacher, M., Azocar, O., Vidalain, P. O., Vidal, M., Lotteau, V. Autophagy induction by the pathogen receptor CD46. Cell Host and Microbe 6, 354-366, 2009.
Jung, C. H., Ro, S. H., Cao, J., Otto, N. M., and Kim, D. H. mTOR regulation of autophagy. Federation of European Biochemical Societies Letters 584, 1287-1295, 2010.
Ke, P. Y., and Chen, S. S. Activation of the unfolded protein response and autophagy after hepatitis C virus infection suppresses innate antiviral immunity in vitro. The Journal of Clinical Investigation 121, 37-56, 2011.
Kihara, A., Noda, T., Ishihara, N., and Ohsumi, Y. Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae. The Journal of Cell Biology 152, 519-530, 2001.
Klionsky, D. J. Autophagy: from phenomenology to molecular understanding in less than a decade. Nature Reviews Molecular Cell Biology 8, 931-937, 2007.
Kroemer, G., Dallaporta, B., and Resche-Rigon, M. The mitochondrial death/life regulator in apoptosis and necrosis. Annual Review of Physiology 60, 619-642, 1998.
Kroemer, G., and Levine, B. Autophagic cell death: the story of a misnomer. Nature Reviews Molecular Cell Biology 9, 1004-1010, 2008.
Kukkula, M., Arstila, P., Klossner, M. L., Maunula, L., Bonsdorff, C. H., and Jaatinen, P. Waterborne outbreak of viral gastroenteritis. Scandinavian Journal of Infectious Diseases 29, 415-418, 1997.
Lambeth, J. D. Nox enzymes, ROS, and chronic disease: an example of antagonistic pleiotropy. Free Radical Biology and Medicine 43, 332-347, 2007.
Lennemann, N. J., and Coyne, C. B. Catch me if you can: the link between autophagy and viruses. PLoS Pathogens 11, e1004685, 2015.
Li, L., Ishdorj, G., and Gibson, S. B. Reactive oxygen species regulation of autophagy in cancer: implications for cancer treatment. Free Radical Biology and Medicine 53, 1399-1410, 2012.
Liu, B., Fang, M., Hu, Y., Huang, B., Li, N., Chang, C., Huang, R., Xu, X., Yang, Z., Chen, Z. Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation. Autophagy 10, 416-430, 2014.
Massey, A. C., Zhang, C., and Cuervo, A. M. Chaperone-mediated autophagy in aging and disease. Current Topics in Developmental Biology 73, 205-235, 2006.
Mizushima, N. Methods for monitoring autophagy. The International Journal of Biochemistry and Cell Biology 36, 2491-2502, 2004.
Mizushima, N. Autophagy: process and function. Genes and Development 21, 2861-2873, 2007.
Mizushima, N., Yoshimori, T., and Levine, B. Methods in mammalian autophagy research. Cell 140, 313-326, 2010.
Mori, K., Nakai, T., Muroga, K., Arimoto, M., Mushiake, K., and Furusawa, I. Properties of a new virus belonging to nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis. Virology 187, 368-371, 1992.
Munday, B. L., Kwang, J., and Moody, N. Betanodavirus infections of teleost fish: a review. Journal of Fish Disease 25, 127-142, 2002.
Nagai, T., and Nishizawa, T. Sequence of the non-structural protein gene encoded by RNA1 of striped jack nervous necrosis virus. The Journal of General Virology 80, 3019-3022, 1999.
Nakatogawa, H., Suzuki, K., Kamada, Y., and Ohsumi, Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nature Reviews Molecular Cell Biology 10, 458-467, 2009.
Nishizawa, T., Furuhashi, M., Nagai, T., Nakai, T., and Muroga, K. Genomic classification of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene. Applied and Environmental Microbiology 63, 1633-1636, 1997.
Novo, E., and Parola, M. Redox mechanisms in hepatic chronic wound healing and fibrogenesis. Fibrogenesis and Tissue Repair 1, 5, 2008.
Nowag, H., Guhl, B., Thriene, K., Romao, S., Ziegler, U., Dengjel, J., and Munz, C. Macroautophagy Proteins Assist Epstein Barr Virus Production and Get Incorporated Into the Virus Particles. EBioMedicine 1, 116-125, 2014.
Nuanualsuwan, S., and Cliver, D.O. Pretreatment to avoid positive RT-PCR results with inactivated viruses. Journal of Virological Methods 104, 217-225, 2002.
Nuanualsuwan, S., and Cliver, D. O. Capsid functions of inactivated human picornaviruses and feline calicivirus. Applied and Environmental Microbiology 69, 350-357, 2003.
Ouyang, L., Shi, Z., Zhao, S., Wang, F. T., Zhou, T. T., Liu, B., and Bao, J. K. Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Proliferation 45, 487-498, 2012.
Pattingre, S., Tassa, A., Qu, X., Garuti, R., Liang, X. H., Mizushima, N., Packer, M., Schneider, M. D., and Levine, B. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 122, 927-939, 2005.
Rubinstein, A. D., and Kimchi, A. Life in the balance - a mechanistic view of the crosstalk between autophagy and apoptosis. Journal of Cell Science 125, 5259-5268, 2012.
Russell, R. C., Tian, Y., Yuan, H., Park, H. W., Chang, Y. Y., Kim, J., Kim, H., Neufeld, T. P., Dillin, A., and Guan, K. L. ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase. Nature Cell Biology 15, 741-750, 2013.
Sabapathy, K. Role of the JNK pathway in human diseases. Progress in Molecular Biology and Translational Science 106, 145-169, 2012.
Saftig, P., Beertsen, W., and Eskelinen, E. L. LAMP-2: a control step for phagosome and autophagosome maturation. Autophagy 4, 510-512, 2008.
Salonen, A., Ahola, T., and Kaariainen, L. Viral RNA replication in association with cellular membranes. Current Topics in Microbiology and Immunology 285, 139-173, 2005.
Sangsanont, J., Katayama, H., Kurisu, F., and Furumai, H. Capsid-Damaging Effects of UV Irradiation as Measured by Quantitative PCR Coupled with Ethidium Monoazide Treatment. Food and Environmental Virology 6, 269-275, 2014.
Shelly, S., Lukinova, N., Bambina, S., Berman, A., and Cherry, S. Autophagy is an essential component of Drosophila immunity against vesicular stomatitis virus. Immunity 30, 588-598, 2009.
Simonsen, A., and Tooze, S. A. Coordination of membrane events during autophagy by multiple class III PI3-kinase complexes. The Journal of Cell Biology 186, 773-782, 2009.
Sir, D., Chen, W. L., Choi, J., Wakita, T., Yen, T. S., and Ou, J. H. Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response. Hepatology 48, 1054-1061, 2008.
Su, Y. C., Chiu, H. W., Hung, J. C., and Hong, J. R. Beta-nodavirus B2 protein induces hydrogen peroxide production, leading to Drp1-recruited mitochondrial fragmentation and cell death via mitochondrial targeting. Apoptosis : An International Journal on Programmed Cell Death 19, 1457-1470, 2014.
Su, Y. C., and Hong, J. R. Betanodavirus B2 causes ATP depletion-induced cell death via mitochondrial targeting and complex II inhibition in vitro and in vivo. Journal of Biological Chemistry 285, 39801-39810, 2010.
Su, Y. C., Wu, J. L., and Hong, J. R. Betanodavirus non-structural protein B2: A novel necrotic death factor that induces mitochondria-mediated cell death in fish cells. Virology 385, 143-154, 2009.
Su, Y. C., Wu, J. L., and Hong, J. R. Betanodavirus up-regulates chaperone GRP78 via ER stress: roles of GRP78 in viral replication and host mitochondria-mediated cell death. Apoptosis : An International Journal on Programmed Cell Death 16, 272-287, 2011.
Sulzer, D., Mosharov, E., Talloczy, Z., Zucca, F. A., Simon, J. D., and Zecca, L. Neuronal pigmented autophagic vacuoles: lipofuscin, neuromelanin, and ceroid as macroautophagic responses during aging and disease. Journal of Neurochemistry 106, 24-36, 2008.
Talloczy, Z., Virgin, H. W. T., and Levine, B. PKR-dependent autophagic degradation of herpes simplex virus type 1. Autophagy 2, 24-29, 2006.
Thiery, R., Cozien, J., de Boisseson, C., Kerbart-Boscher, S., and Nevarez, L. Genomic classification of new betanodavirus isolates by phylogenetic analysis of the coat protein gene suggests a low host-fish species specificity. The Journal of General Virology 85, 3079-3087, 2004.
Ullman, E., Fan, Y., Stawowczyk, M., Chen, H. M., Yue, Z., and Zong, W. X. Autophagy promotes necrosis in apoptosis-deficient cells in response to ER stress. Cell Death and Differentiation 15, 422-425, 2008.
Vernon, P. J., and Tang, D. Eat-me: autophagy, phagocytosis, and reactive oxygen species signaling. Antioxidants and Redox Signaling 18, 677-691, 2013.
Wong, J., Zhang, J., Si, X., Gao, G., Mao, I., McManus, B. M., and Luo, H. Autophagosome supports coxsackievirus B3 replication in host cells. Journal of Virology 82, 9143-9153, 2008.
Wu, H. C., Chiu, C. S., Wu, J. L., Gong, H. Y., Chen, M. C., Lu, M. W., and Hong, J. R. Zebrafish anti-apoptotic protein zfBcl-xL can block betanodavirus protein alpha-induced mitochondria-mediated secondary necrosis cell death. Fish and Shellfish Immunology 24, 436-449, 2008.
Wu, H. C., Wu, J. L., Chu, H. L., Su, Y. C., and Hong, J. R. RGNNV induces mitochondria-mediated cell death via newly synthesized protein dependent pathway in fish cells. Fish and Shellfish Immunology 29, 451-463, 2010.
Yang, T., O'Keefe, D., and Chance, B. The oxidation-reduction potentials of cytochrome o + c4 and cytochrome o purified from Azotobacter vinelandii. The Biochemical Journal 181, 763-766, 1979.
Yoshikoshi, M., and Osawa, F. Simple methods to check intraocular lenses. Insight 15, 19-21, 1990.
Yousefi, S., Perozzo, R., Schmid, I., Ziemiecki, A., Schaffner, T., Scapozza, L., Brunner, T., and Simon, H. U. Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nature Cell Biology 8, 1124-1132, 2006.
Zalckvar, E., Berissi, H., Eisenstein, M., and Kimchi, A. Phosphorylation of Beclin 1 by DAP-kinase promotes autophagy by weakening its interactions with Bcl-2 and Bcl-XL. Autophagy 5, 720-722, 2009.
Zhou, Y. Y., Li, Y., Jiang, W. Q., and Zhou, L. F. MAPK/JNK signalling: a potential autophagy regulation pathway. Bioscience Reports 35, 2015.
Zhou, Z., Jiang, X., Liu, D., Fan, Z., Hu, X., Yan, J., Wang, M., and Gao, G. F. Autophagy is involved in influenza A virus replication. Autophagy 5, 321-328, 2009.
Zou, S., Guo, J., Gao, R., Dong, L., Zhou, J., Zhang, Y., Dong, J., Bo, H., Qin, K., and Shu, Y. Inactivation of the novel avian influenza A (H7N9) virus under physical conditions or chemical agents treatment. Virology Journal 10, 289, 2013.