臺灣博碩士論文加值系統

日本腦炎(Japanese encephalitis)在亞洲許多國家，是很重要的蟲媒病毒傳染病，由日本腦炎病毒(Japanese encephalitis virus，簡稱JEV)感染造成。而日本腦炎病毒屬於Flaviviriade，傳染途徑是藉由蚊子傳播。
此病毒不僅能感染蚊子細胞，也能感染哺乳動物細胞。很多研究報告都已知道日本腦炎病毒感染哺乳動物細胞是經由receptor-mediated endocytosis的方式進入。在1998年Nawa以專一性抑制V-ATPase的bafilomycin A1處理蚊蟲細胞，發現日本腦炎病毒生長會受其限制，因而猜測日本腦炎病毒進入蚊蟲細胞，也是透過receptor-mediated endocytosis的方式。
所以此研究中，我們利用RNAi技術，抑制哺乳動物細胞(BHK-21) V-ATPase基因的表現，結果顯示siRNA確實造成V-ATPase mRNA表現量減少，也使得病毒複製受到限制，證實V-ATPase與日本腦炎病毒感染哺乳動物細胞上確實有一定的相關性。
隨後抑制蚊蟲細胞(C6/36)V-ATPase基因的表現，觀察其V-ATPase與病毒複製之間的關係，推測日本腦炎病毒進入蚊蟲細胞路徑是否與進入哺乳動物細胞相同。結果可以看出dsRNA確實造成了V-ATPase mRNA及protein表現量減少，但日本腦炎病毒複製能力僅微幅下降。
綜合結果顯示，日本腦炎病毒在感染哺乳動物細胞或是蚊蟲細胞，可能皆是藉由V-ATPase-regulated endocytosis。且利用RNAi技術干擾V-ATPase，影響endosome酸化能力，來抑制日本腦炎病毒在蚊蟲細胞內的表現與複製，此結果也提供我們一個從病媒方向控制日本腦炎病毒的遠景。

Japanese encephalitis is an important arboviral disease in Asia. It is caused by Japanese encephalitis virus (JEV) infection. The JEV is a member of the virus family Flaviviridae, which is transmitted to human through the bite of mosquitoes.
In addition to mosquito cells, JE virus also infects mammalian cells. Many researches have reported that JEV enters mammalian cells by receptor-mediated endocytosis. In 1998, Nawa treated mosquito cells with bafilomycin A1, a specific inhibitor of vacuolar type H+-ATPase (V-ATPase) and observed inhibited growth of JEV in the cells. These results indicate that JEV may also enter C6/36 cells through the endocytic pathway involving intracellular acidic compartments.
In this study, we first designed specific siRNAs to inhibit V-ATPase of mammalian cells (BHK-21). The results indicated that siRNA may reduce V-ATPase mRNA expression as well as virus replication. This study also demonstrated that the V-ATPase plays a role in the JEV infection to mammalian cells.
Subsequently, we used dsRNAs to knock down the V-ATPase expression of C6/36 cells based on the hypothesis that JEV takes the same route to enter the mosquito cells as does in mammalian cells. The results showed that the dsRNA could inhibit the expression at both mRNA and protein levels of the V-ATPase and reduce virus replication as well.
These results have revealed that JEV actually enters mosquitoe and mammalian cells by the V-ATPase-regulated endocytosis. In addition, functional to inhibition of V-ATPase by RNAi also blocks the JEV replication in mosquito cells. Results shown in this study have provided a promising way for the control of JEV infection in future.

指導教授推薦書
口試委員審定書
授權書
長庚大學博碩士紙本論文著作授權書
致謝……………………………………………………………………………………v
中文摘要 ……………………………………………………………………………vi
英文摘要 …………………………………………………………………………viii
第一章 文獻探討
第一節 日本腦炎及其流行病學…………………………………………1
第二節 日本腦炎病毒……………………………………………………3
第三節 RNA interference (RNAi)簡介 ………………………………6
第四節 V-ATPase (vacuolar H＋-ATPase)……………………………10
第五節 具有套膜之病毒感染進入細胞的模式 ………………………12
第二章 研究動機與設計
第一節 研究目的 ………………………………………………………13
第二節 實驗設計 ………………………………………………………13
第三章 材料與方法
一. 實驗材料……………………………………………………………15
二. 病毒培養……………………………………………………………17
三. BHK-21細胞的V-ATPase subunit A之基因全長定序……………17
四. 基因選殖……………………………………………………………18
五. 構築V-ATPase RNAi construct……………………………………20
六. 細胞外轉錄及dsRNA的螢光標幟…………………………………22
七. 轉染作用……………………………………………………………23
八. 細胞存活率計算……………………………………………………23
九. 細胞內total RNA之萃取…………………………………………24
十. 即時聚合酶鍊反應…………………………………………………24
十一. 西方墨點法………………………………………………………25
十二. 免疫螢光抗體技術………………………………………………27
十三. 流式細胞儀分析…………………………………………………28
十四. 溶斑檢定法………………………………………………………28
十五. 吖啶橙染色試驗…………………………………………………29
第四章 結果………………………………………………………………………30
第五章 討論………………………………………………………………………40
參考文獻……………………………………………………………………………47

Adelman ZN, Sanchez-Vargas I, Travanty EA, Carlson JO, Beaty BJ, Blair CD, Olson KE. (2002) RNA silencing of dengue virus type 2 replication in transformed C6/36 mosquito cells transcribing an inverted-repeat RNA derived from the virus genome. J. Virol. 76, 12925-12933.
Brandt WE. (1990) From the World Health Organization. Development of dengue and Japanese encephalitis vaccines. J. Infect. Dis. 162, 577-583.
Burke DS, Monath TP. (2001) Flaviviruses. In “Fields Virology”, 4th ed., pp.1043-1125. Lippincott Williams & Wilkins publishers, Philadelphia.
Caplen NJ, Zheng Z, Falgout B, Morgan RA. (2002) Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference. Mol. Ther. 6, 243-251.
Chambers TJ, Hahn CS, Galler R, Rice, CM. (1990) Flavivirus genome organization, expression, and replication. Annu. Rev. Microbiol. 44, 649-688.
Choudhury N, Saxena NBL, Dwivedi SR, Khamre JS. (1983) Study of the outbreak of Japanese encephalitis in Goa. J. Commun. Dis. 15, 111-120.
Clerc S, Barenholz Y. (1998) A quantitative model for using acridine orange as a transmembrane pH gradient probe. Anal. Biochem. 259, 104-111.
Detels R, Cross JH, Huang WC, Lien JC, Chen S. (1976) Japanese encephalitis virus in northern Taiwan, 1967-1973. Am. J. Trop. Med. Hyg. 25, 477-485.
Dschida WJ, Bowman BJ. (1992) Structure of the vacuolar ATPase from Neurospora crassa as determined by electron microscopy. J. Biol. Chem. 15, 18783-18789.
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498.
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.

Futai M, Oka T, Sun-Wada G, Moriyama Y, Kanazawa H, Wada Y. (2000) Luminal acidification of diverse organelles by V-ATPase in animal cells. J. Exp. Biol. 203, 107-116.
Gaunt MW, Sall AA, de Lamballerie X, Falconar AK, Dzhivanian TI, Gould EA. (2001) Phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography. J. Gen. Virol. 82, 1867-1876.
Grabe M, Wang H, Oster G. (2000) The mechanochemistry of V-ATPase proton pumps. Biophys. J. 78, 2798-2813.
Guo S, Kemphues KJ. (1995) par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81, 611-620.
Hamilton AJ and Baulcombe DC. (1999) A species of small antisense RNA in post-transcriptional gene silencing in plants. Science 286, 950-952.
Hannon GJ. (2002) RNA interference. Nature 418, 244-251.
Heinz FX, Allison SL. (2000) Structure and mechanisms in flavivirus fusion. Adv. Virus Res. 55, 231-269.
Huang CG, Tsai KH, Wu WJ, Chen WJ. (2005) Intestinal expression of H+ V-ATPase in the mosquito Aedes albopictus is tightly associated with gregarine infection. J. Eukaryot. Microbiol. (accepted)
Ishak R, Tovey DG, Howard CR. (1988) Morphogenesis of yellow fever virus 17D in infected cell cultures. J. Gen. Virol. 69, 325-335.
Johansen CA, van den Hurk AF, Pyke AT, Zborowski P, Phillips DA, Mackenzie JS, Ritchie SA. (2001) Entomological investigations of an outbreak of Japanese encephalitis virus in the Torres Strait, Australia, in 1998. J. Med. Entomol. 38, 581-588.
Jorgensen RA, Cluster PD, English J, Que Q, Napoli CA. (1996) Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences. Plant Mol. Biol. 31, 957-973.
Kahana R, Kuznetzova L, Rogel A, Shemesh M, Hai D, Yadin H, Stram Y. (2004) Inhibition of foot-and-mouth disease virus replication by small interfering RNA. J. Gen. Virol. 85, 3213-3217.

Kane PM, Parra KJ. (2000) Assembly and regulation of the yeast vacuolar H(+)-ATPase. J. Exp. Biol. 203, 81-87.
Kennerdell JR, Carthew RW. (1998) Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95, 1017-1026.
Kennerdell JR, Carthew RW. (2000) Heritable gene silencing in Drosophila using double-stranded RNA. Nat. Biotechnol. 18, 896-898.
Kitano T, Suzuki K, Yamaguchi T. (1974) Morphological, chemical, and biological characterization of Japanese encephalitis virus virion and its hemagglutinin. J. Virol. 14, 631-639.
Kobayashi H. (1940) On the virus of encephalitis epidemics isolated in Taihoku and Sintiku provinces in the summer 1938. Acta Jpn. Med. Trop. 2, 55-62.
Kumar R, Mathur A, Kumar A, Sharma S, Chakraborty S, Chaturvedi UC. (1990) Clinical features and prognostic indicators of Japanese encephalitis in children in Lucknow (India). Indian J. Med. Res. 91, 321-327.
Kuzuhara S, Nakamura H, Hayashida K, Obata J, Abe M, Sonoda K, Nishiyama K, Sugawara K, Takeda K, Honda T, Matsui H, Shigaki T, Kino Y, Mizokami H, Tanaka M, Mizuno K, Ueda K. (2003) Non-clinical and phase I clinical trials of a Vero cell-derived inactivated Japanese encephalitis vaccine. Vaccine 21, 4519-4526.
Li YX, Farrell MJ, Liu R, Mohanty N, Kirby ML. (2000) Double-stranded RNA injection produces null phenotypes in Zebrafish. Dev. Biol. 217, 394–405.
Liao CL, Lin YL, Wang JJ, Huang YL, Yeh CT, Ma SH, Chen LK. (1997) Effect of enforced expression of human bcl-2 on Japanese encephalitis virus-induced apoptosis in cultured cells. J. Virol. 71, 5963-5971.
Lindenbach BD, Rice CM. (2001) Flaviviridae: The viruses and their replication. In “Fields Virology”, 4th ed., pp.991-1041. Lippincott Williams & Wilkins publishers, Philadelphia.
Lipardi C, Wei Q, Paterson BM. (2001) RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107, 297-307.
Mackenzie JS, Gubler DJ, Petersen LR. (2004) Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. Nat. Med. 10, S98-109.
Merzendorfer H, Reineke S, Zhao XF, Jacobmeier B, Harvey WR, Wieczorek H. (2000) The multigene family of the tobacco hornworm V-ATPase: novel subunits a, C, D, H and putative isoforms. Biochim. Biophys. Acta 1467, 369–379.
Mitamura T, Kitaoka M, Watanabe M. (1936) Study of Japanese encephalitis virus animal experiments and mosquito transmission experiment. Kansai Iji 1, 260.
Monath TP. (2002) Japanese encephalitis vaccines: current vaccines and future prospects. Curr. Top. Microbiol. Immunol. 267, 105-138.
Murray PR, Rosenthal KS, Kobayashi GS, Pfaller MA. (1998) Togaviruses and Flaviviruses. In “Medical Microbiology”, 3rd ed., pp.492-502. Mosby, St. Louis.
Myers M, Forgac M. (1993) Assembly of the peripheral domain of the bovine vacuolar H(+)-adenosine triphosphatase. J. Cell Physiol. 156, 35-42.
Naito Y, Yamada T, Ui-Tei K, Morishita S, Saigo K. (2004) siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference. Nucleic Acids Res. 32(Web Server issue), W124-129.
Napoli C, Lemieux C, Jorgensen R. (1990) Introduction of a chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans. Plant cell 2, 279-289.
Nawa M. (1998) Effects of bafilomycin A1 on Japanese encephalitis virus in C6/36 mosquito cells. Arch. Virol. 143, 1555-1568.
Nawa M, Takasaki T, Yamada K, Kurane I, Akatsuka T. (2003) Interference in Japanese encephalitis virus infection of Vero cells by a cationic amphiphilic drug, chlorpromazine. J. Gen. Virol. 84, 1737-1741.
Nelson N. (1992) The vacuolar H＋-ATPase, one of the most fundamental ion pumps in nature. J. Exp. Biol. 172, 19-27.
Ogata A, Nagashima K, Yasui K, Matsuura T, Tashiro K. (1998) Sustained release dosage of thyrotropin-releasing hormone improves experimental Japanese encephalitis virus-induced parkinsonism in rats. J. Neurol. Sci. 159, 135-139.

Olson KE, Adelman ZN, Travanty EA, Sanchez-Vargas I, Beaty BJ, Blair CD. (2002) Developing arbovirus resistance in mosquitoes. Insect Biochem. Mol. Biol. 32, 1333-1343.
Ota Z. (1965) Electron microscope study of the development of Japanese B encephalitis virus in porcine kidney stable(PS) cell. Virology 25, 372-378.
Palmgren MG. (1991) Acridine orange as a probe for measuring pH gradients across membranes: mechanism and limitations. Anal. Biochem. 192, 316-321.
Park K, Lee K, Lee J, Yeo S, Lee S, Cheon DS, Choi W, Ahn J, Kim S, Jee Y. (2006) Acute hemorrhagic conjunctivitis epidemic caused by coxsackievirus A24 variants in Korea during 2002-2003. J. Med. Virol. 78, 91-97.
Pelkmans L, Helenius A. (2003) Insider information: what viruses tell us about endocytosis. Curr. Opin. Cell Biol. 15, 414-422.
Plasterk RH. (2002) RNA silencing: the genome's immune system. Science 296, 1263-1265.
Poveda E, Briz V, Soriano V. (2005) Enfuvirtide, the first fusion inhibitor to treat HIV infection. AIDS Rev. 7, 139-147.
Pradhan S, Pandey N, Shashank S, Gupta RK, Mathur A. (1999) Parkinsonism due to predominant involvement of substantia nigra in Japanese encephalitis. Neurology 53, 1781-1786.
Rao PN. (2001) Japanese encephalitis. Indian Pediatr. 38, 1252-1264.
Rodhain F. (1996) Recent data on the epidemiology of Japanese encephalitis. Bull. Acad. Natl. Med. 180, 1325-1340.
Rust MJ, Lakadamyali M, Zhang F, Zhuang X. (2004) Assembly of endocytic machinery around individual influenza viruses during viral entry. Nat. Struct. Mol. Biol. 11, 567-573.
Sanchez-Vargas I, Travanty EA, Keene KM, Franz AW, Beaty BJ, Blair CD, Olson KE. (2004) RNA interference, arthropod-borne viruses, and mosquitoes. Virus Res. 102, 65-74.

Sankian M, Varasteh A, Pazouki N, Mahmoudi M. (2005) Sequence homology: a poor predictive value for profilins cross-reactivity. Clin. Mol. Allergy. 10, 3-13.
Schenborn ET, Mierendorf RC Jr. (1985) A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure. Nucleic Acids Res. 13, 6223-6236.
Scherer WF, Kitaoka M, Okuno T. (1959) Ecological studies of Japanese Encephalitis virus in Japan. VII. Human infections. Am. J. Trop. Med. Hyg. 8, 707-715.
Schwarz DS, Hutvagner G, Haley B, Zamore PD. (2002) Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol. Cell. 10, 537-548.
Shuey DJ, McCallus DE, Giordano T. (2002) RNAi：gene-silencing in the therapeutic intervention. Drug Discov. Today 7, 1040-1046.
Sieczkarski SB, Whittaker GR. (2002) Dissecting virus entry via endocytosis. J. Gen. Virol. 83, 1535-1545.
Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. (1998) How cells respond to interferons. Annu. Rev. Biochem. 67, 227-264.
Stevens TH, Forgac M. (1997) Structure, function and regulation of the vacuolar (H+)-ATPase. Annu. Rev. Cell Dev. Biol. 13, 779-808.
Su CM, Liao CL, Lee YL, Lin YL. (2001) Highly sulfated forms of heparin sulfate are involved in Japanese encephalitis virus infection. Virology 286, 206-215.
Sui G, Soohoo C, Affar E-B, Gay F, Shi Y, Forrester WC, Shi Y. (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 5515-5520.
Sumner JP, Dow JA, Earley FG, Klein U, Jager D, Wieczorek H. (1995) Regulation of plasma membrane V-ATPase activity by dissociation of peripheral subunits. J. Biol. Chem. 270, 5649-5653.
Svoboda P, Stein P, Schultz RM. (2001) RNAi in mouse oocytes and preimplantation embryos: Effectiveness of hairpin dsRNA. Biochem. Biophys. Res. Commun. 287, 1099–1104.
Tomashek JJ, Sonnenburg JL, Artimovich JM, Klionsky DJ. (1996) Resolution of subunit interactions and cytoplasmic subcomplexes of the yeast vacuolar proton-translocating ATPase. J. Biol. Chem. 271, 10397-10404.
Tomochika K, Shinoda S, Kumon H, Mori M, Moriyama Y, Futai M. (1997) Vacuolar-type H＋-ATPase in mouse bladder epithelium is responsible for urinary acidification. FEBS Lett. 404, 61-64.
Umata T, Moriyama Y, Futai M, Mekada E. (1990) The cytotoxic action of diphtheria toxin and its degradation in intact Vero cells are inhibited by bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase. J. Biol. Chem. 265, 21940–21945.
Wanger EK, Hewlett MJ. (1999) Basic virology. pp.70-71. Blackwell Science, Inc. Massachusetts U.S.A.
Waterhouse PM, Graham MW, Wang MB. (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Plant Biol. 95, 13959-13964.
Westaway EG, Brinton MA, Gaidamovich SYa, Horzinek MC, Igarashi A, Kaariainen L, Lvov DK, Porterfield JS, Russell PK, Trent DW. (1985) Flaviviridae. Intervirology 24, 183-192.
Wieczorek H, Brown D, Grinstein S, Ehrenfeld J, Harvey WR. (1999) Animal plasma membrane energization by proton-motive V-ATPases. BioEssays 21, 637-648.
Wieczorek H, Grber G, Harvey WR, Huss M, Merzendorfer H, Zeiske W. (2000) Structure and regulation of insect plasma membrane H＋ V-ATPase. J. Exp. Biol. 203, 127-135.
Wu YC, Huang YS, Chien LJ, Lin TL, Yueh YY, Tseng WL, Chang KJ, Wang GR. (1999) The epidemiology of Japanese encephalitis on Taiwan during 1966-1997. Am. J. Trop. Med. Hyg. 61, 78-84.
Yu JY, DeRuiter SL, Turner DL. (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA. 99, 6047-6052.
Zamore PD, Tuschl T, Sharp PA, Bartel DP. (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 31, 25-33.
Zhou Y, Ching YP, Kok KH, Kung HF, Jin DY. (2002) Post-transcriptional suppression of gene expression in Xenopus embryos by small interfering RNA. Nucleic Acids Res. 30, 1664-1669.
董志峰。1998。白腹叢蚊在小琉球地區傳播日本腦炎病毒可能性之探討，74頁。國立台灣大學公共衛生學院流行病學研究所碩士論文

翟維君。2005。日本腦炎病毒藉由與V-ATPase有關的內噬作用進入蚊蟲細胞之研究，62頁。長庚大學基礎醫學研究所碩士論文