臺灣博碩士論文加值系統

登革病毒為一單股、正向RNA病毒，屬於黃病毒屬，是具膜病毒。病毒的傳播是經由埃及斑蚊以及白線斑蚊。利用血清學的分類方法，登革病毒可以分成四個血清型。
在病毒進入細胞的過程當中，登革病毒的外套膜蛋白質扮演了一個重要的角色。它是一個由四百九十五個胺基酸所構成的醣蛋白質，位於病毒脂質雙層膜的外側，是一個穿膜蛋白質，會兩兩成對，在膜上形成一穩定的雙體構型。外套膜蛋白質外露在病毒膜外的部分，被認為可以和其受體結合。
本研究的主要目的，在於建立一套細胞融合測試方法，以探討外套膜蛋白質所媒介的登革病毒進入宿主細胞的過程。
在本研究的第一個部分，我們以五個不同的外套膜蛋白質表現質體：pD2ME, pD2ME His, pE His, pCB8D2J2 以及pCB8D2J2VSV，分別轉染哺乳動物細胞株（293細胞），以檢測何者可有效地表現外套膜蛋白質。
本研究的第二個目標，是針對登革病毒發展一套細胞融合測試。將共同轉染pCB8D2J2VSV（來自第二型登革病毒，16681 strain）以及報導質體pET-21a-GFP的NPCTW04細胞，與感染重組痘苗病毒的BHK細胞（幼倉鼠腎細胞株）共同培養。以螢光顯微鏡觀察的結果，顯示NPCTW04細胞確實可以與BHK細胞或K562（自然殺手細胞株）細胞融合（這兩個細胞株是已知的登革病毒宿主細胞），與H9(T細胞株)或Hut78(T細胞株)共同培養則否。在分別對四個目標細胞株進行登革病毒感染力的測試之後，發現登革病毒可以有效感染BHK細胞及K562細胞，對H9細胞及Hut78細胞則否。這些結果顯示本實驗所建立的細胞融合測試，可以反映登革病毒對宿主細胞的選擇性。
本研究的第三個目標，則針對第二型及第三型登革病毒高度保守的Capsid基因設計引子與探針，發展即時反轉錄聚合酵素連鎖反應，以定量登革病毒。以此方法檢測不同目標細胞株對於第二型登革病毒的感染力的結果，可以與用傳統的溶斑試驗或免疫螢光反應得到的方法相互比較。此方法方便，靈敏，正確性高，可應用在未來的研究。

Dengue virus is a positive, single-stranded RNA enveloped flavivirus that is transmitted by mosquitoes, Aedes aegypti and Aedes albopictus. The virus is divided into four serotypes, DEN-1, DEN-2, DEN-3 and DEN-4.
In the process of virus entry, the envelope protein, E protein, plays an important role. It is a glycoprotein of 495 amino acids, with a transmembrane domain at the C-terminal. It is believed to form a stable, non-covalently linked homodimer. The extracellular domains of E glycoprotein are thought to interact with the host cell receptor.
The overall objective of this study is to investigate the dengue virus entry by establishing an E protein-mediated cell fusion assay.
In the first aim, we examined the expression of five DEN-2 E protein constructs, including pD2ME, pD2ME His, E His, pCB8D2J2 and pCB8D2J2VSV, in the mammalian cell line, 293 cells, to identify the ideal dengue E protein expression construct.
In the second aim, we developed a dengue cell fusion assay by cotransfecting NPCTW04 cells with pCB8D2J2VSV (from DEN-2, 16681 strain) and a reporter construct, pET-21a-GFP, followed by coculturing with target cells which were infected with recombinant vaccinia virus. Examination by fluorescent microscope revealed that NPCTW04 cell can fuse with BHK and K562 cells, two known dengue target cells, but not with H9 or Hut78 cells. The infectivity assay in different target cells revealed that DEN-2 (16681 strain) virus can replicate in BHK and K562 cells efficiently, but not in H9 or Hut78 cells. This finding indicated that our cell fusion assay correlated with cellular tropism of dengue virus.
In the third aim, we developed a real-time RT-PCR assay for quantification of DEN-2 and DEN-3 viruses, using the probe and primers targeting a highly conserved region in the capsid gene. This assay was used to study replication kinetics, in comparison with traditional plaque assay and immunofluorescence assay. It is a convenient, sensitive and accurate method of quantification, and has potential for future application.

中文摘要 --------------------- 1
英文摘要 --------------------- 2
導論 --------------------- 3
材料與方法 --------------------- 10
結果 --------------------- 18
討論 --------------------- 25
圖表 --------------------- 29
參考文獻 --------------------- 46

Allison, S.L., Schalich, J., Stiasny, K., Mandl, C.W., Kunz, C., and Heinz, F.X. (1995). Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH. J. Virol. 69, 695-700.
Amberg, S.M., Nestorowicz, A., McCourt, D.W., and Rice, C.M. (1994). NS2B-3 proteinase-mediated processing in the yellow fever virus structural region : in vitro and in vivo studies. J. Virol. 68, 3794-3802.
Amberg, S.M., and Rice, C.M. (1999). Mutagenesis of the NS2B-NS3-mediated cleavage site in the flavivirus capsid protein demonstrates a requirement for coordinated processing. J. Virol. 73, 8083-8094.
Arias, C.F., Preugschat, F., and Strauss, J.H. (1993). Dengue 2 virus NS2B and NS3 form a stable complex that can cleave NS3 within the helicase domain. Virology 193, 888-899.
Bazan, J.F., and Fletterick, R.J. (1989). Detection of a trypsin-like serine protease domain in flaviviruses and pestivirus. Virology 171, 637-639.
Bazan, J.F., and Fletterick, R.J. (1990). Structural and catalytic models of trypsin-like viral proteases. Semin Virol. 1, 311-322.
Brandt, W.E., McCown, J.M., Gentry, M.K., and Russell, P.K. (1982). Infection enhancement of dengue-2 virus in the U937 human monocyte cell line by antibodies to flavivirus cross-reactive determinants. Infect. Immun. 36,1036-1041.
Brinton, M.A. (1986). Replication of flaviviruses. In Schlesinger S., Schlesinger M.J., eds. The Togaviridae and Flaviviridae. New York, Plenum. 162, 290-299.
Chambers, T.J., Hahn, C.S., Galler, R., and Rice, C.M. (1990). Flavivirus genome organization, expression, and replication. Annu. Rev. Microbiol. 44, 649-688.
Crise, B., Ruusala, A., Zagouras, P., Shaw, A., and Rose, J.K. (1989). Oligomerization of glycolipid-anchored and soluble forms of the vesicular stomatitis virus glycoprotein. J. Virol. 63, 5328-5333.
Diamond, M.S., Edgil, D., Roberts, T.G., Lu, B., and Harris E. (2000). Infection of human cells by dengue virus is modulated by different cell types and viral strains. J. Virol. 74, 7814-7823.
Dietz, V., Gubler, D.J., Ortiz, S., Kuno, G., Casta-Velez, A., Sather, G.E., Gomez, I., and Vergne, E. (1996). The 1986 dengue and dengue hemorrhagic fever epidemic in Puerto Rico: epidemiologic and clinical observations. P. R. Health Sci. J. 15,201-210.
Fries, E., and Rothman, J.E. (1980). Transport of vesicular stomatitis virus glycoprotein in a cell-free extract. Proc. Natl. Acad. Sci. USA 77, 3870-3874.
Gorbalenya, A.E., Donchenko, A.P., Koonin, E.V., and Blinov, V.M. (1989). N-terminal domains of putative helicases of flavi- and pestiviruses may be serine proteases. Nucleic Acids Res. 17, 3889-3897.
Gubler, D.J. (1987). Dengue and dengue hemorrhagic fever in the Americas. P. R. Health Sci. J. 6,107-111.
Gubler, D.J. (1988). Dengue, p223-260. In T. P. Monath (ed.), Epidemiology of arthropod-borne viral diseases. CRC Press, Inc., Boca Raton, Fla.
Gubler, D.J. (1989). Aedes aegypti and Aedes aegypti-borne disease control in the 1990s: top down or bottom up. Am. J. Trop. Med. Hyg. 40,571-578.
Gubler, D.J. (1997). Dengue and dengue hemorrhagic fever: its history and resurgence as a global public health problem, p.1-22. In D.J. Gubler and G. kuno (ed.), Dengue and dengue hemorrhagic fever. CAB International, London, United Kingdom.
Gubler, D. (1998). Dengue and hemorrhagic fever. Clin. Microbiol. Rev. 11, 480-496.
Gubler, D.J., Reed, D., Rosen, L., and Hitchcock, J.C.J. (1978). Epidemiologic, clinical and virologic observations on dengue in the Kingdom of Tonga. Am. J. Trop. Med. Hyg. 27, 581-589.
Halstead, S.B., Nimmannity, S., and Cohen, S.N. (1970). Observations related to pathogenesis of dengue hemorrhagic fever. Relation of disease severity to antibody response and virus recovered. Yale J. Biol. Med. 42, 311-328.
Halstead, S.B., and O'Rourke, E.J. (1977a). Dengue viruses and mononuclear phagocytes. Infection enhancement by non-neutralizing antibody. J. Exp. Med. 146, 201-217.
Halstead, S.B., and O'Rourke, E.J. (1977b). Antibody-enhanced dengue virus infection in primate leukocytes. Nature (London) 265, 739-741.
Halstead, S.B. (1988). Pathogenesis of dengue: challenges to molecular biology. Science 239, 476-481.
Hayes, E.B., and Gubler, D.J. (1992). Dengue and dengue hemorrhagic fever. Pediatr. Infect. Dis. J. 11, 311-317.
Heinz, F.X. (1986). Epitope mapping of flavivirus glycoproteins. Adv. Virus Res. 31, 103-168.
Heinz, F.X., Stiasny, K., Puschner-auer, G., Holzmann, H., Allison, S.L., Mandl, C.W., and Kunz, C. (1994). Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prM. Virology 198, 109-117.
Henchal, E.A., and Putnak, J.R. (1990). The dengue viruses. Clin. Microbiol. Rev. 3, 376-396.
Hirsch, A. (1883). Dengue, a comparative new disease: its symptoms. p. 55-81. In Handbook of geographical and historical pathology, vol. 1. Syndenham Society, London, United Kingdom.
Holzmann, H., Stiasny, K., York, H., Dorner, F., Kunz, C., Heinz, F.X. (1995). Tick-borne encephalitis virus envelope protein E-specific monoclonal antibodies for the study of low pH-induced conformational changes and immature virions. Arch. Virol. 140, 213-221.
Houng, H.-S.H., Hritz, D., and Kanesa-thasan N. (2000). Quantitative detection of dengue 2 virus using fluorogenic RT-PCR based on 3'-noncoding sequence. J. Virol. Meth. 86, 1-11.
Howe, G.M. (1977). A world geography of human diseases. Academic Press Inc., New York, N. Y.
Kurane, I., and Ennis, F.A. (1997). Immunopathogenesis of dengue virus infections, p.273-290. In D.J. Gubler and G. Kuno (ed.), Dengue and dengue hemorrhagic fever. CAB International, London, United Kingdom.
Lagging, L.M., Meyer, K., Owens, R.J., and Ray, R. (1998). Functional role of hepatitis C virus chimeric glycoproteins in the infectivity of pseudotyped virus. J. Virol. 72, 3539-3546.
Laue, T., Emmerich, P., and Schmitz, H. (1999). Detection of dengue virus RNA in patients after primary or secondary dengue infection by using the TaqMan automated amplification system. J. Clin. Microbiol. 37, 2543-2547.
Lee, E., Stocks, C.E., Amberg, S.M., Rice, C.M., and Lobigs, M. (2000). Mutagenesis of the signal sequence of yellow fever virus prM protein : enhancement of signalase cleavage in vitro is lethal for virus production. J. Virol. 74, 24-32.
Lewin, S.R., Vesanen, M., Kostrikis, L., Hurley, A., Duran, M., Zhang, L., Ho, D.D., and Markowitz, M. (1999). Use of real-time PCR and molecular beacons to detection virus replication in human immunodeficiency virus type 1-infected individuals on prolonged effective antiretroviral therapy. J. Virol. 73, 6099-6103.
Lin, Y.-L., Liu, C.-C., Lei, H.-y., Yeh, T,-M., Lin, Y.-S., Chen, R.M.-Y., and Liu, H.-S. (2000). Infection of five human liver cell lines by dengue-2 virus. J. Med. Virol. 60, 425-431.
Livak, K.J., Flood, S.A.J., Marmaro, J., Giusti, W., and Deetz, K. (1995). Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods and Applications 4, 357-362.
Mandl, C.W., Guirakhoo, F., Holzmann, H., Heinz, F.X., and Kunz, C. (1989). Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model. J. Virol. 63, 564-571.
Monath, T.P. (1994). Dengue: the risk to developed and developing countries. Proc. Natl. Acad. Sci. USA 91, 2395-2400.
Monath, T.P., and Heinz, F.X. (1996). Flaviviruses. In Fields Virology, B.N. Fields, D.M. Knipe, and P.M. Howley, eds. (Philadelphia: Lippincott-Raven), pp. 961-1023.
Moore, C.G., and Mitchell, C.J. (1997). Aedes albopictus in the United States: ten-year presence and public health implications. Emerg. Infect. Dis. 3, 329-334.
Morens, D.M., Venkateshan, C.N., and Halstead, S.B. (1987). Dengue 4 virus monoclonal antibodies identify epitopes that mediate immune enhancement of dengue 2 viruses. J. Gen. Virol. 68, 91-98.
Pinheiro, F.P. (1989). Dengue in the Americas, 1980-1987. Epidemiol. Bull. 10,1.
Rey, F.A., Heinz, F.X., Mandl, C., Kunz, C., and Harrison, S.C. (1995). The envelope glycoprotein from tick-borne encephalitis virus at 2 amstron resolution. Nature 375, 291-298.
Rice, C.M. (1996). Flaviviridae: the viruses and their replication. In Fields Virology, B.N. Fields, D.M. Knipe, and P.M. Howley, eds. (Philadelphia: Lippincott-Raven), pp. 931-960.
Rice, C.M., Lenches, E.M., Eddy, S.R., Shin, S.J., and Strauss, J.H. (1985). Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution. Science 229, 726-733.
Roehrig, J.T., Hunt, A.R., Johnson, A.J., and Hawkes, R.A. (1989). Synthetic peptides derived from the deduced amino sequence of the E-glycoprotein of Murray Valley encephalitis virus elicit antiviral antibody. Virology 171, 49-60.
Roehrig, J.T., Johnson, A.J., Hunt, A.R., Bolin, R.A., and Chu, M.C. (1990). Antibodies to dengue 2 virus E-glycoprotein synthetic peptides identify antigenic conformation. Virology 177, 668-675.
Roehrig, J. T., Bolin, R. A., and Kelly, R. G. (1998). Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica. Virology 246, 317-328.
Rose, J.K., Welch, W.J., Sefton, B.M., Esch, F.S., and Ling, N.C. (1980). Vesicular stomatitis virus glycoprotein is anchored in the viral membrane by a hydrophobic domain near the COOH terminus. Proc. Natl. Acad. Sci. USA 77, 3884-3888.
Rothman, A.L., and Ennis, F.A. (1999). Immunopathogenesis of dengue hemorrhagic fever. Virology 257, 1-6.
Rush, A.B. (1789). An account of the bilious remitting fever, as it appeared in Philadelphia in summer and autumn of the year 1780. Medical enquiries and observations, p. 104-117. Prichard and Hall, Philadelphia, Pa.
Sabin, A.B. (1952). Research on dengue during World War Ⅱ. Am. J. Trop. Med. Hyg. 1,30-50.
Siler, J.F., Hall, M.W., Hitchens, A. (1926). Dengue, its history, epidemiology, mechanism of transmission, etiology, clinical manifestations, immunity and prevention. Philipp. J. Sci. 29,1-304.
Takikawa, S., Ishii, K., Aizaki, H., Suzuki, T., Asakura, H., Matsuura, Y., and Miyamura, T. (2000). Cell fusion activity of hepatitis C virus envelope proteins. J. Virol. 74, 5066-5074.
Wang, S., He, R., and Anderson, R. (1999). PrM- and cell-binding domains of the dengue virus E protein. J. Virol. 73, 2547-2551.
Waterman, S.H., and Gubler, D.J. (1989). Dengue fever. Clin. Dermatol. 7,117-122.
Wengler, G., Nowak, T., and Castle, E. (1990). Description of a procedure which allows isolation of viral nonstructural proteins from BHK vertebrate cells infected with the West Nile flavivirus in a state which allows their direct chemical characterization. Virology 177, 795-801.
WHO. (1986). Dengue hemorrhagic fever: diagnosis, treatment, control. World Health Organization, Geneva.