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系統識別號 U0026-0812200910343008
論文名稱(中文) 單純疱疹病毒毒力的探討
論文名稱(英文) Investigation of HSV strains varied in their virulence
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 91
學期 1
出版年 92
研究生(中文) 曹昌暉
研究生(英文) Chuang-Hui Tsao
學號 s4689111
學位類別 碩士
語文別 英文
論文頁數 46頁
口試委員 口試委員-吳鴻明
口試委員-許桂森
指導教授-陳舜華
中文關鍵字 單純疱疹病毒第一型 
英文關鍵字 HSV-1 
學科別分類
中文摘要   單純疱疹病毒第一型(HSV-1)為一種DNA病毒,根據血清學統計,世界上成年人口中有90%以上曾被此病毒感染過。此病毒可以在哺乳類動物的神經系統中複製與擴散,並造成致死性腦炎。然而,病毒的毒力致病機轉仍不清楚,先前的研究指出當小鼠感染了KOS病毒株,其半致死劑量 (LD50) 大於2 × 107 PFU。當小鼠感染另一病毒株294.1,其LD50為1.38 × 104 PFU。因此我設計實驗去了解這兩個病毒株在感染小鼠後,為何其毒力有如此大的差異。首先我分析這兩株病毒在周邊組織與神經組織生長複製的情形,結果顯示這兩個病毒複製的能力並無明顯差異,然而不論是在周邊或是神經組織,294.1這株病毒存在的時間都比KOS持久。其次,我也藉由分析血腦屏障(blood-brain barrier;BBB)的通透性來做為病毒引起腦部損害的指標。根據伊凡氏藍(Evans blue extraction assay)萃取法與M4 tracer法,顯示不論是在KOS或是294.1感染的腦部都表現出BBB通透性的增加。此外我選用一個只能在周邊組織複製而不能在神經組織複製之突變病毒株294.1△tk,來分析被該病毒感染的小鼠BBB的通透性之改變,結果發現該組小鼠的通透性並無增加,此結果確定了BBB通透性的增加是因為病毒在腦部複製所引起,然而免疫組織化學染色的結果說明了,病毒影響BBB的通透性並不是以直接地對內皮細胞或是其他構成BBB的細胞造成傷害,而是以一種間接的方式。許多的研究指出發炎反應的細胞激素(pro-inflammatory cytokines)會改變BBB的通透性,同時我發現到在病毒感染的腦部中有前發炎反應之細胞激素的產生。另外在BBB的通透性增加後,我發現病毒感染的腦部裡,免疫細胞有增加的現象,其中以單核球及巨噬細胞為主。同時在病理染色切片發現到294.1感染的小鼠腦部其神經細胞所受到的傷害都較KOS嚴重,根據上述的結果,294.1其毒力之所以會比KOS強,可能是因為本身病毒基因上的差異與所引起不同程度的免疫反應所導致。
英文摘要   Herpes simplex virus type 1 (HSV-1), a member of Herpesviridae with double-stranded DNA genome, is able to multiply and spread in multiple organs, including nervous system, to cause death. However, the pathogenesis of HSV virulence is not clearly understood. Previous and my studies showed that different HSV-1 strains varied in their virulence to cause death in mice. My results showed that these two viruses replicated to the same degree but 294.1 persisted longer than KOS in peripheral and nervous tissues. How these two viruses affect the infected brains was also investigated and compared. I found the permeability of blood-brain barrier (BBB) was altered in infected mice. The change of BBB permeability is due to viral replication in the brain because 294.1Δtk, which cannot replicate in nervous tissues, failed to open BBB. The expression of pro-inflammatory cytokines were detected in infected brains. The number of inflammatory cells increased and the major type of inflammatory cells switched from lymphocytes to monocyte/marcopgage in the brain after infection. The change of BBB permeability and degree of cytokine expression as well as inflammatory responses observed in 294.1-infected brains were greater than those in KOS-infected brains. Moreover, histological examination and Nissl staining showed the neuron damage and loss, particularly in 294.1-infected brains. Taken together, the higher virulence of 294.1 in mice might result from neuron damage in the CNS caused by both viral as well as immune activities.
論文目次 CHINESE ABSTRACT………………………………………………………………V
ENGLISH ABSTRACT……………………………………………………………VII
LIST OF TABLES…………………………………………………………………VIII
LIST OF FIGURES…………………………………………………………………IX

INTRODUCTION……………………………………………………………………1

MATERIALS AND METHODS………………………………………………………5
 Viruses and cells…………………………………………………………………5
 Infection of mice and tissue collection……………………………………………5
 Assay of BBB permeability………………………………………………………6
 Immunohistochemistry staining…………………………………………………7
 Western blotting…………………………………………………………………8
 Quantification of immune cells in HSV-1-infected mouse brain…………………8
 Histological examination…………………………………………………………9
 Nissl staining………………………………………………………………………9
 TUNEL assay……………………………………………………………………10
 Statistical analysis………………………………………………………………10

RESULTS……………………………………………………………………………11
 HSV-1 strains varied in their virulence in mice in vivo…………………………11
 Virus growth in peripheral tissues………………………………………………11
 Virus growth in nervous tissues…………………………………………………12
 Change of BBB permeability during infection…………………………………12
 Increase of immune cells and change of immune cell type in infected brains……15
 Damages of HSV-1-infected brains………………………………………………15

DISCUSSION……………………………………………………………………17

REFERENCES……………………………………………………………………23
參考文獻 1. Abbott, N.J. 2000. Inflammatory mediators and modulation of blood-brain barrier permeability. Cellular and Molecular Neurobiology. 20,131-147.

2. Aggarwal, B.B., and Puri, R.K. 1995. Human cytokines: Their role in disease and therapy. In Sharief, M.K. ed. Role of cytokines in blood-brain barrier damage. Blackwell Science: Cambridge, MA

3. Allan, S.M., and Rothwell, N.J. 2001. Cytokines and acute neurodengeneration. Nature Neuroscience Review. 2,734-744.

4. Aloisi, F. 2001. Immune function of microglia. GLIA. 36,165-179.

5. Anderson, JR. 2001. The mechanisms of direct, virus-induced destruction of neuron. Current Topics in Microbiology and Immunology. 253,15-33.

6. Bauer, J., Rauschka, H., and Lassmann, H. 2001. Inflammation in the nervous system: the human perspective. GLIA. 36,235-243.

7. Beers, D.R., Henlel, J.S. Schaefer, D.C., Rose, J.W. and Stroop, W.G. 1993. Neuropathology of herpes simplex virus encephalitis in a rat seizure model. Journal of Neuropathology and Experimental Neurology. 52,241-252.

8. Ben-Hur, T., Cialic, R., Itzik, A., Yirmiya, R., and Weidenfeld, J. 2001. Actue effects of purified and UV-inactivated herpes simplex virus type 1 on the hypothalamic-pituitary-adrenocortical axis. Neuroendocrinology. 74,160-166.

9. Ben-Hur, T., Cialic, R., Itzik, A., Bsrsk, O., Yirmiya, R., and Weidenfeld, J. 2001. A novel permissive role for glucocrticoids in induction of febrile and behavioral signs of experimental herpes simplex virus encephalitis. Neuroscience. 108,119-127.

10. Ben-Hur, T., Conforti, N., Itzik, A., and Weidenfeld, J. 1995. Effects of HSV-1, a neurotropic virus, on the hypothalamic-pituitary-adrenocortical axis in rat. Brain Research. 702, 17-22.

11. Ben-Hur, T., Rosenthal, J., Itzik, A., and Weidenfeld, J. 1996. Adrenocortical activation by herpes virus: involvement of IL-1b and central noradrenergic system. Neuroreport. 7,927-931.

12. Benn, T., Halfpenny, C., and Scolding, N. 2001. Glial cells as target for cytotoxic immune mediators. GLIA. 36,200-211.

13. Bower, J.R., Mao, H., Durishin, C., Rozenbom, E., Detwiler, M., Rempinski, D., Karban, T.L., and Rosenthal, K.S. 1999. Intrastrain variants of herpes simplex virus type 1 isolated from a neonate with fatal disseminated infection differ in the ICP34.5 gene, glycoprotein processing, and neuroinvasiveness. Journal of Virology. 73,3843-3853.

14. Brankin, B., Hart, M.N., Cosby, S.L., Fabry, Z., and Allen, I.V. 1995. Adhesion molecule expression and lymphocyte adhesion to cerebral endothelium: effects of measles virus and herpes simplex 1 virus. Journal of Neuroimmunology. 56,1-8.

15. Campanella, M., Sciorati, C., Tarozzo, G., and Beltramo, M. 2002. Flow cytometric analysis of inflammatory cells in ischemic rat brain. Stroke. 33,586-592.

16. Chan, W.L., Javanovic, T., and Lukic, M.L. 1989. Infiltration of immune T cells in the brain of mice with herpes simplex virus-induced encephalitis. Journal of Neuroimmunology. 23,195-201.

17. Chaturvedi, U.C., Dhawan, R., Khanna, M., and Mathur, A. 1991. Breakdown of the blood-brain barrier during dengue virus infection of mice. Journal of General Virology. 72,859-866.

18. Chen, N., Restivo, A., and Reiss, C. K. 2001. Leukotrienes play protective roles early during experimental VSV encephalitis. Journal of Neuroimmunology. 120,94-102

19. Chrisp, C.E., Sunstrum, J.C., Averill, JR, D.R., Levine, M., and Glorioso, J.C. 1989. Characterization of encephalitis in adult mice induced by intracerevral inoculation of herpes simplex virus type 1 (KOS) and comparison with mutants showing decreased virulence. Laboratory Investigation. 60,822-830.

20. Dong, Y., and Benveniste, E.N. 2001. Immune function of astrocytes. GLIA. 36,180-190.

21. Erälinna, J.P., Soilu-Hänninen, M., Röyttä, M., Hukkanen,V., Salmi, A.A., and Salonen, R. 1996. Blood-brain barrier breakdown and increased intercellular adhesion molecule (ICAM-1/CD54) expression after Semliki Forest (A7) virus infection facilitates the development of experimental allergic encephalomyelitis. Journal of Neuroimmunology. 66,103-114.

22. Esiri, M.M., Drummond, C.W.E., and Morris, C.S. 1995. Macrophage and microglia in HSV-1 infected mouse brain. Journal of Neuroimmunology. 62,201-205.

23. Havenith, C.E.G., Askew, D., and Walker, W.S. 1998. Mouse resident microglia: isolation and characterization of immunoregulatory properties with naïve CD4+ and CD8+ T-cells. GLIA. 22,348-359.

24. Hickey,W.F. 2001. Basic principles of immunological surveillance of the normal central nervous system. GLIA. 36,118-124.

25. Huber, J.D., Egleton, R.D., and Davis, T.P. 2001. Molecularphysiology and pathophysiology of tight junctions in the blood-brain barrier. TRENDS in Neurosciences. 24,719-725.

26. Kastruckoff, L.F., Lau, A.S., Leung, G.Y., Walker, D., and Thomas, E.E. 1992. Herpes simplex virus type 1 (HSV 1)-induced multifocal central nervous system (CNS) demyelination in mice. Journal of Neuropathology and Experimental Neurology. 51,432-439.

27. Khanna, M., Chaturvedi, U.C., Sharma, M.C., Pandey, V.C. and Mathur, A. 1990. Increased capillary permeability mediated by a dengue virus-induced lymphokine. Immunology. 69,449-453. .

28. Kleinschmidt-DeMasters, B.K., and Gilden, D.H. 2001. The Expanding spectrum of herpesvirus infections of the nervous system. Brain Pathology. 11,440-451

29. Komatsu, T., Ireland, D.D.C., Chung, N., Dore, A., Yoder, M., and Reiss, C.S. 1999. Regulation of the BBB during viral encephalitis: roles of IL-12 and NOS. Nitric Oxide. 3,327-339.

30. Leib, D.A., Coen, D.M., Bogard, C.L., Hicks, K. A., Yager, D. R., Knipe, D. M., Tyler, K.L., and Schaffer, P.A. 1989. Immediate-early regulatory gene mutants define different stages in the establishment and reactivation of herpes simplex virus latency. Journal of Virology. 63, 759-768.

31. Lambertsen, K.L., Gregersen, R., Drojdahl, N., Owens, T., and Finsen, B. 2001. A specific and sensitive method for visualization of tumor necrosis factor in the murine central nervous system. Brain Reasearch Protocols. 7,175-191.

32. Liang, X-H, Goldman, J. E., Jiang, H-H, and Levine, B. 1999. Resistance of IL-1b-deficient mice to fatal sindbis virus encephalitis. Journal of Virology. 73,2563-2567.

33. Lindsberg, P.J., Launes, J., Tian, L., Välimaa, H., Subramanian, V., Siren, J., Hokkanen, L., Hyypiä, T., Carpén, O., and Gahmberg, C.G. 2002. Release of soluble ICAM-5, a neuronal adhension molecule, in acute encephalitis. Neurology. 58,446-451.

34. Lusting, S., Danenberg, H. D., Kafri, Y., Kobiler, D., and Ben-Nathan, D. 1992. Viral neuroinvasion and encephalitis induced by lipopolysaccharide and its mediators. Journal of Experimental Medicine. 176,707-712.

35. Mathur, A., Khanna, N., and Chaturvedi, U.C. 1992. Breakdown of blood-brain barrier by virus-induced cytokine during Japanese encephalitis virus infection. International Journal of Experimental Pathology. 73,603-611.

36. Nakajima, H., Kobayashi, M., Pollard, R., and Suzuki, F. 2001. Monocyte chemoattractant protein-1 enhances HSV-induced encephalomyelitis by stimulating TH2 response. Journal of Leukocyte Biology. 70,374-380.

37. Päivärinta, M.A., Röyttä, M., Hukkanen, V., Marttila, R.J., and Rinne, U.K. 1994. Nervous system inflammatory lesions and viral nucleic acids in rabbit with herpes simplex virus encephalitis-induced rotational behaviour. Acta Neuropathologica. 1994. 87,259-268.

38. Pelosi, E., Rozenberg, F., Coen, D.M., and Tyler, K.L. 1998. A herpes simple virus DNA polymerase mutation that specifically attenuates neuroviruence in mice. Virology. 252, 364-372.

39. Prat, A., Biernacki, K., Wosik, K., and Antel, J.P. 2001. Glial cell infiuence on the human blood-brain barrier. GLIA. 36,145-155.

40. Renis, H.E., Edison, E.E., Mathews, J., and Gray, J.E. 1976. Pathogenesis of herpes simplex virus type 1 and 2 in mice after various routes of inoculation. Infection and Immunity. 14,571-578.

41. Richards, J.T., Kern, E.R., Overall Jr, J.C., and Glasgow, L.A. 1981. Differences in neurovirulence among isolates of herpes simplex virus type 1 and 2 in mice using four routes of infection. The Journal of Infectious Disease. 144,464-471.

42. Schmutzhard, E. 2001. Viral infections of CNS with special emphasis on herpes simplex infections. Journal of Neurology. 248,469-477.

43. Sciammas, R., Kodukula, P., Tang, Q., and Hendricks, R.L. 1997. T cell rcepetor-g d cells protect mice from herpes simplex virus type 1-induced lethal encephalitis. Journal of Experimental Medicine. 185,1969-1975.

44. Schmued, L., and Slikker, JR, W. 1999. Black-Gold: a simple, high-resoultion histochemical label for normal and pathological myelin in brain tissue sections. Brain Reasearch. 837, 289-297.

45. Takikita, S., Takano.T., Narita, T., Takikita, M., Ohno, M., and Shimada, M. 2001. Neuronal apoptosis mediated by IL-1b expression in viral encephalitis caused by a neuroadapted strain of the mumps virus (Kiham strain) in hamsters. Experimental Neurology. 172,47-59.

46. Tsao, N., Hsu, H.P., and Lei, H.Y. 1999. TNF-a-induced cyclooxygenase 2 not only increase the vasopermeability of blood-brain barrier but also enhances the neutrophil survival in Escherichia coli-induced brain inflammation. Prostaglandins and other Lipid Mediators. 57,371-382.

47. Tsao, N., Kanakamma, P.P., Luh, T.Y., Chou, C.K., and Lei, H.Y. 1999. Inhibition of Escherichia coli-induced meningitis by carboxyfullerence. Antimicrobial Agents and Chemotherapy. 43,2273-2277.

48. Whitely, F.J., Kimberlin, D.W., and Roizman, B. 1998. Herpes simplex virus. Clinical Infectious Disease. 26,541-555.

49. Fields, B.N., Knipe, D.M., Howley, P.M., Chanock, R. M., and Melnick, J.L. et al. (2001) Fields Virology. In Whitley, R.J. ed. Herpes simplex viruses. Lippincott-Raven, Philadelphia.
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