博尔纳病病毒ORFⅠ基因中部片段的检测
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摘要
目的：博尔纳病病毒(Borna disease virus，BDV)是一种嗜神经性、非节段性、负股单链的RNA病毒。该病毒所致的Borna病(Bornadisease,BD)则是一种以明显的精神、行为异常和脑炎为特征的神经综合征。该病毒内含一条8.9Kb的基因组，有六个开放阅读框，分别编码核蛋白(P40)，磷蛋白(P24)，非糖基化蛋白(P10)，糖基化基质蛋白(P16，gp18)，糖蛋白(P57,gp84)以及含有18kDa大小分子团的L-多聚酶。目前为证实是否存在BDV感染一般采用对其ORFⅡ基因片段(P24)的检测，研究结果表明此序列较保守、变异性小，是检测病毒感染的特异性标志物。但对另一编码P40蛋白序列的检测结果的特异性和稳定性还不肯定，我们通过对先前检测BDV ORFⅡ基因片段为阳性或阴性的神经精神病人和健康献血者进行了BDV ORFⅠ基因中部片段的检测。以帮助确定在国内健康人群和某些神经精神疾病患者中如：病毒性脑炎、多发性硬化、精神分裂症以及情感障碍等疾病中是否存在BDV的感染，更为重要的是了解感染人和动物的该病毒片段是否存在差异，从而找出另一检测BDV感染的敏感性和稳定性的标记物，为下一步的研究奠定良好的基础。
重庆医科大学硕士学位论文
方法：提取从2000年12月至2001年6月在重庆医科大学第一附
属医院神经科和精神科住院及门诊的60例健康献血者、30例包括原因
未明的病毒性脑炎、多发性硬化、帕金森综合征，以及30例包括精神
分裂症和情感障碍患者PBMC中的总KN’A，采用套式逆转录聚合酶链
反应＊ested RTICR）技术进行了 BDV ORF基因中部片段的检测，
并对 PCR产物进行电泳分析及 Southern blot杂交分析。
结果：1．PCR产物电泳结果：20例病毒性脑炎病例中有1例阳性
u性率 5％人 17例精神分裂症病例中有 2例阳性叩性率 11．8％人
13例抑郁症病例中有 1例阳性u性率 7．7％人健康献血者中未有阳性
结果出现。其中精神分裂症组与对照组相比其结果有显着性差异0
＜0＊5）。
2．所有标本进行 Southern blot杂交分析，结果予以证实。
结论：1．感染人和动物的BDV ORFI基因中部片段具有高度的保
守性和一致性。
2．BDV ORFI基因中部片段可能是证实BDV感染的另一具有敏
感性和稳定性的指标。
3．中国的某些神经精神病人中存在BDV的感染。
Objective: Borna disease virus (BDV) is a neurotropic nonsegmented negative-stranded RNA virus that caused the illness calling Borna disease (BD). The illness is a nervous syndrome characterized by profound behavirol and consciousness abnormalities as well as encephalitis. BDV contanins a 8.9Kb antigenome that exists of six open reading frames correspond to encoding the nucleoprotein (p40), the phosphoprotein (p24), the unglycoslated protein (p10), the glycoslated matrixprotein (p16, p18), the glycoprotein (p57, gp94), and the L-polymerase with a molecular mass of approximately 180kDa. At present we usually use detecting BDV ORF II genome to cofirm whether the people have a BDV infection or not,the research suggests that BDV ORF II is more conservative and less variability,thus it is a good marker for detecting BDV infection. But it is doubt that the speciality and stability for the outcome of detecting BDV ORF I genome.We will detect the indviduals'(include the neuropsychiatric
4
patients with BDV ORFII postive or not and healthy blood donator) middle fragment of BDV ORF I to help defmiting whether internal healthy people and the certain neuropsychiatric patient such as with viral encephalitis,multiple,schizophrenia,affective disorder have catch a BDV infection or not, most of all, the research is for understanding if there is any difference in the fragment from animal and human beings infected with BDV, thereby to find another sensitive and stably marker for confirming BDV infection, and establish a solide bottom for advanced study.
Methods: Extracting the total RNA of human PBMC, the objective include 60 healthy blood donator, 30 patient with viral encephalitis and multiple sclerosis and Parkinsonian syndrome, 30 patient with schizophrenia and affective disorder, this indviuals were inpatients or outpatients of the first hospital of Chongqing University of Medical Science from December, 2000 to June, 2001. Using nested RT-PCR techique to detect Borna disease virus' middle fragment in ORF I, and using southern blot hybridization to analyze the PCR product.
Results: 1. The electrophoresis results of PCR product: one of the 20 patient with viral encephalitis had BDV positive (5%), two of the 17 patient with schizophrenia had BDV positive (11.8%), one of the 13 patient with affective disorder had BDV positive (7.7%), none of healthy blood donators
had BDV positive (0.0%). The positive results have a marked discrepancy between the patients with schizophrenia and the healthy blood donators(P< 0.05).
2. The analysis results of southern blot hybridization among all of specimens are coincidence with the detecting results of PCR products electrophoresis.
Conclusions: 1. The middle fragment derived from ORF I of Borna disease virus which infected human being and animal has a very high conversation and concordance.
2. Detection the middle fragment derived from ORF I of Borna disease virus is another probably sensitive and stably marker for confirming Borna disease virus infection.
3.There is a Borna diseae virus infection in the certain neuropsychiatric patients of China.
重庆医科大学硕士学位论文
方法：提取从2000年12月至2001年6月在重庆医科大学第一附
属医院神经科和精神科住院及门诊的60例健康献血者、30例包括原因
未明的病毒性脑炎、多发性硬化、帕金森综合征，以及30例包括精神
分裂症和情感障碍患者PBMC中的总KN’A，采用套式逆转录聚合酶链
反应＊ested RTICR）技术进行了 BDV ORF基因中部片段的检测，
并对 PCR产物进行电泳分析及 Southern blot杂交分析。
结果：1．PCR产物电泳结果：20例病毒性脑炎病例中有1例阳性
u性率 5％人 17例精神分裂症病例中有 2例阳性叩性率 11．8％人
13例抑郁症病例中有 1例阳性u性率 7．7％人健康献血者中未有阳性
结果出现。其中精神分裂症组与对照组相比其结果有显着性差异0
＜0＊5）。
2．所有标本进行 Southern blot杂交分析，结果予以证实。
结论：1．感染人和动物的BDV ORFI基因中部片段具有高度的保
守性和一致性。
2．BDV ORFI基因中部片段可能是证实BDV感染的另一具有敏
感性和稳定性的指标。
3．中国的某些神经精神病人中存在BDV的感染。
Objective: Borna disease virus (BDV) is a neurotropic nonsegmented negative-stranded RNA virus that caused the illness calling Borna disease (BD). The illness is a nervous syndrome characterized by profound behavirol and consciousness abnormalities as well as encephalitis. BDV contanins a 8.9Kb antigenome that exists of six open reading frames correspond to encoding the nucleoprotein (p40), the phosphoprotein (p24), the unglycoslated protein (p10), the glycoslated matrixprotein (p16, p18), the glycoprotein (p57, gp94), and the L-polymerase with a molecular mass of approximately 180kDa. At present we usually use detecting BDV ORF II genome to cofirm whether the people have a BDV infection or not,the research suggests that BDV ORF II is more conservative and less variability,thus it is a good marker for detecting BDV infection. But it is doubt that the speciality and stability for the outcome of detecting BDV ORF I genome.We will detect the indviduals'(include the neuropsychiatric
4
patients with BDV ORFII postive or not and healthy blood donator) middle fragment of BDV ORF I to help defmiting whether internal healthy people and the certain neuropsychiatric patient such as with viral encephalitis,multiple,schizophrenia,affective disorder have catch a BDV infection or not, most of all, the research is for understanding if there is any difference in the fragment from animal and human beings infected with BDV, thereby to find another sensitive and stably marker for confirming BDV infection, and establish a solide bottom for advanced study.
Methods: Extracting the total RNA of human PBMC, the objective include 60 healthy blood donator, 30 patient with viral encephalitis and multiple sclerosis and Parkinsonian syndrome, 30 patient with schizophrenia and affective disorder, this indviuals were inpatients or outpatients of the first hospital of Chongqing University of Medical Science from December, 2000 to June, 2001. Using nested RT-PCR techique to detect Borna disease virus' middle fragment in ORF I, and using southern blot hybridization to analyze the PCR product.
Results: 1. The electrophoresis results of PCR product: one of the 20 patient with viral encephalitis had BDV positive (5%), two of the 17 patient with schizophrenia had BDV positive (11.8%), one of the 13 patient with affective disorder had BDV positive (7.7%), none of healthy blood donators
had BDV positive (0.0%). The positive results have a marked discrepancy between the patients with schizophrenia and the healthy blood donators(P< 0.05).
2. The analysis results of southern blot hybridization among all of specimens are coincidence with the detecting results of PCR products electrophoresis.
Conclusions: 1. The middle fragment derived from ORF I of Borna disease virus which infected human being and animal has a very high conversation and concordance.
2. Detection the middle fragment derived from ORF I of Borna disease virus is another probably sensitive and stably marker for confirming Borna disease virus infection.
3.There is a Borna diseae virus infection in the certain neuropsychiatric patients of China.
引文
1 A.L.Berg, R.Dorries,and M.Berg. Borna disease virus infection in racing horses with behavioral and movement disorders. Arch Virol, 1999, 144:547-559.
2 Lnndgren AL, Zimmermann W, Bode L, et al. Staggering disease in cat: isolation and characterization of the feline Borna disease virus. J Gen Virol, 1995,76:2215-2222.
3 Taakehiro Kohno, Toshiyuki Goto, Tomohiko Takasaki, et al. Fine structure and morphogenesis of Borna disease virus. J virol, 1999,73:760-766.
4 Lebelt J and Hagenau K.Distribution of Borna disease virus in naturally infected animals with clinical disease. Berl Munch Tierarztl Wochenschr, 1996,109:178-183.
5 Wormser U, Weisman Y, Ashash E, et al. In vitro cytotoxicity of sera of paralysed ostriches showing serological positivity for Borna disease virus. Vet Rec, 1996,138:418-419.
6 Rott R, Herzog S, Fleischer B, et al. Borna disease, a possible hazard for man? Arch Virol, 1991, 118:143-149.
7 Bechter K, Schuttler R, Herzog S. Possible significance of Borna disease for humans. Neurol Psychiat, Brain Res, 1992, 1:23.
8 Yurie Nakamura,Takahashi H,Shoya Y, et al. Isolatiion of Borna disease virus from human brain tissue. J Virol, 2000,74:4601-4611.
9 Bode L,Durrwald R,Rantam FA,et al. First isolates of infectious human Borna disease virus from patients with mood disorders. Mol Psychiatry, 1996,1:200-212.
10 W. Lan Lipkin,Anette Schneemann and Marylou V. Solbrig. Borna disease virus:implications for human neuropsychiatric illiness. Trend In microbial, 1995,3:64-69.
11 Oliver Planz,Christine Rentzsch,Anil Batra, et al. Pathogenesis of Borna disease virus: Granulocyte fractions of psychiatric patients harbor infectious virus in the absence of antiviral antibodies. J Virol, 1999,73:6251-6256.
12 L.Stitz, K.Noske, et al. A functional role for neutralizing antibodies in Borna disease: Influence on virus outside the central nervous system. J Virol. Nov. (1998). P. 8884-8892
13 Hanns Ludwig. The neuropathogenesis of Borna disease virus infections. Intervirology. 1997; 40:185-197.
14 Mikael B, Christian E, Jonas B, et al. Two domain of the Borna disease virus p40 protein are required for interaction with the p23 protein. J Gen Virol. 1998.79:2957-2963.
15 Takeshi K, Yuko S, Toshiaki K, et al. Nuclear targeting activity associated with the amino terminal region of the Borna disease virus nucleoprotein. Virology. 1998:243:188-197.
16 Oliver P, Lothar S. Borna disease virus nucleoprotein(p40) is a major target for CD8~+-T-Cell-Mediated immune response. J Virol, 1999,p. 1715-1718.
17 Planz O, Rentzsch C, Batra A, et al. Pathegenesis of Borna disease virus:granulocyte fraction of psychiatric patients harbors infectious virus in the absence of antiviral antibodies. J Virol, 1999,73(8): 6251-6256.
18 Kishi M, Arimura Y. Sequence variability of Borna disease virus RNA transcription and replication. J Virol, 1998,72(9): 7697-702.
19 Cubitt B, Oldstone C, Torre JC. Sequence and genome organization of Borna disease virus. J Virol, 1994,68:1382-96.
20 Briese T, Schneemann, Lewis AJ,et al. Sequence organization of Borna disease virus. Proc Natl Acad Sci USA, 1994,91:4362-66.
21 Schneider P, Briese T, Zimmermann W, et al. Sequence conservation in field and experimental isolates of Borna disease virus. J Virol, 1994,68:63-8.
22 谢鹏，岩田太秀，高桥和郎等。感染人体的博尔纳病病毒ORFⅠ基因前部片段的检测。中华微生物和免疫学杂志，1998，18(3)：172-75。
23 Sauder C, Muller A, Cubitt B, et al. Dection of Borna disease virus(BDV) antibodies and BDV RNA in psychiatric patients: evidence for high sequence conservation of human blood-derived BDV RNA. J Virol, 1996,70:7713-24.
24 Bechter K, Schuttler R, Herzog S. Case of neurological and behavioral abnormalities: due to Borna disease virus encephalitis? Psychiary Res, 1992,42:193-196.
25 Haas B, Becht H, Rott R. Purification and properties of an intranuclear virus-specific antigen from tissues infected with Borna disease virus. J Gen Virol 1986:67:235-47.
26 Bode L,Riegel S,Lange W, et al.Human infections with Borna disease virus:seroprevalence in patients with chronic diseases and healthy individuals. J Med Virol, 1992, 36:309-315.
27 Michael Deuschle, Liv Bode, Isabelia Heuser, et al. Borna disease vires and neuropsychiatric disorders. Lancet, 1998,352:1828-1829..
28 Mirella Sslvatore,Sergei Morzunov, Martin Schwemmle, et al. Borna disease virus in brains of North American and European people with schizophrenia and bipolar disorder. Lancet, 1997,349:1813-1814.
29 Bode L and Riegel S. Immune response to Borna disease vires infections in animals and man(abstract). 2nd world conference on viruses, immunity and mental health,4-7 Oct 1998, Montreal,Canada.
30 Iwahashi K,et al. Clinical investigation of the relaionship between Borna disease virus infection and schizophrenia in 67 paients in Japan. Acta Psychiatr Scand, 1997, 96:412-415.
31 Chen CH,Chiu YL,Shaw CK,et al. Detction of Borna disease virus RNA from peripheral blood cells in patients and mental health works. Mol psychiatry, 1999,4:566-571.
32 谢鹏，岩田泰秀，高桥和郎等．精神病病人Borna病病毒P23基因片段的检测．中华精神科杂志，1997，30：136-138．
33 Sambrook J, Fritsch E, Maniatis T. Molecular Cloning A Laboratory Manual.2nd ed. Cold Spring Harbor Laboratory Press, 1989.
2 Lnndgren AL, Zimmermann W, Bode L, et al. Staggering disease in cat: isolation and characterization of the feline Borna disease virus. J Gen Virol, 1995,76:2215-2222.
3 Taakehiro Kohno, Toshiyuki Goto, Tomohiko Takasaki, et al. Fine structure and morphogenesis of Borna disease virus. J virol, 1999,73:760-766.
4 Lebelt J and Hagenau K.Distribution of Borna disease virus in naturally infected animals with clinical disease. Berl Munch Tierarztl Wochenschr, 1996,109:178-183.
5 Wormser U, Weisman Y, Ashash E, et al. In vitro cytotoxicity of sera of paralysed ostriches showing serological positivity for Borna disease virus. Vet Rec, 1996,138:418-419.
6 Rott R, Herzog S, Fleischer B, et al. Borna disease, a possible hazard for man? Arch Virol, 1991, 118:143-149.
7 Bechter K, Schuttler R, Herzog S. Possible significance of Borna disease for humans. Neurol Psychiat, Brain Res, 1992, 1:23.
8 Yurie Nakamura,Takahashi H,Shoya Y, et al. Isolatiion of Borna disease virus from human brain tissue. J Virol, 2000,74:4601-4611.
9 Bode L,Durrwald R,Rantam FA,et al. First isolates of infectious human Borna disease virus from patients with mood disorders. Mol Psychiatry, 1996,1:200-212.
10 W. Lan Lipkin,Anette Schneemann and Marylou V. Solbrig. Borna disease virus:implications for human neuropsychiatric illiness. Trend In microbial, 1995,3:64-69.
11 Oliver Planz,Christine Rentzsch,Anil Batra, et al. Pathogenesis of Borna disease virus: Granulocyte fractions of psychiatric patients harbor infectious virus in the absence of antiviral antibodies. J Virol, 1999,73:6251-6256.
12 L.Stitz, K.Noske, et al. A functional role for neutralizing antibodies in Borna disease: Influence on virus outside the central nervous system. J Virol. Nov. (1998). P. 8884-8892
13 Hanns Ludwig. The neuropathogenesis of Borna disease virus infections. Intervirology. 1997; 40:185-197.
14 Mikael B, Christian E, Jonas B, et al. Two domain of the Borna disease virus p40 protein are required for interaction with the p23 protein. J Gen Virol. 1998.79:2957-2963.
15 Takeshi K, Yuko S, Toshiaki K, et al. Nuclear targeting activity associated with the amino terminal region of the Borna disease virus nucleoprotein. Virology. 1998:243:188-197.
16 Oliver P, Lothar S. Borna disease virus nucleoprotein(p40) is a major target for CD8~+-T-Cell-Mediated immune response. J Virol, 1999,p. 1715-1718.
17 Planz O, Rentzsch C, Batra A, et al. Pathegenesis of Borna disease virus:granulocyte fraction of psychiatric patients harbors infectious virus in the absence of antiviral antibodies. J Virol, 1999,73(8): 6251-6256.
18 Kishi M, Arimura Y. Sequence variability of Borna disease virus RNA transcription and replication. J Virol, 1998,72(9): 7697-702.
19 Cubitt B, Oldstone C, Torre JC. Sequence and genome organization of Borna disease virus. J Virol, 1994,68:1382-96.
20 Briese T, Schneemann, Lewis AJ,et al. Sequence organization of Borna disease virus. Proc Natl Acad Sci USA, 1994,91:4362-66.
21 Schneider P, Briese T, Zimmermann W, et al. Sequence conservation in field and experimental isolates of Borna disease virus. J Virol, 1994,68:63-8.
22 谢鹏，岩田太秀，高桥和郎等。感染人体的博尔纳病病毒ORFⅠ基因前部片段的检测。中华微生物和免疫学杂志，1998，18(3)：172-75。
23 Sauder C, Muller A, Cubitt B, et al. Dection of Borna disease virus(BDV) antibodies and BDV RNA in psychiatric patients: evidence for high sequence conservation of human blood-derived BDV RNA. J Virol, 1996,70:7713-24.
24 Bechter K, Schuttler R, Herzog S. Case of neurological and behavioral abnormalities: due to Borna disease virus encephalitis? Psychiary Res, 1992,42:193-196.
25 Haas B, Becht H, Rott R. Purification and properties of an intranuclear virus-specific antigen from tissues infected with Borna disease virus. J Gen Virol 1986:67:235-47.
26 Bode L,Riegel S,Lange W, et al.Human infections with Borna disease virus:seroprevalence in patients with chronic diseases and healthy individuals. J Med Virol, 1992, 36:309-315.
27 Michael Deuschle, Liv Bode, Isabelia Heuser, et al. Borna disease vires and neuropsychiatric disorders. Lancet, 1998,352:1828-1829..
28 Mirella Sslvatore,Sergei Morzunov, Martin Schwemmle, et al. Borna disease virus in brains of North American and European people with schizophrenia and bipolar disorder. Lancet, 1997,349:1813-1814.
29 Bode L and Riegel S. Immune response to Borna disease vires infections in animals and man(abstract). 2nd world conference on viruses, immunity and mental health,4-7 Oct 1998, Montreal,Canada.
30 Iwahashi K,et al. Clinical investigation of the relaionship between Borna disease virus infection and schizophrenia in 67 paients in Japan. Acta Psychiatr Scand, 1997, 96:412-415.
31 Chen CH,Chiu YL,Shaw CK,et al. Detction of Borna disease virus RNA from peripheral blood cells in patients and mental health works. Mol psychiatry, 1999,4:566-571.
32 谢鹏，岩田泰秀，高桥和郎等．精神病病人Borna病病毒P23基因片段的检测．中华精神科杂志，1997，30：136-138．
33 Sambrook J, Fritsch E, Maniatis T. Molecular Cloning A Laboratory Manual.2nd ed. Cold Spring Harbor Laboratory Press, 1989.
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