关于禽脑脊髓炎病毒内蒙分离株基因组的研究
|
摘要
禽脑脊髓炎病毒(Avian Encephalomyelitis Virus,AEV)是一种小RNA病毒。侵害鸡的中枢神经系统和其它实质性器官，该病毒通过口-粪途径传播，具有水平和垂直传播的能力。由于它极大的稳定性，被污染的区域可能长期保持传染性。鸡胚适应株Van Roekel是高度嗜神经的，并且在鸡的肠道内不能有效的生长，而野毒株却是嗜肠道型的。尽管这样，野毒株和Van Roekel株的病毒多肽具有一致的抗原性。幼鸡感染该病毒后，引起麻痹、头颈震颤甚至共济失调，而成鸡常呈亚临床感染或导致产蛋量和孵化率下降。病毒的感染性不受氯仿、低pH、胃蛋白酶、胰酶和脱氧核糖核酸酶的影响，镁离子可增强病毒对热的稳定性，病毒的浮密度为1.31g／ml，直径为22-30nm，该病毒主要在鸡胚中增殖，在大多数细胞培养物中不生长。
1932年Jones首次报道了在1930年美国新英格兰地区幼鸡群爆发的禽脑脊髓炎。随后十年中，在欧洲、加拿大、日本和澳大利亚都有此病的报道。八十年代，该病传入我国。AEV一直被认为是最重要的禽病病原之一，尽管它很重要，但有关其基础生物学，发病机理的了解却较少。本项研究，旨在利用分子生物学的技术手段，搞清楚AEV基因组的序列和结构，从而探讨基因结构与功能的关系。
本研究共分四个部分：第一部分为AEV的增殖，纯化和电镜观察，用1:5倍稀释的AEV-NH937株和阴性对照PBS分别经卵黄囊接种于6dSPF鸡胚，继续孵化10d后，收集尿囊液。比较接种组和健康对照组鸡胚的大小，结果显示，健康对照组鸡胚明显大于接种组。分离、提纯AEV，把纯化的病毒在电镜下观察，证明确有大量AEV病毒粒子存在，说明AEV在鸡胚中成功扩增；第二部分是AEV-NH937基因组的序列测定工作。根据Calnek疫苗株序列设计了9对引物，利用反转录聚合酶链式反应，成功地扩增了AEV-NH937毒株的全基因。把它们分段克隆在pUCm-T载体上，经序列分析，获得了AEV-NH937毒株的基因组全序列及推导的氨基酸序列，基因组全长为7055个核苷酸，与calnek疫苗株具有94.3％的同源性，编码一个含2134个氨基酸的多聚蛋白。这是国内首次对AEV基因组全序列的分析；第三部分：AEV外壳蛋白VP_1基因在大肠杆菌中的融合表达、纯化及活性研究。将VP_1基因编码区的810bp片段插入载体pGEX-4T1构建表达重组质粒，然后转化大肠杆菌BL_(21)，经IPTG诱导后，用聚丙稀酰氨凝被电泳分析。结果表明，表达产物为53KD的融合蛋白。经超声波裂解，用尿素溶解包涵体，电泳纯化后，利用ELISA检测VP_1
外壳蛋白，表明具有一定抗原性；第四部分：应用原位杂交检测 AEV i用 D i g
标记的探针与sd、10d、20d的攻毒鸡脑组织杂交，来检测那V的RNA。结果
显示，sd、10d、20d的攻毒脑组织切片均有阳性杂交信号，阴性对照却没有
杂交信号，该方法与其它检测方法具有良好的平行关系。
Avian encephalomyelitis virus (AEV) is a picornavirus with a predilection for the central nervous system and other parenchymous organs of chickens that is transmited by the oral-faecal route.The virus may be spread by the vertical and horizontalroutes, and because of its great stability,contaminated areas may remain infectious for long periods.The egg-adapted Van Roekel strain is highly neurotropic and does not grow efficiently in the enteric tract of the chicken,and the field isolates of AEV is usually enterotropic .Despite this.the virion polypeptides of both naturally-occurring strains and the Van Roekel strain are antigenically identical. In young chickens AEV induces paralysis, ataxia and muscular dystrophy, while in older chickens, infection is usually subclinical,resulting in a decline in egg production and hatchability.Infectivity was shown to remain unaffected by chloroform,low pH, pepsin, trypsin and deoxyribonuclease.Magnesium cations were shown to stabilise preparations of the virus against heat
inactivation.The buoyant density of virions are 1.31g/ml.The diameter of the virion was estimated to be 22 to 30nm.The AEV can be adapted to grow in chicken embryo.The inability of AEV to grow effeciently in most cell cultures.
In 1932,outbreaks of a disease causing neurotropic dysfunction in young chickens in the New England region of the USA in 1930 were first reported by Jones.Over the next decade,outbreaks of the disease were reported in Europe, Canadajapan and Australia.In 1980s,The disease was transmited into China. AEV has long been recognised as the aetidaogical agent of one of the most significant disease of poultry.Despite its importance,relatively little progress had been made towards an understanding of its basic biology and pathogenesis.
The purpose of this study was to learn the sequence and structure of the AEV by technology of the molecular biology.to reveal the relation between genome features and function. The research consist of four parts.The first part is multiplication,purification and electron microscope examination of the avian encephalomyelitis virus.A 1:5 dilution of isolate-NH937 of AEV and control group of PBS were inoculated to susceptible 6-day-old chickens
embryos.respectively.After incubation for 10 days,the urinay vesicle liquid was collected.Making a comparison the size of the chickens embryos between the test group and the control group,the results showed that the size of the control group is bigger than that of the test group.Purified virions were examined under the electron microscope,the result revealed that there are a lot of virions and the AEV-NH937 was multiplicated in embryos.The second part was seguence analysis of the genome of the AEV-NH937.Nine pairs of primers were designed according to published Calnek vaccine strain of AEV. RT-PCR was used to amplify the cDNA of the genome .These cDNA fragments were cloned into the plasmid pUCm-T.The result indicated that the seguence of the genome was obtained. The genome of AEV-NH937 composed of 7055 nucleotides, potentially encodes a polyprotein of 2134 amino acids. The genome of AEV-NH937 has 94.3% nucleotides identity with the Calnek vaccine strain of AEV. it was firstly reported in China.The third part
rthe capsid protein VP| gene was expressed.810 bp fragment was inserted into vector pGEX-4Tl,and then transformed into E.coli BL21. SDS-PAGE analysis showed that the expressed product is a 53KD fusion protein aftor induction with IPTG. The expressed product was lysised with supersonic wave and purified by SDS-PAGE.ELISA analysis revealed that the antigenicity of the VPi protein has been detected. The forth part-detection of AEV-NH937 strain by In Situ Hybridization(1SH)-Probe was labelled with digoxigenin(Dig).Then the probe hybrid with 5d,10d,and 20d postinfection brain tissue of chicken.The results of the ISH showed that the positive signal was found in 3 cases,while control group was negative.there has been a reasonable correlatien between this method and other detection test.
1932年Jones首次报道了在1930年美国新英格兰地区幼鸡群爆发的禽脑脊髓炎。随后十年中，在欧洲、加拿大、日本和澳大利亚都有此病的报道。八十年代，该病传入我国。AEV一直被认为是最重要的禽病病原之一，尽管它很重要，但有关其基础生物学，发病机理的了解却较少。本项研究，旨在利用分子生物学的技术手段，搞清楚AEV基因组的序列和结构，从而探讨基因结构与功能的关系。
本研究共分四个部分：第一部分为AEV的增殖，纯化和电镜观察，用1:5倍稀释的AEV-NH937株和阴性对照PBS分别经卵黄囊接种于6dSPF鸡胚，继续孵化10d后，收集尿囊液。比较接种组和健康对照组鸡胚的大小，结果显示，健康对照组鸡胚明显大于接种组。分离、提纯AEV，把纯化的病毒在电镜下观察，证明确有大量AEV病毒粒子存在，说明AEV在鸡胚中成功扩增；第二部分是AEV-NH937基因组的序列测定工作。根据Calnek疫苗株序列设计了9对引物，利用反转录聚合酶链式反应，成功地扩增了AEV-NH937毒株的全基因。把它们分段克隆在pUCm-T载体上，经序列分析，获得了AEV-NH937毒株的基因组全序列及推导的氨基酸序列，基因组全长为7055个核苷酸，与calnek疫苗株具有94.3％的同源性，编码一个含2134个氨基酸的多聚蛋白。这是国内首次对AEV基因组全序列的分析；第三部分：AEV外壳蛋白VP_1基因在大肠杆菌中的融合表达、纯化及活性研究。将VP_1基因编码区的810bp片段插入载体pGEX-4T1构建表达重组质粒，然后转化大肠杆菌BL_(21)，经IPTG诱导后，用聚丙稀酰氨凝被电泳分析。结果表明，表达产物为53KD的融合蛋白。经超声波裂解，用尿素溶解包涵体，电泳纯化后，利用ELISA检测VP_1
外壳蛋白，表明具有一定抗原性；第四部分：应用原位杂交检测 AEV i用 D i g
标记的探针与sd、10d、20d的攻毒鸡脑组织杂交，来检测那V的RNA。结果
显示，sd、10d、20d的攻毒脑组织切片均有阳性杂交信号，阴性对照却没有
杂交信号，该方法与其它检测方法具有良好的平行关系。
Avian encephalomyelitis virus (AEV) is a picornavirus with a predilection for the central nervous system and other parenchymous organs of chickens that is transmited by the oral-faecal route.The virus may be spread by the vertical and horizontalroutes, and because of its great stability,contaminated areas may remain infectious for long periods.The egg-adapted Van Roekel strain is highly neurotropic and does not grow efficiently in the enteric tract of the chicken,and the field isolates of AEV is usually enterotropic .Despite this.the virion polypeptides of both naturally-occurring strains and the Van Roekel strain are antigenically identical. In young chickens AEV induces paralysis, ataxia and muscular dystrophy, while in older chickens, infection is usually subclinical,resulting in a decline in egg production and hatchability.Infectivity was shown to remain unaffected by chloroform,low pH, pepsin, trypsin and deoxyribonuclease.Magnesium cations were shown to stabilise preparations of the virus against heat
inactivation.The buoyant density of virions are 1.31g/ml.The diameter of the virion was estimated to be 22 to 30nm.The AEV can be adapted to grow in chicken embryo.The inability of AEV to grow effeciently in most cell cultures.
In 1932,outbreaks of a disease causing neurotropic dysfunction in young chickens in the New England region of the USA in 1930 were first reported by Jones.Over the next decade,outbreaks of the disease were reported in Europe, Canadajapan and Australia.In 1980s,The disease was transmited into China. AEV has long been recognised as the aetidaogical agent of one of the most significant disease of poultry.Despite its importance,relatively little progress had been made towards an understanding of its basic biology and pathogenesis.
The purpose of this study was to learn the sequence and structure of the AEV by technology of the molecular biology.to reveal the relation between genome features and function. The research consist of four parts.The first part is multiplication,purification and electron microscope examination of the avian encephalomyelitis virus.A 1:5 dilution of isolate-NH937 of AEV and control group of PBS were inoculated to susceptible 6-day-old chickens
embryos.respectively.After incubation for 10 days,the urinay vesicle liquid was collected.Making a comparison the size of the chickens embryos between the test group and the control group,the results showed that the size of the control group is bigger than that of the test group.Purified virions were examined under the electron microscope,the result revealed that there are a lot of virions and the AEV-NH937 was multiplicated in embryos.The second part was seguence analysis of the genome of the AEV-NH937.Nine pairs of primers were designed according to published Calnek vaccine strain of AEV. RT-PCR was used to amplify the cDNA of the genome .These cDNA fragments were cloned into the plasmid pUCm-T.The result indicated that the seguence of the genome was obtained. The genome of AEV-NH937 composed of 7055 nucleotides, potentially encodes a polyprotein of 2134 amino acids. The genome of AEV-NH937 has 94.3% nucleotides identity with the Calnek vaccine strain of AEV. it was firstly reported in China.The third part
rthe capsid protein VP| gene was expressed.810 bp fragment was inserted into vector pGEX-4Tl,and then transformed into E.coli BL21. SDS-PAGE analysis showed that the expressed product is a 53KD fusion protein aftor induction with IPTG. The expressed product was lysised with supersonic wave and purified by SDS-PAGE.ELISA analysis revealed that the antigenicity of the VPi protein has been detected. The forth part-detection of AEV-NH937 strain by In Situ Hybridization(1SH)-Probe was labelled with digoxigenin(Dig).Then the probe hybrid with 5d,10d,and 20d postinfection brain tissue of chicken.The results of the ISH showed that the positive signal was found in 3 cases,while control group was negative.there has been a reasonable correlatien between this method and other detection test.
引文
1 Jones, E.E. An encephalomyelitisinthechicken. Science, 1932,76,331-33
2 Jones, E.E. Epidemic tremor and encephalomyelitis affecting young chickens. Journal of Experimental Medicine, 1934,59,781-798
3 Van Roekel, H. Bullis,K.L.&Clarke, M.K. Preliainary report on infectious avian encephalomyelitis. Journal of The American Veterinary Medical Association, 1938,93,372-375
4 Schaaf, k.& Lamoreux, W.F. control of avian encephalomyelitis by vaccination. American Journal of Veterinary Research, 1995,16,627-633
5 Taylor, J.R.E.& Schelling, E.P. The distribution of avian encephalomyelit in North America as indicated by an immunity test. Avian Diseases, 1960,4,122-123
6 Hoekstra, J. Experiments with avian encephalomyelitis. British Veterinary Journal, 1964,120,322-335
7 Albiston, H.E. Diseases of Domestic Animals in Australia. Part 4, Protozoan and virus Diseases seddon, H.R. (Ed),Znd edn. commonwealth Department of Health, 1960,201
8 Mathey, W.J. Avian encephalomyelitis in pheasant. Cornell Veterinarian, 1995,45,89-93
9 Hill,R.N.& Raymond, R.G. Apparent natural infectious of corturnix quail hens with the virus of avian encephalomyelitis-a case report. Avian Diseases, 1962,6,226-227
10 Hohlstein, W.M, Reshmukh, D.R. An epornitic of avian encephalomyelitis in turkeys in Minnesota. American Journal of veterinary Research, 1970,31,2233-2234
11 Jungherr, E.& Minard, E.L. The preseut status of avian encephalomyelitis. Journal of the American veterinary Medical Association, 1942,100,38-46
12 Taylor, L.W, Lowry, D.C. Effects of an outbreak of avian encephalomyelitis(epidemic tremor) in a breeding flock. Poultry Science, 1955,34,1036-1045
13 Calnek, B. W. & Taylor, P.J. Studies on avian encephalomyelitis. Immune response tO beta-propiolactone inactivated virus. Avian Diseases, 1960,4,116-121
14 Butterfield, W.K, Luginbuhl,R.E. Characterization of avian encephalomyelitis virus (an avian enterovirus).Avian Diseases, 1969,13,363-378
15 Gosting, L.H, Grinnell,B.W. Physico-chemical characteristics of avian encephalomyelitis virus Veterinary Microbiology, 1980,5,87-100
16 shafren, D.R & Tannak, G.A. Further evidence that the nucleic acid of avian encephalomyelitis virus consist of RNA, Avian Diseases, 1992,36,1031-1033
17 Tannock, G.A, Shafren, D.R.A rapid procedure for purification of avian encephalomyelitis virus. Avian Diseases, 1985,29,312-321
18 Cooper, P.D, Agol,V.I. Picornaviridae: Secondreport. Intervirology, 1978,10,16 5-180
19 Cheville, N.F. The influence of the thymic and bursal lymphoid systems in the pathogenesis of avian encephalomyelitis. American Journal of pathology, 1970,58,105-125
20 Hishida, n.,Odagiri,Y. Morphological changes in neurons in experimental avian encephalomyelitis. Japanese Journal of Veterinary Science, 1986,48,169-172
21 Krauss, H.& Ueberschar, C. Zur ultrastruktur des virus der aviaeren encephalomyelitis. Berliner and Muenchener Tierazliche Wochenschrift, 1996,79,480-482
22 赵振华，顾玉芳，李润清等．禽脑脊髓炎发病机理的研究．中国畜禽传染病，1995，3：42—48
23 杨启明．禽脑脊髓炎研究概况．中国兽医科技，1993，11：18—19
24 刘思当，匡宝晓，范伟兴等．禽脑脊髓炎的病理学研究．山东农业大学学报，1998，3：391—394
25 赵振华，林曦，禹旺盛等．禽脑脊髓炎的病理学研究．中国兽医杂志，1995，21(3)：5—7
26 Willis,E.K.& Moulthrop, I.M. Propagation of avian encephalomyelitis virus in the chicken embryo. Southwest Veterinarian, 1956,10,39-42
27 Sumner, F.W.,Jungherr, E.L.& Luginbuhl,R.E. Studies on avian encephalomyelitis. I Egg adaptation of the virus. American Journal of Veterinary Research, 1957,18,717-719
28 Miyamae, T. Emergence pattern of egg-adapted avian encephalomyelitis virus by alternating passage in chickens and embryo. Avian
Diseases, 19?6,20,425-428
29 Jungherr, E.,Sumner, F.&Luginbuhl,R.E. Pathology of egg-adapted avianencephalomyelitis virus. Science, 1956,124,80-81.
30 Springer,W.T.& Schmittle, S.C. Avian encephalomyelitis. A chronological study of the histopathogenesis in selected tissues. Avian Diseases, 1968,12,229-239
31 Burke, C.N.,Krauss, H. & Luginbuhl,R.E. The multiplication of avian encephalomyelitis in chicken embryo tissues. Avian Diseases, 1965,9,104-108
32 Braune, M.O.& Gentry, R.F. Avian encephalomyelitis virus. I Pathogenesis in chicken embryos. Avian Diseases, 1971,15,638-647
33 Ikeda, S. Imaunodiffusion test in avian encephalomyelitis. I Standardization of procedure and detection of antigen in infected chickens and embryos. National Institute of Animal Health Quarterly, 1976a, 17,81-87
34 Calnek, B.W.& Jehnich, H. Studies on avian encephalomyetitis. I The use of a serum-neutralization test in the detection of immunity levels. Avian Diseases, 1959a, 3,95-104
35 Ikeda, S.&Matsuda, K. Susceptibility of chickens to avian encephalomyelitis virus. Ⅳ Behaviour of the virus in laying hens. National Institue of Animal Health Quarterly, 1976a,16,83-89
36 Westbury, H.A.& Sinkovic, B. The pathogenesis of infectious avian encephalomyelitis. 1 The effect of the age of the chicken and the route of administration of the virus. Australian Veterinary Journal, 1978a, 54,68-71
37 Westbury, H.A.& Sinkovic, B. The pathogenesis of infectious avian encephalomyelitis. 3 The relationship brtween viraemia, invasion of the brain by the virus, and the development of specific serum neutralising antibody. Australian Veterinary Journal, 1978b, 54,76-80
38 Ikeda, S.& Matsuda, K. Susceptibility of chickens to avian encephalomyelitis virus. V Behaviour of a field strain in laying hens. National Institute of Animal Health Quarterly, 1976b, 16,90-96
39 Calnek, B.W.,& Taylor, P.J. Studies on avian encephalomyelitis. Ⅳ Epizootiology. Avian Diseases, 1960,4,325-347
40 Shafren, D.R.& Tannock, G.A. The pathogenesis of avian encephalomyelitis viruses. Journal of General Virology, 1991,72,2713-2719
41 Miyamae, T. Immunofluorescent study on egg-adapted avian encephalomyelitis virus infection in chickens. American Journal of Veterinary Research, 1977,38,2009-2012.
42 Oirtsky, P.K. (1939).Experimental studies of the virus of infectious avian encephalomyelitis. Journal of Experimental Medicine, 1939,70,565-582
43 Ikeda, S.,Asahi,O.& Oka, M. (1966).Outbreaks of avian encephalomyelitis in chicks raised in Japan. National Institute of Animal Health Quarterly, 1966,6,127-135
44 Shafren, D.R.& Tannock, G.A. Development and application of an improved infectivity assay for the standardization of avian encephalomyelitis vaccines. Vaccine, 1990,8,283-285
45 Westbury, H.A.& Sinkovic, B. The immunisation of chickens against infectious avian encephalomyelitis. Australian Veterinary Journal, 1976,52,374-378
46 Gaunt,C.J.,Jones. D.C.,& De Shambo, R.M. Murine forebrain anomalies induced by Coxsackie B3 variants. Journal of Medical Virology, 1984,14,341-355
47 Sumner, F.W.,Luginbuhl,R.E.& Jungherr, E.L. Studies on avian encephalomyelitis. Ⅱ Flock survey for embryo susceptibility to the virus. American Jurnal of Veterinary Research, 1957b, 18,720-723
48 Calnek, B.W.& Jehnich, I. Studies on avian encephalomyelitis. Ⅱ Immune responses to vaccination procedures. Avian Diseases, 1959b, 3,225-239
49 Matsukura, T. (1970).A study of maternal immunity in chicks derived from flocks naturally infected with avian encephalomyelitis virus (AEV).Avian Diseases, 18,93-94
50 Westbury, H.A.& Sinkovic, B. The pathogenesis of infectious avian encephalomyelitis. 2 The effect of immunosuppression on the disease. Australian Veterinary fournal, 54,72-75
51 Westbury, H.A.& Sinkovic, B. The pathogenesis of infectious avian encephalomyelitis. 2 The effect of immunosuppression on the disease. Australian Veterinary Journal, 1978b, 54,72-75
52 Shafren, D.R.,Tannock, G.A.& Groves, P.J. Antibody responses to avian encephalomyelitis virus vaccines when administered by different routes. Australian Veterinary Journal, 1992,69,272-275
53 Sato, G.,Watanabe, H.& Miura, S. An attempt to produce a complement-fixation antigen for the avian encephalomyelitis virus form chick embryo brains. Avian
Diseases, 1969,13,461-469
54 Choi,W.P.& Miura, S. Indirect fluorescent antibody technique for the detection of avian encephalomyelitis antibody in chickens. Avian Diseases, 1972,16,949-951
55 赵振华，禹旺盛，郝先谱等．禽脑脊髓炎的发生与诊断，内蒙古畜牧科学，1994，3：34-37
56 秦爱建，崔治中，段玉友等．间接免疫荧光染色法和琼脂扩散试验在禽脑脊髓炎流行病学调查中的应用．中国畜禽传染病，1994，76(3)：49-50
57 Lukert,P.D.& Davis, R.B.:An antigen used in the agar-gel precipitin reaction to detect avian encephalomyelitis virus antibodies. Avian Diseases, 1971,15,935-938
58 Ahmed, A.A.S., Ayoub, N.N.K.& E1-Toukhi, B.I.M. Studies on the serological detection of antibodies to avian encephalomyelitis virus. Avian Pathology, 1982,11,253-262
59 Girshick, T.& Crary, C.K, Jr. Preparation of an agar-gel-precipitating antigen for avian encephalomyelitis and its use in evaluating the antibody status of poultry, Auian Diseases, 1982,26,798-804
60 何水林，陈杰，蔡玉书，禽脑脊髓炎病毒的分离与初步鉴定．上海畜牧兽医通讯，1998，1：27-29
61 李增光，杜元钊，王树伦等．青岛地区发生禽脑脊髓炎的诊断报告。动物检疫，1992，9(2)：16—17
62 Garrett, J.W.,David, R.B.& Ragland, W.L. Enzyme-linked immunosorbent assey for the detection of antibodies to avian encephalomyelitis viruses in chickens. Avian Diseases, 1984,28,117-129
63 Malkinson, M.,Weisman, Y.,Staviniski,A. Application of ELISA to study avian encephalomyelitis in a flock of turkeys. Veterinary Record, 1986,119,503-504
64 Richter, R. Von, Kosters, J.&guhavanta-Kalkosol,S. Vergleichende untersuchungen zur anwendung eines enzyme-linked immunosorbent-assay(ELISA) zum antikorpernachweis gagen der erreger der aviaeren encephalomyelitis. Zentralblatt fur ueterinarmedizin, 1985, B 32,116-127
65 Smart, I.J.& Grix, D.C. Measurement of antibodies to infectious avian encephalomyelitis virus by ELISA. Avian Pathology, 1985,14,314-352
66 Smart, I.J.,Grix, D.C.& Barr, D.A. The application of ELISA to the diagnosis and control of avian encephalomyelitis. Australian Veterinary
Journal, 1986,63,297-299
67 Shafren, D.R.& Tannock, G.A. An enzyme-linked immunosorbent assay for the detection of avian encephalomyelitis antigens. Avian Diseases, 1988,32,209-214
68 Shafren, D.R.& Tannock, G.A.& Morrow, C.J. Diagnosis of a field outbreak of avian encephalomyelitis by enzyme-linked immunosorbent assay. Autralian Veterinary Journal, 1989a, 66,224-225
69 Shafren, D.R.,Tannock, G.A.& Roberts, T.K. Development and application of an ELISA technique for the detection of antibody to avian encephalomyelitis viruses. Research in veterinary Science, 1989b, 46,95-99
70 Hwang, J.,Luginbuhl,R.E.& Helmboldt,C.F. The in vivo and in vitro isolation of avian encephalomyelitis virus(AEV).Avian Diseases, 1960,4,545-546
71 Mancini,L.O.& Vates,V.J. Cultivation of avian encephalomyelitis virus in vitro. I In chicken embryo neuroglial cell culture. Avian Diseases, 1967,11,672-679
72 Nicholas,R.A.J.,Ream, A.J.& Thornton, D.H. Replication of avian encephalomyelitis virus in chick embryo neurological cell cultures. Archives of Virology, 1987,96,283-287
73 Schaaf, K. Immunization for the control of avian encephalomyelitis. Avian Diseases, 1958,2,279-289
74 Calnek, B.W.,Taylor, P.J.& Sevoian, M. Studies on avian encephalomyelitis. V Development and application of an oral vaccine. Avian Diseases, 1961,5,297-312
75 罗开健，廖明，任涛等。禽脑脊髓炎的防治要点．中国家禽，1999，21(7)：44—45
76 田兆菊，李庆官．禽脑脊髓炎的防制．山东畜牧兽医，2000，1：16
77 Butterfield, W.K.,Luginbuhl,R.E.& Sumner, F.W. Studies on avian encephalomyelitis. Ⅲ Immunization with an inactivated virus. Avian Diseases, 1961,5,445-450
78 秦爱建，段玉友，崔治中等．禽脑脊髓炎油剂灭活苗的现场应用试验．中国家禽，1994，2：22—23
79 姚永秀，秦卓明，刘红等．禽脑脊髓炎油乳剂灭活苗的田间免疫试验．山东家禽，1996，4：3—4
80 Glisson, J.R.& Fletcher, O.J. Clinical encephalitis following avian encephalomyelitis vaccination in broiler breeder pullets. Avian
Diseases,1987, 31,383—385
81 朱静，范维珂．人类基因组计划概况．中国检验医学与临床，2000，1(4)：167—168
82 Gaffeau, A. Science, 1996, 274: 546—548
83 蒋圣娟，张红雨．人类基因组计划及其发展简史．淄博学院学报(自然科学与工程版)，2001，3(3)：81—88
84 Blattner, F.R. Science, 1997, 277: 1453
85 张春霆．生物信息学的现状与展望．世界科技研究与发展，2000，22(6)：17—20
86 俞利荣，曾嵘，夏其昌．蛋白质组研究技术及其进展．生命化学，1998，18(6)：4—9
87 李伯良，李林，吴家睿．功能蛋白质组学．生物工程进展，1999，19(4)：15—16
88 Agvanovsky AA. Koenin E V. Boykovp et al. Beet yellows closterovirus:Complete genome structure and identification of a papain-like thiol protease. Virology, 1994, 198:311-324
89 Klaassen V A.B oeshore ML, Koenin EV et al. Genome Structure and phylogenetic analysis of lettuce infectious yellows virus, a whitefly-trasmitted, bipartite closterovirus. Virology, 1995, 208: 99—110
90 Karasev A V. Boyko VP. Gowda S et al. Complete seguence of the citrus tristeza virus RNA genome. Virology, 1995, 208:511—520
91 Murphy FA. Fauguet CM. Bishop BHL et al. Classification and nomen cluture of viruses:Sixth Report of the International Committee on Taxonomy of Viruses. Arch Virol, 1995, Suppl, 10
92 Karasev AV. Agranovsky AA. Rogov VV et al. Virion RNA of beet yellows closterovirus:cell-free translation and some properties. J Gen Virol, 1989, 70:241—245
93 Pappu HR,Karasev AV, Auderson EJ et al. Nucleotide Seguence and organization of eight 3' open reading fragment of the citrus tisteza closterovirus genome. Virology, 1994, 199:35—46
94 Lomakina, N.F.,Lomakin, A.I.,Drygin, V.V. et al. Detection of avian
encephalomyelitis Virus genome by nested PCR. European Society for Veterinary Virology, 2001, 5:348—349
95 Todd, D.,Weston, J.H.,Mawhinney, K.A. et al. Characterization of the genoae of avian encephelomyelitis virus with cloned cDNA fragm ents Avian Dis, 1999, 43(2), 219—226
96 赵心力，赵振华，马学恩等．禽脑脊髓炎病毒的分离鉴定．中国畜禽传染病，1995，81(2)：3—7
97 叶满红，崔浩中，秦爱建．禽脑脊髓炎病毒单克隆抗体制备．中国兽医学报，1999，19(3)：25-27
98 韦莉，刘爵，张杰等．用蔗糖密度梯度离心法提纯禽脑脊髓炎病毒．北京农业科学，1999，17(6)：35-36
99 马学恩，赵振华。禽脑脊髓炎病毒的提纯和电镜观察．中国兽医科技，1995，25(7)：3-4
100 卢中华，张红英．禽脑脊髓炎病毒的分离与疫苗研制，河南农业科学，1996，46-48
101 刘思当，范伟兴．禽脑脊髓炎病毒的分离与初步鉴定研究．山东畜牧兽医，1999，5：3-4
102 赵心力，郭金贵．禽脑脊髓炎琼脂扩散沉淀抗原制备与应用的研究．中国兽医科技，1995，25(2)：6-8
103 Marvil,p. Nick, J. Avian encephalomyelitis virus is a picornavirus and is most closely related to hepatitis A virus. Journal of General virology, 1999,80:653-662
104 Shafaren, D.R, Tannock, G.A. Further evidence that the necleic acid of avian cncephalomyelitis virus consists of RNA. Avian Diseases, 1992,36:1031-1033
105 殷震．动物病毒学[M]，第二版，科学出版社，1997
106 侯云德主编．分子病毒学[M]．学苑出版社，1990
107 Todd, D. Weston, J.H. Characterization of the genome of avian encephalomyelitis virus with cloned cDNA fragments. Avian Diseases, 1999,43(2):219-226
108 徐耀辉，卢中华．禽脑脊髓炎病毒河南株VPO基因的克隆与序列分析．河南畜牧兽医，2002，23(9)：6—7
109 彭勇，黄庆．枯草杆菌DC-2纳豆激酶基因的克隆及其融合蛋白在E. coli的表达．国生物化学与分子生物学报，2002，18(5)：559-563
110 尹崇，李朝飞．新基因Splt137在大肠杆菌中的表达及抗体制备．中山大学学
报，2002，41(4)：75—77
111 李武平，吕宏亮。干扰素-β1b的高效表达、纯化及抗病毒活性研究．病毒学报，2002，18(3)：240-243
112 杨兴文，刘俭．抗凝活性肽PGD226基因构建、表达，产物纯化及活性分析．中国生物化学与分子生物学报，2002，18(5)：583-587
113 王立霞，张珠强．Cre基因在大肠杆菌中的表达及表达蛋白活性的检测。生物工程学报，2002，18(4)：497—500
114 Shadren，D．R．检测禽脑脊髓炎病毒抗体的ELISA技术的建立和应用．国外兽医学—畜禽传染病，1989，9(3)：28-30
115 曹永长，毕英佐．用酶联免疫吸附实验检测禽脑脊髓炎病毒抗体．中国兽医杂志，1997，23(1)：13-14
116 武东亮，郭三堆．融合基因研究进展．生物技术通报，2001，2：5-7
117 萨姆布鲁克，丁．费里奇 E．F．分子克隆实验指南[M]，金冬雁，黎孟枫等译．第二版，科学出版社，1995
118 阎惠平，郎振为．原位杂交法检测肝组织中输血传播病毒．首都医科大学学报，2001，2：116—118
119 吕玲，向建国．核酸探针原位杂交检测白斑综合症病毒的组织特异性．热带海洋，2000，4：86-90
120 Wagner, E.F. Prenatel lethatities in mice homozygous for human growth hormore gene seguences integratel in the germ line. Cell,1983,35:637-655.
121 周世力．DNA疫苗的研究进展．江汉大学学报(自然科学版)，2002，19(2)：80—83
2 Jones, E.E. Epidemic tremor and encephalomyelitis affecting young chickens. Journal of Experimental Medicine, 1934,59,781-798
3 Van Roekel, H. Bullis,K.L.&Clarke, M.K. Preliainary report on infectious avian encephalomyelitis. Journal of The American Veterinary Medical Association, 1938,93,372-375
4 Schaaf, k.& Lamoreux, W.F. control of avian encephalomyelitis by vaccination. American Journal of Veterinary Research, 1995,16,627-633
5 Taylor, J.R.E.& Schelling, E.P. The distribution of avian encephalomyelit in North America as indicated by an immunity test. Avian Diseases, 1960,4,122-123
6 Hoekstra, J. Experiments with avian encephalomyelitis. British Veterinary Journal, 1964,120,322-335
7 Albiston, H.E. Diseases of Domestic Animals in Australia. Part 4, Protozoan and virus Diseases seddon, H.R. (Ed),Znd edn. commonwealth Department of Health, 1960,201
8 Mathey, W.J. Avian encephalomyelitis in pheasant. Cornell Veterinarian, 1995,45,89-93
9 Hill,R.N.& Raymond, R.G. Apparent natural infectious of corturnix quail hens with the virus of avian encephalomyelitis-a case report. Avian Diseases, 1962,6,226-227
10 Hohlstein, W.M, Reshmukh, D.R. An epornitic of avian encephalomyelitis in turkeys in Minnesota. American Journal of veterinary Research, 1970,31,2233-2234
11 Jungherr, E.& Minard, E.L. The preseut status of avian encephalomyelitis. Journal of the American veterinary Medical Association, 1942,100,38-46
12 Taylor, L.W, Lowry, D.C. Effects of an outbreak of avian encephalomyelitis(epidemic tremor) in a breeding flock. Poultry Science, 1955,34,1036-1045
13 Calnek, B. W. & Taylor, P.J. Studies on avian encephalomyelitis. Immune response tO beta-propiolactone inactivated virus. Avian Diseases, 1960,4,116-121
14 Butterfield, W.K, Luginbuhl,R.E. Characterization of avian encephalomyelitis virus (an avian enterovirus).Avian Diseases, 1969,13,363-378
15 Gosting, L.H, Grinnell,B.W. Physico-chemical characteristics of avian encephalomyelitis virus Veterinary Microbiology, 1980,5,87-100
16 shafren, D.R & Tannak, G.A. Further evidence that the nucleic acid of avian encephalomyelitis virus consist of RNA, Avian Diseases, 1992,36,1031-1033
17 Tannock, G.A, Shafren, D.R.A rapid procedure for purification of avian encephalomyelitis virus. Avian Diseases, 1985,29,312-321
18 Cooper, P.D, Agol,V.I. Picornaviridae: Secondreport. Intervirology, 1978,10,16 5-180
19 Cheville, N.F. The influence of the thymic and bursal lymphoid systems in the pathogenesis of avian encephalomyelitis. American Journal of pathology, 1970,58,105-125
20 Hishida, n.,Odagiri,Y. Morphological changes in neurons in experimental avian encephalomyelitis. Japanese Journal of Veterinary Science, 1986,48,169-172
21 Krauss, H.& Ueberschar, C. Zur ultrastruktur des virus der aviaeren encephalomyelitis. Berliner and Muenchener Tierazliche Wochenschrift, 1996,79,480-482
22 赵振华，顾玉芳，李润清等．禽脑脊髓炎发病机理的研究．中国畜禽传染病，1995，3：42—48
23 杨启明．禽脑脊髓炎研究概况．中国兽医科技，1993，11：18—19
24 刘思当，匡宝晓，范伟兴等．禽脑脊髓炎的病理学研究．山东农业大学学报，1998，3：391—394
25 赵振华，林曦，禹旺盛等．禽脑脊髓炎的病理学研究．中国兽医杂志，1995，21(3)：5—7
26 Willis,E.K.& Moulthrop, I.M. Propagation of avian encephalomyelitis virus in the chicken embryo. Southwest Veterinarian, 1956,10,39-42
27 Sumner, F.W.,Jungherr, E.L.& Luginbuhl,R.E. Studies on avian encephalomyelitis. I Egg adaptation of the virus. American Journal of Veterinary Research, 1957,18,717-719
28 Miyamae, T. Emergence pattern of egg-adapted avian encephalomyelitis virus by alternating passage in chickens and embryo. Avian
Diseases, 19?6,20,425-428
29 Jungherr, E.,Sumner, F.&Luginbuhl,R.E. Pathology of egg-adapted avianencephalomyelitis virus. Science, 1956,124,80-81.
30 Springer,W.T.& Schmittle, S.C. Avian encephalomyelitis. A chronological study of the histopathogenesis in selected tissues. Avian Diseases, 1968,12,229-239
31 Burke, C.N.,Krauss, H. & Luginbuhl,R.E. The multiplication of avian encephalomyelitis in chicken embryo tissues. Avian Diseases, 1965,9,104-108
32 Braune, M.O.& Gentry, R.F. Avian encephalomyelitis virus. I Pathogenesis in chicken embryos. Avian Diseases, 1971,15,638-647
33 Ikeda, S. Imaunodiffusion test in avian encephalomyelitis. I Standardization of procedure and detection of antigen in infected chickens and embryos. National Institute of Animal Health Quarterly, 1976a, 17,81-87
34 Calnek, B.W.& Jehnich, H. Studies on avian encephalomyetitis. I The use of a serum-neutralization test in the detection of immunity levels. Avian Diseases, 1959a, 3,95-104
35 Ikeda, S.&Matsuda, K. Susceptibility of chickens to avian encephalomyelitis virus. Ⅳ Behaviour of the virus in laying hens. National Institue of Animal Health Quarterly, 1976a,16,83-89
36 Westbury, H.A.& Sinkovic, B. The pathogenesis of infectious avian encephalomyelitis. 1 The effect of the age of the chicken and the route of administration of the virus. Australian Veterinary Journal, 1978a, 54,68-71
37 Westbury, H.A.& Sinkovic, B. The pathogenesis of infectious avian encephalomyelitis. 3 The relationship brtween viraemia, invasion of the brain by the virus, and the development of specific serum neutralising antibody. Australian Veterinary Journal, 1978b, 54,76-80
38 Ikeda, S.& Matsuda, K. Susceptibility of chickens to avian encephalomyelitis virus. V Behaviour of a field strain in laying hens. National Institute of Animal Health Quarterly, 1976b, 16,90-96
39 Calnek, B.W.,& Taylor, P.J. Studies on avian encephalomyelitis. Ⅳ Epizootiology. Avian Diseases, 1960,4,325-347
40 Shafren, D.R.& Tannock, G.A. The pathogenesis of avian encephalomyelitis viruses. Journal of General Virology, 1991,72,2713-2719
41 Miyamae, T. Immunofluorescent study on egg-adapted avian encephalomyelitis virus infection in chickens. American Journal of Veterinary Research, 1977,38,2009-2012.
42 Oirtsky, P.K. (1939).Experimental studies of the virus of infectious avian encephalomyelitis. Journal of Experimental Medicine, 1939,70,565-582
43 Ikeda, S.,Asahi,O.& Oka, M. (1966).Outbreaks of avian encephalomyelitis in chicks raised in Japan. National Institute of Animal Health Quarterly, 1966,6,127-135
44 Shafren, D.R.& Tannock, G.A. Development and application of an improved infectivity assay for the standardization of avian encephalomyelitis vaccines. Vaccine, 1990,8,283-285
45 Westbury, H.A.& Sinkovic, B. The immunisation of chickens against infectious avian encephalomyelitis. Australian Veterinary Journal, 1976,52,374-378
46 Gaunt,C.J.,Jones. D.C.,& De Shambo, R.M. Murine forebrain anomalies induced by Coxsackie B3 variants. Journal of Medical Virology, 1984,14,341-355
47 Sumner, F.W.,Luginbuhl,R.E.& Jungherr, E.L. Studies on avian encephalomyelitis. Ⅱ Flock survey for embryo susceptibility to the virus. American Jurnal of Veterinary Research, 1957b, 18,720-723
48 Calnek, B.W.& Jehnich, I. Studies on avian encephalomyelitis. Ⅱ Immune responses to vaccination procedures. Avian Diseases, 1959b, 3,225-239
49 Matsukura, T. (1970).A study of maternal immunity in chicks derived from flocks naturally infected with avian encephalomyelitis virus (AEV).Avian Diseases, 18,93-94
50 Westbury, H.A.& Sinkovic, B. The pathogenesis of infectious avian encephalomyelitis. 2 The effect of immunosuppression on the disease. Australian Veterinary fournal, 54,72-75
51 Westbury, H.A.& Sinkovic, B. The pathogenesis of infectious avian encephalomyelitis. 2 The effect of immunosuppression on the disease. Australian Veterinary Journal, 1978b, 54,72-75
52 Shafren, D.R.,Tannock, G.A.& Groves, P.J. Antibody responses to avian encephalomyelitis virus vaccines when administered by different routes. Australian Veterinary Journal, 1992,69,272-275
53 Sato, G.,Watanabe, H.& Miura, S. An attempt to produce a complement-fixation antigen for the avian encephalomyelitis virus form chick embryo brains. Avian
Diseases, 1969,13,461-469
54 Choi,W.P.& Miura, S. Indirect fluorescent antibody technique for the detection of avian encephalomyelitis antibody in chickens. Avian Diseases, 1972,16,949-951
55 赵振华，禹旺盛，郝先谱等．禽脑脊髓炎的发生与诊断，内蒙古畜牧科学，1994，3：34-37
56 秦爱建，崔治中，段玉友等．间接免疫荧光染色法和琼脂扩散试验在禽脑脊髓炎流行病学调查中的应用．中国畜禽传染病，1994，76(3)：49-50
57 Lukert,P.D.& Davis, R.B.:An antigen used in the agar-gel precipitin reaction to detect avian encephalomyelitis virus antibodies. Avian Diseases, 1971,15,935-938
58 Ahmed, A.A.S., Ayoub, N.N.K.& E1-Toukhi, B.I.M. Studies on the serological detection of antibodies to avian encephalomyelitis virus. Avian Pathology, 1982,11,253-262
59 Girshick, T.& Crary, C.K, Jr. Preparation of an agar-gel-precipitating antigen for avian encephalomyelitis and its use in evaluating the antibody status of poultry, Auian Diseases, 1982,26,798-804
60 何水林，陈杰，蔡玉书，禽脑脊髓炎病毒的分离与初步鉴定．上海畜牧兽医通讯，1998，1：27-29
61 李增光，杜元钊，王树伦等．青岛地区发生禽脑脊髓炎的诊断报告。动物检疫，1992，9(2)：16—17
62 Garrett, J.W.,David, R.B.& Ragland, W.L. Enzyme-linked immunosorbent assey for the detection of antibodies to avian encephalomyelitis viruses in chickens. Avian Diseases, 1984,28,117-129
63 Malkinson, M.,Weisman, Y.,Staviniski,A. Application of ELISA to study avian encephalomyelitis in a flock of turkeys. Veterinary Record, 1986,119,503-504
64 Richter, R. Von, Kosters, J.&guhavanta-Kalkosol,S. Vergleichende untersuchungen zur anwendung eines enzyme-linked immunosorbent-assay(ELISA) zum antikorpernachweis gagen der erreger der aviaeren encephalomyelitis. Zentralblatt fur ueterinarmedizin, 1985, B 32,116-127
65 Smart, I.J.& Grix, D.C. Measurement of antibodies to infectious avian encephalomyelitis virus by ELISA. Avian Pathology, 1985,14,314-352
66 Smart, I.J.,Grix, D.C.& Barr, D.A. The application of ELISA to the diagnosis and control of avian encephalomyelitis. Australian Veterinary
Journal, 1986,63,297-299
67 Shafren, D.R.& Tannock, G.A. An enzyme-linked immunosorbent assay for the detection of avian encephalomyelitis antigens. Avian Diseases, 1988,32,209-214
68 Shafren, D.R.& Tannock, G.A.& Morrow, C.J. Diagnosis of a field outbreak of avian encephalomyelitis by enzyme-linked immunosorbent assay. Autralian Veterinary Journal, 1989a, 66,224-225
69 Shafren, D.R.,Tannock, G.A.& Roberts, T.K. Development and application of an ELISA technique for the detection of antibody to avian encephalomyelitis viruses. Research in veterinary Science, 1989b, 46,95-99
70 Hwang, J.,Luginbuhl,R.E.& Helmboldt,C.F. The in vivo and in vitro isolation of avian encephalomyelitis virus(AEV).Avian Diseases, 1960,4,545-546
71 Mancini,L.O.& Vates,V.J. Cultivation of avian encephalomyelitis virus in vitro. I In chicken embryo neuroglial cell culture. Avian Diseases, 1967,11,672-679
72 Nicholas,R.A.J.,Ream, A.J.& Thornton, D.H. Replication of avian encephalomyelitis virus in chick embryo neurological cell cultures. Archives of Virology, 1987,96,283-287
73 Schaaf, K. Immunization for the control of avian encephalomyelitis. Avian Diseases, 1958,2,279-289
74 Calnek, B.W.,Taylor, P.J.& Sevoian, M. Studies on avian encephalomyelitis. V Development and application of an oral vaccine. Avian Diseases, 1961,5,297-312
75 罗开健，廖明，任涛等。禽脑脊髓炎的防治要点．中国家禽，1999，21(7)：44—45
76 田兆菊，李庆官．禽脑脊髓炎的防制．山东畜牧兽医，2000，1：16
77 Butterfield, W.K.,Luginbuhl,R.E.& Sumner, F.W. Studies on avian encephalomyelitis. Ⅲ Immunization with an inactivated virus. Avian Diseases, 1961,5,445-450
78 秦爱建，段玉友，崔治中等．禽脑脊髓炎油剂灭活苗的现场应用试验．中国家禽，1994，2：22—23
79 姚永秀，秦卓明，刘红等．禽脑脊髓炎油乳剂灭活苗的田间免疫试验．山东家禽，1996，4：3—4
80 Glisson, J.R.& Fletcher, O.J. Clinical encephalitis following avian encephalomyelitis vaccination in broiler breeder pullets. Avian
Diseases,1987, 31,383—385
81 朱静，范维珂．人类基因组计划概况．中国检验医学与临床，2000，1(4)：167—168
82 Gaffeau, A. Science, 1996, 274: 546—548
83 蒋圣娟，张红雨．人类基因组计划及其发展简史．淄博学院学报(自然科学与工程版)，2001，3(3)：81—88
84 Blattner, F.R. Science, 1997, 277: 1453
85 张春霆．生物信息学的现状与展望．世界科技研究与发展，2000，22(6)：17—20
86 俞利荣，曾嵘，夏其昌．蛋白质组研究技术及其进展．生命化学，1998，18(6)：4—9
87 李伯良，李林，吴家睿．功能蛋白质组学．生物工程进展，1999，19(4)：15—16
88 Agvanovsky AA. Koenin E V. Boykovp et al. Beet yellows closterovirus:Complete genome structure and identification of a papain-like thiol protease. Virology, 1994, 198:311-324
89 Klaassen V A.B oeshore ML, Koenin EV et al. Genome Structure and phylogenetic analysis of lettuce infectious yellows virus, a whitefly-trasmitted, bipartite closterovirus. Virology, 1995, 208: 99—110
90 Karasev A V. Boyko VP. Gowda S et al. Complete seguence of the citrus tristeza virus RNA genome. Virology, 1995, 208:511—520
91 Murphy FA. Fauguet CM. Bishop BHL et al. Classification and nomen cluture of viruses:Sixth Report of the International Committee on Taxonomy of Viruses. Arch Virol, 1995, Suppl, 10
92 Karasev AV. Agranovsky AA. Rogov VV et al. Virion RNA of beet yellows closterovirus:cell-free translation and some properties. J Gen Virol, 1989, 70:241—245
93 Pappu HR,Karasev AV, Auderson EJ et al. Nucleotide Seguence and organization of eight 3' open reading fragment of the citrus tisteza closterovirus genome. Virology, 1994, 199:35—46
94 Lomakina, N.F.,Lomakin, A.I.,Drygin, V.V. et al. Detection of avian
encephalomyelitis Virus genome by nested PCR. European Society for Veterinary Virology, 2001, 5:348—349
95 Todd, D.,Weston, J.H.,Mawhinney, K.A. et al. Characterization of the genoae of avian encephelomyelitis virus with cloned cDNA fragm ents Avian Dis, 1999, 43(2), 219—226
96 赵心力，赵振华，马学恩等．禽脑脊髓炎病毒的分离鉴定．中国畜禽传染病，1995，81(2)：3—7
97 叶满红，崔浩中，秦爱建．禽脑脊髓炎病毒单克隆抗体制备．中国兽医学报，1999，19(3)：25-27
98 韦莉，刘爵，张杰等．用蔗糖密度梯度离心法提纯禽脑脊髓炎病毒．北京农业科学，1999，17(6)：35-36
99 马学恩，赵振华。禽脑脊髓炎病毒的提纯和电镜观察．中国兽医科技，1995，25(7)：3-4
100 卢中华，张红英．禽脑脊髓炎病毒的分离与疫苗研制，河南农业科学，1996，46-48
101 刘思当，范伟兴．禽脑脊髓炎病毒的分离与初步鉴定研究．山东畜牧兽医，1999，5：3-4
102 赵心力，郭金贵．禽脑脊髓炎琼脂扩散沉淀抗原制备与应用的研究．中国兽医科技，1995，25(2)：6-8
103 Marvil,p. Nick, J. Avian encephalomyelitis virus is a picornavirus and is most closely related to hepatitis A virus. Journal of General virology, 1999,80:653-662
104 Shafaren, D.R, Tannock, G.A. Further evidence that the necleic acid of avian cncephalomyelitis virus consists of RNA. Avian Diseases, 1992,36:1031-1033
105 殷震．动物病毒学[M]，第二版，科学出版社，1997
106 侯云德主编．分子病毒学[M]．学苑出版社，1990
107 Todd, D. Weston, J.H. Characterization of the genome of avian encephalomyelitis virus with cloned cDNA fragments. Avian Diseases, 1999,43(2):219-226
108 徐耀辉，卢中华．禽脑脊髓炎病毒河南株VPO基因的克隆与序列分析．河南畜牧兽医，2002，23(9)：6—7
109 彭勇，黄庆．枯草杆菌DC-2纳豆激酶基因的克隆及其融合蛋白在E. coli的表达．国生物化学与分子生物学报，2002，18(5)：559-563
110 尹崇，李朝飞．新基因Splt137在大肠杆菌中的表达及抗体制备．中山大学学
报，2002，41(4)：75—77
111 李武平，吕宏亮。干扰素-β1b的高效表达、纯化及抗病毒活性研究．病毒学报，2002，18(3)：240-243
112 杨兴文，刘俭．抗凝活性肽PGD226基因构建、表达，产物纯化及活性分析．中国生物化学与分子生物学报，2002，18(5)：583-587
113 王立霞，张珠强．Cre基因在大肠杆菌中的表达及表达蛋白活性的检测。生物工程学报，2002，18(4)：497—500
114 Shadren，D．R．检测禽脑脊髓炎病毒抗体的ELISA技术的建立和应用．国外兽医学—畜禽传染病，1989，9(3)：28-30
115 曹永长，毕英佐．用酶联免疫吸附实验检测禽脑脊髓炎病毒抗体．中国兽医杂志，1997，23(1)：13-14
116 武东亮，郭三堆．融合基因研究进展．生物技术通报，2001，2：5-7
117 萨姆布鲁克，丁．费里奇 E．F．分子克隆实验指南[M]，金冬雁，黎孟枫等译．第二版，科学出版社，1995
118 阎惠平，郎振为．原位杂交法检测肝组织中输血传播病毒．首都医科大学学报，2001，2：116—118
119 吕玲，向建国．核酸探针原位杂交检测白斑综合症病毒的组织特异性．热带海洋，2000，4：86-90
120 Wagner, E.F. Prenatel lethatities in mice homozygous for human growth hormore gene seguences integratel in the germ line. Cell,1983,35:637-655.
121 周世力．DNA疫苗的研究进展．江汉大学学报(自然科学版)，2002，19(2)：80—83