NDV强毒株的分离鉴定与复合新城疫蜂胶灭活疫苗的研究
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摘要
正文：本研究从山东流行烈性传染病的鸡群中分离出7株具有血凝活性的病毒，该7株病毒的血凝活性均能被新城疫LaSota株标准阳性血清所抑制，但病毒不能被中和，仍能致死鸡胚。经分离鉴定，分离毒均为新城疫病毒株，通过鸡胚半数致死量(ELD_(50))、最小致死量致死鸡胚的平均时间(MDT)、1日龄雏鸡脑内致病指数(ICPI)、6周龄雏鸡静脉内致病指数(IVPI)、血凝解脱及血凝素稳定性试验等表明：7株分离毒株均为新城疫病毒强毒株，其毒力与标准强毒株F_(48)E_9株相似。
对4株具有一定代表性的NDV分离株的F基因进行RT-PCR扩增和序列分析，根据基因裂解位点的氨基酸序列推测，其中1株属于弱毒株，3株属于强毒株；核苷酸序列及其推导的氨基酸序列比较结果表明：3株强毒株与Clone30基因核苷酸序列的同源性在83.6％-84.0％之间；与F_(48)E_9典型NDV强毒株同源性在86.5.6％-88.3％之间，推导的氨基酸序列同源性与Clone-30株在85.9％-87.0％之间；与F_(48)E_9典型NDV强毒株在89.1％-91.3％之间；利用MegAlign软件绘制了NDV的系统发育进化树，其结果表明，3株分离强毒株为Ⅶ基因型，弱毒株为基因Ⅱ型。
通过鸡胚接种的方法研究了蜂胶乙醇浸出物(EEP)对NDV Clone-30株的灭活作用，结果表明，0.1-10mg／ml的蜂胶乙醇浸出物(EEP)对NDV Clone-30株的灭活作用，并且这种灭活作用与温度、乙醇浓度成正比，在3-30min内与作用时间无关。
用NDVClone-30株和分离毒株分别接种非免疫鸡胚，制备抗原液，经甲醛灭活后与纯化的天然免疫增强剂蜂胶乳化，研制成功复合新城疫蜂胶灭活疫苗，对制品的安全性和免疫原性进行了测定，对3-6周龄易感雏鸡以2倍使用剂量肌肉注射，安全性良好，对1日龄雏鸡接种1个使用剂量，进行急性毒性试验观测，15天生长曲线表明，对雏鸡无影响，对3-6周龄雏鸡注射1个使用剂量，每批疫苗免疫10只鸡，免疫5-32天鸡血清中ND血凝抑制(HI)抗体的几何平均滴度(GMT)分别为4.8-9.8 log2，对鸡新城疫免疫攻毒试验表明，3批疫苗，每羽份含有的半数保护量(PD_(50))分别为≥131和≥127。注射部位的动态病理组织学比较观察研究结果表明，蜂胶疫苗免疫后对局部组织学影响明显小于油乳剂苗。蜂胶疫苗的免疫期暂定为6个月；疫苗的保存期为-10℃不结冰，可保存18个月以上，4-8℃为12个月，15-20℃为6个月。田间试验与2亿羽份现场应用结果表明，疫苗安全可靠，效果良好。
Content: Seven viral strains which have heagglutination activity were isolated from 7 chicken flocks with high morbidity and mortality contagious disease. These strains were able to agglutinate chicken red blood cell which could be inhibited by Newcastle disease virus LaSota strain standard antisera, but could not be neutralized by the antisera, and were still able to kill chicken embryos. These strains were confirmed to be Newcastle disease virus based on the test of 50% embryo lethal dose, mean death time of chicken embryos, intracerebral pathogenicity index in 1-day-old chicken, intravenous pathogenicity index in 6-week-old chicken, dehaemagglutination and haemagglutinin heat-stability. The results demonstrated that these strains isolated were high virulent Newcastle disease virus, which were virulent as similar as F48E9 strain.
Gene fragment with F protein splitting site of 4 typical NDV strains isolated was amplified by RT-PCR and analyzed by sequences. Maybe one strain was low mortality virus and three strains were high mortality virus according to amino acid sequence of splitting site of gene. The result of the sequencing and comparison of sequence demonstrated that the homogeneity of nucleoside sequence among the 3 high mortality virus strains and Clone30 was 83.6%-84.0%, among typical high mortality F48E9 strain was 86.5-88.3%.The homogeneity of amino acid sequence among the 3 high mortality virus strains and Clone30 was 85.9%-87.0%, among typical high mortality F48E9 strain was 89.1-91.3%. The NDV systemic evolution tree was depicted by MegAlign software and showed that the three high mortality viral strains were gene type VII and the low mortality one was gene type II.
The inactivation effect of ethanol extract of propolis on Newcastle Disease virus Clone-30 strain was studied through chicken embryo inoculation. The result demonstrated that, 0.1-10mg/ml ethanol extract of propolis could inactivate NDV clone-30 and the inactivation effect directed ratio the temperature inactivated and concentrate of ethanol and had correlation with the reaction time within 3-30min.
Egg embryo from hen which hadn't been immunized were inoculated by NDV Clone-30 strain and virus strain isolated and the antigen was gathered and inactivated with formaldehyde solution and mixed with natural immunity intensifier propolis,so the compositive NDV proplis inactivated vaccine was made successfully. The safety and immunity of vaccines were detected and the result demonstrated that the safety was good when 3-6 week old chicken were immunized through muscle with twice dose; the 15 days growth curve demonstrated the vaccine hadn't bad effect to chicken when they were immunized with one dose and studied the actuate toxicity test. The 3-6 week old chicken were immunized through muscle with one dose used and 10 chicken every batch vaccine, the GMT of HI antibodies of ND in the 5-32 days old chicken sera were 4.8 log2 and 9.81og2,respectively.The result of ND immunity and challenge to chicken demonstrated that every dose of three batches vaccine contain PD50 more than or equal to 131 and 127,respectively. The result of dynamic pathology tissue comparison of muscle injected demonstrated that the effect on the injection tissue of propolis vaccines was apparently less than that of oil vaccines. The immunity period of proplis vaccine was temporary as 6 months, the stored period of this vaccine was more than 18 months in -10℃ and didn't ice up in this temperature, 12 months in 4-8 ℃ and 6 months in 15- 20℃. The result of field test and the effect of immunity 2 billions fowls demonstrated that the vaccine was safe and reliable, meanwhile had good immunity effect.
对4株具有一定代表性的NDV分离株的F基因进行RT-PCR扩增和序列分析，根据基因裂解位点的氨基酸序列推测，其中1株属于弱毒株，3株属于强毒株；核苷酸序列及其推导的氨基酸序列比较结果表明：3株强毒株与Clone30基因核苷酸序列的同源性在83.6％-84.0％之间；与F_(48)E_9典型NDV强毒株同源性在86.5.6％-88.3％之间，推导的氨基酸序列同源性与Clone-30株在85.9％-87.0％之间；与F_(48)E_9典型NDV强毒株在89.1％-91.3％之间；利用MegAlign软件绘制了NDV的系统发育进化树，其结果表明，3株分离强毒株为Ⅶ基因型，弱毒株为基因Ⅱ型。
通过鸡胚接种的方法研究了蜂胶乙醇浸出物(EEP)对NDV Clone-30株的灭活作用，结果表明，0.1-10mg／ml的蜂胶乙醇浸出物(EEP)对NDV Clone-30株的灭活作用，并且这种灭活作用与温度、乙醇浓度成正比，在3-30min内与作用时间无关。
用NDVClone-30株和分离毒株分别接种非免疫鸡胚，制备抗原液，经甲醛灭活后与纯化的天然免疫增强剂蜂胶乳化，研制成功复合新城疫蜂胶灭活疫苗，对制品的安全性和免疫原性进行了测定，对3-6周龄易感雏鸡以2倍使用剂量肌肉注射，安全性良好，对1日龄雏鸡接种1个使用剂量，进行急性毒性试验观测，15天生长曲线表明，对雏鸡无影响，对3-6周龄雏鸡注射1个使用剂量，每批疫苗免疫10只鸡，免疫5-32天鸡血清中ND血凝抑制(HI)抗体的几何平均滴度(GMT)分别为4.8-9.8 log2，对鸡新城疫免疫攻毒试验表明，3批疫苗，每羽份含有的半数保护量(PD_(50))分别为≥131和≥127。注射部位的动态病理组织学比较观察研究结果表明，蜂胶疫苗免疫后对局部组织学影响明显小于油乳剂苗。蜂胶疫苗的免疫期暂定为6个月；疫苗的保存期为-10℃不结冰，可保存18个月以上，4-8℃为12个月，15-20℃为6个月。田间试验与2亿羽份现场应用结果表明，疫苗安全可靠，效果良好。
Content: Seven viral strains which have heagglutination activity were isolated from 7 chicken flocks with high morbidity and mortality contagious disease. These strains were able to agglutinate chicken red blood cell which could be inhibited by Newcastle disease virus LaSota strain standard antisera, but could not be neutralized by the antisera, and were still able to kill chicken embryos. These strains were confirmed to be Newcastle disease virus based on the test of 50% embryo lethal dose, mean death time of chicken embryos, intracerebral pathogenicity index in 1-day-old chicken, intravenous pathogenicity index in 6-week-old chicken, dehaemagglutination and haemagglutinin heat-stability. The results demonstrated that these strains isolated were high virulent Newcastle disease virus, which were virulent as similar as F48E9 strain.
Gene fragment with F protein splitting site of 4 typical NDV strains isolated was amplified by RT-PCR and analyzed by sequences. Maybe one strain was low mortality virus and three strains were high mortality virus according to amino acid sequence of splitting site of gene. The result of the sequencing and comparison of sequence demonstrated that the homogeneity of nucleoside sequence among the 3 high mortality virus strains and Clone30 was 83.6%-84.0%, among typical high mortality F48E9 strain was 86.5-88.3%.The homogeneity of amino acid sequence among the 3 high mortality virus strains and Clone30 was 85.9%-87.0%, among typical high mortality F48E9 strain was 89.1-91.3%. The NDV systemic evolution tree was depicted by MegAlign software and showed that the three high mortality viral strains were gene type VII and the low mortality one was gene type II.
The inactivation effect of ethanol extract of propolis on Newcastle Disease virus Clone-30 strain was studied through chicken embryo inoculation. The result demonstrated that, 0.1-10mg/ml ethanol extract of propolis could inactivate NDV clone-30 and the inactivation effect directed ratio the temperature inactivated and concentrate of ethanol and had correlation with the reaction time within 3-30min.
Egg embryo from hen which hadn't been immunized were inoculated by NDV Clone-30 strain and virus strain isolated and the antigen was gathered and inactivated with formaldehyde solution and mixed with natural immunity intensifier propolis,so the compositive NDV proplis inactivated vaccine was made successfully. The safety and immunity of vaccines were detected and the result demonstrated that the safety was good when 3-6 week old chicken were immunized through muscle with twice dose; the 15 days growth curve demonstrated the vaccine hadn't bad effect to chicken when they were immunized with one dose and studied the actuate toxicity test. The 3-6 week old chicken were immunized through muscle with one dose used and 10 chicken every batch vaccine, the GMT of HI antibodies of ND in the 5-32 days old chicken sera were 4.8 log2 and 9.81og2,respectively.The result of ND immunity and challenge to chicken demonstrated that every dose of three batches vaccine contain PD50 more than or equal to 131 and 127,respectively. The result of dynamic pathology tissue comparison of muscle injected demonstrated that the effect on the injection tissue of propolis vaccines was apparently less than that of oil vaccines. The immunity period of proplis vaccine was temporary as 6 months, the stored period of this vaccine was more than 18 months in -10℃ and didn't ice up in this temperature, 12 months in 4-8 ℃ and 6 months in 15- 20℃. The result of field test and the effect of immunity 2 billions fowls demonstrated that the vaccine was safe and reliable, meanwhile had good immunity effect.
引文
1 李慧姣．我国新城疫病毒分离株生物学特性的研究．病毒学报，1985，1：354—359
2 Alexander, d.j.Newcastle disease in countries of the European Union. Avian Pathoi 1995,24: 545-551
3 Parede,L.,and P.L.Young.The pathogenesis of velogenic Newcastle disease virus infection of chickens of different ages and different levels of immunity. Avian Dis. 1990, 34:803-808
4 Kohama T, Garten W and Klenk H-D.Changes in conformation and charge paralleling proteolytic activation of Newcastle disease virus glycoprotein.Virology,1981,111 (2):364-376
5 Nagai Y, et al.The spread of a pathogenic and an apathogenic strain of Newcastle disease virus in the chick embryo as depending on the protease sensitivity of the virus glycoprotein. J Gen Virol, 1989,45(2):263-272
6 Somson ACR,Chambers P and Dickinson J H. Location of post-translational cleavage events within F and HN glycoproteins of Newcastle disease Virus. J Gen Virol,1980,47(1):19-27
7 McMillar B C,Hanson R P.RNA oligonucleotide fingerprinting:A proposed method of identifying strains of Newcastle disease virus. Avian Dis, 1980,24:1016-1020
8 Palmirei S,Perdue M L.An alternative method of oligonucleotide finger-printing for resolving NDV-specific RNA fragments. Avian Dis,1989,33:343-350
9 Jarecki-Black J C,Benuett J D,Palmieri S.A novel oligonucleotide probe for the detection of NDV. Avian Dis,1992,36:134-138
10 Hodder AN, Liu ZY, Selleck PW et al, Characterization of field isolates of Newcastle disease virus using antipeptide antibodies. Avian Disease, 1994;38:103-118
11 Niikura M,Matsuura Y, et al.Characterization of HN of NDV expressed by a recombinant baculovirus.Virus Res,1991,20:31～43
12 Taylor, J.,C.Edbauer, A.rey-Senelonge et al, Newcastle disease virus fusion protein expressed in a foxlpox virus recombinant confers protection in chickens J Virol 1990,64:1441-1450
13 Miers.L.A.,R.A.Bankowski,andY.C.Zee,Optimizing the enzyme-linked immunosorbent assay for evaluating immunity in chickens to Newscastle disease. Avian Dis. 1983 27:1112-1125
14 Meulemans,G.,C.Letellier, M.Gonze,et al,Newcastle disease virus F glycoprotein expressed form a recombinant vaccina virus vector protects chickens against live virus challenge. Avian Pathol. 17:821-827
15 Boursnell ME, Green PF, Samson AC et al. A recombinant fowlpox virus expressing the hemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) protects chickens against challenge by NDV.Virology, 1990; 178:297-300
16 Morgan R W, Gelb J J,Schreurs C S,et al. Protection of chickens from NDV and Mariks disease with a recombinant herpesvirus of turkeys vaccine expression the NDV fusion protein.Avian Dis,1992,36:858-870
17 H.Mori, H.Tawara, H.Nakazawa et al. Expression of the Newcastle Disease Virus(NDV) Fusion Glycoprotein and Vaccination against NDV Challenge with a Recombinant Baculovirus. Avian Disease, 1994,38:772-777
18 Smith G E,Summers M D and Fraser M J. Production of human beta interferon in insect cells
infected with a baculovirus expression vector. Mol Cell Biol, 1983, 3:2156-2165
19 Taylor J,Edbauer C, Rey-Semelonge A,et al. Newcastle disease virus Fusion protein expressed in a fowlpox virus recombinant confers protection in chickens.J Virol, 1990,64:1441—1450
20 殷震，刘景华主编．动物病毒学．第2版．北京：科学出版社，1997．351-355
21 BW卡尔尼克主编．禽病学．第10版．高福，苏敬良主译．北京：中国农业出版社，1999．691-726
22 Millar N S, Chambers P, Emmerson P N.Nucleotide sequence of the fusion and hemagglutinin-neuraminidase glycoprotein genes of Newcastle disease virus, strain Ulster: molecular basis for variations in pathogenicity between Strais. J Gen Virol,1988,69:613—620
23 范根成，朱万光，王永玲等．新城疫病毒强毒株的分离与鉴定．中国兽医学报，1999，19：114—117
24 Collins PL, Wertz GW, Ball LA, Hightower LE.Coding assignments of the five smaller mRNAs of Newcastle disease virus. J Virol 1982 Sep;43 (3):1024-1031
25 Alexander D J. Newcastle Disease. London; Kluwer Academic Publishers, 1988, P. 79-97
26 Toyoda T, Sakaguchi T, Hirote H, et.al. Newcastle disease virus evolution: Ⅱ. Lack ofgene recombinant in generating virulent and avirulent strain. Virology, 1989, 169:273-282
27 Boursnell M E, Green P F, Campbell J I, et al. Insertion of the fusion gene from NDV into a non-essential region in the terminal repeats of fowlpox virus and demonstration of protective immunity indued by the recombinant. J Gen Virol, 1990, 71: 621-628
28 Murakami Y, Kagino T, Niikura M, et al. Claracterization of Newcastle disease virus envelope glycoprotein expressed in insect cells. Virus Res, 1994, 33:123-137
29 Van Oers M M,Flipson J T M,Reushen C B E M, et al. Functional domains of the pl0 protein of Autographa califonia nuclear polyhedrosis virus.J Gen Virol, 1993,94:563-574
30 常维山，一株新城疫病毒强毒株的F基因序列测定研究，变异性新城疫病毒分子生物学的研究鉴定材料．
31 王兴龙，新城疫病毒分子生物学．动物科学与动物医学，2001，18(3)，28-30．
32 许正鼎主编 蜂胶的神奇疗法[M] 科学普及出版社，2002．1-90．
34 陆承平，最新脊椎动物病毒分类简介[J]，中国兽医学报，1996，16(1)：94～100．
35 Lomniczi B, Wehmann E, Herczeg J, et al. Newcastle disease outbreaks in recent years in Western Europe were caused by an old (Ⅵ)and a novel genotype(Ⅶ)[J].Arch Virol,1998,143:49-64
36 黄小波，新城疫病毒强弱毒株鉴别方法的研究进展．中国兽医科技，2002，32(10)：20-21．
37 成大荣，徐建生等，新城疫病毒的F蛋白与遗传学分群．预防兽医学进展，2001，3(1)：37-39．
38 Yang C Y,Chang P C, Jyh M H, et al. Nucleotide sequence and phylogenetic analysis of Newcastle disease vrius isolated from recent outbreakes in Taiwan[J].Avian Dis, 1997,41:326-328.
39 王兴龙，四株新城疫病毒流行株F基因的遗传变异分析．中国预防兽医学报，2002，24(2)：123-125．
40 曹殿军，李祥瑞，等(译)．国外兽医学-畜禽传染病，1997，17(2)：8-10．
41 Khayyal MT, el-Ghazaly MA,el-Khatib AS.Mechanisms involved in the anti-inflammatory effect of proplis extrat.Drugs Exp Clin Res 1993,19(5):197-203.
42 Dimov V, Ivanovska N, Bankova V, Popov S. Immunomodulatory action of propolis:Ⅳ. Prophylactic activity against gram-negative infections and adjuvant effect of the water-soluble derivative. Vaccine 1992,10(12):17-23.
43 唐桂运，武华译，禽病原分离鉴定实验室手册，第三版，北京：北京农业大学出版
社，1993，140-150．
44 蔡宝祥等，动物传染病学诊断，南京：江苏科学技术出版社，1992，177-180．
45 B W卡尔尼克主编．禽病学．高福，苏敬良主译．北京：中国农业出版社，1999．691-726．
46 贺东生等，新城疫病毒系统发育分析及强弱毒株的鉴别诊断．动物医学进展，2000，21(2)：40-41．
47 农业部．中华人民共和国兽用生物制品规程，北京：农业部，1992版，47-55．
48 Schsper U M, Fuller F J, Ward M D, et al. Virology, 1998,165:291-294. Peeters B P, de Leeuw OS, Koch G. Rescue of Newcastle disease virus from cloned Cdna:Evidence that cleavability of the Fusion protein is a major determinant for virulence [J].J Vrrol, 1999,73(6):5001-5009..
49 曹殿军等，我国部分地区NDV的分子流行病学研究．中国预防兽医学报，2001，23(1)：29-32．
50 巴德年，当代免疫学技术与应用，第一版，北京医科大学中国协和医科大学联合出版，1998．
51 卢锦汉，医学生物制品学，第一版，人民卫生出版社，1995．
52 余传霖，现代医学免疫学，第一版，上海医科大学出版社，1998．
53 [美]F．奥斯伯，精编分子生物学实验技术，第一版，科学出版社，1998．
54 Chambers P, Millar NS, et al. Molecular cloning of complementary DNA to Newscastle disease virus, and nucleotide sequence analysis of the junction between the genes encoding the haemagglutinin-neuraminidase and the large protein. Journal of General Virlogy, 1986,67:475～486.
55 曹殿军，郭鑫，苑纯秀，闫丽辉等，新城疫病毒V4株F基因的克隆与核苷酸序列分析．中国预防兽医学报，2000，22(1)：39～43．
56 刘伟忠，姜焱，吴艳涛，张如宽，刘秀梵，新城疫病毒特异性糖蛋白基因探针的制备及应用．畜牧兽医学报，1999，30(1)，44～49．
57 吴艳涛，刘秀梵，张如宽，新城疫病毒F_(48)E_8株融合蛋白基因序列分析．病毒学报，1999，15(2)：143～146．
58 严维巍，王永坤等，一株鸡副粘病毒F基因重要片段的扩增、克隆与分析．中国预防兽医学报，2000，22(5)：368-370．
59 梅双双等，鸡新城疫病毒强毒株的分离及生物学特性鉴定．中国预防兽医学报，2002，24(5)：368-370．
60 田夫林，新城疫流行病学调查及山东分离株遗传变异性研究．中国农业大学博士论文，2002。
61 Lomniczi B, Wehmann E,Herczeg J,et al.Newcastle disease outbreaks in recent years in Western Europe were caused by an old (Ⅵ) and a novel genotype(Ⅷ)[J].Arch Virol, 1998,143:49～44.
62 Yang C Y, Chang P C,Jyh M H,et al.Nucleotide sequence and phylogenetic analysis of Newscastle disease virus isolated from recent outbreaks in Taiwan[J].Avian Dis,1997,41:326～328.
63 曹殿军，刘春丽，王丽林等，新城疫病毒F_(48)E_9株HN基因的克隆与酶切分析[J]．中国兽医学报，1997，17(4)：326～328．
64 王红宁等，新城疫病毒强弱毒株检测方法研究进展．禽病防制，2001，18(6)：28-29．
65 苑纯秀，曹殿军等新城疫病毒F_(48)E_9株基因主要功能区的核苷酸序列分析．中国预防兽医学报，1999，21(1)：31～34．
66 梁德勇等，RNA快速提取一步法的建立．中国医科大学学报，1999，5(2)：35-40．
67 梁荣，陈杰等，禽痘病毒载体在新城疫病毒重组疫苗研究的应用．动物医学进展 2000 21(1)：13-16
68 Lomniczi B,Wehmann E,Herczeg J,et al.Newcastle disease outbreaks in recent years in Western Europe were caused by an old (Ⅵ)and a novel genotype(Ⅶ)[J].Arch Virol, 1998,143:49-64.
69 Yang C Y, Chang P C, Jyh M H, et al. Nucleotide sequence and phylogenetic analysis of Newcastle disease vrius isolated from recent outbreakes in Taiwan[J].Avian Dis, 1997,41:326-328.
70 范纯秀等，鸡新城疫东北地区融合蛋白基因的克隆与鉴定[J]．中国预防兽医学报，1999,21(1)：31-34．
71 分子克隆，金冬雁等译，第二版，北京科学出版社．1999
72 Lomniczi B, Wehmann E, Herczeg J, et al. Newcastle disease outbreaks in recent years in Western Europe were caused by an old (Ⅵ)and a novel genotype(Ⅶ)[J].Arch Virol,1998,143:49-64.
77 Yang C Y, Chang P C, Jyh M H, et al. Nucleotide sequence and phylogenetic analysis of Newcastle disease vrius isolated from recent outbreakes in Taiwan[J].Avian Dis, 1997,41:326-328.
78 沈志强．蜂胶——兽医领域应用前景广阔的天然药物．中国兽药杂志．1992．26(2)：56-59
79 沈志强，杨永福．禽霍乱蜂胶菌苗的研究．中国畜禽传染病．1989．5：1-3
80 黄文诚．蜂胶的化学成分和生物学活性(一)．蜜蜂杂志．1998．9：12
81 奥霍茨基(俄)．蜂胶在疾病临床上的应用．养蜂科技．1994．6：13
82 沈志强，刘吉山，李峰等．鸡新城疫蜂胶灭活疫苗制苗工艺、疫苗安全性和免疫原性的研究．中国预防兽医学报．2000．4，94-96
83 李峰，沈志强，刘吉山等．ND蜂胶灭活疫苗大剂量预防注射紧急防治鸡新城疫效果观察．中国动物保健．2000．11．31-33
84 李强，PRRS蜂胶灭活疫苗的制备及其安全性和免疫原性研究，中国农业大学硕士学位论文，2001。
85 王明俊主编，《兽医生物制品学》，中国农业出版社，1997。
86 赵德明主编，《兽医病理学》，中国农业大学出版社，1998。
87 沈志强，杨永福，佐剂新秀—蜂胶。中国畜禽传染病。1989，5：53-55
88 沈志强，鸡减蛋综合症蜂胶灭活疫苗的研究与应用，畜牧兽医学报，1997，28(6)，535-541
89 沈志强，蜂胶的免疫学作用，中国禽业导刊，2000，13，15-16
90 沈志强，李峰等，蜂胶对NDV的灭活作用效果观察，河南畜牧兽医，2002，1，1-3
91 沈志强，刘吉山等，值得警惕的强毒鸡新城疫的识别与防治对策探讨，中国禽业导刊，2000，9，14-18
92 沈志强，动物疫苗用蜂胶的质量要求及工业化提取技术的研究，中国兽药杂志，1997，4，13-15
93 沈志强，刘吉山等，新城疫病毒山东强毒株的分离鉴定，中国预防兽医学报，2002，2，
94 管宇，沈志强等，sdbz-s99分离株的生物学特性及其F基因的克隆与序列分析，中国预防兽医学报，2002，6，
95 赵蕾，沈志强等，复合新城疫蜂胶灭活疫苗抗体消长规律的研究，中国预防兽医学报，2002，增
96 王玲，蜂胶乙醇提取物增强机体免疫功能机理的研究，山东农业大学硕士研究生毕业论文，1998。
97 庞昕，鸡新城疫蜂胶灭活疫苗免疫作用机理的研究，山东农业大学硕士研究生毕业论文，2001。
98 陈洪涛，田惠芳，朱国强等，蜂胶免疫佐剂的研究，江苏农学院学报，1996，17(1)73-75
99 王守智，李景水，张国领等，用蜂胶佐剂提高抗鸡法氏囊病和新城疫二联苗血清抗体效价的试验，中国兽医科技，1997，27(8)，24
100 唐传核，孟岳成， 蜂胶的生理功能以及开发研究，食品工业科技，1999，20(2)，30-33
101 沈志强，蜂胶及其在农牧业中的应用进展，农牧产品开发，2000，3，18-20
102 庞万勇，沈志强，刘吉山等，禽霍乱与大肠杆菌病多价蜂胶二联疫苗与油乳剂疫苗注射部位动态病理组织学比较观察，山东家禽，2003，7，1-3
2 Alexander, d.j.Newcastle disease in countries of the European Union. Avian Pathoi 1995,24: 545-551
3 Parede,L.,and P.L.Young.The pathogenesis of velogenic Newcastle disease virus infection of chickens of different ages and different levels of immunity. Avian Dis. 1990, 34:803-808
4 Kohama T, Garten W and Klenk H-D.Changes in conformation and charge paralleling proteolytic activation of Newcastle disease virus glycoprotein.Virology,1981,111 (2):364-376
5 Nagai Y, et al.The spread of a pathogenic and an apathogenic strain of Newcastle disease virus in the chick embryo as depending on the protease sensitivity of the virus glycoprotein. J Gen Virol, 1989,45(2):263-272
6 Somson ACR,Chambers P and Dickinson J H. Location of post-translational cleavage events within F and HN glycoproteins of Newcastle disease Virus. J Gen Virol,1980,47(1):19-27
7 McMillar B C,Hanson R P.RNA oligonucleotide fingerprinting:A proposed method of identifying strains of Newcastle disease virus. Avian Dis, 1980,24:1016-1020
8 Palmirei S,Perdue M L.An alternative method of oligonucleotide finger-printing for resolving NDV-specific RNA fragments. Avian Dis,1989,33:343-350
9 Jarecki-Black J C,Benuett J D,Palmieri S.A novel oligonucleotide probe for the detection of NDV. Avian Dis,1992,36:134-138
10 Hodder AN, Liu ZY, Selleck PW et al, Characterization of field isolates of Newcastle disease virus using antipeptide antibodies. Avian Disease, 1994;38:103-118
11 Niikura M,Matsuura Y, et al.Characterization of HN of NDV expressed by a recombinant baculovirus.Virus Res,1991,20:31～43
12 Taylor, J.,C.Edbauer, A.rey-Senelonge et al, Newcastle disease virus fusion protein expressed in a foxlpox virus recombinant confers protection in chickens J Virol 1990,64:1441-1450
13 Miers.L.A.,R.A.Bankowski,andY.C.Zee,Optimizing the enzyme-linked immunosorbent assay for evaluating immunity in chickens to Newscastle disease. Avian Dis. 1983 27:1112-1125
14 Meulemans,G.,C.Letellier, M.Gonze,et al,Newcastle disease virus F glycoprotein expressed form a recombinant vaccina virus vector protects chickens against live virus challenge. Avian Pathol. 17:821-827
15 Boursnell ME, Green PF, Samson AC et al. A recombinant fowlpox virus expressing the hemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) protects chickens against challenge by NDV.Virology, 1990; 178:297-300
16 Morgan R W, Gelb J J,Schreurs C S,et al. Protection of chickens from NDV and Mariks disease with a recombinant herpesvirus of turkeys vaccine expression the NDV fusion protein.Avian Dis,1992,36:858-870
17 H.Mori, H.Tawara, H.Nakazawa et al. Expression of the Newcastle Disease Virus(NDV) Fusion Glycoprotein and Vaccination against NDV Challenge with a Recombinant Baculovirus. Avian Disease, 1994,38:772-777
18 Smith G E,Summers M D and Fraser M J. Production of human beta interferon in insect cells
infected with a baculovirus expression vector. Mol Cell Biol, 1983, 3:2156-2165
19 Taylor J,Edbauer C, Rey-Semelonge A,et al. Newcastle disease virus Fusion protein expressed in a fowlpox virus recombinant confers protection in chickens.J Virol, 1990,64:1441—1450
20 殷震，刘景华主编．动物病毒学．第2版．北京：科学出版社，1997．351-355
21 BW卡尔尼克主编．禽病学．第10版．高福，苏敬良主译．北京：中国农业出版社，1999．691-726
22 Millar N S, Chambers P, Emmerson P N.Nucleotide sequence of the fusion and hemagglutinin-neuraminidase glycoprotein genes of Newcastle disease virus, strain Ulster: molecular basis for variations in pathogenicity between Strais. J Gen Virol,1988,69:613—620
23 范根成，朱万光，王永玲等．新城疫病毒强毒株的分离与鉴定．中国兽医学报，1999，19：114—117
24 Collins PL, Wertz GW, Ball LA, Hightower LE.Coding assignments of the five smaller mRNAs of Newcastle disease virus. J Virol 1982 Sep;43 (3):1024-1031
25 Alexander D J. Newcastle Disease. London; Kluwer Academic Publishers, 1988, P. 79-97
26 Toyoda T, Sakaguchi T, Hirote H, et.al. Newcastle disease virus evolution: Ⅱ. Lack ofgene recombinant in generating virulent and avirulent strain. Virology, 1989, 169:273-282
27 Boursnell M E, Green P F, Campbell J I, et al. Insertion of the fusion gene from NDV into a non-essential region in the terminal repeats of fowlpox virus and demonstration of protective immunity indued by the recombinant. J Gen Virol, 1990, 71: 621-628
28 Murakami Y, Kagino T, Niikura M, et al. Claracterization of Newcastle disease virus envelope glycoprotein expressed in insect cells. Virus Res, 1994, 33:123-137
29 Van Oers M M,Flipson J T M,Reushen C B E M, et al. Functional domains of the pl0 protein of Autographa califonia nuclear polyhedrosis virus.J Gen Virol, 1993,94:563-574
30 常维山，一株新城疫病毒强毒株的F基因序列测定研究，变异性新城疫病毒分子生物学的研究鉴定材料．
31 王兴龙，新城疫病毒分子生物学．动物科学与动物医学，2001，18(3)，28-30．
32 许正鼎主编 蜂胶的神奇疗法[M] 科学普及出版社，2002．1-90．
34 陆承平，最新脊椎动物病毒分类简介[J]，中国兽医学报，1996，16(1)：94～100．
35 Lomniczi B, Wehmann E, Herczeg J, et al. Newcastle disease outbreaks in recent years in Western Europe were caused by an old (Ⅵ)and a novel genotype(Ⅶ)[J].Arch Virol,1998,143:49-64
36 黄小波，新城疫病毒强弱毒株鉴别方法的研究进展．中国兽医科技，2002，32(10)：20-21．
37 成大荣，徐建生等，新城疫病毒的F蛋白与遗传学分群．预防兽医学进展，2001，3(1)：37-39．
38 Yang C Y,Chang P C, Jyh M H, et al. Nucleotide sequence and phylogenetic analysis of Newcastle disease vrius isolated from recent outbreakes in Taiwan[J].Avian Dis, 1997,41:326-328.
39 王兴龙，四株新城疫病毒流行株F基因的遗传变异分析．中国预防兽医学报，2002，24(2)：123-125．
40 曹殿军，李祥瑞，等(译)．国外兽医学-畜禽传染病，1997，17(2)：8-10．
41 Khayyal MT, el-Ghazaly MA,el-Khatib AS.Mechanisms involved in the anti-inflammatory effect of proplis extrat.Drugs Exp Clin Res 1993,19(5):197-203.
42 Dimov V, Ivanovska N, Bankova V, Popov S. Immunomodulatory action of propolis:Ⅳ. Prophylactic activity against gram-negative infections and adjuvant effect of the water-soluble derivative. Vaccine 1992,10(12):17-23.
43 唐桂运，武华译，禽病原分离鉴定实验室手册，第三版，北京：北京农业大学出版
社，1993，140-150．
44 蔡宝祥等，动物传染病学诊断，南京：江苏科学技术出版社，1992，177-180．
45 B W卡尔尼克主编．禽病学．高福，苏敬良主译．北京：中国农业出版社，1999．691-726．
46 贺东生等，新城疫病毒系统发育分析及强弱毒株的鉴别诊断．动物医学进展，2000，21(2)：40-41．
47 农业部．中华人民共和国兽用生物制品规程，北京：农业部，1992版，47-55．
48 Schsper U M, Fuller F J, Ward M D, et al. Virology, 1998,165:291-294. Peeters B P, de Leeuw OS, Koch G. Rescue of Newcastle disease virus from cloned Cdna:Evidence that cleavability of the Fusion protein is a major determinant for virulence [J].J Vrrol, 1999,73(6):5001-5009..
49 曹殿军等，我国部分地区NDV的分子流行病学研究．中国预防兽医学报，2001，23(1)：29-32．
50 巴德年，当代免疫学技术与应用，第一版，北京医科大学中国协和医科大学联合出版，1998．
51 卢锦汉，医学生物制品学，第一版，人民卫生出版社，1995．
52 余传霖，现代医学免疫学，第一版，上海医科大学出版社，1998．
53 [美]F．奥斯伯，精编分子生物学实验技术，第一版，科学出版社，1998．
54 Chambers P, Millar NS, et al. Molecular cloning of complementary DNA to Newscastle disease virus, and nucleotide sequence analysis of the junction between the genes encoding the haemagglutinin-neuraminidase and the large protein. Journal of General Virlogy, 1986,67:475～486.
55 曹殿军，郭鑫，苑纯秀，闫丽辉等，新城疫病毒V4株F基因的克隆与核苷酸序列分析．中国预防兽医学报，2000，22(1)：39～43．
56 刘伟忠，姜焱，吴艳涛，张如宽，刘秀梵，新城疫病毒特异性糖蛋白基因探针的制备及应用．畜牧兽医学报，1999，30(1)，44～49．
57 吴艳涛，刘秀梵，张如宽，新城疫病毒F_(48)E_8株融合蛋白基因序列分析．病毒学报，1999，15(2)：143～146．
58 严维巍，王永坤等，一株鸡副粘病毒F基因重要片段的扩增、克隆与分析．中国预防兽医学报，2000，22(5)：368-370．
59 梅双双等，鸡新城疫病毒强毒株的分离及生物学特性鉴定．中国预防兽医学报，2002，24(5)：368-370．
60 田夫林，新城疫流行病学调查及山东分离株遗传变异性研究．中国农业大学博士论文，2002。
61 Lomniczi B, Wehmann E,Herczeg J,et al.Newcastle disease outbreaks in recent years in Western Europe were caused by an old (Ⅵ) and a novel genotype(Ⅷ)[J].Arch Virol, 1998,143:49～44.
62 Yang C Y, Chang P C,Jyh M H,et al.Nucleotide sequence and phylogenetic analysis of Newscastle disease virus isolated from recent outbreaks in Taiwan[J].Avian Dis,1997,41:326～328.
63 曹殿军，刘春丽，王丽林等，新城疫病毒F_(48)E_9株HN基因的克隆与酶切分析[J]．中国兽医学报，1997，17(4)：326～328．
64 王红宁等，新城疫病毒强弱毒株检测方法研究进展．禽病防制，2001，18(6)：28-29．
65 苑纯秀，曹殿军等新城疫病毒F_(48)E_9株基因主要功能区的核苷酸序列分析．中国预防兽医学报，1999，21(1)：31～34．
66 梁德勇等，RNA快速提取一步法的建立．中国医科大学学报，1999，5(2)：35-40．
67 梁荣，陈杰等，禽痘病毒载体在新城疫病毒重组疫苗研究的应用．动物医学进展 2000 21(1)：13-16
68 Lomniczi B,Wehmann E,Herczeg J,et al.Newcastle disease outbreaks in recent years in Western Europe were caused by an old (Ⅵ)and a novel genotype(Ⅶ)[J].Arch Virol, 1998,143:49-64.
69 Yang C Y, Chang P C, Jyh M H, et al. Nucleotide sequence and phylogenetic analysis of Newcastle disease vrius isolated from recent outbreakes in Taiwan[J].Avian Dis, 1997,41:326-328.
70 范纯秀等，鸡新城疫东北地区融合蛋白基因的克隆与鉴定[J]．中国预防兽医学报，1999,21(1)：31-34．
71 分子克隆，金冬雁等译，第二版，北京科学出版社．1999
72 Lomniczi B, Wehmann E, Herczeg J, et al. Newcastle disease outbreaks in recent years in Western Europe were caused by an old (Ⅵ)and a novel genotype(Ⅶ)[J].Arch Virol,1998,143:49-64.
77 Yang C Y, Chang P C, Jyh M H, et al. Nucleotide sequence and phylogenetic analysis of Newcastle disease vrius isolated from recent outbreakes in Taiwan[J].Avian Dis, 1997,41:326-328.
78 沈志强．蜂胶——兽医领域应用前景广阔的天然药物．中国兽药杂志．1992．26(2)：56-59
79 沈志强，杨永福．禽霍乱蜂胶菌苗的研究．中国畜禽传染病．1989．5：1-3
80 黄文诚．蜂胶的化学成分和生物学活性(一)．蜜蜂杂志．1998．9：12
81 奥霍茨基(俄)．蜂胶在疾病临床上的应用．养蜂科技．1994．6：13
82 沈志强，刘吉山，李峰等．鸡新城疫蜂胶灭活疫苗制苗工艺、疫苗安全性和免疫原性的研究．中国预防兽医学报．2000．4，94-96
83 李峰，沈志强，刘吉山等．ND蜂胶灭活疫苗大剂量预防注射紧急防治鸡新城疫效果观察．中国动物保健．2000．11．31-33
84 李强，PRRS蜂胶灭活疫苗的制备及其安全性和免疫原性研究，中国农业大学硕士学位论文，2001。
85 王明俊主编，《兽医生物制品学》，中国农业出版社，1997。
86 赵德明主编，《兽医病理学》，中国农业大学出版社，1998。
87 沈志强，杨永福，佐剂新秀—蜂胶。中国畜禽传染病。1989，5：53-55
88 沈志强，鸡减蛋综合症蜂胶灭活疫苗的研究与应用，畜牧兽医学报，1997，28(6)，535-541
89 沈志强，蜂胶的免疫学作用，中国禽业导刊，2000，13，15-16
90 沈志强，李峰等，蜂胶对NDV的灭活作用效果观察，河南畜牧兽医，2002，1，1-3
91 沈志强，刘吉山等，值得警惕的强毒鸡新城疫的识别与防治对策探讨，中国禽业导刊，2000，9，14-18
92 沈志强，动物疫苗用蜂胶的质量要求及工业化提取技术的研究，中国兽药杂志，1997，4，13-15
93 沈志强，刘吉山等，新城疫病毒山东强毒株的分离鉴定，中国预防兽医学报，2002，2，
94 管宇，沈志强等，sdbz-s99分离株的生物学特性及其F基因的克隆与序列分析，中国预防兽医学报，2002，6，
95 赵蕾，沈志强等，复合新城疫蜂胶灭活疫苗抗体消长规律的研究，中国预防兽医学报，2002，增
96 王玲，蜂胶乙醇提取物增强机体免疫功能机理的研究，山东农业大学硕士研究生毕业论文，1998。
97 庞昕，鸡新城疫蜂胶灭活疫苗免疫作用机理的研究，山东农业大学硕士研究生毕业论文，2001。
98 陈洪涛，田惠芳，朱国强等，蜂胶免疫佐剂的研究，江苏农学院学报，1996，17(1)73-75
99 王守智，李景水，张国领等，用蜂胶佐剂提高抗鸡法氏囊病和新城疫二联苗血清抗体效价的试验，中国兽医科技，1997，27(8)，24
100 唐传核，孟岳成， 蜂胶的生理功能以及开发研究，食品工业科技，1999，20(2)，30-33
101 沈志强，蜂胶及其在农牧业中的应用进展，农牧产品开发，2000，3，18-20
102 庞万勇，沈志强，刘吉山等，禽霍乱与大肠杆菌病多价蜂胶二联疫苗与油乳剂疫苗注射部位动态病理组织学比较观察，山东家禽，2003，7，1-3