鹅腺病毒的分离鉴定、E3区序列分析和人工致病性初步研究
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摘要
本研究以养殖场、临床病料、采集雏鹅或成年鹅的泄殖腔分泌物或肠道、肝脏、脾脏作为分离样品，通过离心和抗生素处理灭菌后，接种鸭胚，以血球凝集(HA)和血球凝集抑制(HI)的试验方法检测病毒。试验结果表明，50份样品中有2份样本分离到腺病毒，分离率为4％。将分离到的两株分离物病毒分别命名为H5J24和N8J8。HA和HI试验结果表明，该2株分离物病毒能够凝集鸡、鸭、鹅的红细胞，且这种凝集作用可被抗Y81G4株鹅腺病毒多抗血清所抑制，但抗新城疫(ND)病毒的单克隆抗体(单抗)、抗H5和H9亚型禽流感病毒(AIV)的单抗对其无抑制作用。透射电镜进行经形态学观察，结果发现大小约为78nm、球形、无囊膜的病毒粒子。理化特性测定结果表明，分离到的病毒血凝素蛋白对50％氯仿、0.25％胰酶、pH3.0和pH9.0处理1小时以及50℃ 30分钟表现稳定，而60℃ 45分钟和100℃ 1分钟可使其失活。根据Ⅲ群腺病毒EDS标准毒株基因组序列，设计并合成一对可扩增出E3区的PCR引物，实验证明可以扩增出特异性核酸片段。这些结果证明该两株分离物病毒为Ⅲ群禽腺病毒。
腺病毒E3区两端分别是保守性较强的pⅧ蛋白基因和纤维蛋白基因。通过特定的引物，用PCR方法从2株鹅腺病毒基因组中扩增出E3区并进行了序列测定。结果表明，这2株病毒分离物E3区的长度较短，全长约为240
扬州大学硕士论文
个核昔酸，不具有明显的编码框。与EDS一76以及其他腺病毒的E3区比较结
果提示，此2株鹅腺病毒应归类于m群禽腺病毒。
为了弄清楚这2株病毒的病原性，我们通过肌肉注射感然8日龄肉鹅，
进行人工致病性实验.结果发现，实验鹅在感染病毒后，未出现任何明显临
床症状，提示两株病毒的病原性较弱。对这些人工感染鹅进一步研究，通过
在不同时间段的排毒、带毒情况及血清中抗体水平检测，证明病毒能够在实
验鹅体内存在并持续排毒，同时实验鹅血清中循环抗体能够较长时间维持在
2”一2”的较高水平.组织病理学切片观察结果表明，病毒可以在内脏组织器
官引起一定的病理变化.如肾小管上皮细胞颗粒变性，有些细胞出现脂肪变
性，有些发生充血或轻微出血;肺三级支气管上皮细胞坏死脱落，管腔内有
脱落的上皮细胞及淋巴细胞，肺房有大量红细胞及少盘的淋巴细胞:脾脏有
轻度充血和出血;小肠部分猫膜层与肌层剥离，肠猫膜上皮细胞坏死、脱落，
小肠官腔内有坏死脱落的上皮细胞，有的肠绒毛脱落，固有层肠腺体上皮细
胞发生水泡性变性，固有层、猫膜下层有淋巴细胞浸润，肌层血管有充血;
肝细胞水泡变性、坏死.这些结果提示，我们分离到的两株分离物病毒对小
鹅没有临床致病作用，但仍有一定的毒力。
Cloacae secretion of healthy geese, livers, spleens and guts of clinical young or adult geese were collected for viruses isolation. These samples were sterilized by centrifuge and antibiotics and then inoculated into duck embryos. Two virus strains named H5J24 and N8J8 respectively were isolated from 50 samples and identified by hemagglutination (HA) and hemagglutination inhibition (HI) tests. The isolation rate is 4%. Both viruses could agglutinate 0.8% red blood cells (RBC) of chicken, duck or goose. The agglutination activity could be inhibited by the serum against virus Y81G4 isolated from goose, but not by the monoclonal antibodies to Newcastle disease virus, Avian influenza virus subtype H5 or H9. The virus particles are non-enveloped, 78nm diameter , and have icosahedrad symmetry visible in the transmission electron microscope(TEM) by negative stain. Based on the morphology, physical and chemical properties of the viruses and PCR analysis, the viruses were located at group III Adenoviruses.
Adenoviruse E3 region locates between the conservative genes pVIII and Fiber. We designed a pair of primers to amplify the fragement from two isolates by PCR. The results showed that E3 regions of the 2 virus strains was about
240bp in size. There was no distinct opening reading frame (ORF) in sequence analysis with DNAstar software. The 2 isolate strains from geese were further classified as Group III Avianadenovirus compared with the E3 region sequences of EDS-76 and other adenovirus.
The 2 isolate virus strains were experimentally inoculated into 10-day-old goslings in order to understand their pathogenicity. During different infection phases, the experimental geese did not exhibit any clinical syndrome. This result suggested that their pathogenicity were very weak for geese. Following detection made it clear that the 2 virus strains could long exist in geese and be expelled into environment for long time. Meanwhile the serum antibodies were maintained at a high level. The tilers of HI of the serum were about 211~212. Based on the tissue pathology, the 2 virus strains still could induce pathological changes in some organs, such as kidney, spleen, small intestine and lung.
腺病毒E3区两端分别是保守性较强的pⅧ蛋白基因和纤维蛋白基因。通过特定的引物，用PCR方法从2株鹅腺病毒基因组中扩增出E3区并进行了序列测定。结果表明，这2株病毒分离物E3区的长度较短，全长约为240
扬州大学硕士论文
个核昔酸，不具有明显的编码框。与EDS一76以及其他腺病毒的E3区比较结
果提示，此2株鹅腺病毒应归类于m群禽腺病毒。
为了弄清楚这2株病毒的病原性，我们通过肌肉注射感然8日龄肉鹅，
进行人工致病性实验.结果发现，实验鹅在感染病毒后，未出现任何明显临
床症状，提示两株病毒的病原性较弱。对这些人工感染鹅进一步研究，通过
在不同时间段的排毒、带毒情况及血清中抗体水平检测，证明病毒能够在实
验鹅体内存在并持续排毒，同时实验鹅血清中循环抗体能够较长时间维持在
2”一2”的较高水平.组织病理学切片观察结果表明，病毒可以在内脏组织器
官引起一定的病理变化.如肾小管上皮细胞颗粒变性，有些细胞出现脂肪变
性，有些发生充血或轻微出血;肺三级支气管上皮细胞坏死脱落，管腔内有
脱落的上皮细胞及淋巴细胞，肺房有大量红细胞及少盘的淋巴细胞:脾脏有
轻度充血和出血;小肠部分猫膜层与肌层剥离，肠猫膜上皮细胞坏死、脱落，
小肠官腔内有坏死脱落的上皮细胞，有的肠绒毛脱落，固有层肠腺体上皮细
胞发生水泡性变性，固有层、猫膜下层有淋巴细胞浸润，肌层血管有充血;
肝细胞水泡变性、坏死.这些结果提示，我们分离到的两株分离物病毒对小
鹅没有临床致病作用，但仍有一定的毒力。
Cloacae secretion of healthy geese, livers, spleens and guts of clinical young or adult geese were collected for viruses isolation. These samples were sterilized by centrifuge and antibiotics and then inoculated into duck embryos. Two virus strains named H5J24 and N8J8 respectively were isolated from 50 samples and identified by hemagglutination (HA) and hemagglutination inhibition (HI) tests. The isolation rate is 4%. Both viruses could agglutinate 0.8% red blood cells (RBC) of chicken, duck or goose. The agglutination activity could be inhibited by the serum against virus Y81G4 isolated from goose, but not by the monoclonal antibodies to Newcastle disease virus, Avian influenza virus subtype H5 or H9. The virus particles are non-enveloped, 78nm diameter , and have icosahedrad symmetry visible in the transmission electron microscope(TEM) by negative stain. Based on the morphology, physical and chemical properties of the viruses and PCR analysis, the viruses were located at group III Adenoviruses.
Adenoviruse E3 region locates between the conservative genes pVIII and Fiber. We designed a pair of primers to amplify the fragement from two isolates by PCR. The results showed that E3 regions of the 2 virus strains was about
240bp in size. There was no distinct opening reading frame (ORF) in sequence analysis with DNAstar software. The 2 isolate strains from geese were further classified as Group III Avianadenovirus compared with the E3 region sequences of EDS-76 and other adenovirus.
The 2 isolate virus strains were experimentally inoculated into 10-day-old goslings in order to understand their pathogenicity. During different infection phases, the experimental geese did not exhibit any clinical syndrome. This result suggested that their pathogenicity were very weak for geese. Following detection made it clear that the 2 virus strains could long exist in geese and be expelled into environment for long time. Meanwhile the serum antibodies were maintained at a high level. The tilers of HI of the serum were about 211~212. Based on the tissue pathology, the 2 virus strains still could induce pathological changes in some organs, such as kidney, spleen, small intestine and lung.
引文
[1] B.W.Calnet et al. Disease of Poultry[M]. Tenth Edition. 1997:607～642
[2] Philipson L. Adenovirus an eternal archetypes[M]. Curr. Top. Microbiol Immunol. 1995, 199 (1): 1～24
[3] 侯云德．分子病毒学[M]．北京：学苑出版社．1990：151～177
[4] 张维维．腺病毒载体及其在基因治疗中的应用[M]．肿瘤分子生物学研究进展．军事医学出版社．1996：30～41
[5] 李茂祥．减蛋综合征病毒基因文库构建及其基因组右侧DNA序列分析[M]．博士学位论文．1998．6
[6] Murphy F.A.. Virus taxonomy: Classification and nomenclature of virus, Sixth report of the International Committee on Taxonomy of virus. New York: Springer-vedag Vien[M],1995
[7] Monreal G. Poultry science.Rev.1992,4:1～27
[8] Domermutch,C.H.,C.R.Westotn,B.S.Cowen, W.M.Colwell,W.B.Gross,and R.T.DuBose.1980 Incidence and distribution of avian Adenovirus group Ⅱ splenomegaly of chickens. Avian Dis 24:591～594
[9] McFerran.J.B.,T.J.Connor, and B.M.Adair.1978 Studies on the antigenic relationship between and isolate(127) from the egg drop syndrome 1976 and a fowl adenovirus. Avian Pathol 7:629～636
[10] 殷震，刘景华主编．动物病毒学(第二版)．科学出版社．1997：1104～1129
[11] Greber U.F.,Willitts M.,Webster P. et al. Stepwise dismantling of adenovirus 2 during entry into cells. Cell. 1993,75:477～486
[12] Bernards R.,and Van de Eb A.J.V. Adenovirns: transformation and oncogenicity. Biochen. Biophys. Acta. 1984,783:187～204
[13] Wold W.S.M. and Gooding L.R, Region E3 of adcnovirus: A cassette of genes involved in host immunosurveillance and virus-cell interactions. Virology 1991,184:1～8
[14] Paabo S., Nilsson T. and Peterson P.A. Adcnoviruscs of subgcncra B,C,D, and E, modulate cell-surface expression of major histocompatibility complex class Ⅰ antigens. Pro.Nalt.Acad.Sci.USA.,1986,83:9665～9669
[15] Gooding L.R.,Elmore L.W.,Tollefson et al.A 14 700MW protein from E3 region of adenovirus inhibits cytolysis by tumor necrosis factor. Cell 1988,53:341～349
[16] Gooding L.R.,Sofola I.O.,Tollefson A.E. et al. The adenovirus E3-14.7K protein is a general inhibitor of tumor necrosis factor mediated cytolysis. J.Immunol. 1990,145:3080～3086
[17] Krajcsi P., Tollefson A.E. et al. The adenovirus E3 14.5K protein ,which is required for down-regulation of the epidermal growth factor receptor and prevemion of tumor necrosis factor cytolysis, is an integral membrance protein oriented with its C-terminals in the cytoplasm. J.Virol. 1992,66:1665～1675
[18] 甘孟侯主编．中国禽病学．中国农业出版社．1999，94～104
[19] ZsakL,Kisary L. Characterization of adenoviruses isolated from geese. Avian Pathology. 1984,13(2):253～264
[20] Bergmarm.V. Pathology and electron microscapical detection of virus in the tissue of goslings with Derzsy's disease (parvovirus infectious). Archive fur Experiment Elle Veterinarmedizin. 1987,42(2):212～221
[21] 郑玉美，王永坤，徐瑗等．鹅腺病毒的初步研究．中国家禽，1992，(6)：23～24
[22] 程安春．一种新发现的雏鹅传染病研究．中国兽医科技．1998，28(7)：3～6
[23] 程安春，汪铭书，陈孝跃，等．四川省家禽及鸟类减蛋综合征血清流行病学调查．中国兽医科技，1997，27(4)：11～13
[24] Engelhardt J.F.,et al. Proc Natl Acad Sci USA. 1994,91(13):6196-6200
[25] Gorziglia M.I.,et al. J Virol.1996,70(6):4173-4178
[26] Brough D.E.,et al. J Virol. 1996,70(9):6497-6501
[27] Yeh P.,et al. J Virol. 1996,70(1):559-565
[28] Chiocca S.,et al. J Virol. 1996,70:2939-2949
[29] Sheppard M.,et al. J.Gen Virol. 1995,76:2595-2600
[30] Jucker M.T.,et al. J.Gen Virol.1996,77:469-479
[31] Michou A.I.,et al. J Virol. 1999 Feb;73(2): 1399-1410
[32] Francois A.,et al. J Virol.2001 Jun;75(11):5288-5301
[33] Sheppard M.,et al. Arch Virol.1998,143(5):915-930
[34] 郑玉美，王永坤等．中国家禽．1992，(6)：23-24
[35] 朱国强，王永坤等．中国畜牧兽医学会禽病学分会第四次代表大会暨第七次学术讨论文集．郑州，1994
[36] 段玉友，崔治中等．1995年全国禽病分子生物学研讨会论文集．扬州，1995．141-148
[37] 王泽霖．中国兽医学报．1994(4)：360-364
[38] 徐福洲，崔治中等．中国病毒学．2000，15(4)：388-394
[39] 刘岳龙，崔治中等．中国预防兽医学报．2000，22(4)：285-288
[40] 胡敬东等．中国兽医学报．1996，16(12)：421-423
[41] 向华等．中国兽医学报．1999，19(3)：226-229
[42] Morikazu Shinagawa, Yoichilida et al. Phylogenetic relationships between adenovirus as inferred from nucleotide sequences of inverted terminal repeats.
Gene. 1987, (55):55～93
[43] J.Pitcovski, M.Mualem, Z.Rei-Koren et al. The complete DNA sequence and genome organization of the avian adenovirus, Hemorrhagic Enteritis Virus. Virology. 1998,(249):307～315
[44] Michael. Hess, Helmut Blocker, Petra Brandt. The complete nucleotide sequence of the EDSV An intermediate between Mastadenoviruses and Avianadenoviruses. Virology. 1997,(238)145～156
[45] Susanna Chiocea, Robert Kurzbauer, et al. The complete DNA sequence and genomic organization of the avian adenovirus CELO. J. Virol. 1996,(70):2939～2949
[2] Philipson L. Adenovirus an eternal archetypes[M]. Curr. Top. Microbiol Immunol. 1995, 199 (1): 1～24
[3] 侯云德．分子病毒学[M]．北京：学苑出版社．1990：151～177
[4] 张维维．腺病毒载体及其在基因治疗中的应用[M]．肿瘤分子生物学研究进展．军事医学出版社．1996：30～41
[5] 李茂祥．减蛋综合征病毒基因文库构建及其基因组右侧DNA序列分析[M]．博士学位论文．1998．6
[6] Murphy F.A.. Virus taxonomy: Classification and nomenclature of virus, Sixth report of the International Committee on Taxonomy of virus. New York: Springer-vedag Vien[M],1995
[7] Monreal G. Poultry science.Rev.1992,4:1～27
[8] Domermutch,C.H.,C.R.Westotn,B.S.Cowen, W.M.Colwell,W.B.Gross,and R.T.DuBose.1980 Incidence and distribution of avian Adenovirus group Ⅱ splenomegaly of chickens. Avian Dis 24:591～594
[9] McFerran.J.B.,T.J.Connor, and B.M.Adair.1978 Studies on the antigenic relationship between and isolate(127) from the egg drop syndrome 1976 and a fowl adenovirus. Avian Pathol 7:629～636
[10] 殷震，刘景华主编．动物病毒学(第二版)．科学出版社．1997：1104～1129
[11] Greber U.F.,Willitts M.,Webster P. et al. Stepwise dismantling of adenovirus 2 during entry into cells. Cell. 1993,75:477～486
[12] Bernards R.,and Van de Eb A.J.V. Adenovirns: transformation and oncogenicity. Biochen. Biophys. Acta. 1984,783:187～204
[13] Wold W.S.M. and Gooding L.R, Region E3 of adcnovirus: A cassette of genes involved in host immunosurveillance and virus-cell interactions. Virology 1991,184:1～8
[14] Paabo S., Nilsson T. and Peterson P.A. Adcnoviruscs of subgcncra B,C,D, and E, modulate cell-surface expression of major histocompatibility complex class Ⅰ antigens. Pro.Nalt.Acad.Sci.USA.,1986,83:9665～9669
[15] Gooding L.R.,Elmore L.W.,Tollefson et al.A 14 700MW protein from E3 region of adenovirus inhibits cytolysis by tumor necrosis factor. Cell 1988,53:341～349
[16] Gooding L.R.,Sofola I.O.,Tollefson A.E. et al. The adenovirus E3-14.7K protein is a general inhibitor of tumor necrosis factor mediated cytolysis. J.Immunol. 1990,145:3080～3086
[17] Krajcsi P., Tollefson A.E. et al. The adenovirus E3 14.5K protein ,which is required for down-regulation of the epidermal growth factor receptor and prevemion of tumor necrosis factor cytolysis, is an integral membrance protein oriented with its C-terminals in the cytoplasm. J.Virol. 1992,66:1665～1675
[18] 甘孟侯主编．中国禽病学．中国农业出版社．1999，94～104
[19] ZsakL,Kisary L. Characterization of adenoviruses isolated from geese. Avian Pathology. 1984,13(2):253～264
[20] Bergmarm.V. Pathology and electron microscapical detection of virus in the tissue of goslings with Derzsy's disease (parvovirus infectious). Archive fur Experiment Elle Veterinarmedizin. 1987,42(2):212～221
[21] 郑玉美，王永坤，徐瑗等．鹅腺病毒的初步研究．中国家禽，1992，(6)：23～24
[22] 程安春．一种新发现的雏鹅传染病研究．中国兽医科技．1998，28(7)：3～6
[23] 程安春，汪铭书，陈孝跃，等．四川省家禽及鸟类减蛋综合征血清流行病学调查．中国兽医科技，1997，27(4)：11～13
[24] Engelhardt J.F.,et al. Proc Natl Acad Sci USA. 1994,91(13):6196-6200
[25] Gorziglia M.I.,et al. J Virol.1996,70(6):4173-4178
[26] Brough D.E.,et al. J Virol. 1996,70(9):6497-6501
[27] Yeh P.,et al. J Virol. 1996,70(1):559-565
[28] Chiocca S.,et al. J Virol. 1996,70:2939-2949
[29] Sheppard M.,et al. J.Gen Virol. 1995,76:2595-2600
[30] Jucker M.T.,et al. J.Gen Virol.1996,77:469-479
[31] Michou A.I.,et al. J Virol. 1999 Feb;73(2): 1399-1410
[32] Francois A.,et al. J Virol.2001 Jun;75(11):5288-5301
[33] Sheppard M.,et al. Arch Virol.1998,143(5):915-930
[34] 郑玉美，王永坤等．中国家禽．1992，(6)：23-24
[35] 朱国强，王永坤等．中国畜牧兽医学会禽病学分会第四次代表大会暨第七次学术讨论文集．郑州，1994
[36] 段玉友，崔治中等．1995年全国禽病分子生物学研讨会论文集．扬州，1995．141-148
[37] 王泽霖．中国兽医学报．1994(4)：360-364
[38] 徐福洲，崔治中等．中国病毒学．2000，15(4)：388-394
[39] 刘岳龙，崔治中等．中国预防兽医学报．2000，22(4)：285-288
[40] 胡敬东等．中国兽医学报．1996，16(12)：421-423
[41] 向华等．中国兽医学报．1999，19(3)：226-229
[42] Morikazu Shinagawa, Yoichilida et al. Phylogenetic relationships between adenovirus as inferred from nucleotide sequences of inverted terminal repeats.
Gene. 1987, (55):55～93
[43] J.Pitcovski, M.Mualem, Z.Rei-Koren et al. The complete DNA sequence and genome organization of the avian adenovirus, Hemorrhagic Enteritis Virus. Virology. 1998,(249):307～315
[44] Michael. Hess, Helmut Blocker, Petra Brandt. The complete nucleotide sequence of the EDSV An intermediate between Mastadenoviruses and Avianadenoviruses. Virology. 1997,(238)145～156
[45] Susanna Chiocea, Robert Kurzbauer, et al. The complete DNA sequence and genomic organization of the avian adenovirus CELO. J. Virol. 1996,(70):2939～2949