鹅出血性坏死性肝炎病毒的生物学特性研究
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摘要
2001年江苏省某养殖场鹅群中爆发了一种新的传染病，该病只引起幼龄鹅发病，死亡率在70％左右，死亡鹅以肝脏出血坏死为主要的病理变化，故我们将其命名为鹅出血性坏死性肝炎。
将发病鹅肝脏无菌研磨液通过肌肉注射接种动物，复制到了与自然发病基本一致的临床症状和病理变化；并制备了发病鹅肝脏、脾脏、肾脏的病理切片，对其微观病变进行了观察。用离子捕捉法对肝脏无菌研磨液进行处理，经过负染和电镜观察，发现病毒粒子呈球型，双层衣壳，外层衣壳直径约75nm，内层约50nm，无囊膜；该病毒粒子对乙醚、氯仿、胰蛋白酶有抵抗作用，耐酸、耐热；病毒不能通过尿囊腔途径生长，通过尿囊膜接种适日龄鸡胚、鸭胚、鹅胚，一周内有80％左右的死亡率；病毒在鸡胚成纤维细胞上盲传5代后，能够诱发鸡胚成纤维细胞出现稳定的病理变化，病理变化主要是细胞融合和合胞体的出现。对病毒将要致死鸡胚器官取材；按时间梯度，对接种病毒的鸡胚成纤维细胞连续取材，分别制成超薄切片在电镜下观察，发现病毒粒子在胞浆中复制，不成熟病毒粒子有时形成包涵体，对病毒所造成的组织损伤也进行了描述。
依照国际病毒委员会病毒分类原则，参考该病毒的性质，初步鉴定该病毒为呼肠孤病毒科正呼肠孤病毒属禽呼肠孤病毒。
A new disease was out breaking in the geese no older than one month in JiangSu province with 70% mortality. We named the disease goose hemorrhagic necrotic hepatitis according to its mainly pathologic changes.
Replicate the disease by inoculating goose the suspension of liver; the desired disease appeared 7 days later, which was consistent with the nature induced one. The liver, spleen and kidney of goose were made out pathologic slices. The virus in the liver suspension dealing with the technology of ion exchange capture showed spherical, no envelope, having double-deck capsid, which range from 75nm-50nm in diameter by TEM. The virus resist to aether, chloroform,typton,heat and acid; the virus give the chicken embryo, duck embryo, goose embryo a 80% mortality inoculating by the way of allantochorion and can't adapt to allantoic cavity. The virus, after continuously propagating in chicken embryo fibroblast for 5 generations, will induce the CEF stable pathogenesis such as fusion cell. The ultrathin sections which were made out from the infected CEF at progressive time and from the chicken embryo infecting 72h later were observed by TEM, the results suggested that the virus exist by single or endobody and replicate in cytoplasm.
According to the ICTV principles, all the information above indicate this virus reoviridae ,orthoreovirus,avian reovirus.
将发病鹅肝脏无菌研磨液通过肌肉注射接种动物，复制到了与自然发病基本一致的临床症状和病理变化；并制备了发病鹅肝脏、脾脏、肾脏的病理切片，对其微观病变进行了观察。用离子捕捉法对肝脏无菌研磨液进行处理，经过负染和电镜观察，发现病毒粒子呈球型，双层衣壳，外层衣壳直径约75nm，内层约50nm，无囊膜；该病毒粒子对乙醚、氯仿、胰蛋白酶有抵抗作用，耐酸、耐热；病毒不能通过尿囊腔途径生长，通过尿囊膜接种适日龄鸡胚、鸭胚、鹅胚，一周内有80％左右的死亡率；病毒在鸡胚成纤维细胞上盲传5代后，能够诱发鸡胚成纤维细胞出现稳定的病理变化，病理变化主要是细胞融合和合胞体的出现。对病毒将要致死鸡胚器官取材；按时间梯度，对接种病毒的鸡胚成纤维细胞连续取材，分别制成超薄切片在电镜下观察，发现病毒粒子在胞浆中复制，不成熟病毒粒子有时形成包涵体，对病毒所造成的组织损伤也进行了描述。
依照国际病毒委员会病毒分类原则，参考该病毒的性质，初步鉴定该病毒为呼肠孤病毒科正呼肠孤病毒属禽呼肠孤病毒。
A new disease was out breaking in the geese no older than one month in JiangSu province with 70% mortality. We named the disease goose hemorrhagic necrotic hepatitis according to its mainly pathologic changes.
Replicate the disease by inoculating goose the suspension of liver; the desired disease appeared 7 days later, which was consistent with the nature induced one. The liver, spleen and kidney of goose were made out pathologic slices. The virus in the liver suspension dealing with the technology of ion exchange capture showed spherical, no envelope, having double-deck capsid, which range from 75nm-50nm in diameter by TEM. The virus resist to aether, chloroform,typton,heat and acid; the virus give the chicken embryo, duck embryo, goose embryo a 80% mortality inoculating by the way of allantochorion and can't adapt to allantoic cavity. The virus, after continuously propagating in chicken embryo fibroblast for 5 generations, will induce the CEF stable pathogenesis such as fusion cell. The ultrathin sections which were made out from the infected CEF at progressive time and from the chicken embryo infecting 72h later were observed by TEM, the results suggested that the virus exist by single or endobody and replicate in cytoplasm.
According to the ICTV principles, all the information above indicate this virus reoviridae ,orthoreovirus,avian reovirus.
引文
[1] 殷震，刘景华主编．动物病毒学[M]．北京：科学出版社，1997，538～548．
[2] 王永坤等．雏鹅出血性坏死性肝炎．[J]．中国家禽，2002，19
[3] Ursula Heffels redmann, Mullerh, Kaletaef. Structural and biological characteristics of reoviruses isolated from Muscovy ducks[J]. Avian Pathology, 1992, 21: 481～491.
[4] 陆承平．禽呼肠孤病毒[J]．中国畜禽传染病，1992，67：60～61．
[5] 崔洪超．鸡病毒性关节炎[J]．吉林畜牧兽医，1995，17：34～35．
[6] Shapourimrs, Frenetted, Larocheller, et al. Characterization of monoclonalantibodies against avian reoviru strain S1133. [J]Avian Pathology, 1996, 25: 57～67.
[7] Wockramasingher, Enriqueee, Meangerj, et al. TypeSpecific antigenieity of avian reoviruses[J]. Avian Pathology, 1995, 24: 121～134.
[8] Vakhariaunan, Nadatam. Comparative Analysis of Virus induced Polypetide of an Avirulent and avirulent strain of avian reovirus [J]. Avian Disease, 1994, 38: 244～250.
[9] Shapourimrs, Kanem, Letartems, etal. Colning, Sequeneing and expression of the Slgene of avian reovirus. Journal of General Virology [J]. 1995, 1515～1520.
[10] Shapourims, Arellam, Silima. Immunogenieity of E. coli expressed protein of avian reovirus in chickens [J]. Avian Pathology, 1997, 26: 419～425.
[11] Clarkfd, Niy, Collissoneww, Clarkfd, et al. Characterization of avian reovirus strainspecific polymorphisms [J]. Avian Disease, 1990, 34: 304～314.
[12] Gouveans, Sehnitertj, Gouveans. Polymorphism of the genomicRNAS
among the avian reoviruses [J]. Journal of General Virology,1982,61:87～91.
[13] Niy, Kempme.Strain specific selection of genome segments in avian reoviru coin feetions [J].Journal of General virology, 1992, 73, 3107～3113.
[14] Niy, Kempme.A Comparative study of avian reovirus Pathogenicity: Virus spread and replication and induction of lesions [J].Avian Disease, 1995,39:554～566.
[15] Niy, Ramingrf, Kempmc.Identification of proteins encode by avian reoviruses and evidence for posttrouslational modification [J].Virology, 1993,193:466～469.
[16] Shapourimrs,Arellam,Silima.Evidence for the multimerie nature and cell binding ability of avian reovirus σ 3 protein[J].Journal of General Virology, 1996,77:1203～1210.
[17] Duneanrd.The low pH-Dependent entry of avian Reovirusis accompanied by two specific cleavages of the major outer capsid protein μ 2e[J].Virology,1996,219:179～189.
[18] XIE Zhixun, et al.Amplifieaetion of avian reovirus RNA using the reversetran seriptase polymerase chain reaetion[J].Avian Disease, 1997,41:654～660.
[19] Liu Hungjen,Giambronejj.eharaeterization of a nonradio active cloned cDNA probe for detecting avian reoviruses[J]. Avian Diseasee, 1997,41:374～378.
[20] Liu Hungjen, Giambronejj. In situ detection of reovirus informalinfixted, paraffin embedded chicken tissues using a digoxigenin Labeled cDNA probe[J]. AvianDisease, 1997,41:447～451.
[21] Yinsh,Leelh.Development and characterization of a nucleicacid
probe fo ravian reoviruses[J].Avian Pathology,1998,27:423～426.
[22] Gustavo Bodelon et al. [J].The Avian reovirus Genome Segment S1 is a Functionally Tricistronic Gene That Expresses One Structural and Two Nonstructural Proteins in Infencted cells.Virology 290,181-191(2001).
[23] Wan Chain Pal, Jui Huang Shien. [J].Characterization of monoclonal antibodies against Avian Reovirus S1133 Synthesized in E.coli.Veterinary Microbiology.www.elsevier.com.
[24] Hsien Sheng Yin,et al. [J].Characterization of Avian Reorirus non structural protein σ NS Synthesized in E.coli.Virus research 67(2000) 1-9.www. elsevier.com.
[25] Hsien Sheng Yin,et al. [J].Characterization of the double-stranded RNA Genome Segment S3 of Avian Reovirus. Journal of Virological methods,67(1997)93-101.
[26] Hung Jen Liu,et al. [J]. Amplication, cloning and sequencing of the σc-encoded Gene of Avian Reovirus. Journal of Virological Methods63 (1997) 203-208.
[27] Hung Jen Liu,et al. [J].Development of an ELISA for Detection of Antibodies to Avian Reovirus in Chickens. Journal of Virological Methods 102 (2002) 129-138.
[28] Hung Jen Liu,et al. [J].The use of Monoclonal antibodies probes for the Detection of Avian Reovirus antigens. Journal of Virological Methods, 86 (2000) 115-119.
[29] Hung Jen Liu,et al. [J]. Sequence and Phylogenctic analysis of the σA-encoding Gene of Avian Reovirus. Journal of Virological Methods 98(2001)99-107.
[30] Hung Jen Liu,et al. [J]. Avian Reovirus σc Protein Contains a
Putative Fusion Sequence and induces Fusion when Expressed in Mammalian cells. Virology. 208,678-684(1995).
[31] Hung Jen Liu, et al. [J]. Comparison of Two Molecular Techniques for the Detection of Avian Reovirus in Formalin-fixed, paraffin -embedded chicken tissues. Journal of Virological Methods, 80(1999) 197-201.
[32] 国外兽医学—畜禽传染病，1 986(14—16)
[33] 栗寿初，[J]．呼肠孤病毒的防制是个复杂的问题。国外兽医学，44-46(1991)。
[34] 陈士友，[J]．一株分离自外观正常鸡胚的禽呼肠孤病毒的鉴定。(1990)畜牧兽医学报，243—246。
[35] M．D．Robertson．[J]．禽呼肠孤病毒。国外兽医学—畜禽传染病第7卷第二期1987年5月。
[36] 吴宝成等，[J]．禽呼肠孤病毒B3分离株感染番鸭的病理组织学研究。福建农业大学学报 30(4)：514—517，2001。
[37] 吴宝成等，[J]．番鸭呼肠孤病毒的分离与鉴定。福建农业大学学报 227—230，2001。
[38] 李关汉等，[J]．HIV—1 SH01分离株在MT4细胞的超微结构特征和形态发生学研究。病毒学报，305—312(1999)。
[39] 邹桂平，方勤。[J]．草鱼呼肠孤病毒在CIK细胞中复制及形态发生的研究。中国病毒学，15卷2期，2000，6。
[40] 王镇等，[J]．猪瘟病毒的形态结构与形态发生。微生物学报237—241(2000)。
[41] 李成等，(M)畜禽病毒学图谱。
[2] 王永坤等．雏鹅出血性坏死性肝炎．[J]．中国家禽，2002，19
[3] Ursula Heffels redmann, Mullerh, Kaletaef. Structural and biological characteristics of reoviruses isolated from Muscovy ducks[J]. Avian Pathology, 1992, 21: 481～491.
[4] 陆承平．禽呼肠孤病毒[J]．中国畜禽传染病，1992，67：60～61．
[5] 崔洪超．鸡病毒性关节炎[J]．吉林畜牧兽医，1995，17：34～35．
[6] Shapourimrs, Frenetted, Larocheller, et al. Characterization of monoclonalantibodies against avian reoviru strain S1133. [J]Avian Pathology, 1996, 25: 57～67.
[7] Wockramasingher, Enriqueee, Meangerj, et al. TypeSpecific antigenieity of avian reoviruses[J]. Avian Pathology, 1995, 24: 121～134.
[8] Vakhariaunan, Nadatam. Comparative Analysis of Virus induced Polypetide of an Avirulent and avirulent strain of avian reovirus [J]. Avian Disease, 1994, 38: 244～250.
[9] Shapourimrs, Kanem, Letartems, etal. Colning, Sequeneing and expression of the Slgene of avian reovirus. Journal of General Virology [J]. 1995, 1515～1520.
[10] Shapourims, Arellam, Silima. Immunogenieity of E. coli expressed protein of avian reovirus in chickens [J]. Avian Pathology, 1997, 26: 419～425.
[11] Clarkfd, Niy, Collissoneww, Clarkfd, et al. Characterization of avian reovirus strainspecific polymorphisms [J]. Avian Disease, 1990, 34: 304～314.
[12] Gouveans, Sehnitertj, Gouveans. Polymorphism of the genomicRNAS
among the avian reoviruses [J]. Journal of General Virology,1982,61:87～91.
[13] Niy, Kempme.Strain specific selection of genome segments in avian reoviru coin feetions [J].Journal of General virology, 1992, 73, 3107～3113.
[14] Niy, Kempme.A Comparative study of avian reovirus Pathogenicity: Virus spread and replication and induction of lesions [J].Avian Disease, 1995,39:554～566.
[15] Niy, Ramingrf, Kempmc.Identification of proteins encode by avian reoviruses and evidence for posttrouslational modification [J].Virology, 1993,193:466～469.
[16] Shapourimrs,Arellam,Silima.Evidence for the multimerie nature and cell binding ability of avian reovirus σ 3 protein[J].Journal of General Virology, 1996,77:1203～1210.
[17] Duneanrd.The low pH-Dependent entry of avian Reovirusis accompanied by two specific cleavages of the major outer capsid protein μ 2e[J].Virology,1996,219:179～189.
[18] XIE Zhixun, et al.Amplifieaetion of avian reovirus RNA using the reversetran seriptase polymerase chain reaetion[J].Avian Disease, 1997,41:654～660.
[19] Liu Hungjen,Giambronejj.eharaeterization of a nonradio active cloned cDNA probe for detecting avian reoviruses[J]. Avian Diseasee, 1997,41:374～378.
[20] Liu Hungjen, Giambronejj. In situ detection of reovirus informalinfixted, paraffin embedded chicken tissues using a digoxigenin Labeled cDNA probe[J]. AvianDisease, 1997,41:447～451.
[21] Yinsh,Leelh.Development and characterization of a nucleicacid
probe fo ravian reoviruses[J].Avian Pathology,1998,27:423～426.
[22] Gustavo Bodelon et al. [J].The Avian reovirus Genome Segment S1 is a Functionally Tricistronic Gene That Expresses One Structural and Two Nonstructural Proteins in Infencted cells.Virology 290,181-191(2001).
[23] Wan Chain Pal, Jui Huang Shien. [J].Characterization of monoclonal antibodies against Avian Reovirus S1133 Synthesized in E.coli.Veterinary Microbiology.www.elsevier.com.
[24] Hsien Sheng Yin,et al. [J].Characterization of Avian Reorirus non structural protein σ NS Synthesized in E.coli.Virus research 67(2000) 1-9.www. elsevier.com.
[25] Hsien Sheng Yin,et al. [J].Characterization of the double-stranded RNA Genome Segment S3 of Avian Reovirus. Journal of Virological methods,67(1997)93-101.
[26] Hung Jen Liu,et al. [J]. Amplication, cloning and sequencing of the σc-encoded Gene of Avian Reovirus. Journal of Virological Methods63 (1997) 203-208.
[27] Hung Jen Liu,et al. [J].Development of an ELISA for Detection of Antibodies to Avian Reovirus in Chickens. Journal of Virological Methods 102 (2002) 129-138.
[28] Hung Jen Liu,et al. [J].The use of Monoclonal antibodies probes for the Detection of Avian Reovirus antigens. Journal of Virological Methods, 86 (2000) 115-119.
[29] Hung Jen Liu,et al. [J]. Sequence and Phylogenctic analysis of the σA-encoding Gene of Avian Reovirus. Journal of Virological Methods 98(2001)99-107.
[30] Hung Jen Liu,et al. [J]. Avian Reovirus σc Protein Contains a
Putative Fusion Sequence and induces Fusion when Expressed in Mammalian cells. Virology. 208,678-684(1995).
[31] Hung Jen Liu, et al. [J]. Comparison of Two Molecular Techniques for the Detection of Avian Reovirus in Formalin-fixed, paraffin -embedded chicken tissues. Journal of Virological Methods, 80(1999) 197-201.
[32] 国外兽医学—畜禽传染病，1 986(14—16)
[33] 栗寿初，[J]．呼肠孤病毒的防制是个复杂的问题。国外兽医学，44-46(1991)。
[34] 陈士友，[J]．一株分离自外观正常鸡胚的禽呼肠孤病毒的鉴定。(1990)畜牧兽医学报，243—246。
[35] M．D．Robertson．[J]．禽呼肠孤病毒。国外兽医学—畜禽传染病第7卷第二期1987年5月。
[36] 吴宝成等，[J]．禽呼肠孤病毒B3分离株感染番鸭的病理组织学研究。福建农业大学学报 30(4)：514—517，2001。
[37] 吴宝成等，[J]．番鸭呼肠孤病毒的分离与鉴定。福建农业大学学报 227—230，2001。
[38] 李关汉等，[J]．HIV—1 SH01分离株在MT4细胞的超微结构特征和形态发生学研究。病毒学报，305—312(1999)。
[39] 邹桂平，方勤。[J]．草鱼呼肠孤病毒在CIK细胞中复制及形态发生的研究。中国病毒学，15卷2期，2000，6。
[40] 王镇等，[J]．猪瘟病毒的形态结构与形态发生。微生物学报237—241(2000)。
[41] 李成等，(M)畜禽病毒学图谱。