| 摘要: |
| 【目的】从临床发病的疑似感染鸡呼肠孤病毒的白羽肉鸡病料中分离鉴定获得1株鸡源呼肠孤病毒SD18，对SD18株进行致病性及传播特性研究，为该病毒的深入研究和综合防治奠定基础。【方法】无菌采集疑似感染鸡呼肠孤病毒发病鸡跗关节处的肌腱和渗出物，分离病原物，在LMH细胞上培养，并连续盲传3代，经无菌生理盐水倍比稀释，采用Reed-Muench法测定病毒鸡胚半数致死量（ELD50）和组织细胞半数感染量（TCID50）；对分离毒株进行理化特性和血凝特性测定；利用设计的鸡呼肠孤病毒L1和S1基因特异性引物，对提取的病毒总RNA进行RT PCR扩增，经PCR鉴定为阳性的菌落进行基因测序以及核苷酸同源性比对和系统遗传进化分析；用该分离株病毒对SPF鸡和白羽肉鸡进行动物回归试验，并用酶联免疫吸附试验（ELISA）测定攻毒后的SPF鸡血清中呼肠孤病毒抗体滴度。【结果】从疑似感染鸡呼肠孤病毒发病鸡跗关节处的肌腱和渗出物中分离获得1株鸡源呼肠孤病毒SD18，其ELD50为10-6.5/0.1 mL，TCID50为10-7.36/0.1 mL。RT-PCR结果显示，目的基因片段长度分别为1 100，750，265 bp，依次为鸡呼肠孤病毒的S1、σC和L1基因片段。核苷酸同源性比对结果显示，该分离株SD18与Ⅴ群鸡源呼肠孤病毒TW 918（台湾株）的S1基因序列同源性达92.7%，与Ⅰ群鸡病毒性关节炎疫苗S1133株的同源性仅为59.0%。核酸序列遗传进化分析表明，该分离株SD18与Ⅴ群鸡源呼肠孤病毒TW-918（台湾株）处于同一进化分支上，与Ⅰ群鸡源呼肠孤病毒S1133株处于不同的进化分支上，说明新分离的肉鸡呼肠孤病毒SD18株属于基因Ⅴ群，可能是Ⅰ群经典鸡源呼肠孤病毒S1133株的变异毒株。SPF鸡和白羽肉鸡回归试验表明，SD18株病毒能够引起鸡病毒性关节炎，与临床发病一致，并能水平传播。接种试验表明，病毒SD18能致死鸡胚，打开死亡鸡胚可见其绒毛尿囊膜增厚、尿酸盐沉积、胚体出血等症状，符合呼肠孤病毒的致病特点。【结论】该分离株SD18为新型肉鸡呼肠孤病毒，能够引起鸡发生病毒性关节炎。 |
| 关键词: 鸡呼肠孤病毒 病毒分离鉴定 病毒致病性 |
| DOI： |
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| 基金项目:青岛市动物保健品行业智库联合基金二期项目（18-6-3-1-jch） |
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| Isolation,identification and pathogenicity of broiler-chicken reovirus |
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ZHANG Yujie,LIU Dong,LIU Hongxiang,et al
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| Abstract: |
| 【Objective】A strain of broiler chicken reovirus was isolated and identification from suspected infectious materials,and its pathogenicity and spreading characteristics were studied.【Method】The joint tendons tissues and exudates of diseased chickens with suspected infection of chicken reovirus were collected and pathogens were separated before being cultured on LMH cell with continuous blind transmission for 3 generations.After aseptic saline ratio dilution,egg median lethal dose (ELD50) and tissue culture median infective dose (TCID50) were determined by the Reed-Muench method,and physicochemical characteristics and hemagglutination characteristics of isolated strains were measured.RT-PCR amplifications of L1 and S1 genes were conducted to extract total RNA using the designed specific primers of chicken revirus gene.The positive colonies of PCR identification were sequenced,nucleotide homology was compared and system genetic evolution was analyzed.The isolate was used for animal regression experiment of SPF chickens and broiler chickens,and the reovirus antibody titrations of SPF chicken serum were determined by ELISA.【Result】A chicken reovirus SD18 strain was isolated from the joint tendons tissues and exudates of diseased chickens with ELD50 of 10-6.5/0.1 mL and TCID50 of 10-7.36/0.1 mL.The fragments of S1 gene,σC gene and L1 gene of the isolated SD18 were 1 100,750 and 265 bp,respectively.The results of nucleotide homology showed that the homology of S1 gene sequence of SD18 with TW-918 (Taiwan strain) of Ⅴ group and S1133 vaccine of Ⅰ group chicken viral arthritis was 92.7% and 59.0%,respectively.The genetic evolution analysis of nucleotide sequences showed that SD18 was on the same evolutionary branch as the Ⅴ group chicken source isolate virus TW 918 (Taiwan strain) but different from the Ⅰ group chicken source isolate virus S1133,indicating that SD18 belonged to the genotype Ⅴ and was a variant strain of S1133.Animal regression experiment showed that SD18 could cause chicken viral arthritis in consistent with clinical morbidity and spread horizontally.The embryo inoculation test showed that the isolate could kill chicken embryo and cause thickness CAM,urate deposition and hemorrhage of chicken embryo,which was consistent with the pathogenic characteristics of reovirus.【Conclusion】The isolated SD18 strain was a new type of reovirus and could cause chicken viral arthritis. |
| Key words: chicken reovirus (REO) isolation and identification of virus pathogenicity of virus |
