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魏燕鸣,龚文孝,杨影,马季,邹忠,金梅林.鸡源禽腺病毒的LMH细胞传代及细胞传代毒株对SPF鸡的致病性[J].华中农业大学学报,2020,39(2):95-101
鸡源禽腺病毒的LMH细胞传代及细胞传代毒株对SPF鸡的致病性
Passage of chicken avian adenoviruses in LMH cells and pathogenicity of cell culture strain to SPF chicken
  
DOI:
中文关键词:  禽腺病毒  鸡肝癌细胞  包涵体  心包积水  病毒传代  细胞感染模型  灭活疫苗  弱毒活疫苗
英文关键词:avian adenovirus  leghorn male hepatocellular cells(LMH)  inclusion body  pericardial hydrops  virus passage  cell infection model  inactivated vaccine  attenuated living avian
基金项目:国家重点研发计划项目(2016YFD0501600)
作者单位E-mail
魏燕鸣 华中农业大学动物医学院武汉 430070 370551650@qq.com 
龚文孝 华中农业大学动物医学院武汉 430070  
杨影 武汉科前生物股份有限公司武汉 430070  
马季 华中农业大学动物医学院武汉 430070  
邹忠 华中农业大学动物医学院武汉 430070  
金梅林 华中农业大学动物医学院武汉 430070 jml8328@126.com 
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中文摘要:
      为建立禽腺病毒4型的禽源细胞感染模型,并考察细胞培养毒株对动物的致病性,将实验室2014-2017年间分离的5株血清4型禽腺病毒接种鸡肝癌细胞(leghorn male hepatocellular cells,LMH)并盲传,选取各病毒的LMH细胞培养物,按一定剂量经腿部肌肉注射接种4周龄SPF鸡;记录试验动物的发病情况,剖检采集发病或死亡鸡的脏器并制作病理切片,观察并分析病毒细胞培养物感染SPF鸡的组织病理变化。结果发现:5株血清4型禽腺病毒在LMH细胞上盲传至第4代时开始出现I群禽腺病毒所致的特异性细胞病变,提取培养物中病毒核酸并进行PCR鉴定,均能扩增出大小约564 bp的特异性扩增片段,表明病毒能在LMH细胞上稳定增殖。选取各病毒第10代细胞培养物进行SPF鸡回归试验,发现细胞培养上清接种4周龄SPF鸡后第3天开始,有部分鸡开始精神沉郁,食欲消退、卧地不起瘫痪并迅速死亡,病理剖检及病理组织切片结果显示HB 1、HB 2、HB 3和SD 1毒株保持对4周龄SPF鸡的致病性,病死鸡中能观察到典型的组织病理学变化,而对照组不出现任何临床症状及病理变化。以上结果表明,LMH细胞可用于禽腺病毒增殖传代,是开展禽腺病毒分子致病机制研究的理想细胞模型。
英文摘要:
      In order to establish an avian original cells infection model of the avian adenovirus type 4,and investigate the pathogenicity of the cell culture viruses to chicken,five serotype 4 avian adenovirus isolated in our laboratory from 2014 to 2017 were inoculated into the LMH cells and transmitted blindly,and supernatants of the LMH cell cultures of each virus were selected and injected into the 4 week old SPF chickens through the leg muscle at a certain dose. The incidence of experimental animals was recorded,the organs of the diseased or dead chickens were collected and pathological sections were made. The histopathological changes of SPF chickens infected by virus were observed and analyzed. The results showed that specific cytopathic effect caused by the group I avian adenovirus began to appear when 5 strains of serotype 4 avian adenovirus were blindly transmitted into the LMH cells. The viral nucleic acid was extracted from the culture and identified by PCR,and a specific amplification fragment of about 564 bp was amplified,indicating that the virus can proliferate stably in the LMH cells. Supernatants of the 10th generation cell culture of each virus were selected for SPF chicken regression test. The results showed that from the 3rd day after the 4 week old SPF chickens were infected,some chickens began to depressed,lost their appetite,became paralyzed and died quickly. Observations of the pathological examination and pathological sections showed that the LMH passaged viruses of HB 1,HB 2,HB 3 and SD 1 maintained the pathogenicity to the 4 week old SPF chickens. Typical histopathological changes were observed in the sick and dead chickens,while there were no clinical symptoms and pathological changes in the control group. These results suggest that the LMH cells can be used for the proliferation and passage of avian adenovirus,and it is an ideal cell model for study of the molecular pathogenesis of avian adenovirus. 
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