H7N9禽流感病毒在鸡多个器官分布的研究

Study on distribution of influenza H7N9 in organs of chickens

目的建立免疫荧光及激光共聚焦检测H7N9禽流感病毒的方法,了解H7N9禽流感病毒在鸡多个器官的分布情况。方法从活禽市场现场宰杀的20只活鸡中筛选H7N9禽流感病毒阳性者,取其气管、肺、肝、肠制作石蜡切片,用抗流感病毒核蛋白单抗孵育结合后与DAPI(4',6-二脒基-2-苯基吲哚)及带有FITC(异硫氰酸荧光素)的二抗孵育结合,阳性样本有蓝色和绿色荧光,阴性样本仅发出蓝色荧光,用荧光显微镜及激光扫描共聚焦显微镜分别进行观察。结果在受检的20只活鸡中,共发现9只活鸡感染H7N9禽流感病毒,感染率为45%。其中5只在气管切片观察到病毒颗粒荧光,2只在肺组织切片观察到病毒颗粒荧光,6只在肝组织切片观察到病毒颗粒荧光,5只在肠组织切片观察到病毒颗粒荧光;其中1只鸡在四种器官中均阳性,1只鸡只在肠组织切片中观察到病毒。结论鸡全身多个器官组织有H7N9禽流感病毒的受体,肺组织较其他三种器官组织受体分布少,病毒在鸡的消化道分布也较广泛,提示粪-口途径是活禽感染H7N9流感病毒的重要途径。免疫荧光及激光共聚焦实验可检测病毒在组织及细胞的分布。

Objective To establish methods of immunofluorescence and confocal laser for detecting influenza H7N9,and understand the distribution of the virus in organs of chickens. Methods The chickens of H7N9 influenza virus positive were selected from 20 chickens which were culled in a market. The paraffin sections were made with the trachea,lungs,livers,intestines of the chickens,with which monoclonal antibody against nucleoprotein of influenza virus was reacted,then DAPI and FITC labeled of the second antibody were applied to react with above paraffin section. The positive samples were blue and green fluorescence,and the negative samples only emitted blue fluorescence. Then they were examined with laser confocal fluorescence microscopy and fluorescence microscopy. Results There were 9 （45%）chickens of H7N9 influenza virus positive,and among which 5 chickens were seen of fluorescence of virus particles in the tracheal tissues,2 chickens were seen of fluorescence of virus particles in the lung tissues,6 chickens were seen of fluorescence of virus particles in the hepatic tissues,and 5 chickens were seen of fluorescence of virus particles in the intestinal tissues. Among which,1 chicken was positive in all of four organs and 1 chicken was positive only in the intestinal tissues. Conclusion Multiple organ tissues of chickens have receptor molecules of influenza H7N9,and the lung tissues have less receptor molecules than others. The distribution of the virus in the digestive tract of chicken is widespread,suggesting that the fecal-oral is an important way of H7N9 influenza virus infection. Immunofluorescence and confocal laser can detect the distribution of influenza H7N9 in tissues and cells.