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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 a method for detecting H7N9 avian influenza virus by immunofluorescence and confocal laser scanning to understand the distribution of H7N9 avian influenza virus in multiple organs of chicken. Methods Twenty live chickens slaughtered in the live poultry market were screened for H7N9 avian influenza virus. The paraffin sections of trachea, lung, liver and intestine were obtained and incubated with anti-influenza virus nucleoside monoclonal antibody (DAPI) , 6-diamidino-2-phenylindole) and secondary antibody with FITC (Fluorescein isothiocyanate), blue and green fluorescence positive samples, negative fluorescence blue fluorescence Fluorescence microscopy and laser scanning confocal microscopy were observed. Results Of the 20 live chickens tested, 9 live chickens were found to be infected with H7N9 bird flu virus at a rate of 45%. Among them, 5 showed fluorescence in the tracheal section, 2 in the lung tissue section, 6 in the liver section, and 5 in the intestinal section showed the fluorescence of virus particles. Among them, 1 Chickens were positive in all four organs, and one chickens was observed virus only in intestinal tissue sections. Conclusion There are many H7N9 avian influenza virus receptors in the whole body of chicken. The lung tissue has fewer receptors than the other three organs and the virus is also widely distributed in the digestive tract of chickens, suggesting that the fecal-oral route is a result of H7N9 Influenza virus is an important way. Immunofluorescence and confocal laser scanning can detect the virus in the tissue and cell distribution.