虎源高致病性禽流感病毒H5N1在实验小鼠体内分布与含量测定

Determining the Distribution and Contents of High Pathogenic Avian influenza virus from Tiger in Mice

将本实验室分离、鉴定的虎源高致病性禽流感病毒A/Tiger/Harbin/01/2003(H5N1)株第3代鸡胚尿囊液进行10-3倍稀释,接种小鼠,待小鼠死亡后无菌采取心、肝、脾、肺、肾、脑六种脏器分别研磨制成乳剂,接种MDCK细胞,以细胞病变为判定指标,计算病死小鼠不同脏器内病毒含量。同时用RT-PCR、血凝-血凝抑制试验以及电镜负染的方法进行鉴定和观察。结果显示,可在小鼠的肺脏、肝脏、肾脏、脑组织中检出所接种的病毒,肺脏中的病毒含量最高,脑中的病毒含量次之,肝脏和肾脏中的病毒含量最少。可在肺、肝、肾、脑组织与感染细胞培养物中扩增出与理论值一致的核酸条带,肺乳剂上清液感染细胞培养物中可检出1∶23的血凝效价,电镜观察在肺乳剂上清液及其感染细胞培养物中可见到典型的流感病毒颗粒。

With the dilution of 10.3 from the third generation of allantoic fluid in embryonated eggs infected by H5N1 high pathogenic avian influenza virus A/Tiger/Harbin/01/2003 strain which was isolated and verified in this laboratory, mice were inoculated. After mouse death, we collected hearts, livers, spleens, lungs, kidneys and brains to prepare emulsion. And meanwhile, MDCK cells were inoculated to observe the cytopathogenic effect as the determinant index. The contents of virus in each organ of dead mouse was calculated by the method of Reed-Muench. Virus was evaluated with the technology of RT-PCR and was observed with the method of electron microscope negative staining. The results demonstrated that all the organs above were infected by HPAIV. The specific nucleate straps of HPAIV were expanded in lungs, kidneys, livers and brains and the infected cell cultures as same as the theory value which was 464bp by evaluation of RT-PCR. We can detect that the hemagglutination valence was 1 ：2^3 and the typical influenza virus-like particles were observed with the electron microscope in the infected cell culture and the supernatant of lung emulsion.