动物狂犬病病毒街毒株的细胞分离鉴定

Isolation and identification of rabies viruses in in vitro cells from animal rabies cases

目的N2A细胞和BHK-21细胞用于我国动物狂犬病流行街毒株的分离培养。方法狂犬病发病动物脑组织悬液首先脑内接种乳鼠,再采取Nonclon Delta Tube内置盖玻片法从鼠脑中进行病毒分离培养,脑组织悬液接种细胞后96h,利用直接荧光抗体实验检测盖玻片上的病毒感染细胞,判定病毒是否在细胞上增殖;通过测定不同毒株细胞培养上清中的病毒滴度对病毒进行鉴定。结果22份狂犬病病犬、猪和牛的脑组织接种乳鼠后均获得了鼠脑分离毒,将鼠脑毒接种N2A细胞分离获得了22株病毒,接种BHK-21细胞却只分离获得20株病毒。不同病毒株第三代细胞培养上清中的病毒滴度从10-1TCID50/100μL到10-3.6TCID50/100μL。结论N2A细胞对狂犬病病毒街毒株的敏感性高于BHK-21细胞,且不同的病毒株对细胞的适应能力不同。实验分离获得的病毒细胞适应株丰富了我国狂犬病病毒毒种资源,为进一步研究不同病毒分离株的抗原性差异奠定基础。

Animal rabies plays a pivotal role in transmission of human rabies in China. In order to characterize rabies viruses, brain tissues of rabid animals were subjected to mouse inoculation test （MIT） followed by viral isolation using N2A and BHK-21 cell lines from mouse brain tissue homogenates in Nonclon Delta Tube with coverslips. Once viral growth was confirmed at 96 hrs p.i. by detection of coverslips using fluorescent antibody test （FAT） the infected cells were propagated three times and the viral titers were determined for each passage. In results, 22 street viruses were isolated in N2A cells from 22 rabid dogs, pigs and cows, while only 20 isolated in BHK-21 cells. The isolated viruses showed a variation of viral titers at passage 3 from 10^-1 TCID50/100μL to 10^-3.6 TCID50/100μL, indicating different adaptive capacities of the viruses in the cell cultures. The result also showed that N2A cell line is more sensitive than BHK-21 in isolation of street viruses. This work provided a possihility for differentially antigenic analysis of Chinese rabies viruses by monoclonal antibodies.