一株人源性轮状病毒的分离及适应性培养

Isolation Human Rotavirus Strains and Adaptive Culture

目的:研究轮状病毒野毒株的分离方法、组织培养适应条件及相应生物学性质。方法:对收集的样品采用胶体金、PCR、PAGE进行轮状病毒定性检测。阳性样品按常规方法处理并进行组织培养分离,对不同传代样品做基因组图谱、基因序列、病毒增殖动力学分析评价,采用轮状病毒P[8]G1型阳性血清进行病毒中和鉴别试验。结果:通过检测为A组轮状病毒,基因组为4∶2∶3∶2排列,基因分型G1P[8]型,在MA104细胞中传代后转至Vero细胞上适应培养可观察到CPE;电镜观察可见典型轮状病毒形态。毒株在Vero细胞上增殖到第10代,复制稳定,且感染性滴度达到7.25 log CCID50/ml,增殖高峰为96h。中和鉴别试验证实病毒培养液只包含轮状病毒,无其他病毒污染。结论:从腹泻样品中分离得到一株人源轮状病毒毒株,命名为ZTR-68株,该分离株具有良好的组织培养适应性,遗传稳定性,为进一步研究其生物学性质和疫苗制备提供了基础。

Objective： To investigate separation method, the condition for culture and biological properties of wild rotavirus strain . Methods： To the qualitative detection of samples collected rotavirus use colloidal gold, PCR, PAGE . Process the positive samples according to the conventional method, and then do the separation of tissue culture. To analyze and evaluate the genome, gene sequences and proliferation kinetics of different transfer of culture samples. Using the positive serum of the GIP [ 8 ] rotavirus to do the virus neutralizing identification test. Results ： By testing, the ZTR-68 rotavirus belong to A group were got, the arrangement of genome is 4： 2：3 ： 2, the genotype is G1P[ 8 ]. The CPE was observed after the adapted culture the rotavirus , proliferated in MA104 cells, in the Veto cells. And the typical rotavirus form can be observed by using electron microscope. The processing is stable, which the strain proliferated in the Vero cells. And the infectious titer reached 7.25 log CCID50/ml, the peak time of proliferation is at 96h. There was not any other virus except the rotavirus in the culture medium of virus neutralizing identification test. Conclusion： The separated strain of human rotavirus, named ZTR-86 ,which was got from the separation of the diarrhea samples, has a good adaptability to the tissue culture and a good genetic stability. It provides a basis for the further study of its biological properties and the making of corresponding vaccine.