脑心肌炎病毒蚀斑纯化及滴度测定方法的建立

Development of methods for plaque purification and titer determination of encephalomyocarditis virus

目的建立脑心肌炎病毒(Encephalomyocarditis virus,EMCV)蚀斑纯化及滴度测定方法。方法将EMCV系列稀释后接种至单层Vero细胞,覆盖甲基纤维素,出斑后挑取蚀斑进行扩增,收获病毒液。选取5个EMCV原液样品感染Vero细胞进行蚀斑滴度测定,经结晶紫染色计数蚀斑,计算病毒滴度。重复测定5次,验证蚀斑法的精密性,并与微量细胞病变法进行比较。结果 EMCV感染Vero细胞后,约48 h出现边缘整齐直径为0.5～1.5 mm的蚀斑,经结晶紫染色后,可见清晰透明空斑,边缘清晰。5个EMCV原液样品经蚀斑法测得的病毒滴度在3.16×106～1×107PFU/ml之间,变异系数为0.88%～2.07%;经微量细胞病变法测得的病毒滴度均大于3.16×107PFU/ml,变异系数为1.63%～2.90%,表明蚀斑法精密性更好;两种方法经直线回归分析,具有线性相关性(r=0.810,P=0.097)。结论建立了简便直观的EMCV蚀斑纯化方法及精密性良好的蚀斑滴度测定方法,为EMCV的生物学特性及疫苗研究奠定了基础。

Objective To develop the methods for plaque purification and titer determination of encephalomyocarditis virus（EMCV）.Methods EMCV was diluted serially and inoculated to Vero cell monolayer which was then covered with methyl cellulose.The formed plaques were selected for amplification,and the virus liquid was harvested.Five test samples of bulk EMCV were infected to Vero cells,based on which plaques were counted by crystal violet staining,and the virus titer was calculated.The precision of plaque method was verified by retest for 5 times and compared with that of micro-cytopathic effect（micro-CPE） method.Results The plaques with regular borders,at diameters of 0.5 ～ 1.5 mm,were observed in Vero cell monolayer 48 h after inoculation with EMCV.Transparent plaques with clear borders were observed by crystal violet staining.The titers of five samples of bulk EMCV determined by plaque method were 3.16 × 106 ～ 1 × 107 PFU / ml,with a coefficient of variation of 0.88% ～ 2.07%.However,all the virus titers determined by micro-CPE method were more than 3.16 × 107 PFU / ml,with a coefficient of variation of 1.63% ～ 2.90%.It indicated that the precision of plaque method was higher than that of micro-CPE method.Linear regression analysis showed linear relationship between the determination results by the two methods（r = 0.810,P = 0.097）.Conclusion A more simple and more intuitive method for plaque purification of EMCV and a precise method for determination of plaque titer were developed,which laid a foundation of study on biological characters of EMCV and development of EMCV vaccine.