森林脑炎病毒滴度检测蚀斑法的建立及验证  

Development and verification of plaque method for detection of tick-borne encephalitis virus titer

作　　者：陈娜娜 陈诗阳 王颖 杜翔宇 苗会 拱小棠 刘双军 李景良 CHEN Na-na;CHEN Shi-yang;WANG Ying;DU Xiang-yu;MIAO Hui;GONG Xiao-tang;LIU Shuang-jun;LI Jing-liang(不详;DepartmentⅢof Vaccine,Changchun Institute of Biological Products,Co.,Ltd.,Changchun 130062,Jilin Province,China)

机构地区：[1]长春生物制品研究所有限责任公司疫苗三室,吉林长春130062 [2]长春理工大学生命科学技术学院,吉林长春130062

出　　处：《中国生物制品学杂志》2023年第2期207-210,216,共5页Chinese Journal of Biologicals

基　　金：吉林省省级医药健康产业发展专项基金(20210204206YY)。

摘　　要：目的建立原代地鼠肾(primary hamster kidney,PHK)细胞适应的森林脑炎病毒(tick-borne encephalitis virus,TBEV)株(简称PHKT株)感染性滴度检测的蚀斑法,并对建立的方法进行验证。方法将主代鼠脑适应株TBEV感染原代地鼠肾PHK细胞,连续传12代。分别采用蚀斑法(将不同代次PHKT以不同稀释倍数感染单层BHK-21细胞,中性红染色,计算蚀斑数)和小鼠脑内攻毒滴定法(将不同代次PHKT以不同稀倍数经脑腔攻击小鼠,0.03 mL/只,连续观察14 d,Reed-Muench方法计算半数致死量)检测PHKT滴度,并对两种方法检测结果的相关性进行分析。对建立的检测TBEV滴度的蚀斑法进行专属性、重复性和中间精密度验证。结果PHKT病毒蚀斑滴度最高达8.9 lgPFU/mL;蚀斑法与小鼠脑内攻毒滴定法检测结果相关性较好(r=0.92);蚀斑法检测PHKT病毒感染性滴度专属性良好,重复性和中间精密度变异系数(CV)均<5%。结论建立了检测PHKT感染性滴度的蚀斑法,可作为小鼠脑内攻毒滴定法的替代方法。Objective To develop and verify a plaque method for detection of infectious titer of tick-borne encephalitis virus(TBEV)strain(PHKT strain for short)adapted to primary hamster kidney(PHK)cells.Methods PHK cells were infected with TBEV,a primary mouse brain adaption strain,and passed consecutively for 12 passages.The titer of PHKT was detected by plaque method(Monolayer BHK-21 cells were infected with PHKT of various passages at different dilution ratios,and the plaque number was calculated by neutral red staining)and challenge titration in mouse brain(Mice were challenged with PHKT of various passages at different dilution ratios through brain cavity,0.03 mL for each,observed continuously for 14 days,and calculated for the median lethal dose(LD50)by Reed-Muench method)respectively,and the correlation between the results of two methods was analyzed.The developed plaque method for the detection of TBEV titer was verified for specificity,repeatability and intermediate precision.Results The plaque titer of PHKT virus was up to8.9 lgPFU/mL;The correlation between the results of plaque method and mouse brain challenge titration method was good(r=0.92);The specificity of plaque method for detecting infectious titer of PHKT virus was good,and the coefficients of variation(CVs)of repeatability and intermediate precision were both less than 5%.Conclusion A plaque method for detecting infectious titer of PHKT virus was developed,which may be used as an alternative method for challenge titration in mouse brain.

关 键 词：森林脑炎病毒 蚀斑法 小鼠脑内攻毒滴定法 感染性滴度 森林脑炎灭活疫苗 

分 类 号：R373.3[医药卫生—病原生物学]