检测痘苗病毒体外感染的病灶形成方法的优化与应用  

Optimization and Performance Evaluation of A Novel Focus-Forming Assay for Titration of the Vaccinia Virus

作　　者：楚巧鸿 冯霞[1] 李佳[2] 张菱芳 单徐畅 舒畅 程雪婷 霍恕婷 邓瑶[1] 谭文杰[1,2] CHU Qiaohong;FENG Xia;LI Jia;ZHANG Lingfang;SHAN Xuchang;SHU Chang;CHENG Xueting;HUO Shuting;DENG Yao;TAN Wenjie(Key Laboratory of Biosafety,National Health Commission of the People′s Republic of China,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Zhejiang Provincial Key Laboratory of Medical Genetics,School of Laboratory Medicine and Life Sciences,Wenzhou Medical University,Wenzhou 325035,China;School of Basic Medical Sciences,Inner Mongolia Medical University,Hohhot 010110,China;School of Public Health,Xinxiang Medical University,Xinxiang 453003,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,国家卫生健康委员会生物安全重点实验室,北京102206 [2]温州医科大学检验医学院,生命科学学院,浙江省医学遗传学重点实验室,温州325035 [3]内蒙古医科大学基础医学院,呼和浩特010110 [4]新乡医学院公共卫生学院,新乡453003

出　　处：《病毒学报》2024年第3期492-499,共8页Chinese Journal of Virology

基　　金：国家重点研发计划(项目号:2023YFD1800405),题目:抗猴痘药物筛选与阻断技术研究;国家重点研发计划(项目号:2022YFC2303401),题目:潜在高危新病毒风险识别的新技术体系研究。

摘　　要：本研究旨在建立一种快速、通用、高通量的病灶形成测定法(Focus-forming assay, FFA),用于痘苗病毒(Vaccinia virus, VACV)的研究。首先采用经典噬斑染色法与免疫噬斑法对不同VACV代表性毒株(Western Reserve strain, WR;Tiantan strain, VTT;Modified Ankara strain, MVA)在BHK-21细胞和Vero细胞上的噬斑特性进行评估,通过实验条件(包括显色液、非特异性染色、细胞培养板和病毒培养时间)的优化,提高病灶的可视化质量和测定的准确性。并基于改进优化的FFA法研究了VACV在BHK-21细胞和Vero细胞上的生长曲线,同时对FFA法的重复性进行了评估。研究发现BHK-21细胞对VACV的感染更加敏感,适宜于多种VACV滴度测定。实验条件的优化显著提高了病灶的可视化质量和测定的准确性。应用FFA法发现WR、VTT和MVA毒株在BHK-21细胞上能够有效复制,而MVA在Vero细胞上的复制受限,且FFA法在同一实验内具有良好的重复性,变异系数在0.17%到3.50%之间。改进后的FFA法是一种快速、通用、高通量的方法,适用于痘苗病毒滴度的测定,为痘苗病毒的研究和应用提供了可靠的技术支持。We aimed to establish a rapid,universal,high-throughput focus-forming assay(FFA)for vaccinia virus(VACV)research.Initially,the plaque characteristics of representative VACV strains(Western Reserve strain,WR;Tiantan strain,VTT;Modified Ankara strain,MVA)in BHK-21 cells and Vero cells were evaluated using classical plaque-staining and immunostaining methods.Experimental conditions(choice of substrate,non-specific staining,cell culture plates,and virus incubation time)were optimized to enhance the visibility and accuracy of focal detection.An improved FFA method,based on these optimizations,was used to study the growth curves of VACV in BHK-21 cells and Vero cells.The repeatability of the FFA method was assessed.BHK-21 cells were sensitive to VACV infection and suitable for the titration of various VACV strains.Optimization of experimental conditions improved the quality of focal visualization and accuracy of measurement significantly.The FFA method demonstrated effective replication of WR,VTT,and MVA strains in BHK-21 cells,whereas replication of MVA in Vero cells was limited.The FFA method showed good repeatability within the same experiment,with a coefficient of variation ranging from 0.17%to 3.50%.The improved FFA method was a rapid,versatile,and high-throughput approach suitable for VACV titration,and provided reliable technical support for the research and application of VACV.

关 键 词：痘苗病毒 噬斑实验 病灶形成实验 滴度测定 

分 类 号：R373.1[医药卫生—病原生物学]