摘要
目的 制备新型冠状病毒野生株(WT)、Beta变异株、Delta变异株、Omicron变异株(BA.2、BA.5、BQ.1和XBB.1分支)等7株单克隆病毒株,用于申请国家标准株.方法 利用蚀斑纯化技术感染Vero细胞后筛选单克隆毒株,通过观察细胞病变特性与病毒形态学特征、病毒滴度与全基因组序列测定、支原体检测等方法,以评价单克隆毒株的基础生物学特征.结果 WT株、Beta变异株、Delta变异株、Omicron变异株(BA.2、BA.5、BQ.1和XBB.1)7株病毒感染细胞后,细胞病变表现为细胞圆缩脱落;蚀斑纯化后的病毒支原体检测均为阴性,2代至5代的病毒滴度在105～108 TCID50/ml之间;电镜下病毒颗粒呈圆形或者椭圆形,直径在60～140 nm之间;全基因组测序与系统进化分析明确了 7种毒株的变异株类别,证实了单克隆毒株连续传代5代基因组特征稳定.结论 制备7种代表性新型冠状病毒变异株的克隆株,具有典型冠状病毒细胞病变效应、形态结构清晰、病毒活性良好、遗传稳定等特性,可作为候选毒株用于申请国家标准株.
Abstract
Objective To prepare seven standard strains of 2019 novel coronavirus(2019-nCoV),including wild type(WT)strain,Beta variant,Delta variant,Omicron variants(BA.2,BA.5,BQ.1,XBB.1 branches),which could be used to apply for national standard strains.Methods According to cytopathic effect(CPE),virus titer,whole-virus-genome-sequencing and detection of mycoplasma,the basic biological characteristics of clonal isolation were determined through plaque purification technology.Results The CPE was mainly characterized by cell shrinkage and exfoliation in Vero cells after infection with seven 2019-nCoV clonal isolations(WT,Beta,Delta,Omicron,BA.2,BA.5,BQ.1 and XBB.1 branches).The result of mycoplasma detection were negative and titers of the clonal isolation from 2nd to 5th generations were stable at 105-108 TCID50/ml;the electron microscope showed that the virions were all round or elliptical,with a diameter between 60 nm and 140 nm.The subtypes of 7 strains were identified by whole-virus-genome-sequencing and phylogenetic analysis,with genomic stability after the fifth successive generations of clonal isolation.Conclusions The series clonal isolation of 2019-nCoV with typical CPE of coronavirus,clear morphological structure,good viral activity and stable genetic characteristics were prepared,and they could be used to apply for national standard strains.
基金项目
广东省重点领域研发计划(N0.2023B1111030001)
卫生健康平台科技创新人才项目(0620220104)
广东省科技计划(2021B1212030007)
NETL
NSTL
万方数据