新型冠状病毒BF.7变异株的分离鉴定与基因序列特征分析  

Isolation and identification of SARS-CoV-2 BF.7 variant strain and analysis of its genomic sequence characteristics

作　　者：宋冬梅 马淑花 杨永娟 韩婧雯 刘倩 张久鑫 汤重发 赵宇星 戴新宪 Song Dongmei;Ma Shuhua;Yang Yongjuan;Han Jingwen;Liu Qian;Zhang Jiuxin;Tang Chongfa;Zhao Yuxing;Dai Xinxian(Etiology Laboratory,National Vaccine&Serum Institute,Beijing 101111,China;Biosafety Level-3 Laboratory,National Vaccine&Serum Institute,Beijing 101111,China)

机构地区：[1]国药中生生物技术研究院(新型疫苗国家工程研究中心)病原学实验室,北京101111 [2]国药中生生物技术研究院(新型疫苗国家工程研究中心)生物安全三级实验室,北京101111

出　　处：《中华微生物学和免疫学杂志》2024年第11期951-957,共7页Chinese Journal of Microbiology and Immunology

摘　　要：目的分离新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)流行株并进行鉴定,对分离株和传代培养物开展序列特征分析。方法对2022年12月收集的11份SARS-CoV-2抗原初筛呈阳性的鼻咽拭子样本进行病毒分离。实时荧光定量PCR检测SARS-CoV-2核酸,核酸阳性样本接种Vero细胞进行病毒分离,Western blot和间接免疫荧光法检测病毒抗原,透射电镜观察病毒形态。提取病毒分离株及后续传代培养物核酸,对病毒基因组测序并进行序列分析。结果11份样本实时荧光定量PCR检测结果均为SARS-CoV-2阳性。成功分离出7株SARS-CoV-2,其在Vero细胞中可连续稳定传代,滴度可达106.25细胞培养半数感染量(50%cell culture infectious dose,CCID 50)/ml。Western blot和间接免疫荧光结果显示病毒分离株可被SARS-CoV-2单克隆抗体和恢复期血清特异性识别;透射电镜观察到病毒颗粒呈球形,直径约为100 nm;通过二代测序技术成功获得病毒全基因组序列,序列分析结果显示,7株分离株均为SARS-CoV-2,与Wuhan-Hu-1株(NC-045512)序列同源性>99.50%,7株分离株之间序列同源性为99.98%,同属于Omicron BF.7进化分支。对BJ-NVSI-20230005分离株连续传代和蚀斑纯化后进行序列分析,结果显示4~6与1~3代病毒相比,ORF1ab基因和E基因分别有1个核苷酸位点发生突变和3个核苷酸的缺失,其中ORF1ab基因C→T突变导致亮氨酸变为苯丙氨酸;E基因3个核苷酸缺失导致缬氨酸缺失。蚀斑纯化克隆序列存在多态性。结论成功分离的7株SARS-CoV-2流行毒株均为BF.7进化分支,与中国内地2022年12月流行趋势一致。Objective To isolate and identify SARS-CoV-2 epidemic strains and analyze the sequence characteristics of the virus strains following serial passages.Methods Eleven nasopharyngeal swabs positive for SARS-CoV-2 antigen were collected from December 2022.Quantitative real-time PCR was used to detect SARS-CoV-2 nucleic acid,and positive specimens were inoculated onto Vero cells for virus isolation.The isolated strains were identified by Western blot and indirect immunofluorescence assay.The morphology of the isolated strains was observed using transmission electron microscopy.Nucleic acid was extracted from the isolates and passaged viruses for further sequencing and analysis.Results All 11 specimens tested positive for SARS-CoV-2 using quantitative real-time PCR.SARS-CoV-2 strains were successfully isolated from seven specimens,and could be adaptively cultured,passaged,and expanded on Vero cells,achieving a peak titer exceeding 106.2550%cell culture infectious dose(CCID 50)/ml.Western blot and indirect immunofluorescence results showed that the isolates could be specifically recognized by monoclonal antibodies and convalescent serum against SARS-CoV-2.Transmission electron microscopy revealed oval-shaped viral particles with diameters of approximately 100 nm.Next-generation sequencing of the viral isolates demonstrated a sequence homology greater than 99.50%with the Wuhan-Hu-1 reference strain(NC_045512)and 99.98%among the seven isolated strains,and all of the isolates belonged to the Omicron BF.7 variant.Sequence analysis after continuous passage and plaque purification of the BJ-NVSI-20230005 isolate showed that compared with passages 1-3,passages 4-6 had one nucleotide site mutation(C→T)in the ORF1ab gene and a deletion of 3 bp in the E gene,which resulted in a change from leucine to phenylalanine and the deletion of valine,respectively.Polymorphisms were observed in the sequences of plaque-purified clones.Conclusions The seven successfully isolated SARS-CoV-2 strains all belong to the SARS-CoV-2 BF.7 variant,w

关 键 词：病毒分离 新型冠状病毒 实时荧光定量PCR 系统进化分析 

分 类 号：R373[医药卫生—病原生物学]