Isolation and identification of SARS-CoV-2 BF.7 variant strain and analysis of its genomic sequence characteristics
10.3760/cma.j.cn112309-20240424-00145
- VernacularTitle: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
- Author:
Dongmei SONG
1
;
Shuhua MA
;
Yongjuan YANG
;
Jingwen HAN
;
Qian LIU
;
Jiuxin ZHANG
;
Chongfa TANG
;
Yuxing ZHAO
;
Xinxian DAI
Author Information
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
- Keywords:
Virus isolation;
SARS-CoV-2;
Quantitative real-time PCR;
Phylogenetic analysis
- From:
Chinese Journal of Microbiology and Immunology
2024;44(11):951-957
- CountryChina
- Language:Chinese
-
Abstract:
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 10 6.25 50% 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, which is consistent with the prevalence trend in mainland China in December 2022.
