Isolation and full-genome phylogenetic analysis of 2019-nCoV in Shandong province
10.3760/cma.j.cn112866-20210520-00082
- VernacularTitle:山东省新型冠状病毒分离与全基因组进化分析
- Author:
Jianxing WANG
1
;
Ti LIU
;
Mingxiao YAO
;
Zexin TAO
;
Ming FANG
;
Yan LI
;
Yuwei ZHANG
;
Julong WU
;
Yujie HE
;
Lei JIANG
;
Zhong LI
;
Xiaolin JIANG
;
Dianming KANG
;
Zengqiang KOU
Author Information
1. 山东省疾病预防控制中心 山东省预防医学科学研究院 山东省传染病预防控制重点实验室 山东大学预防医学研究院，济南 250014
- Keywords:
(2019 novel coronavirus;
Isolation;
Full-genome sequencing
- From:
Chinese Journal of Experimental and Clinical Virology
2021;35(6):669-674
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish virus culture method and full genome sequencing method for severe acute respiratory syndrome coronavirus (2019-nCoV), and to illuminate the variation of 2019-nCoV.Methods:The pharyngeal swab specimens were inoculated into Vero-E6 cells for isolation, and the cytopathic effect were observed day by day, and the result of virus isolation were confirmed by Real-Time RT-PCR. Some isolated viruses were diluted 10 times to detect the virus titer. Full-genome of 2019-nCoV was sequenced with the whole genome capture technology and next generation sequencing technology. Phylogenetic analysis and molecular characterization for obtained 2019-nCoV sequences were undertaken.Results:Twenty-two 2019-nCoV strains were isolated with Vero-E6 cell line in the Biosafety Level-3 Laboratory (BSL-3). Eighteen full-genome sequences obtained (almost 29 000 nucleotide) were analyzed with 99.94%~100% identity and 28 nucleotides and 20 amino acids variations. Phylogenetic analysis showed that the 2019-nCoV sequences belonged to different clades, including clade S and clade L with Europe lineage L. I and Europe lineage L. II.1. Furthermore, 5 of 18 2019-nCoV harbor the D614G mutation.Conclusions:2019-nCoV were successfully isolated with Vero-E6 cells, and partially illuminated the phylogenetic characteristics, which provided a basis for subsequent drug screening and biological characteristics research. Continuous monitoring and analysis of the sequences of new cases would be vital to understand the genetic evolution and rates of substitution of the 2019-nCoV.
