Construction, characterization and stability analysis of infectious clone of live attenuated dengue virus type 4 Ban18HK20 strain
10.3760/cma.j.issn.0254-5101.2019.11.004
- VernacularTitle: 登革病毒4型Ban18HK20株感染性克隆的构建、鉴定及稳定性分析
- Author:
Enyue FANG
1
,
2
;
Ling WANG
3
;
Danhua ZHAO
3
;
Ming LI
3
;
Minglei LIU
3
;
Yuhua LI
3
Author Information
1. Laboratory 2 of Viral Vaccine, Wuhan Institute of Biological Products Co., LTD., Wuhan 430070, China
2. Arboviruses Vaccine Division, National Institutes for Food and Drug Control, Beijing 102629, China
3. Arboviruses Vaccine Division, National Institutes for Food and Drug Control, Beijing 102629, China
- Publication Type:Journal Article
- Keywords:
Dengue virus;
Reverse genetics;
Infectious clone;
Virus recovery
- From:Chinese Journal of Microbiology and Immunology
2019;39(11):827-834
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct a stable infectious clone of live attenuated dengue virus (DENV) type 4 Ban18HK20 strain for better understanding the virulence determinants of DENV and improving the development of chimeric vaccines.
Methods:Specific primers were constructed according to the genome of Ban18HK20 strain and used to subclone six cDNA fragments, which were linked into a high-copy plasmid pSPTM to obtain a stable full-length cDNA clone of DENV. RNA was transcribed from the full-length cDNA in vitro and electrotransfected into Vero cells to recover the virus. Biological characteristics of the recovered virus were identified using plaque assay, indirect immunofluorescence assay, growth kinetics test and pathogenicity study in mouse brain. Genetic stability of the virus passaged 15 generations was studied using RT-PCR amplification.
Results:Restriction enzyme digestion and sequencing analysis indicated that the infectious clone was constructed successfully. The recovered virus was consistent with the parental virus in terms of plaque morphology, DENV E protein expression, growth characteristics and pathogenicity in mouse brain. Sequencing analysis showed that the recovered virus had the same genome sequence as the parental virus with good hereditary stability.
Conclusions:A stable infection clone of Ban18HK20 was constructed and a reverse genetics technology platform for DENV research was established.
