Construction and rescue of Chikungunya virus infectious clone
10.3969/j.issn.1672-8467.2025.06.005
- VernacularTitle:基孔肯雅病毒感染性克隆构建及拯救
- Author:
Heng CAO
1
;
Na LIU
1
;
Yong-kang WANG
1
;
Man-xi WANG
1
;
Gang LONG
1
Author Information
1. 复旦大学上海市重大传染病和生物安全研究院 上海 200032;复旦大学基础医学院病原生物学系教育部/卫健委/中国医科院医学分子病毒学重点实验室 上海 200032
- Publication Type:Journal Article
- Keywords:
Chikungunya virus(CHIKV);
infectious clone;
virus rescue
- From:
Fudan University Journal of Medical Sciences
2025;52(6):811-818
- CountryChina
- Language:Chinese
-
Abstract:
Objective To rescue a strain of Chikungunya virus(CHIKV)using reverse genetics system.Methods Molecular cloning experiments,including PCR,agarose gel electrophoresis and homologous recombination were employed to generate an infectious cDNA clone(pFK-CHIKV-LN)derived from the isolated Caribbean Chikungunya strain(Caribbean strain,isolate M100,Genbank LN898083.1).The mRNA obtained from vitro transcription were transfected into BHK-21 to rescue the CHIKV.The viral titer was determined by 50%tissue culture infection dose(TCID50),with calculations performed using the Reed-Muench method.Western blot(WB)and real time quantitative polymerase chain reaction(RT-qPCR)were employed to assess the levels of viral protein and mRNA at various time points during infection.Results The full-length cDNA clone of CHIKV was successfully constructed,enabling the rescue of CHIKV progeny.RT-qPCR and WB confirmed the significant increasing expression levels of NS1 mRNA and proteins(NS3,E1)of BHK-21 during the infection process.Conclusion The full-length infectious clone of CHIKV has been constructed successfully,which provides a good tool for subsequent studies on the gene structure,protein function and pathogenic mechanism of CHIKV.
