A dark-to-bright reporter cell for classical swine fever virus infection.
Current methods to quantitate classical swine fever virus (CSFV) infectivity in cell culture are time-consuming and labor-intensive. This study described the generation of a dark-to-bright fluorescent reporter cells to facilitate in vitro studies of CSFV infection and replication. This assay was based on a novel reporter cell stably expressing the enhanced green fluorescent protein (EGFP) fused in-frame to a quenching peptide via a special recognition sequence of the CSFV NS3 protease. Chromophore maturation of EGFP can be prevented by quenching peptide until the quenching peptide was specifically cleaved by NS3 protease during CSFV infection, making it a dark-to-bright reporter of CSFV infection. The result demonstrated that the CSFV-infected cells were clearly distinguishable from mock-infected cells and cells infected with other viruses. There was a strong correlation between the fluorescence intensity and viral RNA replication in CSFV-infected cells. The cell enabled rapid and sensitive detection of CSFV infection and viral replication in cell culture. The best time to examine the fluorescence in CSFV-infected cells was at 48h post-inoculation. These data suggested that the cells can be used as a reporter cell in CSFV infection assays. This reporter cell provides a sensitive method for the detection and isolation of CSFV and it will be useful for the screening of antiviral drugs or neutralizing antibody assays.
Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun, Jilin 130062, China.
Fuwang
F
Chen
Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun, Jilin 130062, China.
Xin
X
Yang
Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun, Jilin 130062, China.
Daxing
D
Pang
Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun, Jilin 130062, China.
Zhiyuan
Z
Peng
Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun, Jilin 130062, China.
Teng
T
Ma
Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun, Jilin 130062, China.
Hongsheng
H
Ouyang
Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun, Jilin 130062, China. Electronic address: renlz@jlu.edu.cn.
Linzhu
L
Ren
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D006692:Classical Swine Fever Virus / Q000302:isolation & purification* / Q000502:physiology*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013552:Swine; D014779:Virus Replication
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
Classical swine fever virus; Dark-to-bright; Fluorescence intensity; GFP (green fluorescent protein); Reporter cell