Construction and application of subgenomic replicon vectors of Japanese encephalitis virus
Huang, Y.; Shao, W.; Jia, L-Li.; Yu, W-Yuan.; Yu, Y-Xin.
Bing du Xue Bao 23(1): 33-38
2007
ISSN/ISBN: 1000-8721 PMID: 17886718 Document Number: 612632
Based on the infectious clone of JEV vaccine SA(14)-14-2, the subgenomic replicons pCTCJEV, pCTMJEV with large deletions in the structural region were constructed. Then they were transfected into BHK-21 cell, the RNA replication of JEV subgenome can be monitored by RT-PCR and the non-structural protein can be found expressed in the cell by IFA. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive-strand and the negative-strand JEV RNA, we inserted an enhanced green fluorescence protein (EGFP) gene into the 3'-UTR of pCTCJEV, pCTMJEV under the control of the internal ribosomal entry site (IRES) of encephalomyelocarditis virus RNA. After transfection, the EGFP fluorescence could be seen under the fluorescence microscope 1 day later, and maintained for more than a week with no apparent cytopathic effect. The constructed JEV replicons would provide valuable tools to provide a possible vector for a long-lasting RNA virus expression system.