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| Construction and Identification of the Cell Line for Detecting Flaviviruses |
| WEI Yan, WANG Huan-qin, WU Meng, ZHANG Feng-juan, LIANG Guo-dong, ZHU Wu-yang |
| National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China |
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Abstract Objective:To generate and identify the cell line for detecting the unknown flaviviruses. Methods: The defective replicon with a deletion of prM-E gene and the insertion of the red fluorescent protein mCherry reporter gene was constructed on the basis of the infectious clone of dengue virus type 4 (P4), which was named P4-△prME-mCherry. To select the packaging cell lines for detecting flaviviruses,The multi-fusion PCR method to construct the defective eukaryotic expression plasmid pCDNA3.1-P4-mCherry on the basis of P4-△prME-mCherry were used. BHK-21cells were transfected with thepCDNA3.1-P4-mCherry, then the G418-resistant BHK-21cellsexpressing the defective replicons were obtained, which were named BHK-Flavivirus.Results: The expression of mCherry reporter gene were detected after the BHK-Flavivirus cells were infected by Flaviviruses,such as Japanese encephalitis virus (JEV,P3) and Dengue virus(DENV,P4). By contrast, mCherry was silent when theBHK-Flavivirus were infected by three related plus-strand RNA viruses, including Sindbis virus (SINV,XJ-160, YN87448), Chikungunya virus (CHIKV,SD08Pan) and Getah virus (GETAV,HB0234),and Tahyna virus (TAHV,XJ0625). Conclusion:These result indicated that the BHK-Flavivirus were specific and effective to detect Flavivirus in cell culture, which is a simple, color-visible detection method could detect corresponding mosquito-borne viruses quickly and specifically without affecting the viral multiplication capacity, making it possible to be used in the detection for clinical examinations and the screening of viral biological warfare agents.
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Received: 12 May 2015
Published: 25 September 2015
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