Publication: Establishment of a stable cell line coexpressing dengue virus-2 and green fluorescent protein for screening of antiviral compounds
Issued Date
2012-03-01
Resource Type
ISSN
1552454X
10870571
10870571
Other identifier(s)
2-s2.0-84859040897
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Mahidol University
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SCOPUS
Bibliographic Citation
Journal of Biomolecular Screening. Vol.17, No.3 (2012), 283-292
Suggested Citation
V. Leardkamolkarn, W. Sirigulpanit Establishment of a stable cell line coexpressing dengue virus-2 and green fluorescent protein for screening of antiviral compounds. Journal of Biomolecular Screening. Vol.17, No.3 (2012), 283-292. doi:10.1177/1087057111426903 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/13779
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Title
Establishment of a stable cell line coexpressing dengue virus-2 and green fluorescent protein for screening of antiviral compounds
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Abstract
This study aimed to generate a stable cell line harboring subgenomic dengue virus replicon and a green fluorescent gene (DENV/GFP) for a cell-based model to screen anti-DENV compounds. The gene-encoding envelope protein of DENV-2 was deleted and then replaced with fragments of the GFP gene and a foot-and-mouth-disease virus 2A-derived cleavage site. The human cytomegalovirus immediate early and antisense hepatitis delta virus ribozyme sequences were added at the 5′- and 3′-ends. An internal ribosome entry site and neomycin resistance genes were placed upstream and next to the NS1 gene. The recombinant plasmids were propagated in a mammalian cell line. A stable cell line with the brightest green fluorescent protein and the highest viral protein and RNA expression was selected from six clones. The clone was then examined for effectiveness in an antiviral drug screening assay with compounds isolated from the local plants using two known antiviral agents as controls. Two novel flavones, PMF and TMF, were discovered having DENV-inhibitory properties. The data were validated by a conventional plaque titration assay. The results indicate that this newly developed cell line is efficient for use as a cell-based model for primary screening of anti-DENV compounds. © 2012 Society for Laboratory Automation and Screening.