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Antimicrob. Agents Chemother. doi:10.1128/AAC.00841-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Real-time Monitoring of Human Enterovirus (HEV)-Infected cells and Anti-HEV 3C^pro Potency by Fluorescence Resonance Energy Transfer

Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taiwan, R.O.C.; Department of Medical Research, Mackay Memorial Hospital, Taiwan, R.O.C.; Department of Clinical Pathology, Cheng Hsin Rehabilitation Medical Center, Taiwan, R.O.C.

^* To whom correspondence should be addressed. Email: szkung{at}ym.edu.tw.

	Abstract

A real-time assay system that allows monitoring of intracellular human enterovirus (HEV) protease activity was established using the principle of fluorescence resonance energy transfer (FRET). It was accomplished by engineering cells to constitutively express a genetically encoded FRET probe. The FRET-based probe was designed to contain an enterovirus 71 3C protease (3C^pro) cleavage motif flanked by the FRET pair composed of the green fluorescent protein 2 (GFP²) and red fluorescent protein 2 (DsRed2). Efficient FRET from the stable line was detected in a real-time manner by the fluorescent microscopy, and the disruption of FRET was readily monitored upon an HEV infection. Level of the repressed FRET was proportional to the input virus titer and the infection duration as measured by the fluorometric method. The FRET biosensor cell line was also responsive to other related HEV serotypes but not to the phylogenetically distant herpes simplex virus, which was then confirmed by Western blot analysis. The FRET biosensor was then utilized to develop a format for determination of the antiviral susceptibility given that the reduced FRET appeared to reflect viral replication. Evaluations of the FRET biosensor system with representative HEV serotypes demonstrated that their drug susceptibilities to a 3C^pro inhibitor, rupintrivir, were all accurately determined. In summary, this novel FRET-based system is a means for rapid detection, quantification and drug susceptibility testing for HEVs, with the potential to the development of a high-throughput screening assay.