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Antimicrobial Agents and Chemotherapy, February 2009, p. 748-755, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.00841-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Real-Time Monitoring of Human Enterovirus (HEV)-Infected Cells and Anti-HEV 3C Protease Potency by Fluorescence Resonance Energy Transfer{triangledown} ,{dagger}

Meng-Tian Tsai,1 Yun-Hsiang Cheng,1 Yu-Ning Liu,2 Nien-Chien Liao,3 Wen-Wen Lu,3 and Szu-Hao Kung1*

Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taiwan, Republic of China,1 Department of Medical Research, Mackay Memorial Hospital, Taiwan, Republic of China,2 Department of Clinical Pathology, Cheng Hsin Rehabilitation Medical Center, Taiwan, Republic of China3

Received 25 June 2008/ Returned for modification 14 August 2008/ Accepted 12 November 2008

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 (3Cpro) cleavage motif flanked by the FRET pair composed of green fluorescent protein 2 and red fluorescent protein 2 (DsRed2). Efficient FRET from the stable line was detected in a real-time manner by fluorescence microscopy, and the disruption of FRET was readily monitored upon HEV infection. The 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 confirmed by Western blot analysis. The FRET biosensor was then utilized to develop a format for the determination of antiviral susceptibility, as the reduced FRET appeared to reflect viral replication. Evaluations of the FRET biosensor system with representative HEV serotypes demonstrated that their susceptibilities to a 3Cpro 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 potential for the development of a high-throughput screening assay.

* Corresponding author. Mailing address: Department of Biotechnology in Medicine and Laboratory Science in Medicine, National Yang-Ming University, 155 Li-Nong St. Section 2, Shih-Pai, Taipei, 112, Taiwan, Republic of China. Phone: 886-2-2826-7034. Fax: 886-2-2826-4092. E-mail: szkung{at}ym.edu.tw

{triangledown} Published ahead of print on 17 November 2008.

{dagger} Supplemental material for this article may be found at http://aac.asm.org/.

Antimicrobial Agents and Chemotherapy, February 2009, p. 748-755, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.00841-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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