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Applied and Environmental Microbiology, April 2008, p. 2239-2243, Vol. 74, No. 7
0099-2240/08/$08.00+0     doi:10.1128/AEM.00259-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Detection of Hepatitis A Virus by Using a Combined Cell Culture-Molecular Beacon Assay{triangledown}

Hsiao-Yun Yeh,1 Yu-Chen Hwang,2 Marylynn V. Yates,2 Ashok Mulchandani,1 and Wilfred Chen1*

Department of Chemical and Environmental Engineering,1 Department of Environmental Sciences, University of California, Riverside, California 925212

Received 29 January 2008/ Accepted 1 February 2008

Rapid and efficient methods for the detection and quantification of infectious viruses are required for public health risk assessment. Current methods to detect infectious viruses are based on mammalian cell culture and rely on the production of visible cytopathic effects (CPE). For hepatitis A virus (HAV), viral replication in cell culture has been reported to be nonlytic and relatively slow. It may take more than 1 week to reach the maximum production and subsequent visualization of CPE. A molecular beacon (MB), H1, specifically targeting a 20-bp 5' noncoding region of HAV, was designed and synthesized. MB H1 was introduced into fixed and permeabilized fetal rhesus monkey kidney (FRhK-4) cells infected with HAV strain HM-175. Upon hybridizing with the viral mRNA, fluorescent cells were visualized easily under a fluorescence microscope. Discernible fluorescence was detected only in infected cells by using the specific MB H1. A nonspecific MB, which was not complementary to the viral RNA sequence, produced no visible fluorescence signal. This MB-based fluorescence assay enabled the direct counting of fluorescent cells and could achieve a detection limit of 1 PFU at 6 h postinfection, demonstrating a significant improvement in viral quantification over current infectivity assays.

* Corresponding author. Mailing address: Department of Environmental Sciences, University of California, Riverside, CA 92521. Phone: (951) 827-2473. Fax: (951) 827-5696. E-mail: wilfred{at}engr.ucr.edu

{triangledown} Published ahead of print on 8 February 2008.

Applied and Environmental Microbiology, April 2008, p. 2239-2243, Vol. 74, No. 7
0099-2240/08/$08.00+0     doi:10.1128/AEM.00259-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Yeh, H.-Y., Yates, M. V., Mulchandani, A., Chen, W. (2008). Visualizing the dynamics of viral replication in living cells via Tat peptide delivery of nuclease-resistant molecular beacons. Proc. Natl. Acad. Sci. USA 105: 17522-17525 [Abstract] [Full Text]  
  • Cliver, D. O. (2008). Shedding light on virus replication. Proc. Natl. Acad. Sci. USA 105: 17213-17214 [Full Text]  


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