Applied and Environmental Microbiology, January 2010, p. 584-588, Vol. 76, No. 2

Detection of Infective Poliovirus by a Simple, Rapid, and Sensitive Flow Cytometry Method Based on Fluorescence Resonance Energy Transfer Technology

Department of Environmental Sciences, University of California, Riverside, California 92521, Department of Chemical and Environmental Engineering, University of California, Riverside, California

Received 31 July 2009/ Accepted 12 November 2009

The rapid and effective detection of virus infection is critical for clinical management and prevention of disease spread during an outbreak. Several methods have been developed for this purpose, of which classical serological and viral nucleic acid detection are the most common. We describe an alternative approach that utilizes engineered cells expressing fluorescent proteins undergoing fluorescence resonance energy transfer (FRET) upon cleavage by the viral 2A protease (2A^pro) as an indication of infection. Quantification of the infectious-virus titers was resolved byusing flow cytometry, and utility was demonstrated for the detection of poliovirus 1 (PV1) infection. Engineered buffalo green monkey kidney (BGMK) cells expressing the cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) substrate linked by a cleavage recognition site for PV1 2A^pro were infected with different titers of PV1. After incubation at various time points, cellswere harvested, washed, and subjected to flow cytometry analysis. The number of infected cells was determined by counting the number of cells with an increased CFP-to-YFP ratio. As early as 5 h postinfection, a significant number of infected cells (3%) was detected by flow cytometry, and cells infected with only 1 PFU were detected after 12 h postinfection. When applied to an environmental water sample spiked with PV1, the flow cytometry-based assay provided a level of sensitivity similar to that of the plaque assay for detecting and quantifying infectious virus particles. This approach, therefore, is more rapid than plaqueassays and can be used to detect other viruses that frequently do not form clear plaques on cell cultures.