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Journal of Virology, January 2006, p. 682-688, Vol. 80, No. 2
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.2.682-688.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Live-Cell Characterization and Analysis of a Clinical Isolate of Bovine Respiratory Syncytial Virus, Using Molecular Beacons

Philip Santangelo,1 Nitin Nitin,1 Leslie LaConte,1 Amelia Woolums,2 and Gang Bao1*

Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332,1 Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, Georgia 306022

Received 8 June 2005/ Accepted 18 October 2005

Understanding viral pathogenesis is critical for prevention of outbreaks, development of antiviral drugs, and biodefense. Here, we utilize molecular beacons to directly detect the viral genome and characterize a clinical isolate of bovine respiratory syncytial virus (bRSV) in living cells. Molecular beacons are dual-labeled, hairpin oligonucleotide probes with a reporter fluorophore at one end and a quencher at the other; they are designed to fluoresce only when hybridizing to a complementary target. By imaging the fluorescence signal of molecular beacons, the spread of bRSV was monitored for 7 days with a signal-to-noise ratio of 50 to 200, and the measured time course of infection was quantified with a mathematical model for viral growth. We found that molecular beacon signal could be detected in single living cells infected with a viral titer of 2 x 103.6 50% tissue culture infective doses/ml diluted 1,000 fold, demonstrating high detection sensitivity. Low background in uninfected cells and simultaneous staining of fixed cells with molecular beacons and antibodies showed high detection specificity. Furthermore, using confocal microscopy to image the viral genome in live, infected cells, we observed a connected, highly three-dimensional, amorphous inclusion body structure not seen in fixed cells. Taken together, the use of molecular beacons for active virus imaging provides a powerful tool for rapid viral infection detection, the characterization of RNA viruses, and the design of new antiviral drugs.

* Corresponding author. Mailing address: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Dr., Suite 2115, Atlanta, GA 30332. Phone: (404) 385-0373. Fax: (404) 894-4243. E-mail: gang.bao{at}bme.gatech.edu.

Journal of Virology, January 2006, p. 682-688, Vol. 80, No. 2
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.2.682-688.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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