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Live-cell characterization and analysis of a clinical isolate of bovine respiratory syncytial virus, using molecular beacons.
- Source :
-
Journal of virology [J Virol] 2006 Jan; Vol. 80 (2), pp. 682-8. - Publication Year :
- 2006
-
Abstract
- 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 10(3.6) 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.
- Subjects :
- Animals
Cattle
Cattle Diseases virology
Cells, Cultured
Fluorescence Resonance Energy Transfer
Genome, Viral
Microscopy, Confocal
Nucleic Acid Hybridization methods
Oligonucleotide Probes
Respiratory Syncytial Virus Infections virology
Respiratory Syncytial Virus, Bovine genetics
Respiratory Syncytial Virus, Bovine growth & development
Sensitivity and Specificity
Staining and Labeling
Cattle Diseases diagnosis
Respiratory Syncytial Virus Infections diagnosis
Respiratory Syncytial Virus Infections veterinary
Respiratory Syncytial Virus, Bovine isolation & purification
Subjects
Details
- Language :
- English
- ISSN :
- 0022-538X
- Volume :
- 80
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Journal of virology
- Publication Type :
- Academic Journal
- Accession number :
- 16378971
- Full Text :
- https://doi.org/10.1128/JVI.80.2.682-688.2006