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 × 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 682-688 |
| Number of pages | 7 |
| Journal | Journal of Virology |
| Volume | 80 |
| Issue number | 2 |
| DOIs | |
| State | Published - Jan 2006 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Immunology
Cite this
Live-cell characterization and analysis of a clinical isolate of bovine respiratory syncytial virus, using molecular beacons. / Santangelo, Philip; Nitin, Nitin; LaConte, Leslie; Woolums, Amelia; Bao, Gang.
In: Journal of Virology, Vol. 80, No. 2, 01.2006, p. 682-688.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Live-cell characterization and analysis of a clinical isolate of bovine respiratory syncytial virus, using molecular beacons
AU - Santangelo, Philip
AU - Nitin, Nitin
AU - LaConte, Leslie
AU - Woolums, Amelia
AU - Bao, Gang
PY - 2006/1
Y1 - 2006/1
N2 - 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 × 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.
AB - 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 × 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.
UR - http://www.scopus.com/inward/record.url?scp=30344469922&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=30344469922&partnerID=8YFLogxK
U2 - 10.1128/JVI.80.2.682-688.2006
DO - 10.1128/JVI.80.2.682-688.2006
M3 - Article
C2 - 16378971
AN - SCOPUS:30344469922
VL - 80
SP - 682
EP - 688
JO - Journal of Virology
JF - Journal of Virology
SN - 0022-538X
IS - 2
ER -