Live-cell characterization and analysis of a clinical isolate of bovine respiratory syncytial virus, using molecular beacons

Philip Santangelo, Nitin Nitin, Leslie LaConte, Amelia Woolums, Gang Bao

Research output: Contribution to journalArticle

64 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)682-688
Number of pages7
JournalJournal of Virology
Volume80
Issue number2
DOIs
StatePublished - Jan 2006
Externally publishedYes

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Bovine respiratory syncytial virus
antiviral agents
Viral Genome
cells
Antiviral Agents
image analysis
oligonucleotide probes
genome
Oligonucleotide Probes
fluorescent dyes
RNA Viruses
Optical Imaging
Inclusion Bodies
inclusion bodies
Signal-To-Noise Ratio
Virus Diseases
viral load
Confocal Microscopy
infection
Disease Outbreaks

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 journalArticle

Santangelo, Philip ; Nitin, Nitin ; LaConte, Leslie ; Woolums, Amelia ; Bao, Gang. / Live-cell characterization and analysis of a clinical isolate of bovine respiratory syncytial virus, using molecular beacons. In: Journal of Virology. 2006 ; Vol. 80, No. 2. pp. 682-688.
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