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

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.

FIG. 1.

Live-cell fluorescence imaging of bRSV viral genome following the progression of infection for 7 days p.i. The left column contains fluorescence images of infected cells and the right column contains overlay images of the fluorescence and white-light observations of cells.

FIG. 2.

Comparison of model prediction and experimental data for spreading of viral infection. The number of infected cells (y) is plotted as a function of time (days), showing the progression of infection over a 13-day period. The parameters for the model were determined either independently (λ) or from the measurements of days 1, 3, and 5 postinfection (α, β, and γ). Comparison between prediction and experimental data for the number of infected cells at day 7 postinfection showed good agreement, indicating the predictive capability of the model. Note that the number of infected cells reached a plateau around day 9 postinfection.

FIG. 3.

Detection sensitivity studies utilizing serial dilutions of virus stock. Virus with a titer of 2 × 10^3.6 TCID₅₀/ml was diluted to dilutions of 10⁰ (A), 10⁻¹ (B), 10⁻² (C), and 10⁻³ (D) and was used to infect four-well plates of bovine turbinate cells at 50% confluence. They were imaged 12 days after infection, at which time there were no more signs of increasing cytopathic effect. Note that viral infection could be detected in single cells even at a 1,000-fold dilution of virus stock.

FIG. 4.

Results of confocal imaging of representative cells showing the structure of cytoplasmic inclusion bodies formed from aggregates of viral genomic RNA in both a live cell (A) and fixed cells (B). Starting from the top left corner of panels A and B, each image represents a slice of the cell with a thickness of 0.5 μm. The images were taken starting from the bottom of the cell with a 0.5-μm gap in between (not all images are shown). The last images in both panels A and B show an overlay of the fluorescence and white-light images of the cell. Live-cell images show large, connected, amorphous inclusion bodies, while the images of inclusion bodies in fixed cell are less connected and spherical shaped.

FIG. 5.

Additional control studies to confirm the composition of inclusion bodies and the specificity of the MB-based method. Staining of all RNA molecules in infected (A) and uninfected (B) living cells with RNA-specific dye SytoRNAselect clearly indicated that the inclusion bodies seen in panel A only are composed predominately of RNA. Further, simultaneous MB and MAb staining of the bRSV genome and the F protein, respectively, exhibited both red (MB) and green (MAb) signals in the infected cells (C) but not in uninfected cells (D), demonstrating the high specificity of the MB-based approach in virus detection. In addition, in cells 5 days postinfection, MBs targeting bRSV showed a strong fluorescence signal (E), whereas the negative-control MB with mismatches gave a very low signal (F), further confirming the high specificity of the MB-based approach.