In addition to detecting viruses, there is a strong need to develop tools to study the pathogenesis of viruses and their response to antiviral agents. There is a definite need for rapid, simple, sensitive, and specific tests for common viruses and for emerging infectious agents, such as severe acquired respiratory syndrome-associated coronavirus, highly virulent viruses such as influenza H5N1 (avian flu) ( 12), and possible bioterror agents such as variola virus (which causes smallpox). Some viruses, such as influenza virus and RSV, cause local respiratory infections, while others, such as varicella-zoster virus, mumps virus, and measles virus ( 22), use the respiratory tract as an entryway to other parts of the body. Even though the respiratory mucosal environment and associated cells have many methods of preventing viral entry, many viruses, such as influenza virus, severe acquired respiratory syndrome-associated coronavirus, parainfluenza virus, and respiratory syncytial virus (RSV), find ways of thwarting the body's many defenses and taking up residence within the cells of the respiratory tract ( 16). One of the predominant routes for viruses to enter a host is via the respiratory tract ( 22). Rapid detection of viral infections and understanding viral pathogenesis are crucial for the prevention of an infectious disease outbreak, development of antiviral drugs, and biodefense.
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.
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. Low background in uninfected cells and simultaneous staining of fixed cells with molecular beacons and antibodies showed high detection specificity. We found that molecular beacon signal could be detected in single living cells infected with a viral titer of 2 × 10 3.6 50% tissue culture infective doses/ml diluted 1,000 fold, demonstrating high detection sensitivity. 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. 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. 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. Understanding viral pathogenesis is critical for prevention of outbreaks, development of antiviral drugs, and biodefense.
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