Santangelo Philip, Nitin Nitin, LaConte Leslie, Woolums Amelia, Bao Gang
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Dr., Atlanta, GA 30332, USA.
J Virol. 2006 Jan;80(2):682-8. doi: 10.1128/JVI.80.2.682-688.2006.
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.
了解病毒发病机制对于预防疫情爆发、开发抗病毒药物和生物防御至关重要。在此，我们利用分子信标直接检测病毒基因组，并对活细胞中的牛呼吸道合胞病毒（bRSV）临床分离株进行表征。分子信标是一种双标记的发夹寡核苷酸探针，一端带有报告荧光团，另一端带有猝灭剂；它们被设计成仅在与互补靶标杂交时才发出荧光。通过对分子信标的荧光信号进行成像，以50至200的信噪比监测bRSV的传播7天，并使用病毒生长的数学模型对测量的感染时间进程进行量化。我们发现，在感染病毒滴度为2×10(3.6) 50%组织培养感染剂量/毫升且稀释1000倍的单个活细胞中可以检测到分子信标信号，这表明检测灵敏度很高。未感染细胞中的低背景以及用分子信标和抗体对固定细胞进行同时染色显示出高检测特异性。此外，使用共聚焦显微镜对活的感染细胞中的病毒基因组进行成像，我们观察到一种在固定细胞中未见的相连的、高度三维的、无定形的包涵体结构。综上所述，使用分子信标进行活性病毒成像为快速病毒感染检测、RNA病毒表征以及新型抗病毒药物设计提供了一个强大的工具。
