Rozelle Daniel K, Filone Claire Marie, Dower Ken, Connor John H
Department of Microbiology, Boston University School of Medicine.
Department of Microbiology, Boston University School of Medicine;
J Vis Exp. 2014 May 15(87):51522. doi: 10.3791/51522.
Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.
痘病毒是一类双链DNA病毒，其中包括活跃的人类病原体，如猴痘病毒、传染性软疣病毒和孔塔加洛病毒。该病毒家族还包括天花病毒，即痘苗病毒。由于痘病毒复制过程的复杂性，关于其基因表达策略仍存在许多问题。在本文中，我们描述了重组痘苗病毒的概念和用途，这些病毒能够以高通量形式实时测量病毒基因表达的单个和多个阶段。这是通过使用光谱上不同的荧光蛋白作为病毒复制三个阶段各自的报告基因来实现的。这些病毒在保留阶段特异性表达模式的同时提供了高信噪比，从而能够进行基于平板的检测以及对病毒传播和复制的显微镜观察。这些工具可用于抗病毒药物研发、病毒与宿主相互作用的研究以及进化生物学研究。
