Institute for Animal Health, Pirbright Laboratory, Woking, Surrey, UK.
J Gen Virol. 2010 Dec;91(Pt 12):2918-27. doi: 10.1099/vir.0.023598-0. Epub 2010 Aug 18.
A full-length DNA clone of a virulent strain of rinderpest virus was constructed with the gene for the enhanced green fluorescent protein (eGFP) inserted as a separate transcription unit between the P and M genes. Rescue of the virus from the modified clone using reverse genetics generated a virus that grew to the same levels as the virus rescued from the unmodified DNA clone in cell culture. The recombinant virus expressed eGFP to a high level and was used to follow virus replication in real-time using live-cell imaging. Cattle infected with both the recombinant wild-type virus and the recombinant eGFP expressing virus developed clinical disease similar to that of the wild-type natural virus isolate. Detection of virus in circulating peripheral blood leukocytes was equivalent to that of the animals infected with the wild-type virus. The high level of expression of soluble eGFP by this virus allowed us to detect viral replication in infected animals by confocal microscopy. Imaging vibrating microtome sections by confocal microscopy provided good preservation of tissue and cellular architecture as well as revealing the sites of replication of the virus in different tissues of infected animals.
构建了一个全长 DNA 克隆，其中包含牛瘟病毒的强毒株，并在 P 和 M 基因之间插入了增强型绿色荧光蛋白（eGFP）基因，作为一个单独的转录单元。使用反向遗传学从修饰后的克隆中拯救病毒产生了一种病毒，其在细胞培养中与从未修饰的 DNA 克隆中拯救的病毒的生长水平相同。重组病毒高水平表达 eGFP，并用于使用活细胞成像实时跟踪病毒复制。感染重组野生型病毒和表达 eGFP 的重组病毒的牛表现出与野生型天然病毒分离株相似的临床疾病。在循环外周血白细胞中检测到的病毒与感染野生型病毒的动物相同。该病毒可溶性 eGFP 的高表达水平使我们能够通过共聚焦显微镜检测感染动物中的病毒复制。通过共聚焦显微镜对振动切片机切片进行成像，为组织和细胞结构的良好保存提供了条件，同时还揭示了病毒在感染动物不同组织中的复制部位。
