Chen Liu, Ni Zheng, Hua Jionggang, Ye Weicheng, Liu Keshu, Yun Tao, Zhu Yinchu, Zhang Cun
Institute of Animal Husbandry and Veterinary Sciences, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China.
Virus Res. 2021 May;297:198393. doi: 10.1016/j.virusres.2021.198393. Epub 2021 Mar 13.
Duck enteritis virus (DEV) can cause an acute, contagious and lethal disease of many species of waterfowl. An infectious bacterial artificial chromosome clone of DEV vaccine strain pE1 (pDEV-EF1) has been constructed in our previous study. Based on pE1, a recombinant mutated clone pDL (pVP26CFP-gCRFP), which carries a red fluorescent protein (mRFP) gene fused to the viral envelope protein gC in combination with a cyan fluorescent protein (CFP) gene fused to the viral capsid VP26, was constructed by two-step Red/ET recombination and the recombinant virus rDL (rVP26CFP-gCRFP) was rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate transfection. Western blot analysis revealed that VP26-CFP and gC-mRFP were both expressed in fusion forms in rDL-infected CEFs, and subcellular localization study showed that gC-mRFP was mainly localized in whole cell at 36, 48 h post infection (p.i.); and then mostly migrated to the cytoplasm after 60 h.p.i., ; whereas VP26-CFP was localized in the nucleus in all stages of virus infection. Additionally, viral particles at different stages of morphogenesis (A capsids, B capsids, C capsids) were observed in virus-infected cells by transmission electron microscopy, indicating that exogenous gene insertion has no effect on virus assembly. This study has laid a foundation for visually studying localization, transportation of DEV capsid proteins and envelope glycoproteins as well as virus assembly, virion movement and virus-cell interaction.
鸭肠炎病毒（DEV）可引起多种水禽的急性、传染性和致死性疾病。在我们之前的研究中构建了DEV疫苗株pE1的感染性细菌人工染色体克隆（pDEV-EF1）。基于pE1，通过两步Red/ET重组构建了一个重组突变克隆pDL（pVP26CFP-gCRFP），其携带与病毒包膜蛋白gC融合的红色荧光蛋白（mRFP）基因以及与病毒衣壳VP26融合的青色荧光蛋白（CFP）基因，并通过磷酸钙转染从鸡胚成纤维细胞（CEF）中拯救出重组病毒rDL（rVP26CFP-gCRFP）。蛋白质免疫印迹分析显示，VP26-CFP和gC-mRFP在rDL感染的CEF中均以融合形式表达，亚细胞定位研究表明，gC-mRFP在感染后（p.i.）36、48小时主要定位于全细胞；然后在感染后60小时大多迁移至细胞质；而VP26-CFP在病毒感染的各个阶段均定位于细胞核。此外，通过透射电子显微镜在病毒感染的细胞中观察到不同形态发生阶段（A衣壳、B衣壳、C衣壳）的病毒颗粒，表明外源基因插入对病毒组装没有影响。本研究为直观研究DEV衣壳蛋白和包膜糖蛋白以及病毒组装、病毒体运动和病毒-细胞相互作用奠定了基础。
