Yu Kexiang, Ma Xiuli, Li Yufeng, Wu Jing, Lin Shuqian, Jiang Yifei, Huang Bing
Institute of Poultry, Shandong Academy of Agricultural Sciences, Key Laboratory of Poultry Disease Diagnose and Immune of Shandong Province, Jinan 250023, China.
Wei Sheng Wu Xue Bao. 2009 Dec;49(12):1650-4.
To construct an infectious clone for studying functions of duck hepatitis virus (DHV) typel genome by reverse genetic technique.
Three fidelity DNA fragments covering the full genome of DHV type 1 CL strain were amplified by RT-PCR, and inserted into pBR322 vector, resulting in the full-length cDNA clone BR-CL. The in vitro-transcribed RNA from BR-CL was transfected into duck embryo renal cells and the rescued virus was identified using RT-PCR, indirect immunofluorescence assay and colloidal gold immunoelectron microscopy after six generations. After inoculating the rescued virus into SPF chick embryos, embryo death and pathological changes were observed.
The results of RT-PCR, indirect immunofluorescence assay and immunoelectron microscope showed that infectious virus was rescued. After inoculating into SPF chick embryos, the rescued virus was able to kill embryos with pathogenic changes.
This is the first report on generation of infectious cDNA clone of DHV, which provides a valuable platform for further research on functions of DHV genome.
通过反向遗传技术构建鸭肝炎病毒1型（DHV-1）基因组功能研究的感染性克隆。
采用RT-PCR扩增覆盖DHV-1 CL株全基因组的3个保真DNA片段，并将其插入pBR322载体，获得全长cDNA克隆BR-CL。将BR-CL体外转录的RNA转染鸭胚肾细胞，传代6次后，用RT-PCR、间接免疫荧光试验和胶体金免疫电镜鉴定拯救病毒。将拯救病毒接种SPF鸡胚，观察鸡胚死亡及病理变化。
RT-PCR、间接免疫荧光试验及免疫电镜结果显示成功拯救出感染性病毒。接种SPF鸡胚后，拯救病毒可致鸡胚死亡并出现病变。
这是关于DHV感染性cDNA克隆构建的首次报道，为进一步研究DHV基因组功能提供了有价值的平台。
