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Published on: January 7, 2019
[Construction of infectious molecular clone of duck hepatitis virus type 1 CL strain]
Kexiang Yu1, Xiuli Ma, Yufeng Li
1Institute of Poultry, Shandong Academy of Agricultural Sciences, Key Laboratory of Poultry Disease Diagnose and Immune of Shandong Province, Jinan 250023, China. yukx1979@163.com
Objective:
To construct an infectious clone for studying functions of duck hepatitis virus (DHV) typel genome by reverse genetic technique.
Methods:
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.
Results:
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.
Conclusion:
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.
