Construction Of Reverse Genetics Research System Of Rabbit Hemorrhagic Disease Virus

Using reverse genetics systems for RNA virus rescue and manipulating in DNA level is a strong technique to study molecular biology and interactions between RNA virus and host. Genetic rescue or constructed infectious clone of RNA virus become the necessary way to study structure and function of virus in molecular virology laboratory. Rabbit hemorrhagic disease is a kind of acute﹑lethal infectious disease caused by rabbit hemorrhagic disease virus and have a great harm to rabbit production all over the world. This study selected rabbit hemorrhagic disease virus which was the pathogen of Rabbit hemorrhagic disease as a model on the basis of obtainment of full-length cDNA clone of RHDV JX/97 strain and attempt to construct RHDV reverse genetics systems and rescue molecular marked RHDV in our country firstly.1. Construction of full-length genome cDNA clone of RHDV JX/CHA/97 strainFive pairs of primers were designed for sequencing the genome of JX/CHA/97.Strain virus total RNA was extracted from rabbit liver tissue died from RHDV infected and each sequence was obtained by RT-PCR .A molecular marker (EcoR V enzyme site) was introduced to distinguish wild-type virus and rescue virus. Each gene amplified fragments and pBluescript II SK(+) vector were digested by restriction enzyme to construct full-length genome cDNA(pBlRHDV)clone of JX/97 strain. The pBlRHDV was identified by PCR﹑restriction enzyme digested and genome sequenced. The results showed that full-length cDNA clone of RHDV JX/97 strain was constructed successfully. The whole genome sequence﹑SP6 promoter sequence﹑a molecular marker(EcoR V enzyme site) and restriction enzyme digestion site added which primer designed were all found in pBlRHDV clone.2.Identification of infection of full-length cDNA clone(pBlRHDV)of RHDV JX/CHA/97 strainpBlRHDV used as a template after linearized with Nru I and virus RNA synthesized in vitro by means of RiboMAXTM Large Scale RNA Production Systems- SP6 polymerase system . RK-13 cell was transfected with transcribed product in vitro using lipofectamine reagent, typical RHDV CPE would be seen after 24 hour. RT-PCR﹑inoculated subcutaneousely and intraperitoneally to unvaccinated sensitive healthy rabbit and immunosorbent electron microscope observation was used for detection. All the detection results showed rescued virus was obtained and constructed infectious cDNA clone of RHDV successfully. The infection of wild-type virus was excluded through detection of molecular marker of infectious clone.In a word, RHDV reverse genetics can be used to study functional genomics, molecular mechanism of pathogenesis and variation of RHDV and show some prospects in research of gene-deleted vaccine and marker vaccine.