Complete Genomic Cloning And Development In The Reverse Genetics System For IBDV B87 Strain

The study of virus and its interactions with host cells and organisms has benefited greatly from the ability to engineer specific mutations into viral genomes, a technique known as reverse genetics.Infectious bursal disease (IBD) is one of the most contagious disease of poultry industry of the world.Infectious bursal disease virus (IBDV) is the pathogen ,which belongs to Birnaviridae family. To develop the reverse genetics system or so called genetic rescue techniques for IBDV, the full-length cDNA of the double segment of the chicken IBDV B87 strain was amplified and cloned by long RT-PCR.And a vaccine strain of IBDV with molecular marker was rescued by reverse genetics manupalution. Sequence analysis showed that A segment of IBDV B87 strain was 3260bp,and B segment was2827bp.And the sequence of A and B segment were submitted into GeneBank (Accession number DQ906922 and DQ906921,respectively).Homologous and bioinformation analysis of IBDV B87 showed that A segment of B87 has highest homologous with Harbin Gt strain,and homologous of B segment of B87 with German P2 strain was 100%,which tell us that B87 may be used for a long time in northeast of China,with A segment mutanted but B segment conserved. Compared with reference sequence showed that 12aa of VP1,11aa of VP2,3aa of VP3 and 5aa of VP4 changed regularity,and VP2 was not the only one gene of IBDV antigen,VP1,VP3,VP5 and VP4 was conserved,which showed virulence of IBDV controlled by polygene.And conserved NCR sequence of different virulence strains of IBDV showed NCR didnot have direct relationship with virulence.In order to develop molecular marker, two recombinant cDNA plasmids of B87,named pMD-18TSA and pMD-18TSB respectively ,was constructed with T7 promoter upstream of each 5′NCR.And genetics marker was inserted into IBDV using synonymous codon by PCR. A2815 and A2817 of A segment changed to C2815 and T2817 and formed a RE site of MluI, A1723 and G1725 of B segment changed to C1723 and C1725 and formed a RE site of SacII. Then the two plasmids was linearized,transcripted in vitro and transfected into Vero cell.The results showed molecular marker vaccine of IBDV was constructed successfully,proved by ELISA,RT-PCR and restriction analysis by MulI and SacII.Furthermore,two recombinant plasmids of IBDV B87,named pEGFP-N1mA and pEGFP -N1mB,was constructed with the two segment of B87 inserted into pEGFP -N1,an eukaryotic expression plasmid. Co-transfection of pEGFP-N1mA and pEGFP -N1mB with Lipofectamine into Vero cells resulted in the expression of IBDV RNA and proteins,which were confirmed by RT-PCR and immunofluorescene assay analysis.The change of cell morphology after co-transfection was similar to the situation cellular pathogenic effects(CPE). The virus-like particles can be found by electron microscopy,confirmed the rescue of IBDV. These genetic markers retained in the recovered progeny virus can be detected by RT-PCR and restriction analysis by MulI and SacII also proved the result that IBDV was rescued successfully.