Isolation And Identification Of Two Very Virulent Infectious Bursal Disease Virus Strains With Natural Reassortment Characteristic And Expression Of The Clonied Genes VP2and VP3in E. Coli

Infectious bursal disease virus (IBDV) is the aetiological agent of an acute and highly contagious disease in young chickens. The disease is of major economic importance because subclinical infections with IBD virus (IBDV) cause losses to the poultry industry worldwide due to their severe, immunosuppressive effect, leading to concurrent or secondary infections. Since2004, several IBD epidemics outbreaks in the layer and broiler farms of East China, manifesting IBD vaccine failures, rapid onset and high mortality in epidemiological and clinical symptoms. Our laboratory isolated a few virus strains from chickens who had the same epidemiological characteristics. And we selected two strain samples to make the virus biological characteristic research. Two gene reassortment strains(QL and ZZ-11) were isolated and their whole genomes were measured and analyzed in this study. The major structural protein VP2and VP3were also expressed using E.coli expressing system.EXP1:Isolation and identification of two very virulent strains of infectious bursal disease virusTwo very virulent strains QL and ZZ-11of infectious bursal disease virus were isolated from vaccined chickens and studied for its mortality in SPF chickens at different age inoculated with bursal suspension of20-2,OOOELD50in repeated experiments. It demonstrated that QL and ZZ-11could induce high mortality between55%-100%in SPF chickens at the age of23-51days; it caused higher mortality of90%at the age of3-7weeks; it still caused some mortality of50%at the age of13-15weeks. The dead chickens showed leg and pectoral muscle bleeding, bursal enlargement, mucosal and serous bleeding, displaying the appearance of the "purple grapes"; Isolates of the virus can directly adapt to grow on chicken embryo but cannot grow on chicken embryo fibroblasts (CEF); VP2genes of QL and ZZ-11have the amino acid residual base features of vvIBDV, including222A,2561,279D,284A,2941,299S and SWSASGS in the heptapeptide sequence region.EXP2:Genomic sequence analyses of two reassortment strains of infectious bursal disease virus in natureTwo isolates of infectious bursal disease virus (IBDV) with new epidemiological characteristics in China were acquired and named as QL and ZZ-11. The mortality rate of specific pathogen free (SPF) chickens inoculated with the isolate QL was94%, and ZZ-11was86%. By sequencing, the genomes of QL and ZZ-11were same in size, and segment A and B were3260bp and2827bp, respectively. The phylogenetic analysis showed that segment-A of the two isolates were96.8%to98.1%and96.9%to98.4%homology to that of the published strains belonging to the very virulent strain branch; while segment-B was in the branch of the attenuated strains, the homologies were from89.7%to90.4%and from90.0%to90.7%. Segment A of the two isolates had molecular characteristics of very virulent strains with222A,249Q,253Q,254G,256I,294I,299S and heptapeptide motif SWSASGS in VP2gene; While segment B owned the sequence characteristics of attenuated strains which had not KpnI restriction sites at their777-782nucleotides (GGTGCC). It indicated that QL and ZZ-11might be the natural reassortment viruses with segment A derived from the very virulent strains and segment B from the attenuated strains, separately. The discovery of reassortment viruses in nature suggests an additional risk of using live IBDV vaccines, which could act as genetic donors for genome reassortment.EXP3:Cloning and expression of VP2and VP3gene of two very virulent infectious bursal disease virus strains with natural reassortment characteristicVP2and VP3gene of gene-reassortant very-virulent infectious bursal disease virus(IBDV) strain(QL) were amplified and analysed. The VP2gene had222A,253Q,2.56I,279D,284A,294I,299S and heptapeptide motif SWSASGS the same as very virulent strains, besides it also had three specific amino acid residues(127A,324R,370S); The homology of VP3gene between QL and other IBDV strains was over97.3%, but there were also799A,849G,931V,976D four specific amino acid residues in the VP3gene. The VP2and VP3gene were cloned into the prokaryotic expression vector pET32a. The expression plasmid pET32a-VP2and pET32a-VP3were constructed and transformed into competent E.coli BL21(DE3), the target protein were expressed under the induction with IPTG. SDS-PAGE analysis showed that the VP2fusion protein was approximately67kDa in molecular weight, and it made up30%of the total bacterial proteins as inclusion bodies in E.coli, and the VP3gene fusion protein was approximately57kDa in molecular weight, and it made up40%of the total bacterial proteins. Western blot showed that the recombinant VP2and VP3protein could be recognized by IBDV positive serum. The indirect ELISA for the detection of antibody against IBDV by using purified recombinant VP2and VP3protein have good specificity.