Comparisions Of The Pathogenicity,Immunogenicity And VP5 Gene Of The Cell-adapted Virus And Chicken-passaged Virus Of VvIBDV GX8/99 Strain

Infectious bursal disease (IBD) is caused by infectious bursal disease virus (IBDV) and causes a highly contagious immunosuppression and death of young chickens, which cause a significant economic loss for the poultry industry.The GX8/99 strain was separated from chicken infected by IBDV naturally in Guangxi Province, which proved to be a strain of very virulent virus by animal experiments.In order to research the characters and the mutation mechanisms of the GX8/99 strain, with the cell cloned virus that has been passaged in CEF for 22 generations, we got a series of passaged strains of the GX8/99 by passaging them on 3 to 5-week-age chicken of SPF for 23 generations. In order to study the relationship between the mutation of VP5 and the pathogenicity of IBDV, we compared the pathogenicity of GX8/99 original virus, cell-adapted virus, SPF-chicken-passaged virus and cloned their VP5 genes.1. The pathogenicity experiments of the cell-adapted virus strain and the SPF-chicken-passaged virus strainsThe results suggested that the GX8/99, vvIBDV strain, have been attenuated after adapting to the CEF cell, the mortality of the 4-week-age SPF chicken infected the cell-adapted virus,GXC23, was 0-5%.And when the cell-cloned virus were returned to SPF-chicken, the pathogenicity did not increased, the mortality was also 0-5%.2. The comparison of the virus dose in the bursal of the chickens infected the GX8/99 original strain and the different passaged virusInfected the 4-week-age SPF-chicken with the same dose of the different passaged virus, diluted the same dose of the suspension of the bursal of the chicken infected the virus for 3 days, 5 days and 7 days at 10×times, examined the virus by immunogold test paper film separately, take the maximum dilution time as the relative amount of the bursal of the chicken. The result demonstrated that the cell-adapted virus reproducted more slowly in the bursal than the GX8/99 original virus and after being passaged on SPF-Chicken for 20 generations, the virus also reproducted more slowly than the GX8/99 original virus.3. The pathogenicity and immunogenicity of the SPF-chicken backpassaged virus of the GX8/99 cell-adapted virusThe mortality in chickens challenged with 3 backpassaged viruses GX-2B23,GX-4B23,GX-5B23 was 0/15,0/15,1/15. In order to research the immunogenicity of the backpassaged virus, we immuned the 2-week-age SPF chicken with GX-2B23,GX-4B23, and infected the vvIBDV GX8/99 after 3 weeks. The result shows that both of them could make the bursal atrophie and decrease the antibody degree of the NDV and AIV (H9-, H5-) vaccine significantly. But they both have very good immunogenicity, the protective degree was 100%.4. The comparison of the VP5 gene of IBDV field strains and the passaged strains of vvIBDV GX8/99 strainThe comparison of the VP5 gene of 25 different virulent IBDV strains showed that the VP5 gene was very conservative, their homologies were 97%-100%. However, there were 8 mutation sites, and 5 of them made the corresponding amino acids change. The sequence comparison of VP5 genes of GX8/99 and GXC23 demonstrated mutations of 8 base sites and 5 of them made the amino acid changes. Especially, the mutation from T to C at bp#2 of VP5 gene made GXC23 to lose its first"ATG"and 4 aa at the N-terminal of VP5 protein. The mutations were very similar to 3 vaccine strains. Furthermore, such mutations did not change in the 3 backpassaged viruses after passaged for 23 generations in chicken as their pathogenicity was still low as GXC23. However, further comparisons indicated that the bases at the sites of VP5 genes in 4 vvIBDV reference strains and 4 vIBDV reference strains were exactly same as primary GX8/99, while another 3 vvIBDV reference strains and 6 vIBDV strains were same as cell-adapted GXC23. The results suggested that the 5 amino acids mutations of VP5 gene from the primary GX8/99 to its cell-adapted GXC23 and backpassaged viruses were not associate with pathogenicity but more related to adaptation processes in cell cultures.The GX-2B23,GX-4B23 could induce the body produce high antibody degree of IBD and could protect the immuned chicken not to be infected by the vvIBDV GX8/99 original virus. It suggested that although some amino acids have mutated in the passaging process, the antigenic determinant connected with the immune defence in different genes did not change significantly.