Immunity Effect Examination Of Cell-cloned Strains Cultivation Of VvIBDV GX8/99 And Relevance Analysis Of High Variation Area On VP2

Infectious bursal disease virus (IBDV), the causative agent of infectious bursal disease (IBD), is one of the major infectious diseases against the world's poultry industry. It causes immunosuppression in chickens by destroying the precursors of B lymphocytes in the bursa of Fabricius and enhances any other pestilence susceptibility. Recent years, molecular biology research of infectious bursal disease virus (IBDV) achieved great advance, but the study of immune and pathogenic mechanism, molecular structure and function of infectious bursal disease virus (IBDV) need further research. Thus, we had identified the epitope of infectious bursal disease virus (IBDV) which is valuable for further research such as the interaction between infectious bursal disease virus (IBDV) and its native host.IBDV is of double RNA virus avian genus in the double RNA virus family. Inactivated or the weak poisonous vaccine which was obtained from local separation's variation and the vaccine strain with the antigenicity similar to the local variation, as well as the multipartial vaccine made from many kinds of strains, provide good immunity protection for the chicken group. Recently, major projects of national natural fund studied more than 70 IBDV in each province. They determined the gene order of the high variation area in VP2 gene, inferred the amino acid sequence and analyzed the molecular foundation of the structure variation and sibship of the gene structures. vvIBDV GX8/99, of all the isolated vvIBDV, induces a comparative high disease incidence and mortality rate and the phenomenon of immune failure occurs frequently in chicken group. In order to grasp the immunogenicity regularity in the whole process of tenuated subculture and the regularity of"individual effect", which lay a foundation for the research of vvIBDV epidemiology and the effective prevention, thus molecular foundation of the evolution rule and toxicity variation. This study takes vvIBDV GX8/99 as the object, studied the immunogenicity regularity and relationship with the nucleotide and the amino acid through subculture for 30 generations from original virus, VanRoekel, cytotoxic, cell cloned to cloned strain; solved the urgent problems, such as how to strengthen toxicity of vvIBDV, whether being attenuated or not and what changes in the sequences of gene and nucleotide occurred in the process, and the relationship between the changes and the biological characteristics of virus, as well as the biological characteristics of weak strain.The research was divided into three parts:1. vvIBDV GX8/99 's cloning and subculture vvIBDV GX8/99 was first subcultured in the SPF chicken embryo for 10 generations, then 22 generations in the the ciliary cell (CEF), and it adapted CEF, for the cell gathers for the cell shrinks, pulls into the filament filiform and finally becomes full and falls off. Then the second clone carried on through the infinite dilution method, 4 strains were obtained. These 4 strains were in attenuated culture on CEF for 30 generations, respectively. Each transfer of generation was examined through the immunity colloid gold indicator paper membrane, IBDV was detected in each transfer of generation except for cytotoxicity before adapting CEF. Acquisition of different generations of vvIBDV GX8/99 strain built a new technological platform for studying the immunogenicity regularity and relationship with the nucleotide and the amino acid, grasping the immunity feature of vvIBDV in the whole circle of subculture, and the regularity of its"individual effect".2.The variation of VP2 region of 4 attenuated cloning virus strains of vvIBDV GX8/99In order to study relevance of high variable region of VP2 gene from vvIBDV GX8/99 to biological characteristics,The study is try to find out the evolution of four strains of cloning's nucleotide sequences and amino acids during the process of attenuation,by Polymerase chain reaction (PCR) ,gene cloning of cloning (GX-IBDVE10C25CL1,GX-IBDVE10C25CL2,GX-IBDVE10C25CL4,GX-IBDVE10C25CL5).Compared with the initial bursa virus ,12 sites in 145 amino acids of high variable region of VP2 from four viruses changed the same ,identical to that of the vaccine D78 virus stain .The chickens were immunized by four strains viruses in the age of 14d.Experiments show that the variation of VP2 region associate with immunogenicity,and had something to do with cell culture or tissue appetency.four strains viruses had transitory tissue damnification ,variations in the virus are regular.conclusion provides theoretical basis.3. Analysis of immune effect of vvIBDV GX8/99's cloning virus generation F10,F20,F25,F30vvIBDV GX8/99's 4 strains immunized to the 14d-chicken with 10000TCID50 dose through oral ingestion, and the second immunization to the 21d-chicken, and the death situation was record. Immune body examination and whole blood analysis were carried on in all the immunity groups with chicken's serum, after the first exempts 3 days and 10 days and 7d, 14d, 20d, 30d after the second immunization. Then the attack virus test was carried on with 40d-chicken. The result showed that 10000TCID50 dose provided 100% protection for F20 and F25, and 14 days after the second immunization, the concentration of immune body achieves the peak. Although F30 caused damage for very few bursa, this kind of damage is reversible. Among the 4 strains, GX-IBDVE10C25CL4-30 in F30 exhibited stable immunity effect and light pathological change, which was expected to be ideal vaccine for the control of vvIBDV infection.