| Infectious bursal disease , an infectious disease characteristic of bursal lesion is caused by infectious bursal disease virus (IBDV) .In 1957,the disease first broke out in broilers in Gumboro,Telahua state,America. The initial strain of virus was of high pathogenicity but low virulence ,it caused less than 5% specific mortality in usual,20-30% occasionaly.It was defined as the standard strain of IBDV(vIBDV) .In the late 1980s,the variation strain of IBDV in sharp contrast with the standard strain was first appeared in America. Acute IBD caused by very virulent infectious bursal disease virus (vvIBDV ) was first reported in Europe at the end of 1980s,causing more than 90% specific mortality.The acute form of the disease were then transmitted to Japan in the early 1990s , and then rapidly spreaded all over Asia and other major parts of the world . It was more severe in industry countries advanced in poultry industry. The vvIBDV has been the cause of significant economic losses in poultry industry for a long time. Because when the chickens are infected by vvIBDV ,it not only causes chickens death directly but also induce immunosuppression and vaccination failure.In the recent years ,along with the popularity of the technic in molecular virology, the RNA and amino acid sequence of VP2.gene of vIBDVs and vvIBDVs have been compared in many different laboratories of the world including china. At the beginning, the researchers found some amino acid mutation in vvIBDVs and they thought it to be associated with pathogenicity. But recently , some researchers considered the mutation more as a common evolutionary symbol than a virulence symbol. Compariso'n between VP2 genes of isolations come from different strains of the same virus which is not cloned can not accurately answere this question. In our research ,we inoculated chicken embryo fibroblast (CEF) witha strain of vvIBDV and get several cell-cloned strains of the virus . And then we injected chickens of SPF with the cell-cloned strains , try to ascertain wether the mutation of VP2 gene has connection with the pathogenicity of vvIBDV.In the project financed by the Country Natural Funds ,our laboratory collected 72 strains of IBDV from 30 different provinces and citys.Based on the examination of the 40 strains in virulence,serology and pathogenicity, the GX8/99 strain was selected. Its virulence is the highest and the mortality is 90%. After the passage of the GX8/99 virus strain,we did research in the cycle of the initial bursa virus (from chicken) → embryo- adapted virus→ cell-adapted virus → cell-cloned virus → SPF -chicken-passage virus (from chickens).We also examined the evolution rules of pathogenicity and the relationship of the pathogenicity with nucleotide and amino acid.Primarily grasped the pathogenic characteristics of the vvIBDV during the circle of passage .We examined the evolution rules of variation of vvIBDV from the colony's pathogenicity of pre-cloned virus to individual ones of cloned virus .Appliation of the method will contribute to revealthe evolution rules of vvIBDV and the molecule basic of virulence changing. It can also solve such problems :how to increase the virulence of vvIBDV,wether the vvIBDV can be attenuated ,what has changed in gene and nucleotide sequences during attenuated process, what is the relationship between the change and the virus biological characteristics , what is the biological characteristics of the attenuated virus strain and so on.The main contents and results of this research:1. The passgage culture and cloning of the GX8/99 strain ofvvIBDV : The passage of the GX8/99 strain of vvIBDV on chicken embryos of specific pathogen free chickens (SPF) first for 10 generations is followed by the blind passage of it on CEF for 22 generations and the virus begin to adapte to CEF and cause cytopathogenic effect( CPE), with the cell gathered, drawn to fibril, then changed to round and shed off finally.Cloning the virus two times by infinite dilution method can get 4 cloned strains of the vv...
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