| In this research, one strain of very virulent infectious bursal disease virus(wIBDV) with high pathogenicity was isolated and identified from south of China. One attenuated strain with high immunity and CEF adaptation was also obtained from this wffiDV through culturing and replicating in SPF embryo and CEF successfully. The changes of VP2 gene, which was identified as the host-protective antigen of BBDV, from this strain of wBDV to attenuated one were investigated during the process of attenuatioa The VP2 gene of this wIBDV strain was high-level expressed in Pichia yeast expression system. VP2 expressed could induce high level of immunity and protected the chickens from lethal attack by highly virulent wIBDV after VP2 was emulsified to be subunit vaccine, all the results showed that this kind of subunit vaccine would provide advanced and reliable technology supporting the effectively control of 1BD in China, As one part of INCO-DC Project (ERBIC18CT980330) named "Acute Infectious Bursal Disease in Poultry: Epidemiology of the Disease, Basis of Virulence and Improvement of Vaccination ", one strain of wIBDV was isolated from a chicken farm of Guangxi Province, China, This strain with the mortality up to 60% in 4 week old SPF chickens was identified to be wIBDV according with European and Offi standard by pathogenicity test, antigenicity test, cloning and analysis of Vp2 gene, the result also showed that the homology of nucleotide sequence, amino acid sequence of VP2 between this strain and wIBDV UK661, which is now considered as the reference strain for European wIBDVs, were 99% and 100% respectively. In this cooperative project, this strain was set up as a standard wIBDV strain of China by scientist coming from China and European Community, it was named wIBDV Gx, being a reference wIBDV strain on the study of IBD and wIBDV, it provides us a standard reagent of cooperating with any other countries.VvIBDV Gx was attenuated through replicating in SPF embryo and CEF, the results of sequencing, analyzing of every generation viruses showed that there wasn't any changes on VP2 gene before 7th generation, there was a few changes on VP2 gene but not on amino acid at 8* generation, from 9th generation amino acid Ala on the position of 222 and Ser on position of 3 in SWSASGS area, which are the characters of wIBDV, changed into Pro and Arg respectively, which are the characters of low virulent EBDV, from 10th to 25* generation there weren't any change on VP2 gene, and the homology of amino acid sequence with European reference IBDV strain was up to 97%. The results of biological test showed that the virus of 5th and 20th generation had been adapted on CEF,the titer was high up 2.0 x l08 PFU/ml, the vims of 5* generation was still typical vvIBDV with the mortality up to 60% in 4 week old SPF chickens, the vims of 20* generation was not pathogenic and couldn't atavistic after propagated 6 generation in SPF chickens. In conclusion, after cultivation on CEF, the wIBDV Gx changed into unstable middle type at first and then changed into stable low virulent strain.Other results of immunology test also showed that the inactivated 5thg generation virus could induce effectively immunity, it could provide SPF chickens 100% protection from challenge by WBDV after SPF chickens were vaccinated with IxlO8 PFU/chicken inactivated virus. The virus of 20th generation without pathogenicity also could induce effectively immunity, it could also induce specific antibody and provide SPF chickens 100% protection from challenge by WIBDV after SPF chickens were vaccinated. In conclusion the viruses of 5* generation and 20* generation could be used to produce infectious bursal disease live and inactivated vaccines respectively wilh excellent effect Oligo primers was designed to amplify wIBDV Gx VP2 in Pud 19, of which the upstream primer was added the yeast consensus sequence, The amplified 1.5Kb VP2 gene fragment was ligated into the smal site of yeast palsmid PpiczA. The transfer plasmid was linearized by SacI and transfected...
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