The Influence Of VP5Genes On IBDV Pathogenic And The Immunogenicity Research Of GX8/99Cells Cloning Strains

The causative agent of infectious bursal disease （IBD）, is one of the majorinfectious diseases infected by infectious bursal disease virus （IBDV）, against theworld’s poultry industry. It causes immunosuppression in chickens by destroying thebursa of Fabricius, so it enhances susceptibility to any other disease and reduces theresponse to other vaccines. Recent years, molecular biology research of infectiousbursal disease virus （IBDV） has achieved great advance in the study of immune andpathogenic mechanism, molecular structure and function.But there is still a lot oninfectious bursal disease virus （IBDV） to need the further research. The emergence ofMutants and vvIBD, antigen variability and diversity, subtypes of the disease makeprevention and clinical diagnosis difficult and cause serious losses to the poultryindustry.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’svariation and the vaccine strain with the antigenicity similar to the local variation, aswell as the multipartial vaccine made from many kinds of strains, provide goodimmunity protection for the chicken group. Recently, major projects of nationalnatural fund have studied more than70IBDV in each province. They determinedvvIBDV GX8/99, of all the isolated vvIBDV, induces a comparative high diseaseincidence and mortality rate and the phenomenon of immune failure occurs frequentlyin chicken group. In order to grasp the immunogenicity regularity in the wholeprocess of tenuated subculture and the regularity of “individual effect”, which lay afoundation for the research of vvIBDV epidemiology and the effective prevention,thus molecular foundation of the evolution rule and toxicity variation, we test thedifferent adjuvants immune effect on four cloned strains to find the bettercombinations of adjuvant and vaccine. This study took vvIBDV GX8/99as the object,studied the immunogenicity regularity and relationship with the nucleotide and theamino acid through subculture for30generations 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 inthe sequences of gene and nucleotide occurred in the process, and the relationshipbetween the changes and the biological characteristics of virus, as well as thebiological characteristics of weak strain.1. The analysis of the relationship between the pathogenicity of different passagevirus of vvIBDV GX8/99strain and the VP5geneIn the study, ELD₅₀of the original virus （chicken virus）, embryo virus, virus ofadapted to cells, cloned virus in cells, the10th passage virus returned SPF chickens（chicken virus）, and the20th passage virus of cloned cells of vvIBDV GX-8/99strainswere detected. By inoculating SPF chicken with virus led the same ELD₅₀, weobserved pathogenic changes of vvIBDV GX-8/99strains during the course ofpassage.RNA were extracted from the certain passage strains of the above virus. ByRT-PCR, PCR amplification, gene cloning, nucleotide sequence determination andanalysis, we compared the VP5gene of the virus IBDV of the32different passages,and we obtained their homology were between94%and100%. However, there are15sites prone to mutation, and the base variation in the sites bp#2、#8、#52、#145、#232、#272、#310、#364、#385、#409caused the changes of corresponding aminoacid, but the base variation in the site bp#18、#285、#331、#354、#397did notcause changes of corresponding amino acid. By comparison, we can see when thedeath rate from86.7%of the original virus reduces to43%of GXE10, four sites ofthe VP5nucleotide mutated, and when the death rate from43%of GXC23reduces to3.3%of GXE10,10sites of the VP5nucleotide mutated. But the pathogenicity havelittle change in the four strains of the cloned GXC23, and only the sites bp#8ofGXCl1-1, the sites bp#364of GXCl2-1and GXCl4-1mutated; the mortality havesome degree of recovery after the four strains of cloned virus returned SPF chickenspassage10generations, and there are two sites mutated; four cloned virus strains inthe cells after20generations of continuous passage of its death rate drops to0, andcloned5strains did not have changes of nucleotide, and cloned1,2,4strains hasonly one nucleotide site mutated. Therefore, we can ascertain that the majorityvariation of amino acid of VP5may have a certain relationship with the pathogenicityof the virus, but the relationships with the cell culture and tissue affinity are moreclosely. This proved the molecular basis of virulence changes in vvIBDV, whichexplains why the vvIBDV appears and causes attenuated in nature. The molecularbasis of vvIBDV in virulence changes, which may explain why vvIBDV appeared andcaused attenuation in nature. 2. The establishment of the attenuated virus VP5gene prokaryotic expressionsystem caused by vvIBDV GX8/99cloned virus strains cellsThe subjects of the study were four cloned cells virus GXCL1、GXCL2、GXCL4、GXCL5of vvIBDV GX8/99strains. Four cloned cells virus which were subcultured20generations consecutive in CEF then subcultured10generations in chicken embryofibroblasts. Sequencing the VP5gene of each cloned virus every four generations.According to the sequencing results of two parts, and selecting VP5gene of threevirus strains （GXCl1-1、GXCl2-10、GXCl4-25） whose amino acid sequences differgreatly as contents of the study of this part. Getting VP5gene amplified from thethree virus strains by RT-PCR, and cloning into the prokaryotic expression vectorpET32a, thus the recombinant prokaryotic expression plasmid pET32a-VP5isconstructed. Transforming Escherichia coli Rosetta to induced expression afterrecombinant expression plasmid is identified correctly. The analysis results ofSDS-PAGE and Western blot showed that IBDV VP5gene expressing correctly inEscherichia coli Rosetta and the fusion protein it expressed had specificantigen-antibody reaction with IBDV positive sera.3. The impact of different immune adjuvants on the immune effects of fourcloned IBDV strainsSelect four strains whose amino acid sequences is significantly different GXCL1-25、GXCL2-30、GXCL4-25、GXCL5–30according to the study of the second partand choose pine pollen polysaccharide as adjuvant （propolis adjuvant as a control） bydifferent immune adjuvants,and analyze its immune effects. After detecting TCID₅₀and fixing quantity of the four cells virus, each strain were set three parallelexperimental group respectively: no immunoadjuvant （1、4、7、10）, pine pollenpolysaccharide adjuvant （2、5、8、11） and propolis adjuvant （3、6、9、12）, and seta blank contraposition group of saline. The first immunization of SPF chickens is onthe14th day and the second on the25th day. Draw blood respectively on0d、3d、7dafter the first immunization and3d、10d、17d、24d after the second; Measure serumantibody titers and IL-2levels by ELISA, and use automated blood cell analyzer todetermine the ratio of peripheral blood lymphocytes on the28th and the42th day, thenkill five chickens to measure index of immune organs on the28th and the42th day ineach group, inject virus on the35th day. We discovered that the antibody and IL-2levels both reached a peak on10day after the second immunization through analyzingexperimental results of each experimental group, the four cloned cell virus immunedchicken had some damage to the bursal, however the group which include immune adjuvants avoided the damage. The result of injecting virus experiments showed thatthe immune effects of each strains was obvious, but there were also some differencesbetween them. Through immune combination of different strains with differentadjuvants, we analyze their immune effects comprehensively to find the bestcombination which is GXCL4-25group with Pine pollen polysaccharide adjuvant andprovide a new pathway for controling vvIBDV infection...