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Immunity Effect Examination of Cell-cloned Strains Cultivation of vvIBDV GX8/99 and Relevance Analysis of High Variation Area on VP2

Author: WangXinJian
Tutor: ZhuRuiLiang
School: Shandong Agricultural University
Course: Preventive Veterinary Medicine
Keywords: vvIBDV Cloned cell passage poison Attenuated strains Immunogenic VP2 hypervariable region
CLC: S852.65
Type: Master's thesis
Year: 2011
Downloads: 7
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Abstract


Infectious bursal disease (Infectious bursal disease, IBD) is an acute, highly contagious disease, mainly against chicken chickens caused by infectious bursal disease virus (Infectious bursal disease virus, IBDV) central immune organs - Law The bursal is to harm one of the major infectious diseases of the world's poultry industry. The virus causes immune suppression, resulting in susceptibility for other diseases of the increase and decrease the reactivity of the remaining vaccine. In recent years, IBDV IBDV immune mechanism of pathogenesis, virus molecular structure and function at the molecular level made great progress but still needs further study, especially mutant the emergence of vvIBDV, antigenic variation and diversity of subtypes of the disease in the clinical diagnosis is difficult, causing serious losses to the poultry industry. IBDV double RNA virus family poultry double RNA viruses of the local separation mutants or use made of vaccine antigen and close to local mutant strains inactivated vaccine or attenuated vaccines, as well as take advantage of a variety of strains joint made of multivalent The vaccine can make flock get good immune protection. In the process of implementation of the major projects of the National Natural Science Foundation, the infection vvIBDV GX8/99 strain chicken morbidity and mortality is relatively high, farms immune failure occur frequently. This project intends to vvIBDV GX8/99 strains for the study, strains vvIBDV GX8/99 passage, research from the original virus (chicken source drugs) → the chick embryo poison → cells adapt to the the drug → cell cloning poison → cloned strains cells passaged to 30 generations immunogenicity of the relationship between the variation of its nucleotide and amino acid. In order to better grasp the vvIBDV the immunogenicity Changes in the entire passage attenuated process, vvIBDV individual effects \virulent evolution and virulence changes in the molecular basis of to solve vvIBDV virulence enhanced whether vvIBDV can cause weak and attenuated gene sequence and the nucleotide sequence changes, this change with the virus biological the relationship between school characteristics, an attenuated strain of the biological characteristics of such a system to provide the basis for an urgent problem. First part vvIBDV GX8/99 strains of virus clone and its subculture of the study will be uploaded in SPF chicken first vvIBDV GX8/99 strain for 10 generations, and then in the chick embryo fibroblasts (CEF) blind passage 22 generations, began to adapt CEF and cause cytopathic effect (CPE), cells showed Condensation, pull into a fibrillar last round off. Then the infinite dilution method 2 virus cloned, screened four cloned virus. The four cloned virus were continuously uploaded in CEF 30 generations. In addition to the adaptation CEF before cytotoxic not detect IBDV immune colloidal gold strip film to detect the passage poison the rest of the pass of IBDV on behalf of the drug can be detected. IBDV in cell proliferation indefinite period, CEF lesions the different CEF appears lesions have increased with increasing cloning strain passaged passages, the results of this study, powerful poison GX8/99 strains from primary drug after the chick embryo, CEF, TCID50 variation: SPF chicken from the primary drug by 10 passages, the virus adapt to the cells increases in cloned strains by CEF passage 30 of each cloned virus passage poison TCID50 were lower. The second part vvIBDV GX8/99 cloned strain cells induced attenuated VP2 hypervariable region variability the analysis vvIBDV GX8/99 strain cloned cytotoxic GX-IBDVE10C25CL1 the GX-IBDVE10C25CL2, the GX-IBDVE10C25CL4, GX-IBDVE10C25CL5 chicken embryo fibroblast cells attenuated four cloned strains were selected for five generations, 10 generations 15 generations 20 generations 25 generations 30 generations attenuated strains extracted RNA by nested-PCR, cloning, nucleotide sequencing and analysis, explore the four clones of the vvIBDV GX8/99 strain in the process of cell passage VP2 nucleotide sequence and deduced amino acid sequence variation. 4 cells adapted virus clone sequencing of VP2 hypervariable region of approximately 145 amino acids, 12 sites in the same mutation, consistent and adapt to the cells of the vaccine virus strain D78. Amino acid variation sites are mainly concentrated in the 222 (A → P), 242 (I to V), 248 (L → F), 253 (Q → H), 256 (I to V), 257 ( Q → L), 270 (A → T or S), 279 (D → N), 284 (A → T), 294 (T → L), 299 (S → N), 330 ( S → R). Experiment further evidence of VP2 hypervariable region amino acid most of the variation cell culture or tissue affinity relationship more closely, the regularity of variation. 270 (A → T) has strains of pathogenic and immunogenic, and is located heptapeptide District 296 (S → R) and the virulence of attenuated strains. The third part vvIBDV GX8/99 strains four cloned poison F10, F20, F25, F30 generation of immune effects analysis vvIBDV GX8/99 strain 4 cloning poison F10, F20, F25, F30 generation 10000TCID50 immunization dose of 14-day-old chicks by the oral first free, Free 21-day-old record of the circumstances of the death. All immunohistochemistry after a free 3D, 10d, 7d two free, 14d, 20d, 30d, respectively, to take the 30 experimental serum antibody detection and analysis of whole blood, and 40d age orally 2000ELD50, vvIBDV GX8/99 The results show that the dose of 10000TCID50 immune vvIBDV GX8/99 cloning strain F20, F25 can provide 100% protection, two free 14d antibody concentration peaked. However, the different generations of the immune effect is obvious, combined with the second experiment showed that VP2 hypervariable region amino acid variation and immunogenicity of an attenuated strain relations. A Free 10d take bursal tissue observed histopathological changes, four cloned strains F30 generation of immune chicken results in very few bursal damage, but this damage is reversible, combined with the second part immune effects and histopathological changes, the the four cloned poison F30 generation GX-IBDVE10C25CL4-30 strains of immune effect, mild pathological changes, is expected to become the ideal vaccine strains to provide a new way to control vvIBDV infection.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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