| Infectious bursal disease(IBD) is caused by infectious bursal disease virus(IBDV), an acute viral infectious disease predominantly affects the Immune organs. IBDV infection can cause disease even dead in chickens, production performance decline, cause the flocks of 70% to 100% mortality especially vvlBDV emerges since the 1980s, causing direct economic loss. IBDV can cause chicks immunosuppressive diseases, so the body immune function is so low that the immune response to other vaccines less effective, enhanced susceptibility to other diseases, increasing the chance of secondary infection diseases. High mutation rate of RNA polymerase hypervariable region cause IBDV antigenic variants and attenuated strains emerge, they may be an important cause of disease after after immunization with IBD frequent in recent years. The disease is a big danger for the poultry industry and widespread attention by the aquaculture industry of the entire world.We isolate a strain of infectious bursal disease virus by using conventional methods and molecular biology methods.The strain was selected from 35 farms suspected of infectious bursal disease virus infection in Henan, named for the number HN12 isolates. We prove the isolated strain is IBDV by the onset of characteristic pathological change, conventional laboratory testing methods and techniques of molecular biology detection methods. After infectioning,4-weeks-old SPF chickens, the incidence of symptoms, serological and molecular biological test data show: HN12 isolate is vvlBDV, can cause high morbidity and mortality, and cause chickens and SPF chicken to produce lesions and incidence of death.After the IBDV isolate cultured four generations in chick embryo, then cultured on DF-lcell, the cytopathic appears when the blind spread fourth generation. Serially passaged strain to the proliferation of stability. The spread eighth cytotoxic strain named HN12C8. Experimental Study of IBDV by adsorption time, amount of inoculum, to maintain serum concentration of the liquid in the IBDV DF-1 cell proliferation, the highest viral titers was obtaine when inoculated IBDV diluted 10-3,2% fetal bovine serum of maintain fluid added. The research results show that cell adapted strains HN12C can not agglutinate red blood cells, sensitive to PH12, is not sensitive to PH2 and organic solvents.RNA is extracted from the original isolate HN12B1, embryo adapted strain HN12E4, cells adapted strain HN12C8. Through gene clone, nucleotide sequence analysis, we can find the variation of amino acid sequence deduced by the isolate strain VP2 nucleotide sequence. There are 11 sites mutated among the hypervariable region sequence of VP2 (206-350), to become consistent with the vaccine virus D78 strain. Amino acid variation sites focused on 222 (A?P), 242 (T?V),249 (Q?K),253 (Q?H),256 (T?V),279 (D?N),284 (A?T),290(M? L),294 (T?L),299 (S?N) and 330 (S?R). On this basis, we compare the VP2 fragment with the reported strain, construct phylogenetic trees. The experiments further prove that VP2 hypervariable region amino acid variation on most cell cultures or tissue affinity relationships with more closely with regularity of variation.The SPF chickens were inoculated with HN12B1, HN12E4, and HN12C8 oil emulsion vaccine. Serum separated respectively after vaccination 7d,14d,21d,28d,35d,42d. The antibody levels using the agar diffusion and cell neutralization were tested. In the third week, the antibody reached 1:16, followed by linear increase to 1:64. The antibody reached highest level in 4 week. The antibody titers generated by cell adapted strain immune can be achieved 10-3.16, i.e., when the 1:1445 dilution of serum that protects 50% of the cells did not appear CPE.This paper tests provide theoretical reference for IBDV prevention by the research of the local IBDV isolate, also provide a theoretical basis for the gene variant characteristics of IBDV strain. |
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