The Study On Molecular Mechanism Of Virulence And The Development Of The Reverse-Genetics System For IBDV

Infectious bursal disease(IBD), mediated by infectious bursal disease virus(IBDV), causes acute and highly contagious immunosuppressive disease in chickens. Two different serotypes of IBDV have been described, serotype Ⅰ strains are pathogenic for chickens, according to the virulent difference, they can be classified into classical, antigenic variant and very virulent isolates,while serotype Ⅱstrains are apathogenetic in chickens, which have been isolated from fowl and turkey.The first outbreak of vv IBDV in China was reported in 1990 s, most of field isolates are also vv IBDV in recent years. By sequencing VP2 gene of 5 strains isolated from different poultry farms in China, there are no mutations in 222A、253Q、256I、279D、284A、294I、299S and SWSASGS. By pathogenic experiment to SPF chickens, we found that the mortality caused by the 5 strains is no less than 47%, which demonstrates that the virulence of vv IBDV has little change in the past 20 years. For the 5 strains, QL&GX, HB96&HBY show a closer nucleotide homology, 99.8% and 99.5%,separately.Which illustrates that strains isolated from different years have little difference.B87 is the most widely used vaccine strain at present, to compare the sequence difference of vv IBDV and B87, the VP2 of different passage strains of B87 in chicken embryo were sequenced and analyzed, the results show that there are 7 amino acid mutations in VP2 between E2 and E6. The two strains also show different cell tropism to CEF, E2 could not adapt to CEF, but E6 could induce CPE. The bursa to body weight ratio of SPF chickens infected with E2 began to decrease from 7 days post inoculation, and the BB Index of E2 was also much lower than that of E6; Bursas of chicks inoculated with E2 after 7 dpi show atrophy, while bursas of that inoculated with E6 had a small part of follicles atrophy, with normal follicular connective tissues. The animal experiment shows that there is virulent difference between E2 and E6. This study suggests that mutations of Q253H/D279N/A284 T may be the molecular basis of virulence and cell tropism.To futher study on the molecular mechanism of virulence and cell tropism of IBDV, t he full-length genome of B87 was cloned by one-step method with high-fidelity DNA polymerase, then the sequences of Hammerhead ribozyme(Ham Rz) and Hepatitis delta ribozyme(Hdv Rz) were introduced into the 5’ends and 3’ends of segments A, B by over-lap PCR. The full-length genome flanked by Ham Rz and Hdv Rz were cloned downstream of the CMV enhancer and the chicken β-actin promoter of the eukaryotic expression vector p CAGGS, the p CAGGSA and p CAGGSB directly co-transfected DF1 by Lipofectamine 2000, 12 h post transfection, IBDV characteristic protein could be tested by indirect immune- fluorescence assay.