Construction Of Infectious Bursal Disease Virus Chimerics With VP5 Knock-out And Attenuated

The infectious bursal disease virus (IBDV), a member of the Birnaviridae family, containing a bisegmented double-stranded RNA genome, encodes four structural viral proteins, VP1, VP2, VP3, and VP4, as well as a non-structural protein, VP5. IBDV is the causative agent of infectious bursal disease (IBD) in chickens. It causes high mortality in young chickens and establishes an immunosuppression state by destroying the precursors of B lymphocytes in the bursa of Fabricius and the postbursal B lymphocytes, leading to vaccination failure against other pathogens.To further evaluate the efficiency and optimize the procedure of previously constructed IBDV reverse genetic system based on plasmids cDNA co-transfection, in the present part, the segment A from two IBDV strains, field isolate ZJ2000 and attenuated strain HZ2, were inserted into one NaeI by site-directed silent mutagenesis, respetively. The two resulting mutants were subcloned into expression plasmid pCI under the control of the human cytomegalovirus (hCMV) immediate early enhancer and promoter to construct the recombinant plasmids pCI-AKZJ2000 and pCI-AKHZ2, respectively. Each of the two recombinants was combined with another recombinant pCI plasmid containing the marked segment B of strain HZ2 (pCI-mBHZ2), and injected intramuscularly into specific-pathogen-free (SPF) chickens. Two chimeric IBDV strains were recovered from the chickens post injection. Two out of eight chickens in each of two groups showed the bursal histopathological change. The reassortant virus derived from pCI-AKZJ2000/pCI-mBHZ2 can infect chicken embryos and shows relatively low virulence. We have developed a novel virus reverse genetic approach for the study of IBDV. The results also form the basis for investigating the role of VP1 in viral replication and pathogenecity.In order to characterize the role of VP5 in IBDV replication, IBDV VP5 gene was amplified and cloned into an N-terminal GST-Tag fusion expression vector, pGEX-4T-2, which was controlled by T7 promoter. The sequencing result showed thatthe VP5 gene was composed of 438 base pairs, and coded 145 amino acids. High VP5 product was expressed in E. coli BL21 induced by IPTG, and the GST-VP5 fusion protein existed in inclusion. High titer anti-VP5 serum was also prepared in New Zealand rabbit immunized with purified fusion protein inclusion.The nonstructural protein VP5 of IBDV has been reported to be associated with virus cell apoptosis and pathogenicity, but its regulator role in viral replication has not been unequivocally identified. In this part, three VP5 deficient IBDV strains were constructed with or without base pairs deletion by a plasmid-based infectious cDNA system. Indirect immuno-fluorescence assay showed that VP5 was not expressed by VP5 deficient strains. The effect of 5' terminal base pairs of VP5 gene on viral replication kinetics was analyzed on chicken embryo fibroblast (CEF) cells. The results showed that VP5 and 5' terminus of its gene is not essential for replication of IBDV. However, the deletion delayed the virus replication on CEF cells and the final titers decreased with increasing of deletion length. Thus, it is indicated that the 5' terminus of VP5 gene contains an expression regulator for virus genome. The regulation mode was further characterized by comparison between virus replication and protein expression. The results showed that the putative factor affects protein translation rather than RNA replication.To characterize the effect of VP5 on IBDV replication, pathogenicity and immunogenicity, cell-derived attenuated strain HZ2 and VP5 deficient strain ANhe2 were oral given to 14-day-old SPF chickens respectively. The bursa/body weight ratios and bursal histopathological lesion were detected at certain intervals post inoculation. Viruses were re-isolated form bursas and titration at the same time intervals for replication characterization. It is showed that mild and transient bursal lesion was occurred post virus inoculation. Both strains could replicate in bursas within a short period after inoculation, although strain ANhe2 replicated a litter slower to peak at 7-d post inoculation.Both strains HZ2 and ANhe2 induced similar antibody response, which could neutralize IBDV strain HZ2. Three weeks after boosting at 4-week-old, all test chickens were challenged with IBDV reference strain BC6/85. Although both strainsprovided immune protection against challenge, the protection rate induced by strain ANhe2 (87.5%) is better than that induced by strain HZ2 (62.5%). Combined with gross and histopathological bursa change, it is showed that the deficiency of VP5 enhanced the immunogenicity of strain HZ2.