Rescue And Evaluation Of A Recombinant IBDV Expressing FLAG-Tagged VP1 As An Attenuated Vaccine Candidate Strain

Infectious bursal disease(IBD)is a highly contagious disease of young chickens caused by infectious bursal disease virus(IBDV),characterised by immunosuppression.Vaccine immunization is the main means of prevention and control of IBD.However,with the emergence of the variant strain and the very virulent IBDV(vv IBDV),it is necessary to carry out the research on an alternative and new vaccine strategy,which will be beneficial to control the disease.IBDV belongs to the Birnaviridae family with a bi-segmented(A and B segments)double-strand RNA(dsRNA)genome.In this study,a highly efficient dual-promoter RNA polymerase(Pol I)-Pol II reverse genetic system was successfully constructed,allowing direct rescue of recombinant IBDV from the viral cDNAs without the aid of VP1 and VP3.We constructed pCI-neo vector-based IBDV infectious plasmids p2-mA and p2-mB that contained the full-length cDNAs of IBDV genomic segment A and segment B,respectively,flanked by both a Pol-I cassette and a CMV promoter(Pol-II)cassette.Transfection of p2-mA and p2-mB into 293 T cells led to a direct rescue of recombinant viruses at 72 hr post transfection,which could infect DF-1 cells with a replication kinetics similar to the parental viruses.Further,we introduced the 8-aa FLAG epitope(DYKDDDDK)sequence at the C-terminus of VP1 to obtain a FLAG-tagged A44/FLAG rescue strain.The rescued IBDV and the fusion-expressed FLAG tag were detected by RT-PCR,indirect immunofluorescence,sequencing,Western-blot,and transmission electron microscopy.Through the viral plaque assay,the replication kinetic assay and the SPF chicken challenge assay,it was shown that despite the similar pattern of viral replication kinetics,there was a significant deduction in the plague phenotype in the DF-1 cells and in the damage to the bursa of Fabricius caused by the rescued FLAG-tagged IBDV as compared to the parental viruses.Moreover,the recombinant FLAG-tagged IBDV retained the ability to elicit neutralizing antibodies against VP2 at comparable levels as the parental virus,and in a single immunization,conferred complete protection against subsequent lethal challenge with virulent IBDV.In conclusion,this study demonstrates that rescued recombinant virus expressing FLAG-tagged VP1 can be used as a live attenuated IBDV vaccine strain;targeted insertion of exogenous epitope sequences in the B-segment gene encoding VP1 is a good strategy for generation of live attenuated IBDV.