Infectious bursal disease virus as a viral vector

Studies were carried out to test the feasibility for infectious bursal disease virus (IBDV) to be employed as a viral vector for chickens. A reverse genetics system for IBDV was first developed by cloning the complete cDNA sequence of segment A and B into separate plasmids, namely SegA and SegB, respectively. Reverse genetic IBDV (RG-IBDV) was recovered after co-transfection of SegA and SegB into Vero cells for 48 hour with titers around 10 5 pfu/ml. The genetic tags which were absent in the parent virus (LP1-IBDV) were maintained in RG-IBDV for serial 6 passages. The plaque size, growth kinetics and morphology of the RG-IBDV by electron microscope (EM) were all similar to those of the LP1-IBDV. RG-IBDV did not cause bursal atrophy in 3 week-old chickens but induced high ELISA and neutralizing antibodies to IBDV. RG-IBDV was further genetically modified to carry the hemagglutinin (HA) epitope at different locations in the original open reading frames (ORF) or at a new ORF in segment A or insert the green fluorescent protein (GFP) at the C-terminus of VP3 to create replication-competent IBDV vectors. IBDVs which express the HA epitope at N-terminus of VP5 (HA5-IBDV) and VP4 (HA4-IBDV) or C-terminus of VP1 (1HA-IBDV) were rescued after co-transfection. These HA-IBDVs had smaller plaque sizes and slower but different patterns of growth kinetics when compared to RG-IBDV. The morphology of HA-IBDV particles was similar to that of RG-IBDV by EM. All three HA-IBDV did not cause bursal atrophy in chickens, but only HA4-IBDV and 1HA-IBDV induced specific antibodies to the HA epitope. Replication-competent vectors were failed to be rescued when the HA epitope was inserted at a new ORF in segment A or the GFP was fused at the C-terminus of VP3. For replication-incompetent vector, GFP was used to replace different viral protein and the missing viral protein was provided in trans. GFP-expressing IBDV was rescued only when the replacement was done for pVP2 protein. This GFP-IBDV was replication-incompetent since it forms small plaques in pVP2-expressing DF-1 cells, but not in normal DF-1 cells. The viral titer obtained after co-transfection into DF-1 cells for 48 hour was about 2 x 103 pfu/ml. The expression of GFP by GFP-IBDV particles in infected cells was confirmed by immunoperoxidase monolayer assay (IPMA) and Western blotting. The morphology of GFP-IBDV particles was similar to that of RG-IBDV by EM. However, propagation of GFP-IBDV in pVP2-expressing DF-1 cells was unsuccessful. In summary, IBDV can be developed both as a replication-competent and replication-incompetent viral vector to carry and express the HA epitope at the N-terminus of VP4 or C-terminus of VP1 or the GFP at the pVP2 region, respectively