title: Infectious bursal disease virus as a replication-incompetent viral vector expressing green fluorescent protein.
abstract: Infectious bursal disease virus (IBDV) has been established as a replication-competent viral vector capable of carrying an epitope at multiple loci in the genome. To enhance the safety and increase the insertion capacity of IBDV as a vector, a replication-incompetent IBDV vector was developed in the present study. The feasibility of replacing one of the viral gene loci, including pvp2, vp3, vp1, or the polyprotein vp243, with the sequence of green fluorescent protein (GFP) was explored. A method combining TCID50and immunoperoxidase monolayer assay (IPMA) determined the most feasible locus for gene replacement to be pvp2. The genomic segment containing gfp at the pvp2 locus was able to be encapsidated into IBDV particles. Furthermore, the expression of GFP in GFP-IBDV infected cells was confirmed by Western blotting and GFP-IBDV particles showed similar morphology and size to that of wildtype IBDV by electron microscopy. By providing the deleted protein in trans in a packaging cell line (pVP2-DF1), replication-incompetent GFP-IBDV particles were successfully plaque-quantified. The gfp sequence from the plaque-forming GFP-IBDV in pVP2-DF1 was confirmed by RT-PCR and sequencing. To our knowledge, GFP-IBDV developed in the present study is the first replication-incompetent IBDV vector which expresses a foreign protein in infected cells without the capability to produce viral progeny. Additionally, such replication-incompetent IBDV vectors could serve as bivalent vaccine vectors for conferring protection against infections with IBDV and other economically important, or zoonotic, avian pathogens.
authors.affiliation: ADDL, Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, 406 South University Street, West Lafayette, IN, 47907-2065, USA.
authors.forename: Yung-Yi C
authors.initials: YC
authors.lastname: Mosley
authors.affiliation: ADDL, Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, 406 South University Street, West Lafayette, IN, 47907-2065, USA.|School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan, ROC.
authors.forename: Ching Ching
authors.initials: CC
authors.lastname: Wu
authors.affiliation: ADDL, Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, 406 South University Street, West Lafayette, IN, 47907-2065, USA. tllin@purdue.edu.
authors.forename: Tsang Long
authors.initials: TL
authors.lastname: Lin
mesh_terms: D017353:Gene Deletion; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D007243:Infectious bursal disease virus / Q000235:genetics* / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D011995:Recombination, Genetic; D014779:Virus Replication
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins