DNA-mediated Immunization Of Chickens Against Infectious Bursal Disease Virus

Infectious bursal disease virus (IBDV), the causative agent of infectious bursal disease (IBD), causes immunosuppression in young chickens by destroying the precursors of B lymphocytes in the bursa of Fabricius. In recent years, antigenic variant IBDV strains and very virulent IBDV strains (wIBDV) have been emerging and caused considerable economic losses to the poultry industry around the world. The traditional inactivated or attenuated vaccines could not provide enough protection against these variants and wIBDV. Therefore, a new generation of more effective and safer vaccines is anticipated. The purpose of this study was to develop a DNA vaccine against IBDV. This research may open a new approach to effectively control of IBDV.Three IBDV strains, designated as ZJ2000, ZJ991 and ZJ992, were isolated from bursa of Fabricius of the sick chicken flocks in Hangzhou, Shenzhou, and Ningbo of Zhejiang province. The isolates could react with IBDV positive reference serum in the agar immune-diffusion test and caused 30-100% mortality when passaged in chicken embryos. Chickens inoculated with these isolates exhibited clinical and pathological signs typical of infectious bursal disease. The viruses were non-enveloped with a diameter of 55 nm, as shown by negative stain electron microscopy. These results showed that the three isolates were infectious bursal disease virus.The full-length cDNA of the genomic segment A of two viruses, one virulent field strain IBDV ZJ2000 and one attenuated strains IBDV JD1, were amplified in a single step procedure by long-accurate reverse-transcription polymerase chain reaction (LA-PCR), cloned into pGEM-T Easy Vector, and sequenced. The cloned segment A contains 3259 nucleotides in full-length and includes two partially overlapping Open Reading Fragments (ORF1 and ORF2) flanked by 5' and 3' noncoding regions (NCR). The two strains shared high sequence homology with each other either at nucleotide or deduced amino acid level. A hairpin structure was predicted in 5'-NCR and 3'-NCR according to their secondary structure. The VP5 protein was composed of 145 amino acids and shared homology of over 98.6% with other strains as well as 100% homology with strains K310, Cu-1, P2 and CEF94. The region coding for polyprotein was from positions 132 to 3146 in the nucleotide sequence, and the deduced amino acids sequence were highly homologous to strains Cu-1, P2 and CEF94. Substitution of four amino acids at positions 253, 279, 284 and 330, a common feature of attenuated and most virulent strains, was observed in the two test strains. Two major hydrophilic peaks were conserved as well. However, there are two amino acid substitutions at positions 280 (N to S) and 290 (M to L) in the second minor hydrophilic peak of the strain ZJ2000, which might have a critical effect on antigenecity. Two amino acid substitutionsnear the VP2-VP4 cleavage site were identified in the virulent strain JZ2000 and might be involved in increased virulence of the virus. Phylogenetic analyses indicated that these two strains were most closely related to some European strains but distinct from wIBDV and variant strains.The complete polyprotein (VP2/VP4/VP3) and VP2 genes of IBDV strains JD1 and ZJ2000 were inserted into the control of human cytomegalovirus (hCMV) immediate early enhancer and promoter of pCI and pcDNA3 respectively, resulting in the construction of pCI-VP2/VP4/VP3, pcDNA3-VP2/VP4/VP3, pCI-VP2 and pcDNA3-VP2. The recombinant plasmid pCI-VP2/VP4/VP3 of IBDV ZJ2000 was co-transfected with the Vero cell mediated by lipofectin. The DNA and RNA dot blotting revealed transcription of the polyprotein gene into mRNA in the Vero cell. Indirect immuno-fluorescent assay and enzyme linked immunosorbent assay (ELISA) showed that the expressed protein was reactive with chicken anti-IBDV serum.Series of DNA vaccines including pCI-VP2/VP4/VP3, pcDNA3-VP2/VP4/VP3, pCI-VP2, pcDNA3-VP2 of IBDV strains ZJ2000 and JD1 were developed using immune-stimulating complexes (ISCOM) as the adjuvant. Fourteen-day-old chickens...