Cloning And Bioinformatics Analysis Of Full-length Genome Of An Infectious Bursal Disease Virus (TL2004) Isolated In Zhejiang Province

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 (vvIBDV) 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 vvIBDV.One IBDV strains, designated as TL2004, was isolated from bursa of Fabricius of the sick chicken flocks in Hangzhou, Zhejiang province. The isolate could react with IBDV positive reference serum in the agar immuno-diffusion test and caused 100% mortality when passaged in chicken embryos. The virus was non-enveloped with a diameter of 55 nm, as shown by negative stain electron microscopy. These results showed that the one isolate was infectious bursal disease virus, the new characterization of the virulence of the stain TL2004 is required to be more research.The full-length genome of TL2004 strain IBDV isolated from immunized chickens,which infected with typical IBD, was amplified by RT-PCR and analysed. The results showed that the segment A contains 3260 nucleotides including 5'and 3'end noncoding region(NCR) and two partially overlapped open reading frames (ORFA1 and ORFA2) .The segment B contains 2827 nucleotides including 5'and 3'end noncoding region and one open reading frames (ORF) . A hairpin structure was predicted in 5'-NCR and 3'-NCR of the segment A and segment B. ORFAland ORFA2 encoded polyprotein VP2-VP4-VP3 composed of 1012 amino acides ,VP5 composed of 145 amino acides.The ORF of segment B encoded VP1 composed of 879 amino acides .Compared with other IBDV strains,the amino acids sequence of VP1,VP2, VP3, VP4 and VP5 shared homology of 94.4%—99.3%,93.4%—99.6%,94.2%—99.2 %,95.9%—99.6% and 94.5%—99.3% at protein level. The amino acides were substituted at 279(S to N) and 290(L to M) in second minor hydrophic peak of stain TL2004 which might have a critical effect on antigenecity. compared with the VP1 amino acids sequence of the very virulent stains, TL2004 contained 8 amino acides at the same position, moreover, the 518(R) and 525(D) are different from the other strains.Phylogentic tree analysis indicated that the segment A of TL2004 is most closely related to CEF94,JD1,HZ2 and NB strains ,however , the segment B of TL2004 is most closely related to very virulent BD3/99 and UK661 strains.The results above indicate that the stain TL2004 is most probably a new virus that comes from the reassortment of the segment A of attenuated stains and the segment B of the very virulent stains in nature.The present studies successfully cloned the full genome of the TL2004 stain and analysed the structure of the genome, anigen and virulence variation, which is important for us to understand the molecule characterization and do cosmically research for molecule epidemic of IBDV in Zhejiang province.