Molecular Studies of a Highly Virulent Strain of Infectious Bursal Disease Virus (IBDV) and Production of VP2 Recombinant Protein
Chong , Lee Kim (2003) Molecular Studies of a Highly Virulent Strain of Infectious Bursal Disease Virus (IBDV) and Production of VP2 Recombinant Protein. PhD thesis, Universiti Putra Malaysia.
The UPM97/61 isolate originated from the field IBD outbreak was identified as a highly virulent IBDV strain based on the sequence and phylogenetic analysis. The VP2 sequence contains amino acid substitutions at positions 222(A), 256(1) and 294(1) which are genetic markers for highly virulent strains. In addition, the serine rich heptapeptide region, S-W-S-A-S-G-S present in all highly virulent strains are also well conserved. Based on the restriction enzyme analysis, UPM97161 has one Sspl, Taql and Styl restriction sites at nucleotide positions 1011, 833, and 888 which correspond to amino acid residues 294, 235 and 254, respectively, but absent for Sacl site which is also one of the characteristic of the highly virulent strain. The sequences alignment of VP3, VP4 and VP5 with other strains of IBDV had showed that the amino acid substitutions at VP4 (685 Asn), VP3 (715 Ser, 761 Asp, 990 Val and 1005 Ala) and VP5 (49 Arg and 78 lie) could also be used to differentiate the highly virulent phenotype from the less virulent phenotype of IBDV. Phylogenetic analysis of the individual VP2, VP3, VP4 and VP5 protein as well as the whole segment A of IBDV has cluster the UPM97/61 together with the highly virulent strains from Japan (OKYM), UK (UK661), HK46 (China) and ks (Isreal). It was speculated that the Malaysian highly virulent strain might have the same origin as the highly virulent strains isolated in Europe, Japan, China and Isreal. VP2 protein was classed under alpha-beta mixed family. The l3-sheet structure formed the main structure of the hypervariable region where the neutralizing epitopes clustered. It was predicted that, this structure allowed intermolecular interaction as well as intra molecular interaction between the viral protein and the antibody via hydrogen bonding. A single-tube RT -peR method that is time saving, less laborious and does not require much samples was developed and it has the potential to be used as a diagnostiC tool. VP2 protein was successfully expressed in a prokaryotic system. The E.coli expressed-VP2 protein was able to induced a significant level of ELISA antibody titer that provided 57% protection to the chicken challenge with highly virulent strain against mortality but not against bursal atrophy. The protection could be improved by a careful evaluation of the vaccine dosage, virus dosage, vaccination timing and route of inoculation in the future studies.
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