Infectious bursal disease in Hong Kong : molecular epidemiology and the development of DNA vaccine
INFECTIOUS BURSAL DISEASE IN BONG KONG:
MOLECULAR EPIDEMIOLOGY AND TBE DEVELOPMENT OF DNA VACCINE
Chung Chau BON
for the degree of Master of Philosophy at The University of Hong Kong
in November 2003
Infectious bursal disease (IBD) is an economically significant viral disease in chickens. Understanding of its epidemiology and development of effective vaccines are the keys for the successful control of the disease. In the first part of this thesis, the global and local molecular epidemiology of mD was investigated using various molecular and bioinformatic techniques. In the second part of this thesis, based on the result of molecular
modeling and structural analysis of the IBD virus (IBDV) capsid proteins, various DNA
vaccine candidates were constructed and characterized.
The molecular epidemiology of IBD in Hong Kong was investigated by a longitudinal study of viral genotypes within a local chicken farm. The hypervariable region of VP2 of 24 samples were amplified and sequenced. Simultaneous infections of multiple strains were detected within a single host, and the possibility of recombination was revealed by the phylogenetic and Simplot analysis of the viral population. Dual infections of mDV with avian influenza were detected in 6 samples, suggesting the possible role of the immunosuppressive mDs in the facilitation of the avian influenza outbreaks. All the sequenced local strains were phylogenetically very close to the previously isolated Chinese strains, implying the possible Chinese origin of the Hong Kong strains. To investigate the
global epidemiology ofIBD, the phylogeny of 412 Genbank sequences was reconstructed. With the validated molecular clock-like behavior of the data-set and its reconstructed
phylogeny, the divergence time of the common ancestors of different lineages were estimated by linear regression. The emergence of the classical strains that had lead to the first American outbreaks was predicted to be at around 1950s. The global epidemiological history of ffiD was reconstructed and described.
To explore the possibility of using different viral antigens in the development of DNA vaccines, the molecular structure and antigenicity of the IBDV capsid proteins were investigated. Fold recognition of VP2 hypervariable region suggested it is a structurally independent all-beta top domain, which exists as a protruding trimer structure on the viral capsid. Putative residues involved in the discontinuous conformational epitopes were identified with this model. Antigenicity analysis of the E. coli expressed recombinant protein of truncated VP2 revealed two synergetic epitopes at its N-terminal. Based on the structural information of the capsid proteins, truncated VP2 fragments and recombinant VP2-4-3 fusion protein were cloned and test as DNA vaccine candidates in mice. N-terminal residues and the putative all-beta domain of VP2 were found to be crucial for anti-ffiDV seroconversion in mice. Significant decrease of IgGl / IgG2a ratio was observed in the antisera of mice immunized with the fragments covering the putative all-beta top domain ofVP2, implying the presence ofT-helper 1 epitope within this region. DNA vaccines coding the recombinant VP2-4-3 polyprotein induced Ab in mice. Removing the putative all-beta top domain of VP2 in the VP2-4-3 protein significantly reduced its efficacy, but replacement of the domain with VP5 significantly increased the production of IgG 1. These results provided valuable information for the rational design of molecular vaccine against the ffiD in future.
School:The University of Hong Kong
School Location:China - Hong Kong SAR
Source Type:Master's Thesis
Keywords:university of hong kong dissertations bursa fabricii diseases molecular epidemiology dna vaccines chickens
Date of Publication:01/01/2004