Infectious bursal disease virus (IBDV) causes an acute and highly contagious disease
affecting young chickens, which is responsible for significant losses in the poultry industry
world-wide. The virus specifically infects and destroys B-cell precursors within the bursa of
Fabricius, an avian lymphoid organ, leading to immunosuppression. IBDV has a bi-segmented,
double-stranded RNA genome. The larger segment encodes a 110-kDa precursor
polyprotein, designated NHâ‚‚-VPX-VP4-VP3-COOH, in a single open reading frame. The
autocatalytic processing of this precursor into mature proteins is a critical step in viral
replication and VP4 is the putative protease responsible for this cleavage. This study
concerns the development of a strategy to clone and express recombinant VP4 and describes
the use of VP4 as a marker for rapid and effective detection of IBDV. VP4 cDNA was
produced and amplified by optimisation of a reverse transcription coupled to the polymerase
chain reaction (RT-PCR), providing a clear and sensitive assay. Anti-peptide antibodies were
raised against a selected peptide from VP4 and were used to probe homogenates of infected
bursae for the native protein to assess their potential for immunological detection. These
antibody-related results are promising though inconclusive, due to the complex nature of the
assayed sample. Amplified VP4 cDNA from KwaZulu-Natal strains of IBDV isolated from
1989 to 1997 was also examined by restriction fragment length polymorphism (RFLP)
analysis to determine the relatedness of local IBDV to global strains. All KwaZulu-Natal
samples produced identical patterns, which were most similar to one of ten international
strains examined, namely, the British strain UK661. Samples infected with IBDV were also
probed for VP4 activity. Double basic amino acid cleavage sites have been proposed for the
putative protease and infected samples were assayed for activity against the fluorogenic
peptide Cbz-Arg-Arg-AMC. Demonstrably higher activity was found in infected versus
uninfected samples, although the origin of this activity is unclear. The findings in this study
suggest that VP4 warrants further attention, both as a marker for infectious bursal disease, and as a novel viral protease. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/10269 |
| Date | 19 December 2013 |
| Creators | Scholfield, Nicola Gillian. |
| Contributors | Coetzer, Theresa Helen Taillefer. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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