Identification of possible infectious bursal disease virus receptors.
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Date
2000
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Abstract
Infectious bursal disease virus (IBDV) is a chicken pathogen that infects the bursa of
Fabricius, an organ involved in the development of the immune system in chickens.
Infection by the virus leads to destruction of the bursa and immunosuppression.
Infection by virulent strains may result in mortality. Current methods to combat the
virus involve the use of vaccines. These are usually a mixture of live attenuated and
oil inactivated virus. Variant strains of the virus are able to escape the vaccine-generated
antibodies. In addition, the vaccines result in damage to the bursa.
Identification of a receptor for IBDV could result in the development of either
treatment for the virus or superior vaccines by interfering with the attachment of the
virus to host cells.
Several methods for identifying IBDV binding proteins from the membranes of cells
from the bursa of Fabricius were examined. Affinity chromatography of IBDV
binding proteins with a matrix consisting of IBDV cross-linked to Sepharose 4B
allowed separation of a number of virus binding proteins. In contrast, virus overlay
protein blot assay (VOPBA) and reversible cross-linking with 2-iminothiolane proved
less; conclusive.
Predominant proteins in the affinity-separated fraction were of 40 and 32 kDa. These
were further examined by N-terminal amino acid sequencing of the whole protein and
N-terminal sequencing of peptides produced by endoproteinase Lys-C digestion of the
protein respectively. The 40 kDa protein showed homology with human synovial
stimulatory protein involved in the formation of autoantibodies in rheumatoid
arthritis. Virus was also shown to bind to a 440 kDa protein complex. This 440 kDa
protein complex appeared to consist primarily of a 40 kDa protein when examined by
reducing Tris-Tricine SDS-PAGE. Analysis of bursal membrane proteins by Western
blots using sera from rheumatoid arthritis patients revealed interactions between
several IBDV proteins and the antibodies from rheumatoid arthritis patients. Using
serum from one of the five patients showed a strong interaction at approximately 80
kDa and a weaker interaction at approximately 40 kDa. This may indicate an immune reaction between a chicken homolog of the synovial stimulatory protein and
antibodies in rheumatoid arthritis sera.
The 32 kDa protein showed homology to a Pseudomonas fluorescens protein. A
section of this sequence was amplified by PCR from chicken DNA and RT-PCR from
chicken RNA using degenerate primers constructed from the established N-terminal
amino acid sequences and chicken codon usage tables. The fragment produced upon
amplification from chicken DNA and RNA did not correspond to the predicted size of
177 bp. In contrast, when the RT-PCR product was heated and snap cooled before
examination by agarose gel electrophoresis, the product consisted of two fragments,
one of approximately 400 bp in size and one of approximately 200 bp in size.
The establishment ofthe 40 and 32 kDa chicken bursal membrane proteins as possible
receptors for the virus could allow for the development of vaccines and/or treatment
strategies for the virus. Treatment strategies or vaccines would be based on blocking
of the interaction between IBDV and chicken host cells. Peptide mimics of the
epitopes involved in such interactions could possibly achieve this.
Description
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.
Keywords
Poultry--Virus diseases., Chickens--Diseases., Membrane proteins., Theses--Biochemistry.