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Identification of possible infectious bursal disease virus receptors.Edwards, Thomas Jonathan. 19 December 2013 (has links)
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. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.
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VP4 : a putative protease encoded by infectious bursal disease virus.Scholfield, Nicola Gillian. 19 December 2013 (has links)
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 NH₂-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.
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Molecular epidemiology of H9N2 avian influenza virus in poultry of southern ChinaButt, Ka-man, Carmen., 畢嘉敏. January 2005 (has links)
published_or_final_version / abstract / Microbiology / Master / Master of Philosophy
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Spatial ecology of the persistence and spread of highly pathogenic avian influenza, H5N1 in Southern China / Ecologie spatiale contribuant à la persistence et la diffusion de la grippe aviaire hautement pathogène, souche H5N1, en Chine du SudMartin, Vincent 09 January 2012 (has links)
Les travaux de recherche effectués dans le cadre de cette thèse ont été guidés par le manque d’information et une compréhension limitée des mécanismes épidémiologiques à l’origine de l’émergence et de la diffusion de la grippe aviaire hautement pathogène, souche H5N1 en Chine du Sud, aussi reconnue comme l’épicentre potentiel de l’émergence des virus influenza aviaires à caractères pandémiques. <p>Dans ce cadre, des données spatio-temporelles relatives aux foyers de la maladie ainsi que des données de surveillance virologiques (isolement du virus effectué dans le cadre du système de surveillance nationale) ont été collectées sur une période de quatre ans et analysées afin d’éxplorer les facteurs de risque relatifs à l’émergence et persistence de la maladie dans certaine zones de production du sud de la Chine. Les analyses ainsi effectuées ont permis d’identifier, à travers l’utilisation de méthodes statistiques robustes ayant fait leur preuve dans le domaine de la santé ou de l’écologie (la régression logistique classique et les arbres de regression logistique), des facteurs de risque liés à certains types de production de volailles (canards élevés en plein air, zones riches en eau et par extension associées à la riziculture) ou des facteurs associés à l’activité humaine. A travers une représentation cartographique des facteurs ainsi identifiés, des cartes de risque ont été produites permettant ainsi de visualiser d’une part les zones à haut risque de persistence de l’infection virale et d’autre part les zones vulnérables à l’apparition de foyers de la maladie, donnant aux autorités nationales la possibilité de mieux cibler leurs politiques de surveillance et de contrôle. <p>Dans un second temps, notre étude s’est portée sur les marchés à volailles traditionnels du sud de la Chine qui représentent un risque permanent de persistence, d’évolution et de diffusion des virus influenza aviaires, ainsi qu’un risque important en matière de santé publique. La dynamique de ces marchés et les liens qui les unissent ont été étudiés à travers des outils d’analyse empruntés à la sociologie tels que l’Analyse des Réseaux Sociaux (Social Network Analysis). Grace à cette approche, l’importance de l’hygiène de ces marchés et notamment du nettoyage et de la désinfection des cages dans la persistence du virus a été mise en évidence. Enfin, des enquêtes effectuées auprès des vendeurs de volailles ont permis d’identifier l’origine et la destination des animaux vendus et de reconstruire des réseaux plus ou moins intriqués de liens commerciaux qui unissent ces marchés entre eux dans trois provinces du sud de la Chine. L’analyse de ces réseaux et de leurs configurations ont permis d’identifier des marchés à plus haut risque de persistence de l’infection du fait de leur position centrale au sein de ces réseaux. De même qu’il est indispensable de cibler la surveillance et le contrôle de la maladie dans des zones écologiquement favorables à la persistence des virus influenza aviaires, cette étude révèle l’importance de certaines pratiques hygiéniques et commerciales dans la persistence de la maladie et la nécessité de cibler la surveillance et le contrôle au niveau de certains de ces marchés situés au centre d’un réseau dense et connecté, pour pouvoir in fine mieux contrôler la maladie au niveau national.<p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished
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