This thesis examines how preparatory, policy and review documents of the Participatory Rangeland Management (PRM) in East Africa, problematize and represent the ‘problems’ which resulted in the design of the development program. The focus is on how these problematizations can hinder or facilitate participation, influence and empowerment of women and marginalized groups in decision-making processes. The findings are based on a gender-based policy analysis undertaken of five documents written by the NGOs involved in the planning and implementation of PRM in Ethiopia, Kenya and Tanzania. A conceptual framework measuring the level of participation, empowerment and influence was constructed to assess the policies and their possible outcomes. The document analysis showed that the implicit ‘problem’ themes identified appeared to be that communities were unmodern, undeveloped, and had under-representation of women and pastoralists in rangeland management. These problematizations seem to originate from a development discourse characterized by solutions focussing on ‘modernization’ and ‘technical fixes’. These pre-conceived ideas of the ’problems’ call for more communication and inclusion of community groups in problem formulation and program design. The analysis further revealed that expert-assisted and gender-mainstreaming initiatives such as the PRM could have a positive impact on the level of participation, influence, and empowerment of women. When training was carried out for both women and men by the PRM to raise awareness of women’s rights, it resulted in an increased number of women participating in activities. However, gender-mainstreaming should not stop with participation, it should be further developed towards influence and empowerment. The PRM could consider promoting a change of power relations by combining efforts to demonstrate the benefits of meaningful consultations to decision-makers and efforts to enhance the knowledge and skills of marginalized groups so that they can better engage with these decision-makers. Furthermore, there is a need to expand the discussion on how to design gender-mainstreaming policies and practices, without labelling women as one.

No description available.

隨著資訊技術的崛起與發展，傳統的商業模式已經完全不同於以往，電子商務的興起已逐年快速蓬勃發展，這樣的新興商業模式無論是在B2B或是B2C的領域都讓企業運作變的更加有效率，並且為企業創造了更多的利潤，但除了資訊科技以外仍需其他革命性變革與創新才能造就如此欣欣向榮的環境，供應鏈管理即是極佳的例子，在供應鏈管理的運作之下的企業相互合作和共享資訊與資源，資訊科技扮演連接整個供應鏈的重要角色，並且讓供應鏈更加有效率的運作，這是B2B電子商務極致表現的一個相當好的範例，但企業之間的合作決計不僅僅只依靠資訊科技的連結而變的更有效率，企業需要的是尋找良好的廠商作為合作夥伴，本研究的目的就在於發掘出企業間建立夥伴關係中重要的因素，並嘗試將夥伴關係分級，如此一來夥伴關係管理將不只是空談，企業可以有效的評估合作的廠商並加以分級管理，夥伴關係管理的運作將為企業創造更多的價值。 在不同的產業中會有不同的夥伴關係出現，當然當中關鍵的結合力因素也將不同，在本研究中將會把目標放在處於資訊科技產業中的買與賣的關係之上，因為這個產業有著強調速度與效率的特性，而且電子商務的應用與發展也最蓬勃，本研究將採用深度的訪談分析，並讓受訪問的企業對重要的結合力因素作評比，如此一來研究者將可以分析瞭解重要的結合力並將夥伴關係做等級上的劃分，以利未來其他研究者能針對不同等級的夥伴關係做各類管理方法之研究。 關鍵字：電子商務、B2B、夥伴關係管理、結合力 / In the wake of information technology (IT), business model is taking a paradigm shift. Electronic commerce is an emerging business model that streamlines the operations of a business and results in efficiency and profitability. In addition to the increased dependency on information technology, changes in management will be needed to maximize the effects of electronic commerce. The concept of Supply Chain Management (SCM) tells management to utilize the new found capabilities of IT integration and information sharing between business partners. The concept reveals that a connection beside the tangible IT link should exist to bind a business with its suppliers and customers tightly. Businesses should have a good relationship with their business partners to increase the total revenue. In this research, we are trying to find the critical bonds between business partners. After identifying the bonds, we can use them to categorize partner relationships and differentiate the degree of integration between business partners. Keywords: EC, B2B, PRM, partner relation management, bond, buyer-supplier relationship

La gestion des risques est un enjeu majeur, mais difficile pour les projets de construction. La difficulté à gérer les risques dans les projets de construction vient de leur complexité. Ils sont composés de nombreuses entités (activités, acteurs, contrats, ressources, etc.) dont le comportement collectif influencent les comportements individuels. Afin de mieux appréhender et comprendre la complexité du système dans son ensemble, il est nécessaire de capitaliser et structurer la connaissance dans le but de proposer un modèle capable de décrire et simuler le comportement du système étudié. Cependant, la formalisation de tels modèles se confronte à de nombreuses difficultés : présence de facteurs humains, raretés de modèles, connaissances souvent expertes et qualitatives difficiles à formaliser, méconnaissance des mécanismes régissant certains processus, données parcellaires, hétérogènes et souvent imparfaites, échelles multiples, etc. L’objectif est de proposer des approches conceptuelles permettant d’assembler des morceaux de connaissances hétérogènes multi-sources et multi-échelles dans le but de proposer un modèle capable de réduire les incertitudes liées au fonctionnement, au devenir, à la conception et au pilotage des projets de construction.Différentes approches et outils ont été proposés pour modéliser et simuler les projets de construction : structure de répartition des risques, réseaux bayésiens, théorie des réseaux, simulation de Monte Carlo, réseau analytique, etc. Ces outils et méthodes sont utilisés pour simuler le comportement de systèmes, mais inadéquats pour représenter des systèmes complexes dynamiques à grandes échelles. Ils sont pour la plupart parcellaires et ne présentent pas ou peu de généricités. Dans ce contexte, les modèles relationnels probabilistes (MRPs) fourniront un formalisme mathématique pratique permettant de représenter et de simuler des systèmes dynamiques complexes entachés d’incertitudes. Les MRPs étendent le formalisme des réseaux bayésiens en ajoutant la notion de paradigme objet où l'incertitude attachée au système est alors prise en compte en quantifiant la dépendance probabiliste entre les propriétés des objets.Pour ce faire, une ontologie du domaine a été développée pour (a) fournir un vocabulaire commun capable de représenter les connaissances sur les projets de construction, (b) identifier les interconnections entre les différentes entités techniques, humaines, économiques à différents niveaux de description. Guidé par cette ontologie unMRP a été élaboré et utilisé pour simuler le comportement des projets de construction tout en prenant en compte les incertitudes. On montrera comment il peut être utilisé pour prédire la réponse incertaine du système ainsi que pour étudier comment la réponse globale du système est sensible aux valeurs ou hypothèses locales. Enfin, le MRP sera utilisé pour deux études de cas (la construction de routes et de ponts à Hue-Vietnam et d’un bâtiment en France). Les résultats montrent que le formalisme des MRPs permet (1) d’instancier tout type de projets de construction, (2) de prendre en compte l'incertitude, (3) de simuler et prédire le comportement du système et (4) d’extraire de la connaissance à partir d’informations partielles. / The difficulty to manage risks in construction projects comes from their complexity. They are composed of many entities (activities, actors, contracts, resources, etc.) among which interactions exist at many levels and influence the system response. In turn, this response can influence the behaviour of some entities. In order to capture the complexity of the system, it is necessary to structure, model and share cross-disciplinary and interdisciplinary knowledge flows in a common and unifying framework. Because of this high complexity, the system response may appear as unpredictable. Uncertainties at all scales are source of risk for the construction project itself. Tackling this complexity could improve our grasp of the whole system, in order to provide more robust and efficient decision alternatives in risk management. It is then essential to propose conceptual approaches able to represent the behaviour and the interactions of system entities over the time.Different approaches and tools have been proposed to model and simulate risk of construction project as Risk Breakdown Structure, Bayesian networks, Network Theory, Monte Carlo Simulation, Analytical Network Process, etc. These tools and methods can be used to simulate the behaviour of the system, but they are inadequate for representing large and complex dynamical system because they are based on case-dependant model (i.e. a specific model has to be built for each studied construction project), the fragmented representation of knowledge, the lack of common vocabulary, the lack of generic character. Hence, an ontology paradigm is developed in order (a) to provide a common vocabulary able to represent the knowledge about construction projects and its risks, (b) to shape the structure (interrelations) between those identified database and (c) to represent construction project integrating as well technical, human, sustainability dimensions at different detailed levels of uncertainty.In this context, by coupling the advantages of ontology and Bayesian network, the framework of probabilistic relational model (PRM) will provide a practical mathematical formalism allowing to represent and simulate complex stochastic dynamical systems. PRMs extend the formalism of Bayesian networks by adding the notion of object paradigm where uncertainty attached to the system is then taken into account by quantifying probabilistic dependence between the properties of objects and other properties of related objects. To the best of our knowledge, this thesis report will be the first application in which PRM have been proposed to model and simulate construction project while accounting uncertainties.Therefore PRM is used to simulate the propagation of uncertainties existing in this complexdynamic and multi-scale system, which lead to construction project risk. A prototypal software framework has been developed to check the consistency and the viability of the concept. It will be shown how it can be used in order to predict the uncertain response of the system as well as to study how the overall response of the system is sensitive to local values or assumptions. Lastly, PRM will be applied for two case-studies (a road and bridge construction in Hue-Vietnam and another building project in France). Results show that the formalism of PRMs allows to (1) implement any kind of construction project, (2) to take uncertainty into account, (3) to simulate and predict the behaviour of system and (4) to derive information from partial knowledge.

Par rapport aux exigences de plus en plus importantes relatives au Maintien en Condition Opérationnelle d'un système industriel, le processus de maintenance joue un rôle fondamental pour l'amélioration de la disponibilité, de la productivité, etc. Pour essayer de contrôler au mieux ces performances, les responsables de maintenance doivent donc être capables de choisir les stratégies de maintenance et les ressources à mettre en œuvre les plus adaptées aux besoins. Dans un objectif d'aide à la prise de décisions en maintenance, les travaux présentés dans ce mémoire ont pour objet de proposer une méthodologie pour l'élaboration d'un modèle support permettant par simulation d'évaluer les différentes stratégies. La valeur ajoutée de la méthodologie réside dans l'unification, à base de modèles relationnels probabilistes (PRM), des différents types de connaissance nécessaires à la construction de ce modèle d'évaluation. Ce dernier est ainsi construit à partir de motifs génériques et modulables représentatifs des variables décisionnels du système industriel (système principal) et de son système de maintenance. Ces motifs, par instanciation, facilitent la construction des modèles d'applications spécifiques. Cette méthodologie, issue du projet ANR SKOOB, est testée sur le cas applicatif de la maintenance d'un système de production de ferment.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.