Background:  The PDZ domain is a peptide sequence of 80-90 amino acids and can be found in e.g. bacteria, animals and plants. These domains are commonly part of the cytoplasmic and membrane adapter proteins and its function are important in protein-protein interactions. The NS1 proteins of influenza A viruses play an important role in inhibiting the IFN-β production in many ways. In the C-terminus of the NS1 protein, a peptide sequence of four amino acids had been demonstrated to bind to the PDZ domain termed as PDZ binding motif (PBM). Objective:  The aim of this study is to determine whether the PBM sequence of the NS1 protein of influenza A virus plays a key roll in the expression of interferon-β. Methods:  The open reading frame of the NS1 protein was amplified and cloned into expressing vector and transfected into A549 cells along with a reporter plasmid containing ISRE promoter, driving expression of firefly luciferase. Dual luciferase reporter assay was performed to measure luciferase activity which represented expression of IFN-β. The assay was performed only once and unfortunately the result can not be trusted since the negative control showed positive value. Therefore, to understand the interaction between the PBM sequence of NS1 proteins and the production of IFN-β, further experiments are needed.

In this study, the expression of HA, a key immunogenic protein of influenza viruses, in insect cells was compared using three baculovirus expression strategies: protein over-expression, surface (GP64) display, and capsid (VP39) display. Further, a recombinant virus expressing NA, another immunogenic influenza virus protein, was generated and fused to an HA epitope-tag. Western immunoblot using various antibodies, including those against HA, demonstrated the expression of HA and NA for all recombinant viruses. HA showed stronger expression when fused to the C-terminus of VP39 than the N-terminus, but unlike other expression methods, there was no observable cleavage of HA in VP39-displayed viruses. Cells infected with only over-expressed and surfaced-displayed HA were biologically active, and capable of hemadsorption and hemagglutination of chicken red blood cells. These results suggest that GP64 display or over-expression are the most efficacious modes of HA-expression for use as antigen to detect anti-HA antibodies in poultry. / NSERC, OGS, OMAFRA, CPRC

De nos jours, le scénario d’une pandémie à l’échelle mondiale est de plus en plus surveillé par l’Organisation mondiale de la santé en plus d’être exploité par le cinéma et les médias à sensations fortes. La réalité est que plusieurs épidémies ont ravagé la population sur terre à travers les époques telle que ce fut le cas lors de l’éclosion de la peste noire au XIV siècle en Europe et en Asie ou encore le VIH en Afrique encore aujourd’hui. La grande majorité des populations étant concentrée dans des mégalopoles et le transport international étant toujours plus développé et accessible rend une simple épidémie susceptible de se transformer rapidement en pandémie. Plus récemment, l’éclosion de la grippe aviaire H5N1 a décimé d’importantes populations d’oiseau en Asie et certains cas de contamination humaine fatale ont même été répertoriés. Comme les virus mutent rapidement, il serait donc pertinent de connaître leurs mécanismes de prolifération et tenter de les maîtriser afin de les neutraliser avant l’éclosion d’une souche se propageant d’humain à humain. Or, il a été découvert qu’une famille d’endoprotéases à sérines, les proprotéines convertases, sont impliquées dans diverses pathologies dont l’influenza-A H5N1. La furine, première proprotéine convertase à avoir été découverte, semble à l’origine de l’activation de ces virus. Nous avons donc choisi d’entreprendre la conception de divers inhibiteurs de furine dans le but de développer un agent antiviral contre cette pathologie. En ayant une approche impliquant la biologie structurelle et en effectuant une conception rationnelle d’inhibiteurs, nous avons développé des inhibiteurs de furine ayant à la fois une bonne affinité pour l’enzyme ainsi qu’une bonne stabilité en milieu biologique, tout en tentant de comprendre les principes fondamentaux de la liaison avec ces enzymes. Nos résultats ont démontré que diverses techniques peuvent être exploitées afin de concevoir des composés pouvant cibler la furine avec une bonne affinité. Le rationnel derrière la conception de ces composés a été démontrée à l’aide d’outils modernes de visualisation tridimensionnels de biomolécules en mettant en évidence les fonctionnalités importantes des complexes enzymes-substrat et enzyme-inhibiteurs afin de bien vulgariser la biologie structurelle inhérente au projet.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico. Programa de Pós-Graduação em Engenharia de Produção. / Made available in DSpace on 2012-10-24T02:48:31Z (GMT). No. of bitstreams: 1 261561.pdf: 897966 bytes, checksum: d25f3cd89afc8d612e50147f5ab78386 (MD5) / Este trabalho foi elaborado com objetivo de utilizar diferentes metodologias de previsão para verificar se é possível predizer, com maior acurácia que a previsão ingênua, os volumes de exportação de Carne de Frango Inteiro e Carne de Frango Corte do Brasil. Utilizaram-se para predição a Suavização Exponencial, a metodologia ARIMA, Regressão Dinâmica e a combinação destas metodologias. Com a aplicação da metodologia de Regressão Dinâmica, busca-se também determinar variáveis independentes que influenciam, no período avaliado, o volume de venda de Carne de Frango Inteiro e Corte brasileiro para o mercado externo. Com a grande queda nas vendas destes produtos no ano de 2006, em relação aos anos anteriores, a busca de um regressor que possa explicar esta queda também é objetivo importante deste trabalho. Como o evento da ocorrência de casos de contaminação de animais e humanos é frequentemente citado por instituições ligadas ao setor de produção de carnes como sendo a causa das quedas de vendas em 2006, este tema também é avaliado cuidadosamente neste trabalho. Todas as predições geradas com os diferentes métodos de previsão apresentaram resultados mais acurados que a previsão ingênua. Os resultados dos modelos de regressão dinâmica gerados mostram que existe correlação entre a ocorrência de reportagens sobre gripe aviária no jornal americano The New York Times e a queda do volume de exportação brasileiro de carne de frango em 2006.

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La rapidité avec laquelle le virus de l’influenza aviaire H5N1 se propage à toutes les échelles spatiales interroge sur les mécanismes qui sous-tendent la diffusion spatiale des épizooties.Ce travail de thèse montre de quelle manière une démarche de géographie de la santé permet de comprendre les mécanismes sociaux et spatiaux associés à la diffusion de l’épizootie d’influenza aviaire H5N1 en Thaïlande. L’emploi de méthodes de géographie quantitative et d’épidémiologie spatiale nous a d’abord permis de révéler l’existence de disparités spatiales dans le niveau de risque d’infection des élevages au niveau national, puis d’identifier et de hiérarchiser les facteurs de risque spatialisés. A l’échelon local, une enquête épidémiologique nous a ensuite conduits à identifier les pratiques d’élevage favorisant ou limitant le risque d’infection dans les basse-cours. Une analyse socio-économique des réseaux commerciaux liés à la volaille de basse-cour nous a permis de délimiter l’espace géographique (principalement local) de diffusion de la maladie ; elle a également montré de quelle manière les relations sociales unissant les acteurs de la filière, le contexte économique dans lequel ces acteurs évoluent, ainsi que les représentations associées aux maladies aviaires, permettent de comprendre la logique de pratiques facilitant la diffusion du virus. Enfin, notre travail a montré comment, alors que les pratiques spatiales des éleveurs de coqs de combat tendent à favoriser la diffusion du virus H5N1 sur de courtes et longues distances, les enjeux financiers et de prestige associés à ces activités conduisent ces éleveurs à mettre en place des pratiques limitant la propagation du virus. / The rapid spread of highly pathogenic avian influenza (HPAI) H5N1 has raised concerns about the mechanisms that underpin the spatial diffusion of epidemics.This thesis shows how an approach of health geography, based on the combined use of quantitative and qualitative methods from social sciences and epidemiology, can make it possible to identify the social and spatial factors which are associated with the spread of the H5N1 epidemic in poultry in Thailand.In a first step, quantitative geography and spatial epidemiology revealed spatial disparities in the risk of infection for poultry farms at a national level; they also helped at identifying and prioritizing spatial risk factors associated with the spread. In a second step, an epidemiological investigation carried out at a local level made it possible to identify the farming practices associated with the infection of backyard poultry by the H5N1 virus. Then, a socio-economic approach of poultry trade networks allowed us to delineate the geographical area (mainly local) which was relevant to the spread of the epidemic. It also showed how the social relations connecting actors in the sector, the economic context in which these actors operate, and the local perception of poultry diseases can together explain the practices facilitating the spread of the virus. At last, this work demonstrated how, while the practices of fighting cocks owners may favour the spread of the H5N1 virus over short and long distances, the financial stakes and the prestige associated with these activities led farmers to implement prevention practices that finally mitigate the spread of the virus.

Avian influenza H5N1 is one kind of important bird flu. Unfortunately, this virus has swiftly evolved and become highly pathogenic to humans and poultry, resulting in 100% of death in infected poultry and over 60% of mortality among infected human population. Moreover, the virus tends to reassort with other influenza viruses, such as the current swine flu H1N1, to establish themselves in environments and further this epidemic all over the world. The World Health Organization (WHO) has in fact warned that highly pathogenic avian influenza H5N1 poses a graver risk of a global human pandemic than at any time since the Hong Kong outbreak (H3N2) in the 1960s. / Finally, avian influenza is an inter-disciplinary issue across virology, medical geography, and spatial epidemiology. How to quantify and integrate knowledge from different disciplines remains a challenge in fully understanding the disease. We propose a method to formally integrate genetic analysis that identifies the evolution of the H5N1 virus in space and time, epidemiological analysis that determines socio-environmental factors associated with H5N1 occurrence and statistical analysis that identifies outbreak dusters. Our integrated results show a significant advance in findings over reports in, for instance, Gilbert et al. (2008) and we believe our findings are more precise and informative in representing the occurrence and the space-time dynamics of H5N1 spread. Overall, unlike traditional influenza studies, our work sets up a solid foundation for the inter-disciplinary study of this and other spatial infectious diseases. / First, we apply multifractal detrended fluctuation analysis to determine the temporal scaling behavior of outbreaks in Asia, Europe, Africa, and the whole of the world between December 2003 to March 2009. Long-range correlation and multifractality, two important properties characterizing the scaling behavior of complex dynamics, are first detected in the outbreak time series. In addition, this study identifies different temporal scaling behaviors of outbreaks of these continents 8,nd specific seasonal patterns in Asia. These findings confirm our perspective that avian-influenza outbreak behaviors are self-similar over time and are spatially heterogeneous. / One key to preventing such a calamity is to obtain a thorough understanding of the mechanisms of avian influenza transmission and its spatio-temporal patterns of dispersal. The issues at stake are outbreaks' spatial and temporal patterns, the interrelationship of these with the evolution of influenza viruses in such a way that geography is understood as a dimension of the disease's virology, and the human and avian behaviors and socio-ecological environments associated with H5Nl spread. This thesis sets out to study these problems in detail and propose solutions. / Second, we conduct a spatial analysis for global trends and local clusters of H5N1 outbreaks at multiple geographical scales. Currently, the local K function used in a point pattern analysis searches outbreak clusters, assuming the disease is spatially homogeneous. The thesis proposes a much more efficient method to measure the degree of clusters accurately. The modified function works by weighting outbreaks through distances, counting the number of the weighted outbreaks for each lattice point no matter whether the disease emerges in a grid. This weighted local K function extends cluster analysis from a point pattern to lattice data. Spatial representation in these terms then seeks to explore local patterns of H5N1 over a continuous space. / Third, we study a set of socio-environmental factors, which are plausibly associated with the occurrence of H5N1. Spatial epidemiological models are built for predicting the disease at both continental and national levels, covering Indonesia, China, and the whole of East-Southeast Asia. We evaluate the statistical models using 1,000 bootstrap replicates, showing a consistently high rate of prediction, assessed by statistics: AUC, Kappa Index, and pseudo R square. / Ge, Erjia. / Advisers: Yee Leung; Tung Fung. / Source: Dissertation Abstracts International, Volume: 73-06, Section: A, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 169-197). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

This thesis addresses issues related to surveillance for disease in commercial and non-commercial poultry populations. The motivation for this work has largely arisen from the unprecedented outbreaks of highly pathogenic avian influenza (HPAI) H5N1 that have occurred in 52 countries in Asia, Africa and Europe since 2003. A series of studies are presented using data derived from two countries, Vietnam and New Zealand. The two Vietnamese studies provide in-depth epidemiological analyses of the outbreak of HPAI H5N1 from December 2003 to March 2004. The three New Zealand studies deal with issues related to the development of effective surveillance strategies for HPAI — informed both directly and indirectly by the findings from the Vietnamese studies. This approach provides an example of how ‘lessons’ learnt from countries that have experienced large scale infectious disease epidemics can be used to assist in the design of surveillance activities in (as yet) unaffected countries. The descriptive analyses of the 2003 – 2004 outbreak of HPAI H5N1 in Vietnam indicate that the epidemic was seeded simultaneously in the north and south of the country in the later part of 2003 with 87% of provinces affected by February 2004. HPAI risk was concentrated around the Mekong and Red River Deltas. The broad scale spatial distribution of disease is likely to have been associated with regional differences in the poultry farming, trade in poultry, and environmental conditions such as the presence of bodies of water which would support reservoir species for the virus. A Bayesian zero-inflated Poisson regression model was used to quantify the influence of environmental and demographic factors on the spatial distribution of HPAI positive communes. In areas where disease was reported, our results show that HPAI risk was positively associated with the presence of irrigation and negatively associated with elevation. After controlling for these fixed effects, a single large area of elevated risk in the Red River Delta area was identified, presumably arising from similarities in the likelihood of reporting disease or the presence of factors increasing disease transmission and spread. Further investigations to elucidate likely transmission mechanisms, targeting this area of the country, would be a profitable area of future research. The second part of this thesis presents three studies that address issues related to the development of effective surveillance strategies for HPAI in New Zealand. The first was a cross-sectional study to enumerate the prevalence of backyard poultry ownership in two areas (one urban and the other rural) close to a large provincial city in the North Island of New Zealand. The prevalence of poultry ownership was 2% (95% CI 1% – 4%) in the urban area and 19% (95% CI 12% – 30%) in the rural area. The relatively low numbers of land parcels where poultry are present indicates that these areas, in the event of an infectious disease incursion, would be unlikely to pose a risk for spread of infectious agent. A cross-sectional survey of all members of the Poultry Industry Association of New Zealand was conducted in the later half of 2007. Respondents were asked to document contacts made with other enterprises related to feed, live birds and hatching eggs, table eggs and poultry product, and waste litter and manure. Patterns of contact were analysed using social network analyses. Each of the four networks had scale-free properties, meaning that for each movement type there were small numbers of enterprises that had contacts with large numbers of enterprises (potential ‘super-spreaders’ of disease). The presence of an undetected infectious disease in enterprises with super-spreader characteristics increases the likelihood that an epidemic will propagate rapidly through the population, assuming there is a directly proportional relationship between the number of contacts an enterprise makes and the probability that disease will be transferred from one location to another. While the finding that feed suppliers had large numbers of poultry farm contacts in the feed network came as no surprise, what was of greater interest was that there were small numbers of poultry farms that reported off-farm movements of feed. This should serve as an important reminder for disease control authorities: movement (and other) restrictions applied during the course of an animal health emergency should be applied across a range of industry sectors, recognising that some industry participants may practice activities that are not entirely typical for their enterprise type (e.g. poultry farms on-selling feed to other farms). In the absence of perfect and up-to-date network data, knowledge of the characteristics of individual enterprises that render them more likely to be atypical (e.g. size, type, and geographic location) would be of value, since this information could be used to inform a risk based approach to disease surveillance and control. A scenario tree model was developed as an approach for evaluating the effectiveness of New Zealand’s passive surveillance system for HPAI. The model was developed in two stages. In the first, factors thought to influence the geographic distribution of NAI risk of introduction and spread (and therefore surveillance strategy) were combined to create a spatial risk surface. In the second stage, a scenario tree model of the passive surveillance system for NAI was developed using the spatial risk surface and the HPAI surveillance strategy prescribed by Biosecurity New Zealand. The model was most sensitive to farmers reporting the presence of suspected cases of disease. This implies that the sensitivity of the system as a whole stands to increase if the importance of reporting suspicious clinical signs is reiterated to poultry producers. The studies presented in this thesis have presented a range of techniques and methodological approaches that are sufficiently generic to be used in any country to inform the design of surveillance strategies for a variety of animal diseases, not just those of poultry. Although epidemiology, as a discipline, is endoured with a vast range of analytical techniques that can be used to enhance the understanding of factors influencing the spread of disease among animal populations, the quality of data used to support these techniques is often lacking. The challenge in the years ahead, for both developed and developing countries, is to set in place the appropriate infrastructures to collect details of animal populations consistent in quality over time and space.

This thesis addresses issues related to surveillance for disease in commercial and non-commercial poultry populations. The motivation for this work has largely arisen from the unprecedented outbreaks of highly pathogenic avian influenza (HPAI) H5N1 that have occurred in 52 countries in Asia, Africa and Europe since 2003. A series of studies are presented using data derived from two countries, Vietnam and New Zealand. The two Vietnamese studies provide in-depth epidemiological analyses of the outbreak of HPAI H5N1 from December 2003 to March 2004. The three New Zealand studies deal with issues related to the development of effective surveillance strategies for HPAI — informed both directly and indirectly by the findings from the Vietnamese studies. This approach provides an example of how ‘lessons’ learnt from countries that have experienced large scale infectious disease epidemics can be used to assist in the design of surveillance activities in (as yet) unaffected countries. The descriptive analyses of the 2003 – 2004 outbreak of HPAI H5N1 in Vietnam indicate that the epidemic was seeded simultaneously in the north and south of the country in the later part of 2003 with 87% of provinces affected by February 2004. HPAI risk was concentrated around the Mekong and Red River Deltas. The broad scale spatial distribution of disease is likely to have been associated with regional differences in the poultry farming, trade in poultry, and environmental conditions such as the presence of bodies of water which would support reservoir species for the virus. A Bayesian zero-inflated Poisson regression model was used to quantify the influence of environmental and demographic factors on the spatial distribution of HPAI positive communes. In areas where disease was reported, our results show that HPAI risk was positively associated with the presence of irrigation and negatively associated with elevation. After controlling for these fixed effects, a single large area of elevated risk in the Red River Delta area was identified, presumably arising from similarities in the likelihood of reporting disease or the presence of factors increasing disease transmission and spread. Further investigations to elucidate likely transmission mechanisms, targeting this area of the country, would be a profitable area of future research. The second part of this thesis presents three studies that address issues related to the development of effective surveillance strategies for HPAI in New Zealand. The first was a cross-sectional study to enumerate the prevalence of backyard poultry ownership in two areas (one urban and the other rural) close to a large provincial city in the North Island of New Zealand. The prevalence of poultry ownership was 2% (95% CI 1% – 4%) in the urban area and 19% (95% CI 12% – 30%) in the rural area. The relatively low numbers of land parcels where poultry are present indicates that these areas, in the event of an infectious disease incursion, would be unlikely to pose a risk for spread of infectious agent. A cross-sectional survey of all members of the Poultry Industry Association of New Zealand was conducted in the later half of 2007. Respondents were asked to document contacts made with other enterprises related to feed, live birds and hatching eggs, table eggs and poultry product, and waste litter and manure. Patterns of contact were analysed using social network analyses. Each of the four networks had scale-free properties, meaning that for each movement type there were small numbers of enterprises that had contacts with large numbers of enterprises (potential ‘super-spreaders’ of disease). The presence of an undetected infectious disease in enterprises with super-spreader characteristics increases the likelihood that an epidemic will propagate rapidly through the population, assuming there is a directly proportional relationship between the number of contacts an enterprise makes and the probability that disease will be transferred from one location to another. While the finding that feed suppliers had large numbers of poultry farm contacts in the feed network came as no surprise, what was of greater interest was that there were small numbers of poultry farms that reported off-farm movements of feed. This should serve as an important reminder for disease control authorities: movement (and other) restrictions applied during the course of an animal health emergency should be applied across a range of industry sectors, recognising that some industry participants may practice activities that are not entirely typical for their enterprise type (e.g. poultry farms on-selling feed to other farms). In the absence of perfect and up-to-date network data, knowledge of the characteristics of individual enterprises that render them more likely to be atypical (e.g. size, type, and geographic location) would be of value, since this information could be used to inform a risk based approach to disease surveillance and control. A scenario tree model was developed as an approach for evaluating the effectiveness of New Zealand’s passive surveillance system for HPAI. The model was developed in two stages. In the first, factors thought to influence the geographic distribution of NAI risk of introduction and spread (and therefore surveillance strategy) were combined to create a spatial risk surface. In the second stage, a scenario tree model of the passive surveillance system for NAI was developed using the spatial risk surface and the HPAI surveillance strategy prescribed by Biosecurity New Zealand. The model was most sensitive to farmers reporting the presence of suspected cases of disease. This implies that the sensitivity of the system as a whole stands to increase if the importance of reporting suspicious clinical signs is reiterated to poultry producers. The studies presented in this thesis have presented a range of techniques and methodological approaches that are sufficiently generic to be used in any country to inform the design of surveillance strategies for a variety of animal diseases, not just those of poultry. Although epidemiology, as a discipline, is endoured with a vast range of analytical techniques that can be used to enhance the understanding of factors influencing the spread of disease among animal populations, the quality of data used to support these techniques is often lacking. The challenge in the years ahead, for both developed and developing countries, is to set in place the appropriate infrastructures to collect details of animal populations consistent in quality over time and space.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2006. / Härtill 5 uppsatser.