PB2 ist ein essentieller Bestandteil der trimeren RNA abhängigen RNA Polymerase von Influenzaviren und ist bekannt für seine Schlüsselrolle in der Bestimmung des Viruswirtsspektrums. Diese Arbeit diente der Identifizierung neuer Interaktionspartner von PB2 eines saisonalen und eines hochpathogenen Influenzavirus Stammes im Kontext infizierter humaner alveolar Epithelzellen (A549) unter Einsatz massenspektrometrischer Analysen. Die anschließende Untersuchung ausgewählter zellulärer Interaktoren hatte zum Ziel, deren Einfluss auf den Replikationszyklus der Influenzaviren zu bestimmen, sowie Unterschiede in ihrer Relevanz für das saisonale und das hochpathogene Virus aufzuzeigen. Die Erzeugung und Nutzung von Influenzaviren die einen Strep-tag an ihrem PB2 Protein tragen ermöglichte eine Anreicherung von PB2 und seiner Interaktionspartner. Die anschließende massenspektrometrische Analyse identifizierte 22 potentielle PB2 Interaktionspartner. Eine Auswahl an 13 Proteinen wurde tiefer gehend analysiert und eine Komplexbildung mit PB2 konnte für 9 Proteine bestätigt werden. Darüber hinaus zeigten 11 Proteine einen Polymerase stimulierenden bzw. hemmenden Effekt. Das Polymerase stimulierende Protein HSPA8 wurde zur weiteren Untersuchung ausgewählt. Während ein Einfluss von HSPA8 auf den hochpathogenen Influenzastamm nicht abschließend geklärt werden konnte, wurde seine Bedeutung für den Vermehrungszyklus des saisonalen Stammes aufgezeigt. Die Überexpression von HSPA8 führte zu einer Steigerung der Polymerase-Aktivität, wohingegen die Erniedrigung des HSPA8 Spiegels in einer Verringerung der viralen Replikation und der Polymerase-Aktivität resultierte. Interessanterweise führte die Erniedrigung des HSPA8 Spiegels auch zu stark verminderter PB2-Expression, jedoch nur im Falle des saisonalen Influenzastammes. Dieser Befund deutet auf eine Rolle von HSPA8 als PB2-Chaperon, notwendig für Proteinstabilität von saisonalen aber nicht hochpathogenen Influenzaviren, hin. / PB2 is an essential component of the influenza virus trimeric RNA dependent RNA polymerase and is known to play a key role in virus host range determination. Here, a combined affinity-purification/mass spectrometric approach was performed to identify novel interaction partners of PB2 of seasonal and highly pathogenic viral strains in infected human alveolar epithelial cells (A549). The subsequent analysis of selected cellular interaction partners aimed to determine the influence of these proteins on the replication cycle, as well as to determine differences in their relevance for the seasonal and the highly pathogenic influenza virus strain. Generation and use of recombinant influenza viruses carrying a Strep-tag at their PB2 protein allowed for enrichment of PB2 and its interaction partners. The subsequent mass spectrometric analysis identified 22 potential PB2 interaction partners. A selection of 13 proteins was further analyzed, and co-precipitation with PB2 was confirmed for 9 proteins. Moreover, an inhibitory or stimulatory effect on polymerase activity was observed for 11 proteins. The polymerase stimulating protein HSPA8 was selected for further investigation. While the influence of HSPA8 on the highly pathogenic strain remained unclear, its importance for seasonal influenza virus life cycle was demonstrated. Overexpression of HSPA8 resulted in increased polymerase activity while HSP8 knock down resulted in reduction of viral replication and viral polymerase activity. Intriguingly, the knock down of HSPA8 led to a strong decrease of PB2 protein expression. However, this was only observed for seasonal PB2. These results indicate a role of HSPA8 as a PB2 chaperone, necessary for protein stability of seasonal but not highly pathogenic influenza virus.

A influenza aviária é uma enfermidade viral causada por vírus influenza A que acomete várias espécies de aves. No Brasil, a influenza aviária é considerada uma doença exótica, uma vez que os subtipos H5 e H7 nunca foram detectados. O Ministério da Agricultura, Pecuária e Abastecimento realiza vigilância permanente para esta enfermidade nos plantéis comerciais de produção avícola e também em aves de subsistência criadas no entorno de sítios de aves migratórias com o intuito de detectar precocemente a introdução dos subtipos H5 e H7 no país. No entanto, se desconhece a situação sanitária das aves de subsistência no que se refere à infecção por outros subtipos do vírus da influenza aviária. Considerando a importância deste tipo de criação como fonte de alimentos e de rendimentos em comunidades de baixa renda e o risco que provavelmente representa para a introdução da influenza aviária no Brasil, o objetivo deste trabalho foi monitorar as criações de aves de subsistência criadas no entorno de sítios de aves migratórias no período de 2011 a 2015, a fim de verificar a circulação de todos os subtipos do vírus da influenza aviária (AIV). Foram colhidas amostras de soro e suabes de traqueia e cloaca de 2816 aves criadas em onze diferentes sítios de aves migratórias localizados em sete estados brasileiros, totalizando 391 explorações de fundo de quintal amostradas. As amostras de soro (n = 2716) foram submetidas a triagem para pesquisa de anticorpos para a proteína NP do influenza A utilizando-se um kit comercial de ELISA competitivo, com posterior subtipificação das amostras positivas pela técnica de inibição da hemaglutinação para os dezesseis subtipos do vírus influenza A. Os suabes de cloaca e traqueia foram submetidos à prova de PCR em tempo real para detecção do RNA do vírus influenza A. Não foram detectados anticorpos para os subtipos H5 e H7 do AIV, mas anticorpos para os subtipos H1, H3, H4, H6, H8, H9, H10, H12, H13 e H16 foram identificados em aves oriundas de nove dos onze sítios. O RNA do AIV foi detectado em apenas três amostras pertencentes a uma mesma propriedade e nenhum vírus hemaglutinante foi isolado a partir deste material. Os resultados obtidos permitem concluir que os vírus de influenza aviária de baixa patogenicidade circulam em aves de subsistência criadas no entorno de sítios de aves migratórias no Brasil e alertam para a importância da ampliação da vigilância ativa nesta população / Avian influenza is a viral disease caused by an influenza A virus and affects various avian species. Avian influenza is considered an exotic disease in Brazil, since H5 and H7 notifiable subtypes have never been detected. The Ministry of Agriculture, Livestock and Food Supply carries out permanent surveillance for avian influenza in commercial poultry production flocks and also in backyard poultry raised in the vicinity of concentration areas of migratory birds with the purpose of detecting the early introduction of H5 and H7 subtypes in the country. However, the health status of backyard poultry in relation to infection by other avian influenza subtypes is unknown. Considering the importance of this kind of family production system as a source of food and revenue for low-income communities and the risk it probably represents for the introduction of avian influenza in Brazil, the aim of this work was monitoring backyard poultry raised in the vicinity of concentration areas of migratory birds from 2011 to 2015 to verify the circulation of all avian influenza virus (AIV) subtypes. Serum samples and cloacal and tracheal swabs were sampled from 2816 birds raised in eleven migratory birds concentration areas located in seven Brazilian states, totaling 391 backyard poultry flocks harvested. Serum samples (n = 2716) were screened using a commercial competitive ELISA kit to detect specific antibodies for the NP protein of the influenza A virus and afterwards the positive samples were subtyped through inhibition hemagglutination assay for the sixteen subtypes of influenza A viruses. Cloacal and tracheal swabs were tested by real-time PCR for detection of the influenza A virus RNA. No antibodies for H5 and H7 subtypes were detected, but antibodies for subtypes H1, H3, H4, H6, H8, H9, H10, H12, H13 e H16 have been identified in birds from nine out of eleven areas. The influenza A virus RNA was detected only in three samples from one flock and no hemagglutinating viruses were isolated from these specimens. Results obtained in this work suggest that low pathogenic avian influenza viruses are circulating in backyard poultry flocks raised in concentration areas of migratory birds in Brazil and alert to the importance of the expansion of active surveillance in this population

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.

Influenza A viruses cause seasonal and pandemic outbreaks that range from mild infections to the disastrous Spanish Flu. Resistance to neuraminidase inhibitors (NAIs) is a growing problem as these drugs constitute a vital part of treatment strategies and pandemic preparedness plans worldwide. Oseltamivir (Tamiflu®) is the mostly used NAI. Its active metabolite, oseltamivir carboxylate (OC), is excreted from treated patients and degrades poorly in sewage treatment plants and surface water. Thus, OC can enter aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance and resistance could develop. If NAI resistance is established in influenza viruses circulating among wild birds, the resistance can form part of a virus re-entering the human population either by reassortment or by direct transmission. In this thesis, evidence is presented that OC is present in the waterways during a seasonal influenza outbreak in Japan, a country in which oseltamivir is liberally used. Furthermore, when mallards were infected with an influenza A/H1N1 virus and subjected to low, environmental-like concentrations of OC, resistance developed through acquisition of the well-known resistance mutation H274Y. The influenza infection in the mallards was mainly intestinal, had a rapid onset and was progressing in a longitudinal fashion in the intestine. Finally, influenza A viruses isolated from wild mallards in Sweden and containing resistance-related mutations were examined by a neuraminidase inhibition assay. The viruses did not have a decreased sensitivity to NAIs, but had mutations with a resistance-enhancing potential. Thus, OC is present in the environment and environmental-like concentrations of OC induce resistance in influenza viruses of dabbling ducks. The present resistance situation among wild birds is not well understood but the existence of H274Y among wild birds, though rare, and the spread of the former seasonal A/H1N1 virus containing H274Y among humans indicate that resistance mutations could establish themselves also among wild birds. An oseltamivir-resistant pandemic or a human-adapted highly-pathogenic avian influenza virus are frightening scenarios as oseltamivir is a cornerstone in the defense in those situations. There is a need for further studies, surveillance in wild birds and for a prudent use of antivirals.

Binder-based assays are employed throughout the life sciences. Powerful signal amplification techniques have enabled detection of very rare molecule species diluted in simple buffers. Unspecific binding of primary binders leads to increased background in more complex samples. By requiring two recognition events, ligation-based molecular analyses provide highly specific detection of biomolecules in complex samples. We developed a highly multiplexed padlock-ligation assay targeting signature sequences in the hemagglutinin and neuraminidase genes. From a panel of 77 avian influenza isolates of all major serotypes, 97% were genotyped correctly in accordance with previous classifications by classical diagnostic methods (Paper I). Alternative splicing is an important mechanism expanding the proteome. Current analysis techniques fail to provide sequences of complete transcripts beyond the read length of sequencing instruments. We devised and implemented a strategy to compress the sequence information contained in the splicing pattern of a transcript into the presence or absence of sequence-blocks. We demonstrate that this assay yields information about the splicing patterns in thousands of transcripts from cellular cDNA (Paper II). Expression changes of mucin proteins and glycosylation structures are frequently observed from the early stages of cancer development. Expression of mucin 2 and sialyl-Tn are common features of intestinal metaplasia and gastric cancer, and are known to co-locate. Here we have developed an in situ proximity ligation assay (PLA) directed against mucin 2 and sialyl-Tn. Our study on intestinal metaplasia and gastric cancer tissue sections identified mucin 2 as a major carrier of sialyl-Tn in these conditions, and demonstrated how conveniently glycosylation of proteins can be studied by in situ PLA (Paper III). This thesis shows how the dual recognition requirement of ligation-based assays can be employed to detect target molecules with high specificity, to analyze several sequence features of nucleic acids or to study the proximity of two antigens in situ.

The aim of this study is to improve our understanding of the ecology, spatiotemporal patterns, and risk of infectious pathogens of migratory waterfowl, using the blue-winged teal (Anas discors, BWTE), as a model. From 2007-2010, 1,869 BWTE were sampled in the prairie provinces (Alberta, Saskatchewan and Manitoba, Canada) to examine infection status and/or evidence of previous exposure to avian influenza virus (AIV), West Nile virus (WNV), and avian paramyxovirus-1 (APMV-1), in relation to host demographic variables (age, sex, body condition, exposure to other pathogens), other ecological variables such as local waterfowl breeding population density and local pond density, and year. The probability of AIV infection depended on an interaction between age and AIV antibody status. Hatch year birds with antibodies to AIV were more likely to be infected, suggesting an antibody response to an active infection. After hatch year birds with antibodies to AIV were less likely to be infected, suggesting immunity resulting from previous exposure. AIV infection was positively associated with local BWTE density, supporting the hypothesis of density dependent transmission. Exposure to WNV and APMV-1 were also associated with age and year. Furthermore, the probability of WNV exposure was positively associated with local pond density rather than host population density, likely because ponds provide suitable breeding habitat for mosquitoes, the primary vectors for transmission. We also investigated large-scale spatiotemporal trends in apparent prevalence of AIV across Canada and the United States throughout the year, using data from national avian influenza surveillance programs in Canada and the US in 2007-2010. Our analyses revealed that age, sex, year of sampling, flyway, latitude, and season (categorized by stages of the BWTE annual life cycle) were all important variables in predicting probability of AIV infection. There was an interaction between age and season. During late summer staging (August) and fall migration (September-October), hatch year birds were more likely to be infected than after hatch year birds, however there was no difference between age categories for the remainder of the year (winter, spring migration, and breeding season). Probability of infection increased non-linearly with latitude, and was highest in summer, corresponding to the beginning of fall migration when densities of birds and the proportion of susceptible hatch year birds in the population are highest. Birds in the Pacific, Central and Mississippi flyways were significantly more likely to be infected compared to those in the Atlantic flyway. Observed trends in seasonal, annual, and geographic patterns of AIV infection in BWTE across Canada and the US were primarily driven by the dynamics of AIV infection in hatch year birds. Our results demonstrate demographic as well as seasonal, latitudinal and flyway trends across Canada and the US. This research provided further evidence for the role of wild dabbling ducks, particularly BWTE, in the maintenance and ecology of AIV. This improved understanding of the role of BWTE as natural hosts, and the geographic, demographic and temporal variables that affect infection and transmission parameters, moves us closer to deciphering the overall ecology of the virus and its transmission and transportation pathways at the individual, population and continental levels. This knowledge, in turn, will permit development of better tools to predict and perhaps to prevent possible outbreaks in domestic animals as well as in humans.

Although wild birds are natural hosts of avian influenza viruses (AIVs), these viruses can be highly contagious to poultry and a zoonotic threat to humans. The propensity of AIV for genetic variation through genetic shift and drift allows virus to evade vaccine mediated humoral immunity. An alternative approach to current vaccine development is induction of CD8+ T cells which responds to more conserved epitopes than humoral immunity and targets a broader spectrum of viruses. Since the memory CD8+ T lymphocyte responses in chickens to individual AIV proteins have not been defined, the modulation of responses of the memory CD8+ T lymphocytes to H5N9 AIV hemagglutinin (HA) and nucleocapsid (NP) proteins over a time course were evaluated. CD8+ T lymphocyte responses induced by intramuscular inoculation of chickens with AIV HA and NP expressing cDNA plasmids or a non-replicating human adenovirus vector were identified through ex vivo stimulation with virus infected, major histocompatibility complex (MHC) matched antigen presenting cells (APCs). The IFN? production by activated lymphocytes was evaluated by macrophage production of nitric oxide and ELISA. MHC-I restricted memory T lymphocyte responses were determined at 10 days and 3, 5, 7 and 9 weeks post-inoculation (p.i). The use of non-professional APCs and APC driven proliferation of cells with CD8+ phenotype correlated with the activation of CD8+ T lymphocytes. The responses specific to nucleocapsid protein (NP) were consistently greater than those to the hemagglutinin (HA) at 5 weeks when the CD8+ T cell responses were maximum. By 8 to 9 weeks p.i., responses to either protein were undetectable. The T lymphocytes also responded to stimulation with a heterologous H7N2 AIV infected APCs. Administration of booster dose induced secondary effector cell mediated immune responses which had greater magnitudes than primary effector responses at 10 days p.i. Flow cytometric analysis (FACS) of the T lymphocytes demonstrated that memory CD8+ T lymphocytes of chickens can be distinguished from naive lymphocytes by their higher expression of CD44 and CD45 surface antigens. CD45 expression of memory lymphocytes further increases upon ex vivo stimulation with APCs expressing AIV. This is the first characterization of avian memory responses following both primary and secondary expression of any individual viral protein.

Today, infectious animal diseases and the propagation speeds of these diseaseshave been threatening the human health. Threats from animal disease outbreakssuch as Avian Influenza have increased in both number and complexity. So, it is extremely important to determine the animal diseases at first appearances and totake precautions according to propagation speeds of the diseases. Geographic Information Systems (GIS) have become an important tool inveterinary epidemiology, surveillance and monitoring of animal diseases. Such approaches can be used for public health planning and predicting disease risks. This study aims to build a GIS web-based animal health surveillance system in Turkey in order to monitor and analyse disease outbreaks. Different sources of data / geographical data, animal holding locations, disease outbreak recordings, reporting information and special GIS functions have been incorporated in the application. It enables to determine the first, second and third degree risk zones of a disease, query the animals, holdings and disease events, create thematic maps and show the results of explored landscape features associated with Avian Influenza outbreak of 2006 and present graphically illustrated reports. This study will make the management of the disease outbreak situation easier, enhance the response mechanism of the decision makers, help to make better decisions, control the disease as quickly as possible, protect both the animals and humans against diseases, also provide a tool to evaluate different strategies to prevent the spread of infectious diseases. So, in an infectious disease case, emergency precautions can be taken and control strategies can be planned.

published_or_final_version / Public Administration / Master / Master of Public Administration

Computational evolutionary analyses, particularly phylogenetics and ancestral reconstruction, have been extensively exploited under different algorithms and evolutionary models to better understand genome evolution from both small- and large-scale perspectives in order to assign genotypes based on assortment, resolve species relationships and gene annotation issues, further understand gene gain/loss within individual gene families, measure functional divergence among homologs, and infer ancestral character states. These evolutionary studies provide us with insights into biologically relevant issues including paleoenvironments inferred from resurrected proteins, developmental physiology associated with functional divergence of duplicated genes, viral epidemics and modes of transmission in attempt to better prepare, prevent and control diseases, evolution of lineage-specific pathogenicity, and attempts to create a synthetic ancient organism that would benefit the field of synthetic biology. Our work also provides us with greater insights into the accuracies and limitations of ancestral sequence reconstruction methods. In total, our work highlights the diverse questions that evolutionary studies attempt to address and the different biological levels that can be studied to answer these questions.