Master of Science / Department of Diagnostic Medicine/Pathobiology / Alison Paige Adams / Avian influenza (AI) is a zoonotic disease that has garnered much attention in recent years due to its detrimental effects on poultry, producers and potentially human health. This disease can be extremely fatal to domestic poultry, killing as high as 90-100% of the flock. This virus has the potential to cause devastation to and loss of entire flocks. AI is typically spread between wild fowl and domestic poultry with a zoonotic potential to also affect human health as well as other animals. Its spread also has a massive economic impact due to the decreased amounts of available poultry products to consumers around the world. This report will examine the worldwide history and epidemiology of highly pathogenic avian influenza (HPAI). In the last ninety-two years, there have been five recorded outbreaks of HPAI in the United States (US). Globally, notable outbreaks have occurred in Italy (1997-2001), the Dutch region of Europe (2003), Canada (2004), and more recently, in Asia. Preventative measures will be examined in this report. In particular, biosecurity, quarantine, surveillance, and eradication are some of the most widely recognized and accepted ways to help prevent and control HPAI outbreaks. However, none of these methods are failsafe strategies to completely prevent or control the spread of HPAI. This report will focus on an additional preventative measure - currently available and potential future vaccination programs. There is a global shift toward procuring poultry that are AI-free as well as unvaccinated for AI. This is, in part, due to the limitations of currently available vaccines in completely ridding poultry of this disease. Vaccinations may reduce the amount of virus in infected birds, but this does not prevent birds from becoming infected. When addressing the control and eradication of HPAI, some future challenges include viral mutations, intermingling of domesticated and wild birds, and vaccine development. Because of the current limitations of vaccines and future challenges in controlling the spread of infection, there is no one single solution to this problem. It will require a multi-faceted approach.

Los virus de influenza aviar de alta patogenicidad (IAAP) causan una enfermedad muy severa en pollos, los cuales frecuentemente inducen lesiones en el sistema nervioso central (SNC). Esta tesis recoge los resultados de tres estudios que se llevaron a cabo para determinar el mecanismo de patogénesis y neurotropismo, así como establecer la ruta de entrada al SNC para un virus H7N1 IAAP. En el primer estudio se estableció un modelo animal de infección en pollos libres de patógenos específicos, que consistía en la inoculación intranasal con el virus H7N1 IAAP. Para establecer este modelo, se utilizaron tres diferentes dosis del virus, obteniendo que las dosis más altas producen una enfermedad similar a la reportada para otros virus de IAAP. Además, se observó que las dosis más bajas causan infección demostrada porque con las dosis más bajas, el virus es hallado en muestras de tejido, muestras de heces y secreciones respiratorias. Adicionalmente, se pudo comprobar el alto neurotropismo del virus, ya que aún en pollos inoculados con bajas dosis el RNA viral es hallado en el CNS. La viremia fue detectada a un día post infección (dpi), sugiriendo que está podría ser la vía de diseminación al SNC. En el segundo estudio, se determinó la distribución topográfica del antígeno viral en el SNC durante las primeras horas post infección, lo cual permitió determinar que el virus se disemina de forma simétrica y bilateral en núcleos neurales del diencéfalo, mesencéfalo y rombencéfalo. La distribución del antígeno viral indica que el bulbo olfatorio y los nervios periféricos están involucrados en el proceso de invasión del SNC. El hallazgo de receptores aviares y humanos en las células endoteliales explica porque estas células son tan sensibles a la infección. El RNA viral fue hallado en el líquido cerebro espinal el primer dpi, lo que indica que el virus atraviesa la barrera hemato-encefálica (BHE). En el tercer estudio, la alteración de la BHE inducido por el virus H7N1 IAAP fue demostrado usando tres diferentes métodos que incluye la perfusión intracardial de Azul de Evans, la detección de la extravasación de la proteína del suero IgY, y evaluación del patrón de tinción con el marcador de las uniones fuertes de la BHE, ZO-1 y claudin-1. El antígeno viral fue observado a las 24 hpi en las células endoteliales, mientras que el daño de la BHE fue observado a las 36 hpi y 48 hpi. En resumen, se puede afirmar que el virus H7N1 IAAP se disemina por la vía hematógena durante las primeras horas pi, posiblemente favorecido por la presencia de receptores en las células endoteliales del sistema nervioso central, y poco después daña la BHE durante las primeras horas de infección como se demuestra por la presencia de extravasación del azul de Evans and IgY del suero. / Highly pathogenic avian influenza viruses (HPAIV) cause a very severe systemic disease in chickens, in which is also frequent to find central nervous system (CNS) lesions. In this thesis, three studies were undertaken in order to determine the mechanism of pathogenesis, the neurotropism and establish the route of entry into the CNS use for a H7N1 HPAI virus. In the first study, an animal model was set up that consisted of SPF chickens inoculated intranasally with the H7N1 HPAI virus. To do that, three different doses were used, obtaining that the highest dose induced a disease similar to the produce by other HPAI viruses, moreover, it was also observed that very low doses also cause infection demonstrated because viral RNA was found in tissues samples, faeces and respiratory secretions. Besides, the high neurotropism of this virus was demonstrated because still in chickens inoculated with low doses, viral RNA is found in the brain. Viremia was detected at one dpi, which indicated that the bloodstream is the pathway of viral spreading to the brain. In the second study, the topographical distribution study of the viral antigen during the first dpi was determined, which allow to determine that the virus disseminates showing a symmetrical and bilateral pattern in the diencephalon, mesencephalon and rhombencephalon, whereas in the telencephalon and cerebellum it was multifocal and random. Viral antigen distribution indicates that the olfactory bulb (OB) and peripheral nerves are not involved in the process of virus invasion into the brain. Avian and human influenza receptors were found in endothelial cells which explain why these cells are so sensitive to the infection. Viral RNA was found in cerebrospinal fluid (CSF) at one dpi, indicating that the virus was able to cross blood brain barrier (BBB). In the third study, the disruption of the BBB induce by the H7N1 HPAI was demonstrated using three different methods that include the intracardial perfusion of the tracer Evans blue (EB), detection of the extravasation serum IgY, and evaluation of the pattern of staining of the tight junction proteins ZO-1 and claudin-1. Viral antigen can be observed as early as 24 hpi in the endothelial cells, whereas disruption was detected at 36 and 48 hpi. In summary, it can be asserted that this H7N1 HPAIV disseminates via the haematogenous route early during the infection, favored by the presence of abundant receptors on the CNS endothelial cells, and soon after it disrupts the BBB during the first hours of infection as demonstrated by the presence of EB and serum IgY extravasation.

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

Periodic introductions of influenza A viruses (IAVs) from wild birds contribute to emergence of novel strains that infect domestic poultry, lower mammals, and humans, but the mechanisms of emergence are unclear. The objectives of this dissertation research are to infer the genesis of two emerging IAVs, low pathogenic avian influenza (LPAI) H10N8 and highly pathogenic avian influenza (HPAI) H7N8 viruses, and to characterize the antigenic diversity and genetic evolution of contemporary H7 avian influenza viruses (AIVs) from North America. First, AIVs that are genetically close to the human H10N8 isolate were recovered at the live poultry market (LPM) visited by the first H10N8 patient. High seroprevalence of H10 virus was observed in ducks and chickens from five LPMs in the region. These findings suggested that LPM was the most probable source of human infection with the H10N8 virus, and this virus appeared to be present throughout the LPM system in the city. Second, the novel H7N8 virus most likely circulated among diving ducks in the Mississippi flyway during autumn 2015 and was subsequently introduced to Indiana turkey, in which it evolved from LPAI into HPAI. H4N8 IAVs from diving ducks possess a gene constellation comprising five H7N8–like gene segments. These findings suggest that viral gene constellations circulating among diving ducks could contribute towards the emergence of IAVs that can affect poultry. Diving ducks may serve as a unique reservoir, contributing to the maintenance, diversification, and transmission of IAVs in wild birds. Third, antigenic and genetic characterization of 93 H7 AIVs from North America showed limited antigenic diversity. Gradual accumulation of nucleotide and amino acid substitutions in the H7 gene of AIVs from wild and domestic birds caused a wide genetic diversity. These findings suggested that continuous genetic evolution has not led to significant antigenic diversity for contemporary H7 AIVs isolated from wild and domestic birds in North America. In summary, these findings not only improve our understanding of the ecology and evolution of IAVs but also provide information for formulation of effective disease prevention and control strategies.

No description available.

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.

<p>Avian Influenza (AI) is a contagious viral zoonotic disease with great public health implications and negative socioeconomic impact (WHO, 2006a). The highly pathogenic avian influenza (HPAI) infection is transmitted from birds to man mostly through contact with contaminated poultry and objects (INFOSAN, 2005), hence people who come in contact with birds such as live bird sellers (LBS) are the more vulnerable population (WHO, 2006a). Inadequate knowledge of AI health risks and poor practice of AI preventive measures amongst LBS increases the risk of spread of the infection in both humans and animals.The aim of this study was to describe and quantify the knowledge and practice of LBS with regards to avian influenza health risks and preventive activities in Agege, an urban area in Lagos State, Nigeria.</p>

<p>Avian Influenza (AI) is a contagious viral zoonotic disease with great public health implications and negative socioeconomic impact (WHO, 2006a). The highly pathogenic avian influenza (HPAI) infection is transmitted from birds to man mostly through contact with contaminated poultry and objects (INFOSAN, 2005), hence people who come in contact with birds such as live bird sellers (LBS) are the more vulnerable population (WHO, 2006a). Inadequate knowledge of AI health risks and poor practice of AI preventive measures amongst LBS increases the risk of spread of the infection in both humans and animals.The aim of this study was to describe and quantify the knowledge and practice of LBS with regards to avian influenza health risks and preventive activities in Agege, an urban area in Lagos State, Nigeria.</p>

Magister Public Health - MPH / Avian Influenza (AI) is a contagious viral zoonotic disease with great public health implications and negative socioeconomic impact (WHO, 2006a). The highly pathogenic avian influenza (HPAI) infection is transmitted from birds to man mostly through contact with contaminated poultry and objects (INFOSAN, 2005), hence people who come in contact with birds such as live bird sellers (LBS) are the more vulnerable population (WHO, 2006a). Inadequate knowledge of AI health risks and poor practice of AI preventive measures amongst LBS increases the risk of spread of the infection in both humans and animals.The aim of this study was to describe and quantify the knowledge and practice of LBS with regards to avian influenza health risks and preventive activities in Agege, an urban area in Lagos State, Nigeria. / South Africa

La surveillance de la santé se définit comme la production et le traitement de données destinées à informer les programmes de mitigation des risques sanitaires. La surveillance des maladies infectieuses animales est généralement considérée comme un bien public, impliquant la responsabilité de l’Etat. La surveillance des maladies émergentes transfrontalières, dont l’influenza aviaire hautement pathogène (IAHP), est même perçue comme un bien public mondial, justifiant un partage d’informations entre Etats. La forme la plus répandue de la surveillance, dite passive ou réactive, repose sur la communication d’acteurs privés ou publics d’informations qu’ils détiennent sur l’état sanitaire des populations animales qu’ils observent aux autorités en charge de la surveillance. La surveillance se trouve donc confrontée à la problématique des biens publics dont la gestion est dépendante de la décision décentralisée d’acteurs privés. Deux questions se posent alors : quels sont les facteurs qui influencent la décision de transmettre une information aux systèmes de surveillance publics ? Ces facteurs sont-ils purement financiers ou impliquent-ils d’autres types d’enjeux, qui font intervenir l’environnement social de l’individu, le territoire dans lequel il s’insère et ses rapports de pouvoirs ? Une autre question est celle de l’existence de réseaux d’information, établis entre acteurs privés et publics permettant d’alerter un maximum d’acteurs de l’apparition d’un risque sanitaire. Comment ces réseaux de constituent-ils ? Dans quelle mesure sont-ils liés aux systèmes de surveillance publics ? Quelles formes de gestion du risque, sous contrôle privé ou public, permettent-ils ? Le cas étudié est celui de l’IAHP due à H5N1 chez les volailles domestiques en Asie du Sud-Est. Une étude a été menée dans quatre zones d’échelle spatiale réduite réparties sur les deux pays, trois au Viet Nam et une en Thaïlande. La théorie des graphs a été appliquée à la diffusion de l’information sur les suspicions d’IAHP entre acteurs privés et publics des territoires avicoles. La structure de ces réseaux d’information est conditionnée par l’organisation politique des territoires ruraux, sous forme de villages, et par les filières dans lesquels s’insèrent les élevages présents dans les territoires. Dans les zones d’étude du Viet Nam présentant un grand nombre d’élevage commerciaux privés, les acteurs amont de la filière avicole commerciale, qui fournissent aliments et produits vétérinaires aux éleveurs, ont un accès privilégié à l’information issue du secteur avicole commercial et villageois. Dans la zone d’étude de Thaïlande, les acteurs impliqués dans les combats de coqs ont un accès privilégié à l’information issue des éleveurs villageois. Ces acteurs centraux dans les réseaux facilitent la diffusion spatiale des informations et l’accès de l’ensemble des éleveurs à ces informations. Les autorités vétérinaires sont présentes dans les réseaux mais la priorité qui leur est accordée est faible en comparaison aux acteurs privés de la filière. En parallèle, des entretiens qualitatifs ou semi-quantitatifs utilisant les outils de l’épidémiologie participative ont été menés afin d’identifier les enjeux associés à la déclaration des suspicions aux autorités vétérinaire. Les enjeux diffèrent selon les territoires et les types de production avicoles qui les composent. Ces enjeux vont au-delà des problématiques purement financières : risques sanitaires et nuisances environnementales pour le voisinage, responsabilité dans les pertes économiques des autres éleveurs et des partenaires commerciaux, et valeur affective et sociale de l’animal sont autant de composantes potentielles de la décision de l’éleveur de déclarer une suspicion aux autorités. Une partie de ces enjeux est liée aux mesures de contrôle mise en place par l’Etat face au risque sanitaire. Cependant, d’autres sont strictement associés à la diffusion de l’information. / Health surveillance is defined as the production and processing of data aimed at informing health risk mitigation programs. Surveillance of infectious animal diseases is usually considered as a public good, involving the responsibility of the state. Surveillance of transboundary emerging diseases, like HPAI, is even perceived as an international public good, justifying information sharing between countries. The most common type of surveillance, i.e. passive or reactive surveillance, is based on communications from private or public actors of the information they hold about the health status of animal populations they observe to authorities in charge of health surveillance. Animal health is therefore confronted with the problematic of public goods whose production depends on decentralized private decision. Two questions may be raised: what are the factors influencing the decision to transmit information to public surveillance systems? Are these factors solely of monetary nature or are they linked with other types of issues, among which the social environment of individuals, the place where they live and their power relationships? Another question relates to the existence of information networks established between private and public actors which enable to a part of the population of sanitary threats. How these are networks constituted? To which extent are they linked with public surveillance systems? Which type of risk management do they allow? The studied case is H5N1 HPAI in domestic poultry in Southeast Asia. A study was conducted in four areas of limited spatial scale distributed in two countries, three in Viet Nam and one in Thailand. Graph theory was applied to the diffusion of information related to HPAI suspicions between actors, private or public. The structure of these networks is shaped by the political organization of rural places, the villages, and by the value chains to which poultry farms belong. In study areas of Viet Nam with widespread commercial poultry farming, upstream actors of the value chain, supplying feed and veterinary products to farmers have better access to information from the commercial and backyard poultry farms. In the Thailand study area, actors participating in cock fighting games have a better access to information from backyard farms. These actors who are central in information networks facilitate the spatial spread of information and access of farmers to information from distant locations. Veterinary authorities are included in the information networks but their attributed priority is week in comparison with private actors of value chains. Besides, qualitative and semi-quantitative interviews were conducted, using tools of participatory epidemiology, in order to identify issues linked with suspicion reporting to veterinary authorities. Those issues differ according to places and types of poultry production. They go beyond purely monetary concerns: sanitary risks and environmental nuisances to the neighborhood, responsibility in economic losses of other farmers and commercial partners and social and affective value of animals are potential components of the decision of farmers to report a suspicion to veterinary authorities. A part of these issues are linked with disease control measures implemented by the state in response to sanitary risks. However, others are strictly associated with information spread. It is the case, for example, of impacts of information on poultry market prices.