Viral sequence data has great potential for answering questions about the epidemiological dynamics and evolution of viruses. Classical approaches have sought amino acid changes that alter pathogenesis or transmissibility by influencing a virus's ability to enter or replicate within cells. However, this approach rarely recognises the fundamental impact of heterogeneous host contact structures and existing immunological responses on viral transmission. This thesis draws heavily on ecological and immunological concepts to explore the epidemiological dynamics, diversity and evolution of viruses using molecular sequence data. A number of different research approaches and study systems are used in this thesis. I begin by describing a novel polyomavirus in a European badger, and apply phylogenetic techniques to analyze the evolutionary history of the Polyomaviridae. I subsequently describe a large metaviromic study in a population of wild mute swans, for which host demographic data are available. I describe nine new viral species and test whether age and season are associated with differences in abundance and prevalence of different viral taxonomic groups. The study highlights the potential of metaviromics for investigating viral epidemiological dynamics in natural populations. Influenza A viruses of avian origin (AIV) threaten human and animal health. Using phylogeographic methods, I reconstruct the spatial spread of an H5N8 virus at a regional scale, and investigate how bird density and migration shaped this dispersal. Despite the importance of acquisition of humoral immunity to different strains throughout the lifespan of wild birds for epidemiological dynamics, this topic is poorly understood. I assess the accumulation of immune responses to AIV with age in mute swans. I consider how ecological factors, including age-structured immunity, might have affected the epidemiology of an H5N8 outbreak in the population.

Influenza aviaire est une maladie infectieuse contagieuse, due à des virus de la famille des Orthomyxoviridae, les virus Influenza A, touchant les oiseaux et susceptible d'entraîner une mortalité extrêmement élevée, notamment chez les volailles. Les souches hautement pathogènes des virus influenza aviaire provoquent une maladie sévère et mortelle chez les volailles et constituent un risque pour la santé publique. En février 2006, la France a été touchée par une épizootie d'influenza aviaire H5N1 hautement pathogène, qui s'est répandu dans l'avifaune sauvage de la zone humide la Dombes. La Dombes est une zone humide, mosaïque de plus de mille étangs, et une zone d'importance ornithologique internationale, abritant des milliers d'oiseaux d'eau. C'est une zone à risque pour la propagation de l'influenza aviaire de part à la fois la présence des populations d'oiseaux sauvages sur les étangs et de la proximité de ces populations aux élevages de volailles qui parsèment la région. L'objectif principal de ce travail est la modélisation du risque influenza aviaire dans la région de la Dombes. Ce travail est axé sur l'avifaune sauvage et sur le risque d'infection et de transmission de l'infection par les oiseaux sauvages. Nous avons, dans un premier temps, identifié les caractéristiques particulières à la Dombes, pouvant influer sur l'épidémiologie de l'influenza aviaire dans la région. Les trois acteurs principaux de l'écosystème de la Dombes sont les étangs, les populations d'oiseaux sauvages et les élevages de volailles. Ensuite, le risque influenza aviaire dans la Dombes a été décliné à deux niveaux : 1) dans l'écosystème d'un seul étang ; et 2) au sein d'un réseau d'étangs comme caractérisés en 1), où la propagation de l'infection peut se faire d'étang à étang. Au niveau étang, le risque influenza aviaire est modélisé, d'une part, par le temps de persistance des virus influenza aviaire dans l'eau de l'étang, et d'autre part, par la dynamique de l'infection au sein des populations d'oiseaux sauvages par transmission directe entre oiseaux et indirecte via l'eau de l'étang. Au niveau réseau d'étangs, l'analyse du risque permet d'identifier des groupements spatiaux d'étangs à risque élevé d'infection aux virus influenza aviaire. Quant au risque d'exposition des élevages aux virus influenza aviaire d'origine de l'avifaune sauvage, il est calculé en tenant compte à la fois de la proximité des élevages aux étangs susceptibles d'être infectés et de la probabilité de ces étangs à être infectés en cas d'épizootie. / Avian influenza is a contagious infectious disease caused by viruses of the family Orthomyxoviridae, influenza A viruses, affecting birds and known to cause extremely high mortality, especially in poultry. Highly pathogenic strains of avian influenza viruses cause severe and fatal disease in poultry and pose a risk to public health. In February 2006, France was hit by a highly pathogenic H5N1 avian influenza outbreak, affecting aquatic wildfowl in Dombes wetland. The Dombes is a wetland mosaic of more than a thousand ponds and an area of international ornithological importance, home to thousands of waterfowl. This is an area at risk for the spread of avian influenza viruses due to both the presence of wild birds on the ponds and the proximity of these populations to poultry farms that are present in the area. The main objective of this work is the modeling of the avian influenza risk in the Dombes region. This work is focused on the wildfowl and the risk of infection and transmission of infection by wild birds. As a first step, we have identified the specific characteristics of the Dombes area, which may affect the epidemiology of avian influenza in the region. The three main players in the Dombes ecosystem are the ponds, the wildfowl populations and the poultry farms. Secondly, the avian influenza risk in the Dombes was declined at two levels: 1) in the ecosystem of a single pond; and 2) within a network of ponds as characterized by 1), where the infection is spread from pond to pond. At the pond level, the avian influenza risk is modeled, on the one hand, by the persistence time of avian influenza viruses in the water of the pond, and, on the other hand, by the infection dynamics within wild bird populations by direct transmission between birds and indirectly through the water of the pond. At the ponds network, the risk analysis identifies spatial clusters of ponds at high risk of infection with avian influenza viruses. Concerning the risk of exposure of poultry farms to avian influenza viruses of wildfowl origin, it is calculated taking into account both the proximity of the farms to ponds likely to be infected and the probability of these ponds to become infected during an outbreak.

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.

Recent World Animal Health Organization (OIE) reports on Avian Influenza (AI) outbreaks in Asia, Europe and Canada suggest that there is a nonzero probability that an outbreak may occur anywhere in the world, including the US. To help evaluate possible policy in the face of such an event, this dissertation does an economic evaluation of the implications of using two mitigation strategies: one corresponding to the currently response strategy; and the other an OIE recommended one utilizing vaccination. To do this, the dissertation develops and uses an integrated economic-epidemic model. In this effort, I first estimate the cost of an AI outbreak under a deterministic disease spread assumption where a new vaccination strategy and the current strategy are compared. Subsequently, I introduce risk in the model and construct 95 percent confidence intervals for the outbreak costs, and I rank the outcomes of the alternative strategies using stochastic dominance criteria. In addition, during both phases, I develop and estimate the breakeven probability for an event where ex-ante fixed costs of vaccine stockpiling are justified by the reduction in disease event damages. Results under deterministic disease spread assumption suggest that the vaccination strategy lowers the cost of outbreaks as opposed to the current strategy. This happens because vaccination reduces the number of culled and quarantined flocks. The study is conducted in three locations, yielding the finding that the costs of an outbreak vary depending on the densities of poultry flocks. I also find that when consumer demand shifts due to the outbreak, the costs are much larger. Finally, I find that ex-ante vaccine stockpiling is justified for all the sub-regions if the probability of outbreak exceeds 0.07. The stochastic disease spread assumption results also show that the vaccination strategy dominates in first degree stochastic dominance sense. Consistent with stochastic dominance results, the 95 percent confidence intervals have narrower ranges under the vaccination strategy than without it. Finally, the distribution of the breakeven probability for vaccine stocking has a mode of 0.07 and that the probability is accurate with 82 percent likelihood. However, the threshold varies with the disease transmission parameters and could reach up to 0.32.

Thesis (M.A.)--University of Canterbury, 2009. / Typescript (photocopy). Includes bibliographical references (leaves 146-158). Also available via the World Wide Web.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Community Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Politics and Public Administration / Master / Master of Public Administration

Highly pathogenic avian influenza H5N1, which is panzootic in poultry, continues to spread and becomes endemic in Asia, Africa, and Europe. It causes human disease with high fatality (about 60%) and continues to pose a pandemic threat. The pathological lesions associated with human H5N1 disease is Acute Respiratory Distress Syndrome (ARDS). The biological basis underlying the development of ARDS in human H5N1 disease remains controversial. Clinical, animal and in vitro studies suggested that the differences between H5N1 influenza viruss and low pathogenic influenza viruses in regard to viral replication, tissue tropism and cytokine dysregulation may contribute to disease pathogenesis. We previously found delayed onset of apoptosis in influenza H5N1 virus infected human peripheral blood monocyte-derived macrophages. This may allow a longer survival time for the virus in target cells for prolonged viral replication, which may contribute to the pathogenesis of H5N1 disease. As human bronchial and alveolar epithelial cells are target cells of influenza virus, I explored if influenza H5N1 and H1N1 virus infected human respiratory epithelial cells displayed differential apoptotic response and dissected the apoptotic pathways triggered by influenza virus infection. In this study, the apoptotic response in highly pathogenic influenza H5N1 viruses, A/HK/483/97 and A/Vietnam/1203/04, their precursor avian influenza H9N2 virus, A/Quail/HK/G1/97, and seasonal H1N1 virus, A/HK/54/98 infected primary human alveolar and bronchial epithelial cells was compared by TUNEL. A delayed onset of apoptosis in influenza H5N1 viruses and avian H9N2 virus infected alveolar epithelial cells was observed; the pattern was similar in bronchial epithelial cells. Concomitantly, by Western blotting, a delay in caspase 3 activation in H5N1 virus (A/HK/483/97) infected alveolar epithelial cells compared to H1N1 virus (A/HK/54/98) infected cells was shown. Also, influenza H5N1 and H1N1 virus induced apoptosis through both intrinsic and extrinsic pathways in human alveolar epithelial cells. Chemokine IP-10 was differentially up-regulated in influenza H5N1 virus infected alveolar epithelial cells, but its relationship to the delayed onset of apoptosis requires further studies. TRAIL, an upstream signaling molecule of extrinsic apoptotic pathway, mRNA was up-regulated in influenza H5N1 infected alveolar epithelial cells but not in influenza H1N1 infected cells. Using recombinant viruses, I showed that the 627 amino acid residue on PB2 of H5N1 virus and mutation of amino acids on 253 and 591 residues on PB2 of H9N2 virus contribute to the TRAIL upregulation. Immunohistochemical staining of physiologically relevant ex vivo model of human bronchus showed that influenza H5N1 (A/Vietnam/3046/04) and H9N2 (A/Quail/HK/G1/97) virus did not infect human bronchi as well as human H1N1 (A/HK/54/98) virus. Profiling of apoptosis related genes showed that TRAIL tends to be up-regulated in H5N1 virus infected bronchi ex vivo. This study demonstrated the delayed onset of apoptosis by H5N1 virus infected respiratory epithelial cells may be a mean for influenza virus to have prolonged replication within the human respiratory tract and contribute to disease severity. The results generated provide a robust research agenda, yielding critical information that elucidate molecular mechanisms, such as TRAIL up-regulation, that may contribute to the virulence and pathogenesis in human H5N1 disease. / HKU 3 Minute Thesis Award, 2rd Runner-up (2011) / published_or_final_version / Pathology / Master / Master of Philosophy