Resuspended floor dust constitutes up to sixty percent of the total particulate matter in indoor air. This fraction may also include virus-laden particles that settle on the floor after being emitted by an infected individual. This research focuses on predicting the concentration of influenza A viruses in resuspended dust, generated by people walking in a room, at various heights above the floor. Using a sonic anemometer, we measured the velocity field from floor to ceiling at 10-cm intervals to estimate the magnitude of turbulence generated by walking. The resulting eddy diffusion coefficients varied between 0.06 m2 s-1 and 0.20 m2 s-1 and were maximal at ~0.75-1 m above the floor, approximately the height of the swinging hand. We used these coefficients in an atmospheric transport model to predict virus concentrations as a function of the carrier particle size and height in the room. Results indicate that the concentration of resuspended viruses at 1 m above the floor is about seven times the concentration at 2 m. Thus, shorter people may be exposed to higher concentrations of pathogens in resuspended dust indoors. This study illuminates the possibility that particle resuspension could be a mode of disease transmission. It also emphasizes the importance of considering resuspension of particulate matter when designing ventilation systems and flooring in hospitals and residences. / Master of Science

This dissertation examines the socio-behavioral determinants of vaccination and their impacts on public health, using a systems approach that emphasizes the interface between population health research, policy, and practice. First, we identify the facilitators and barriers of parental attitudes and beliefs toward school-located influenza vaccination in the United States. Next, we examine current vaccine sentiment on social media by constructing and analyzing semantic networks of vaccine information online. Finally, we estimate the health benefits, costs, and cost-effectiveness of influenza vaccination strategies in Seattle using a dynamic agent-based model. The underlying motivation for this research is to better inform public health policy by leveraging the facilitators and addressing potential barriers against vaccination; by understanding vaccine sentiment to improve health science communication; and by assessing potential vaccination strategies that may provide the greatest gains in health for a given cost in health resources. / PHD / Public health decisions are ultimately left to those in policy, however these decisions are often subjective and rarely informed by data. This dissertation comprises three studies that, individually, examine various public health aspects of vaccination, and collectively, aim to help inform decision makers by bridging the gaps that persist between scientific evidence and the implementation of relevant health policy. First, we identify the facilitators and barriers of parental attitudes and beliefs toward school-located influenza vaccination in the United States. Next, we examine current vaccine sentiment on social media by constructing and analyzing semantic networks of vaccine information online. Finally, we estimate the health benefits, costs, and cost-effectiveness of influenza vaccination strategies in Seattle using a dynamic agent-based model. The work presented here demonstrates a systems approach to public health by way of computational modeling and interdisciplinary perspectives that describe vaccination behavior at the intersection of public health research, policy, and practice. The motivation for this research is to better inform public health policy: by leveraging the facilitators and addressing potential barriers against vaccination; by understanding vaccine sentiment to improve health science communication; and by assessing potential vaccination strategies that may provide the greatest gains in health for a given cost in health resources.

The natural reservoir of influenza A virus (IAV) is wild waterfowl, and all human IAVs have their genetic origins from avian viruses. Neuraminidase inhibitors (NAIs) are currently the best drugs for treatment of human influenza; therefore, the orally available NAI oseltamivir (Tamiflu®) has been stockpiled worldwide as part of pandemic preparedness planning. Re-sistance to NAIs is related to worse clinical outcomes and if a new pandemic influenza virus would be oseltamivir-resistant its public health impact would be substantially worsened. The active metabolite oseltamivir carboxylate (OC) is not removed by sewage treatment and ends up in river water, where OC-concentrations up to 0.86µg/L have been detected. We hypothesize that occasional OC exposure of wild waterfowl carrying IAVs may result in circulation of resistant variants that may potentially evolve to become human-pathogenic. We tested the hypothesis in an in vivo Mallard (Anas platyrhynchos) model in which birds were infected with avian IAVs and exposed to OC. Excreted viruses were analyzed regarding genotypic and phenotypic resistance by neuraminidase (NA) sequencing and a functional NA inhibition assay. Two viruses with NAs of the phylogenetic N2-group, H6N2 and H7N9, acquired the NA substitutions R292K and I222T when host ducks were exposed to 12µg/L and 2.5µg/L of OC, respectively. Drug susceptibilities were at previously described levels for the substitutions. To test persistence of resistance, an OC resistant avian H1N1/H274Y virus (with a group N1 NA-protein) from a previous study, and three resistant H6N2/R292K variants were allowed to replicate in Mallards without drug pressure. Resistance was entirely maintained in the H1N1/H274Y virus, but the H6N2/R292K variants were outcompeted by wild type virus, indicating retained fitness of the resistant H1N1 but not the H6N2 variants. We conclude that OC in the environment may generate resistant IAVs in wild birds. Resistant avian IAVs may become a problem to humans, should the resistance trait become part of a new human pathogenic virus. It implies a need for prudent use of available NAIs, optimized sewage treatment and resistance surveillance of avian IAVs of wild birds.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Recently, new strains of influenza such as bird flu and swine flu have emerged. These strains have the capacity to infect people on a quite large scale and are characterized by their resistance to existing influenza treatment and their high mortality rates. In this thesis, we consider two models for influenza transmission dynamics that include both sensitive and resistant strains and accounts for disease induced mortality. The first model allows for immigration/migration and does not include any control measure. The second one explores the effects of vaccination and treatment of the sensitive strain but ignores immigration/migration. We studied the two models mathematically and numerically. We started with the model without any control measures; we calculated the basic reproductive numbers, determined the equilibrium points and investigated their stability. Our analysis showed that when the basic reproduction numbers of both strains are less than one then the two strains will die out. When at least one of the basic reproduction numbers is greater than one, then the strain with the higher basic reproduction number is the one that will persist. Numerical simulations were carried out to confirm the stability results and a bifurcation diagram was given. We also studied numerically the impact of the mortality rate of influenza on the dynamics of the disease. Especially, we investigated the effect of the mortality rate on the time needed for the pandemic to reach its peak, the value at the peak for each strain and, when eradication is possible, the time it takes for the disease to be eradicated. For the model with control, we also calculated the control reproductive number and the equilibrium points. The stability analysis was carried out numerically and bifurcation diagrams with vaccination and treatment parameters were given to determine the regions where eradication of the disease is possible. Our results suggest that in the presence of a resistant strain, treating more infected individuals will not eradicate the disease as the resistant strain will always persist. In such a case vaccination and antiviral treatment should be implemented simultaneously. / AFRIKAANSE OPSOMMING: Geen opsomming

published_or_final_version / Community Medicine / Doctoral / Doctor of Philosophy

The influenza virus, a member of the Orthomyxoviridae family, causes regular, large-scale morbidity and mortality in birds and humans and significant human suffering and economic loss. The primary aim of this study was to develop a novel influenza vaccine. Vaccines are an essential tool for the control of influenza because they increase resistance to infection, prevent illness and death and help to limit virus transmission to other birds and mammals, including humans. By reducing the environmental contamination of influenza virus in global poultry stocks, the risk of a new pandemic virus being generated by the human-avian link is diminished. Marek’s Disease is a common lymphoproliferative disease of poultry that is readily controlled worldwide using the live attenuated vaccine, CVI988. The Marek’s Disease Virus (MDV) CVI988 viral genome, available as a Bacterial Artificial Chromosome (BAC), forms viable infectious viral particles when transfected into Chicken Embryo Fibroblast (CEF) cells. Using BAC mutagenesis, two non-essential genes in the MDV CVI988 BAC (UL41 and US10), were identified and replaced by the low pathogenic influenza haemagglutinin 10 (H10) gene. These live recombinant MDV-H10 vectors will allow simultaneous vaccination against both pathogens. In addition, the non-essential genes were also replaced with GFP creating MDV-GFP constructs. Both genes were expressed initially using a CMV promoter, although this disrupted the MDV CVI988 BAC; a second promoter, PGK-1, proved more successful. A third MDV gene (UL50) was deleted, but severe attenuation prevented the incorporation of H10 into this open reading frame. Future work to test the MDV-HA constructs in vivo will be carried out in collaboration with the Istituto Zooprofilattico Sperimentale delle Venezie in Italy. In addition, development of MDV constructs containing multiple HA genes (H10 and H5) linked by the 2A polyprotein can be developed with the goal of establishing heterosubtypic immunity.

Chaque année, les épidémies de grippe, dont les principaux agents étiologiques sont les virus influenza de type A, ont un impact considérable sur la population en terme de morbidité et de mortalité. Le virus influenza A comporte à sa surface deux glycoprotéines, la neuraminidase et l’hémagglutinine. Ces deux protéines ont des fonctions antagonistes : l’hémagglutinine permet l’entrée du virus dans la cellule hôte et la neuraminidase, par son activité sialidase, libère les nouveaux virions formés. Bien que la prophylaxie du virus grippal repose essentiellement sur la vaccination, les antiviraux jouent un rôle important dans la lutte contre les épidémies de grippe et dans la stratégie développée en prévision d'une pandémie grippale. Les inhibiteurs de la neuraminidase (INAs) sont des antiviraux efficaces contre la grippe. Ils inhibent l’activité enzymatique de la neuraminidase et empêchent la libération des nouveaux virions formés. La démarche méthodologique qui a conduit à l’élaboration de molécules ciblant la neuraminidase laissait espérer une apparition limitée de résistance. Cependant, des cas de résistances aux INAs ont été mis en évidence lors d’études cliniques. Outre la nécessité d’une surveillance étroite, il est donc important d’étudier et de comprendre les diverses mécanismes susceptibles d’induire une résistance aux INAs. Le travail de cette thèse s’est ainsi porté sur la compréhension de la diversité des mécanismes de résistance. Dans un premier temps, nous avons étudié l’impact de mutations sur l’ensemble des résidus structuraux du site actif de la neuraminidase. Nous avons observé que la plupart de ces mutations n’altéraient pas les caractéristiques du virus et induisaient une légère baisse de sensibilité aux INAs. Par la suite, nous avons cherché à explorer les possibilités de synergie dans la résistance aux INAs par la combinaison de deux mutations structurales du site actif de la neuraminidase. Sur quatre virus produits, seul le virus possédant la double mutation E119V+I222L était viable, malgré une capacité réplicative in vitro altérée. La combinaison de ces deux mutations induit une synergie dans la résistance à l’oseltamivir. Enfin, nous avons voulu intégrer l’interaction fonctionnelle de la neuraminidase avec l’hémagglutinine. Nous avons montré que la combinaison d’une hémagglutinine de faible affinité pour les récepteurs sialylés permettait de restaurer un virus possédant une neuraminidase déficiente. Ainsi, un virus influenza peut se libérer de la fonction de la neuraminidase, cible des seuls antiviraux efficaces disponibles à l’heure actuelle. Les mécanismes de résistances aux inhibiteurs de la neuraminidase sont multiples. L’émergence durant les deux dernières saisons hivernales de virus résistants aux INAs sans pression de sélection a remis en question les hypothèses développées sur l’infectivité et la transmissibilité de souches résistantes, ouvrant de nouvelles perspectives quant à l’étude des mécanismes permettant l’obtention de virus épidémiogènes résistants aux INAs / Each winter, influenza epidemics have a considerable impact on the population in terms of morbidity and mortality. Influenza A virus is the main etiologic agents of influenza. They present at their surface two glycoproteins, the neuraminidase and the hemagglutinin. These two proteins have antagonist functions : the hemagglutinin allows the virus to enter the host cell and the neuraminidase, through its sialidase activity, releases progeny virions from host cells. Although prophylaxis of influenza is mainly based on vaccination, antiviral drugs play a very important role in the fight against epidemics of influenza and the strategy developed in anticipation of a flu pandemic. The neuraminidase inhibitors are effective antiviral against influenza. Through the inhibition of the neuraminidase enzymatic activity, they prevent the release of new virions formed. The introduction into clinical practice of new drugs requires monitoring in order to detect the potential emergence of resistance. Although the approach to the design of neuraminidase inhibitors has provided hope that resistance will be limited, resistance to NAIs already been observed in clinical, encouraging close monitoring. It is therefore important to continue to study and understand the various mechanisms of resistance to neuraminidase inhibitors. The work of this thesis has thus focused on understanding the diversity of resistance mechanisms. Initially, we studied the impact of mutations in all structural residues of the active site of neuraminidase. We observed that most of these mutations did not alter the characteristics of the virus and induced very limited resistance to antivirals. Subsequently, we then sought to explore opportunities for synergy in resistance by the combination of two structural mutations of the active site of neuraminidase. On four viruses produced, only the virus with the double mutation E119V+I222L in the active site of neuraminidase was viable, although its in vitro replicative capacity was impaired. The combination of these two mutations induced a synergistic resistance to oseltamivir. Finally, we wanted to integrate the functional interaction of neuraminidase with hemagglutinin. We have shown that the combination of a hemagglutinin low affinity for sialylated receptors allowed to rescue a virus with a deficient neuraminidase. Thus an influenza virus may discharge the function of neuraminidase, the target of the only available effective antivirals. The mechanisms of resistance to neuraminidase inhibitors are numerous. Plus, the circulation in the last two seasons of resistant viruses without selective pressure challenges the assumptions developed on the possible emergence of resistance in clinic. This opens new issues to consider in order to understand the mechanisms that allowed this emergence and transmission

Les virus influenza provoquent chaque année la grippe saisonnière qui peut toucher 5 à 15 % de la population. Les médicaments antiviraux sont un moyen important complémentaire à la vaccination pour le traitement et la prévention de la grippe. Actuellement, deux classes d’antiviraux ont été approuvées, l'une pour inhiber l'étape de décapsidation (l’inhibiteur du canal ionique M2), et l'autre pour empêcher la libération de néo-virions (l’inhibiteur de la neuraminidase). Cependant, de plus en plus de virus sont nativement résistants aux inhibiteurs de la protéine M2. Des virus résistants aux inhibiteurs de la neuraminidase ont également circulé durant les hivers 2008 – 2009. Le développement de nouveaux médicaments afin de substituer ou de compléter ces inhibiteurs est donc crucial dans la lutte contre les virus de la grippe. L’accent mis ces dernières années sur l’activité biologique des sucres (oligosaccharides/polysaccharides) montre une voie pour l’étude de l’activité antivirale d’une des plus importantes biosources. Dans le but d'évaluer le potentiel antigrippal des molécules dérivées de sucres, nous avons effectué un criblage à partir d'une bibliothèque de 245 composés de polysaccharides et d'oligosaccharides, dont la plupart proviennent d’algues et de végétaux supérieurs. Plusieurs molécules actives réparties dans différentes familles de sucres ont été mises en évidence. Parmi les candidats d'intérêt, l’oligosaccharide sulfaté 152, appartenant à la famille des arabinogalactanes de l'espèce Codium fragile, a présenté une activité inhibitrice vis-à-vis des deux virus influenza de type A et de type B in vitro. Le mécanisme d’action de cet oligosaccharide a été caractérisé. Il montre que les deux glycoprotéines de surface, l’hémagglutinine et la neuraminidase, sont les cibles virales de cette molécule / The annual seasonal flu caused by influenza viruses can affect 5 to 15 % of the population. In addition to vaccination, the use of antiviral drugs in the treatment and prevention allows the control of influenza virus infection. So far, two classes of antiviral drugs have been approved for influenza treatment, one to inhibit the uncoating step (M2 inhibitors), and the other to prevent the release of progeny virions (NA inhibitors). However, the emergence and circulation of M2 inhibitor resistant viruses at high frequencies have restricted the use of these inhibitors. Neuraminidase inhibitor resistance among circulating influenza viruses has emerged since the 2008 – 2009 season. The development of new classes of antiviral agents is crucial in the fight against influenza virus. In recent years, many molecules belonging to a large group of compounds known as carbohydrates (oligosaccharides/polysaccharides) have been revealed essential for various biological activities. The establishment of carbohydrate-based antiviral agents is, therefore, a highly promising strategy. In order to evaluate the potentially anti-influenza molecules derived from carbohydrates, we have performed a screening from a library of 245 polysaccharides and oligosaccharides. These compounds were extracted mainly from plants and algae. Several active molecules of different families have been identified. Among them, the sulphated oligosaccharide 152, belonging to the family of arabinogalactane, was found to be highly active toward both influenza virus A and B in vitro. This oligosaccharide was purified from the green algal species Codium fragile. The study of the 152 mechanism suggests that this oligosaccharide can cooperatively inhibit both viral HA binding activity and NA catalytic activity

O vírus de influenza aviária H5N1 tem se disseminado rapidamente por diversos países e continentes nos últimos anos, gerando grandes perdas econômicas e de vidas humanas. Existe a possibilidade de a doença chegar ao Brasil, o que provocaria elevado impacto sobre a economia, especialmente sobre o setor avícola. A ausência e a necessidade de estimativas de impacto econômico no país, derivadas de surtos de gripe aviária em território brasileiro, motivaram a presente pesquisa. Visando gerar tais estimativas e analisá-las, foram simulados três cenários utilizando-se de um modelo aplicado de equilíbrio geral inter-regional, denominado TERM-BR. O primeiro cenário (Cenário I) contempla um foco da doença no Rio Grande do Norte, região Nordeste do país. O segundo (Cenário II) simula a presença de diversos focos da doença no estado de São Paulo. O terceiro (Cenário III) considera o surgimento de diversos focos presentes nos estados Rio Grande do Norte, Rondônia, São Paulo e Rio Grande do Sul. Os resultados sinalizam impactos de maior dimensão na medida em que os focos da doença surgem próximos aos mercados produtores, exportadores e consumidores, como é o caso da região Sul e Sudeste. Na região Sul, em função da dimensão da avicultura na economia local, a crise do setor avícola acaba refletindo negativamente e acentuadamente sobre o conjunto da economia local. Nas regiões Norte e Nordeste, as restrições sobre a aquisição de aves vivas pelas famílias têm grande peso na retração da atividade econômica do setor avícola, já que a atividade de abate é pouco representativa e parcela importante das aves é adquirida diretamente pelas famílias, o que não acontece na mesma dimensão nas demais regiões do país. Observa-se também que o aumento do consumo de carne bovina e suína contribui para reduzir a crise na economia estadual nos estados produtores, e é também a fonte de crescimento para os estados em que a bovinocultura se destaca. O choque de demanda doméstica de produtos avícolas, comparado com os demais choques, revela-se como o principal responsável pelo comportamento na produção em quase todos os estados. Já a redução das exportações tem grande peso sobre o comportamento da produção avícola quando o foco da doença é na região exportadora ou próxima a ela. No estado de Santa Catarina, o choque de exportações prepondera sobre a queda do consumo doméstico quando do fechamento quase total dos mercados externos para carne de aves. Por último, o choque de oferta, via mortalidade das aves e destruição de ovos, pouco influencia a magnitude da queda na produção dos produtos da avicultura. / In the past few years, the bird flue virus H5N1 spread rapidly through various countries and continents, causing great economic and human losses. There is also the possibility of the disease arriving in Brazil, which would have a substantial impact on the country\'s economy, particularly on its poultry sector. The present study addresses the lack of estimates of the potential economic consequences of a bird flu outbreak on commercial poultry production in Brazil. The analysis consists of three simulations using a interregional general equilibrium model called TERM-BR. The first scenario focuses on an outbreak in the state of Rio Grande do Norte in the Northeastern part of the country. The second scenario simulates an outbreak at various places in the State of Sao Paulo, and the third scenario assesses the consequences of a bird flue outbreak in various states simultaneously, Rio Grande do Norte, Rondônia, São Paulo and Rio Grande do Sul. The results indicate greater economic impact when the outbreak occurs close to points of production and consumption, which is the case in the Southern and Southeastern regions of Brazil. In the South, where the poultry sector constitutes a larger share of the local economy, a potential avian flu outbreak will also have a greater negative economic impact. In the Northern and Northeastern region, live birds are generally purchased and slaughtered directly by individual families living in suburban and small town settings rather then by large scale processors and packinghouses. This characteristic explains why potential restrictions on these small purchases of live birds will have a very large negative economic impact on the poultry sector in the affected states. A possible reduction in poultry supply could however be offset by an increase in beef and pork consumption, thereby softening the economic affect of a bird flue outbreak by promoting growth of the beef and pork industry. In most states, this fall of domestic poultry demand is the primary cause for a shift in production. In contrast, the fall in export demand only weighs heavily on the local economy when the outbreak occurs close to exporting regions. In the state of Santa Catarina, for example, the effect of a fall in export demand dominates the effect of a fall in domestic demand, as export markets are almost completely shutdown. Finally, the reduction in poultry supply through death of infected birds and destruction of eggs, has little affect on the decrease of poultry production.

O Vírus de Influenza aviária pertence à família Orthomyxoviridae. Nos últimos anos vários subtipos da gripe aviária de baixa patogenicidade têm causado surtos e epidemia em humanos e aves domésticas. As aves selvagens e migratórias participam da manutenção e transmissão interespécie dos 16 subtipos de hemaglutinina e 9 subtipos de Neuraminidase na natureza. Nosso estudo teve como objetivo subtipar as amostras positivas através de teste sorológico de inibição da hemaglutinação (HI) e técnica de Biologia Molecular. Foram positivas as amostras das espécies: Elaenia mesoleuca (2), Sporophila lineola (1) Sporophila caerulescen (1), Vireo olivaceus (3), Columbina talpacoti (3), Paroaria dominicana (2), foram coletadas das reservas experimentais de campo localizadas no estado de São Paulo-Brasil,durante os anos de 1997 e 1998. Tais amostras foram identificadas por teste preconizado HI(de acordo com WHO) usando 20 soros imunes anti-influenza do tipo A e um do tipo B e análise de RT-PCR com sequenciamento do gene Hemaglutinina e Neuraminidase. O teste HI demonstrou que 12 amostras apresentou estreita afinidade com com soros imune A/HongKong/1/68 (H3N2), A/Equine/Miami/63 (H3N8) and A/Duck/Ukraine/63 (H3N8). As análises de sequenciamento do gene hemaglutinina e Neuraminidase desses isolados revelaram uma alta homologia com os do subtipos H3N2. As análise filogenética e variabilidade genética em comparação com as seqüências de Genbank representando diversos países, mostraram que nossas amostras apresentou estreita homologia com os vírus do subtipos que circularam em Victoria (1990), Siena (1991) e Beijim (1989). A análise de amino ácido indicou que há mutações não sinônimas no gene da Hemaglutinina exclusiva de nossas amostras e as mutações da Neuraminidase são sinônimos, quando comparadas com amostras de AIV de outros paises. Nossas Amostras quando análisadas a NA, não apresentaram mutações nos aminoácidos y285t e r297n que conferem resistencia aos inibidores da Neuminidase. / Avian Influenza virus belongs to Orthomyxoviridae family. The last years several low pathogenic avian influenza subtypes have caused outbreaks and epidemic in human and poultry. The wild and migrating birds may be participating of maintenance and interspecies transmission of the sixteen subtypes of the Hemagglutinina and nine Neuraminidase in nature. Our study was aimed to subtype the positive samples for influenza A isolated from migrating and wild birds by molecular technique and Haemagglutination inhibition test (HI). The samples from species Elaenia mesoleuca (2), Sporophila lineola (1) Sporophila caerulescens (1), Vireo olivaceus (3), Columbina talpacoti (3), Paroaria dominicana (2), were collected in reserves and experimental field stations located in the São Paulo State - Brazil, during the years 1997 and 1998. The samples were identified by HI test (according WHO) using the 20 antibody patterns anti-influenza A type and one for the influenza type B and RT-PCR and Sequence analysis of Hemaglutinina and Neuraminidase gene. The HI test demonstrated that 12 samples presented an antigenic close relationship with A/HongKong/1/68 (H3N2), A/ Equine/Miami /63 (H3N8) and A/Duck/ Ukraine/ 63 (H3N8) antiserum.The sequencing analyses of Hemaglutinin and Neuraminidase gene of these 12 isolates revealed a high homology with H3N2. Phylogenetic analysis and genetic variability compared with genbank sequence representing several countries, showed that our current samples submitted close homology to that circulated in Victoria (1990), Siena (1991) and Beijim (1989). Amino acid analysis compared our samples with representative genbank samples all of mutation are synonymous.