The genetic requirements for Influenza virus to infect and adapt to new species is largely unknown. To understand the evolutionary steps required by a virus to become virulent, a human virus (A/HK/1/68) (HK), avirulent in mice, was subjected to 20 and 21 serial lung-to-lung passages in mouse. Sequence analysis revealed the emergence of eleven mutations within the NS1 gene of the new virulent strains, many of which occurred in binding sites for transcriptional and translational cellular factors. In the present study we have rescued viruses containing each of the NS1 mouse adapted mutations onto A/PR/8/34 (PR8) backbone. We found 9 of 16 NS1 mutants were adaptive by inducing mortality, body weight loss in BALB/c mice and enhanced virus replication in MDCK cells with properties of host cell interferon transcription inhibition. Sequence comparisons with the highly pathogenic A/Hong Kong/156/1997 (H5N1) and the most severe pandemic A/Brevig Mission/1/1918 (H1N1) NS1 genes showed convergent evolution with some of the mouse adapted viruses for F103L plus M106I and V226I plus R227K mutations respectively. The F103L and M106I mutations in the HK NS1 gene were shown to be adaptive by assessment with respect to replication, early viral protein synthesis, interferon-β antagonism and tropism in the mouse lung. We extended the study and proved increased virulence associated with F103L+M106I mutations in their respective H5N1 NS1 gene on the PR8 and HK backbones, as well as the PR8 NS1 gene and the H9N2 (A/Ck/Bj/1/95) gene in the PR8 and A/WSN/33 backbones respectively. However the V226I and R227K mutations in their respective HK and 1918 NS1 genes slightly enhanced virulence and viral growth at later stages of infection. This study demonstrates that NS1 is a virulence factor; involved in multiple viral processes including interferon antagonism and viral protein synthesis. Furthermore, NS1 mutations acquired during mouse adaptation are proven to be adaptive in human, mouse and avian NS1 genes.

This dissertation considers the economics of public health in the context of respiratory disease, a leading cause of morbidity and mortality. The pervasive nature of respiratory illness represents a significant reduction to health and longevity, but private actions to prevent illness may not consider the full-scale benefit of societal health improvement. In this thesis, I consider two determinants of respiratory illness: (1) the spread of influenza disease and (2) air pollution. In both cases, public policy aims to attenuate the effects of these factors by incentivizing or mandating preventative action. Because such interventions come at a cost, it is important to consider the magnitude of benefits associated with these actions. I consider each determinant in turn. First, I provide causal evidence on the health and economic consequences of an ongoing broad-scope vaccination program. The Ontario Influenza Immunization Campaign expanded the scope of vaccine coverage leading to a 20-percent increase in vaccination. Using the timing of this campaign and exogenous variation in vaccine quality, I link higher vaccination rates to decreases in lost-work-time, hospitalization, and death. Results indicate that, when vaccine quality is high, the program leads to higher gains for Ontario relative to other provinces and in short, an ounce of prevention is worth a pound of cure. Second, I provide evidence of the impact of air pollution on respiratory health. Recent changes in standards for air pollution are highly contentious and represent stringent constraints on economic activity. Evidence from this dissertation directly informs this debate. By linking daily pollution to hospital admissions for municipalities across Ontario, I study the impact of air pollution at levels below those historically considered. Results indicate that particulate matter has a significant effect on respiratory health of children but that ozone and carbon monoxide have little effect on respiratory hospitalizations for all age groups.

This dissertation considers the economics of public health in the context of respiratory disease, a leading cause of morbidity and mortality. The pervasive nature of respiratory illness represents a significant reduction to health and longevity, but private actions to prevent illness may not consider the full-scale benefit of societal health improvement. In this thesis, I consider two determinants of respiratory illness: (1) the spread of influenza disease and (2) air pollution. In both cases, public policy aims to attenuate the effects of these factors by incentivizing or mandating preventative action. Because such interventions come at a cost, it is important to consider the magnitude of benefits associated with these actions. I consider each determinant in turn. First, I provide causal evidence on the health and economic consequences of an ongoing broad-scope vaccination program. The Ontario Influenza Immunization Campaign expanded the scope of vaccine coverage leading to a 20-percent increase in vaccination. Using the timing of this campaign and exogenous variation in vaccine quality, I link higher vaccination rates to decreases in lost-work-time, hospitalization, and death. Results indicate that, when vaccine quality is high, the program leads to higher gains for Ontario relative to other provinces and in short, an ounce of prevention is worth a pound of cure. Second, I provide evidence of the impact of air pollution on respiratory health. Recent changes in standards for air pollution are highly contentious and represent stringent constraints on economic activity. Evidence from this dissertation directly informs this debate. By linking daily pollution to hospital admissions for municipalities across Ontario, I study the impact of air pollution at levels below those historically considered. Results indicate that particulate matter has a significant effect on respiratory health of children but that ozone and carbon monoxide have little effect on respiratory hospitalizations for all age groups.

Vaccination is one of the major strategies available for combating viral infections in humans. However, currently available vaccines are not without pitfalls; they are laborious to produce, could potentially be unsafe, and in the case of the highly variable influenza virus need to be reformulated each season. The use of synthetic peptides thus represents an exciting alternative to traditional vaccines. However, these synthetic peptides are not highly immunogenic without the use of potent adjuvants. The lack of immunogenicity might be addressed by conjugation between T or B cell epitopes with universal or immunodominant T-helper epitopes. The construction of branched peptides, lipidated peptides, or designs combining both of these elements might also enhance the immunogenicity, as they might target Toll-like receptors and/or mimic the 3-dimensional structure of epitopes within the native protein. In this study, a recognized T-B peptide based on the hemagglutinin protein of the A/Puerto Rico/8/34 influenza virus was chosen as a backbone and modified to evaluate if the construction of branched peptides, lipidation, the addition of cysteine residues, or mutations could indeed alter reactivity. Screening the different designs with various antibody binding and cellular assays revealed that combining a branched design with the addition of lipid moieties leads to a greatly enhanced activity as compared to other similar T-B diepitope constructs.

There are many pathogens that infect birds and perhaps many more that researchers have not yet identified. Of all potential pathogens, the research presented in this manuscript focuses on two avian RNA viruses. First, a serodiagnostic test for newly described Avian Borna Virus (ABV), which has been recently identified as the etiological agent of Proventricular Dilatation Disease (PDD), was developed. PDD is a deadly disease which affects many birds, but to this point, has mainly been a concern of psittacines. The need for a diagnostic test is imperative. An antigen associated with PDD was identified from the brains of affected birds by use of the Western blot assay. This antigen was subsequently purified using various protein purification protocols, including a modification of reverse-phase chromatography. The antigen was then identified as the ABV nucleoprotein according to tandem mass spectroscopy analysis and protein database search. A serodiagnostic assay was developed and standardized using infected cell culture as an antigen source. Over 100 avian serum samples were submitted by veterinarians to test for the presence of antibodies against ABV nucleoprotein. This serodiagnostic assay was found to have 90% sensitivity and 82% specificity for the diagnosis of ABV in infected birds. Second, the ability of a carbohydrate epitope to enhance the humoral immune response to an influenza vaccine was tested in chickens. Influenza is a serious infection that causes 36,000 deaths annually in the United States. The need for a more efficacious is addressed by incorporating a carbohydrate antigen targeted by natural antibodies that are produced by chickens as well as humans. Therefore, chickens may be a suitable animal model to test this hypothesis. Influenza vaccines with alpha-gal antigen are prepared from cell culture. The antigen is then enzymatically removed from some vaccines and the nature of the ensuing humoral immune response to these vaccines in chickens is attempted. Though ABV is not known to be zoonotic at this time, zoonotic infections pose the highest risk as new and emerging infectious diseases in the human population. The following research contains applications relative to challenges faced by researchers and clinicians in infectious disease containment.

Avian influenza virus (AIV) is a type A virus of the family Orthomyxoviridae and its outbreaks not only cause economic losses in poultry, but also are worldwide threats to human health. The phenotypic changes in host cells induced by pathogens are always accompanied by remarkable changes in gene expression. Therefore understanding the gene expression profile of infected cells at the global level is important to get insights into interactions between hosts and viruses. Different genomic approaches have been utilized in the current study to investigate the host-AIV interactions in chickens. The Ser to Asn mutation on position 631 in the chicken Mx1 protein was reported to result in a positive antiviral function in vitro. With AIV infection, the Mx1 mRNA expression levels in heterozygous birds were significantly up-regulated. Additional mutations on the chicken Mx1 coding region were identified by sequencing. The results showed that most identified mutations were co-segregated with S631N mutation except one insertion in the position of 1544bp in the heterozygous birds. We speculate this insertion might be related to the up-regulation of mRNA expression of heterozygous birds with AIV infection. The miRNAs play critical roles in biological processes and are important effectors in host-pathogen interactions. The miRNA deep sequencing was used to profile miRNAs in AIV infected or non-infected chickens. Differentially expressed miRNAs identified have expanded our knowledge in the functions of these potential immune related chicken miRNAs regulating host response to AIV infection. Both microarray and transcriptome analysis by RNA-Seq were used in the current study to investigate the global gene expression of host response to AIV infection. Through the comprehensive analysis, a list of strong candidate miRNAs such as miR-32 and their host target genes including Mx1 were identified for further elucidating the regulatory mechanism of host-AIV interaction. In summary, we have identified many important candidate host genes and miRNAs which play important roles in the modulation of host response to AIV infection using genomic approaches. Further investigation of underline regulatory mechanisms of these genes, miRNAs or related pathways, followed by functional analysis, could lay solid foundation for understanding cellular and molecular mechanisms of the host-AIV interactions, thereby, pave a way for the development of novel protective strategies against AIV infection in chickens.

Objective¡G Influenza is an important public health problem, and may seriously damage the elderly population. Although influenza vaccination is a safe, and can significantly reduces morbidity and influenza -related complication mortality in elderly people, however, the influenza vaccination rate did not reach expected goals. Therefore, to explore the potential factors that affect the vaccination rate becomes a critical research question. The purpose of this study was to apply the Health Belief Model, which includes demographic, health state and healthy behaviour, knowledge about influenza and influenza vaccine, perceived susceptility, perceived seriousness, perceived benefit and barrier, and cues to action variables, to understand factors motivating the community elderly people to receive influenza vaccination. Design: The sampling designs were stratified and systematic sampling with total 700 sample. During June 2007 , a structured questionnaire was sent to the elderly people¡]¡Ù65 years¡^in Kaohsiung city¡C Result: The male and female sample distribution rates were equally in 50%, average age was 72.87 years old and 59.6% elderly people had chronic disease (such as hypertension, heart disease and diabetes). Chi-square tests showed that ¡§chronic disease¡¨, ¡§health examination¡¨, ¡§always seek care from specific physician¡¨, ¡§exercise ¡§, ¡§ recognize about influenza and influenza vaccination¡¨, ¡§ perceived seriousness¡¨, ¡§ perceived benefit and barrier¡¨, were significantly associated with having ever been vaccinated. Logistic regression results indicated that ¡§think the influenza vaccination can effectively prevent influenza¡¨, ¡§regular health examination¡¨, ¡§cues to action¡¨ and ¡§perceived barriers¡¨ were found to be the significant predictors of acceptance of influenza vaccination. Conclusions¡GThe study results indicated that health care worker¡¦s attitude about influenza and influenza vaccination will directly influence community elderly people's cognition. Therefore, by educating the health care worker about influenza seriousness and the vaccination effectiveness may successfully promote vaccination rate. Through health education activities in the community, it may increase correct cognition about influenza and the benefit of vaccination. In addition, the health department needs to strengthen the health policy marketing in order to increase influenza vaccinations rate, reduce morbidity and influenza -related complication mortality.

Foreign and domestic researches reveal that influenza vaccine is safe and can effectively reduce related healthy trouble caused by influenza in the elderly, infant and high-risk population, etc. The main purpose of ¡§Influenza vaccine policy¡¨ is public service. To promote concepts of citizenism and consumer-right brings the concept of marketing. With the progress of democratic trend, when policies are discussed, planned, established and executed, the government should consider policies as ¡§products¡¨ to sale and emphasize policy-marketing concept to gain public approval and acceptance. This study was based on influenza vaccine policy carried out by Centers for Disease Control, R.O.C. (Taiwan). In accordance with the marketing framework of public policy advocated by Snavely (1991), we designed a questionnaire investigation on surveying citizen and medical staff of Kaoshiung to see if government vaccine policies can grasp the important path as well as positive effectiveness of policy marketing, and issued our research outcomes on promoting prevention and cure of vaccine to be part of references for government health center. The results of descriptive statistics displayed that 80% of survey candidates regardless of common people or medical practitioners possessing professional knowledge agreed that influenza vaccine was an outstanding policy and approved the government¡¦s response and clarification in facing medial negative report. 60% of survey candidates were affected and worried about safety of influenza vaccine, after receiving these medial negative reports. Former experience of vaccination would considerably affect the willingness of general public to receive a flu shot. Survey candidates mainly received information about influenza vaccine from television media and preferred to approve either experts or endorsers. Results of Chi-square test showed that there was a significant correlation between ¡§influenza vaccination¡¨ and common people¡¦s experience of suffering from chronic disease or unhealthy inoculable response. Results of independent-samples T test and one-way ANOVA indicated the factors correlating significantly with the analytic variables of approval of degree ¡§service¡¨ in marketing mix program. In the case of common people, these factors included the following variables such as ¡§those who had ever been vaccinated¡¨, ¡§those who had ever been vaccinated and suffered from bad inculable response¡¨, and ¡§those who had ever participated health education during the past year¡¨. In respect of medical practitioners, these factors included the following variables such as ¡§those who had ever been vaccinated and suffered from bad inculable response¡¨ and ¡§those who had ever taken the thematic speech, related websites or webpages¡¨. With regard to analytic variables of approval of degree ¡§cost¡¨ in marketing mix program included following factors. For common people, the factors contained ¡§those who had ever been vaccinated and suffered from bad inculable response¡¨, and ¡§those who had ever participated health education, related bulletin board and scrolling text marquee during the past year¡¨. For medical practitioners, the factors comprised ¡§those who had ever been vaccinated and suffered from bad inculable response¡¨ and ¡§those who had ever taken thematic speech, related vedio, websites or webpages¡¨ As for analytic variables of approval of degree ¡§supply of information and education¡¨ in marketing mix program included following factors. For common people, the factors contained ¡§those who had ever been vaccinated¡¨, and ¡§those who had ever participated related bulletin board, scrolling text marquee, thematic speech, TV vedio, broadcasts, websites or webpages during the past year¡¨. For medical practitioners, the factors comprised ¡§occupation¡¨ and ¡§those who had ever perused related books, journals, newspaper, websites or webpages¡¨. Policy marketing concept should not be neglected in the application of executing public policy. For influenza vaccination policy marketing, first of all, public mental barriers of safety of influenza vaccine must be overcome, therefore the most critical issue is to enhance advertisement and education about safety of vaccine in order to improve confidence of public for influenza vaccine and to prevent from the loophole of influenza prophylaxis and treatment. This study suggests that we must to adopt consumer-driven marketing concept for the influenza vaccine policy marketing mix program. Government public relations should establish harmonious media relations and use higher acceptance of promotion way to focus on the limited resources to target market. We should make concise and rememberable messages and integration marketing communication way to achieve the comprehensive effect. We expect that the government performs policy marketing to promote the public approval of ¡§influenza vaccine policy¡¨ and to raise influenza vaccination rate on diminishing influenza morbidity and mortality rate.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 174-194). Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 169-186). Also available in print.