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Influenza virus belongs to the Orthomyxovirus family of viruses that causes respiratory infection in humans, leading to morbidity and mortality. The mature influenza A virion has an envelope that contains two major surface glycoproteins proteins – hemagglutinin (HA) and neuraminidase (NA). HA is a highly antigenic molecules and is responsible binding to host cell surface receptors (Sialic acid), and membrane fusion between the viral membrane and the host endosomal membrane. Most of the antibody response generated against influenza virus either by vaccination or by natural infection is directed against HA. Influenza virus has segmented negative–sense RNA genome which gives the virus the ability to evade the host immune response by incorporating mutations (antigenic drift) and/or by reassotment with other subtypes of influenza A viruses (antigenic shift). Currently licensed vaccines which include an inactivated vaccine, a live attenuated vaccine, and recombinant subunit vaccine are beneficial for providing protection against seasonal influenza viruses that are closely related to the vaccine strain but fail to provide protection against drifted strains. This limits their breadth of protection and thus requires annual revaccination with reformulated vaccines. Also, because selection of a vaccine strain for the next season is purely based on surveillance and prediction, sometimes mismatches do happen between the selected vaccine strains and circulating viruses, resulting in a drastic decrease in vaccine efficacy and thus high morbidity and mortality. Furthermore, the production of these seasonal vaccines takes 6-8 months on an average, and does not guarantee protection against infection with novel reassortant viruses which can cause pandemics. To overcome the draw-backs of seasonal influenza virus vaccines and to enhance our pandemic preparedness, there is an increasing need for game-changing influenza virus vaccines that can confer robust, long-lasting protection against a broad spectrum of influenza virus isolates. Influenza hemagglutinin (HA) is highly immunogenic and thus a major target for vaccine design. HA is synthesized as a precursor polypeptide (HA0), assembles into a trimer, matures by proteolytic cleavage along the secretory pathway and is transported to the cell surface. Mature HA has a globular head domain, primarily composed of the HA1 subunit, which mediates receptor binding, while the stem domain, predominantly comprises of the HA2 subunit, and houses the fusion peptide. At neutral pH, the HA stem is trapped in a metastable state but undergoes an extensive conformational rearrangement at low pH in the late endosome (host-cell endosome) to trigger the fusion of virus and host membranes. Clusters of ‘antigenic sites’ have been identified in the head domain of HA, indicating that it harbors an almost continuous carpet of epitopes that are targeted by antibodies. However, these immunodominant sites constantly accumulate mutations to escape immune pressure, and thereby narrow the breadth of head-directed neutralizing antibodies (nAbs). In contrast to the highly-variable head domain, the membrane-proximal HA stem subdomain has much less sequence variability and, thus, is a desirable target for influenza vaccine development. In the recent past, several broadly neutralizing antibodies (bnAbs) targeting this subdomain with neutralizing activity against diverse influenza A virus subtypes have been isolated from infected people, further proving that this subdomain of HA can be targeted as a vaccine candidate. Steering the immune response towards this conserved, subimmunodominant stem subdomain in the presence of the variable immunodominant head domain of HA has been quite challenging. Alternatively, mimicking the epitome of these stem-directed bnAbs in the native, pre-fusion conformation in a ‘headless’ stem immunogenic capable of eliciting a broadly protective immune response has been difficult because of the metastable nature of HA. Addressing the aforementioned challenges, here we describe the design, stabilization and characterization of novel stem derived immunogens from HA of influenza A viruses using a protein minimization approach. Chapter 1 gives an overview of the influenza virus life cycle, nomenclature and classification of influenza virus; outlines the structural organization and functional properties of different viral proteins. An introduction to the kind of immune responses generated during vaccination or natural infection with the virus is discussed. The conventional vaccines that are currently used and their limitations, recent progress in the field of novel vaccine developmental approaches targeting the conserved epitopes on HA, is also described in this chapter. This chapter also gives a broad overview of bnAbs that have been isolated in the recent past, which target the novel antigenic signatures on HA. The design of a stem domain construct from an H3N2 virus (A/HK/68) is described in Chapter 2. In order to ensure that HA2 folds into the neutral pH conformation, regions of HA1 interacting with it were included in the design. Additionally, two Asp mutations were introduced in the B loop of HA2 to destabilize the low pH conformation and stabilize the desired native, neutral pH conformation. Studies using small peptides (57-98 of HA2) indicated that Asp mutations at positions 63 and 73 destabilized the low pH conformation. Studies on mutants with additional pairs of introduced Cys residues showed that the designed protein H3HA6 was folded into the neutral pH form. Immunization studies using mice showed that the protein was highly immunogenic and provided complete protection against a lethal dose of a homologous virus. Two constructs H3HA6a and H3HA6b, designed from the stem region of drifted H3N2 viruses (A/Phil/2/82 and A/Bris/10/07) were tested for protection against HK/68 to determine the extent of cross-strain protection provided by HA6. While HA6a (from A/Phil/2/82) provided near complete protection against HK/68, HA6b could protect against challenge only partially, possibly because of lower titers of antibodies elicited by this antigen. Studies using FcRγ chain knockout mice indicated that majority of the protection mediated by anti-HA6 antibodies was because of antibody mediated effectors functions, although neutralization as a mechanism of protection was also likely to contribute. In all the 18 subtypes of HA, the B loop contains residues that form the hydrophobic core of the extended coiled coil of the low pH form. As in the case of H3HA6, we suggest that these residues could be mutated to Asp to destabilize the low pH conformation. Two circularly permuted stem domain constructs from an H1N1 virus (A/PR/8/34) and an H5N1 virus (A/Viet/1203/04) were made. The design and characterization of these proteins is described in Chapter 3. H1HA6, H1HA0HA6 and H5HA6 were purified from inclusion bodies and refolded. The proteins H1HA6 and H1HA0HA6 were highly immunogenic and provided protection against a lethal challenge with homologous PR/8/34 virus. Anti-H1HA6 sera had higher titres of antibodies against heterogonous HAs as compared to convalescent sera. Stem derived immunogens from drifted H1N1 viruses (A/NC/20/99 and A/Cal/7/09) have been made and tested for cross-protection with PR/8/34 challenge. While H5HA6 also elicited high titers of antibodies, it could only protect partially against PR/8/34 challenge probably because high enough titers of cross-reactive protective antibodies were not elicited by this protein. These stem immunogens conferred robust subtype specific and modest heterosubtypic protection in vivo against lethal virus challenge. However, the immunogens, especially H1HA6, a stem immunogen from group 1 (PR8) virus is aggregation prone when expressed in E.coli. The strategy used to improve the biophysical and biochemical properties and thus the immunogenicity of these stem derived immunogens is discussed in Chapter 4. A random mutagenesis library of H1HA6 was constructed by error prone PCR using modified nucleotide analogues. The library was displayed on the yeast cell surface to isolate mutants showing better surface expression and improvement in binding to the broadly neutralizing antibody CR6261 compared to the wild-type protein. We isolated few clones, of which one mutant (H1HA6P2) dominated the enriched population. The other mutants differed slightly from H1HA6P2. This mutant differs from the wild-type by two mutations K314E and M317T (H1 numbering) which are close to the CR6261 binding site but outside the antibody foot-print (epitope). This mutant showed improved binding to CR6261 and exhibited significant improvement in surface expression. Improvement was also observed in binding of this mutant to F16v3-ScFv (another broadly neutralizing antibody). Two cysteine mutations were also introduced to further stabilize the trimeric form of the protein. Chapter 5 describes the biophysical and biochemical characterization of the high affinity isolated mutant at the protein level. We expressed this affinity matured mutant gene in E.coli and purified the protein from inclusion bodies. The stabilized mutant protein showed remarkable improvement in biophysical and biochemical properties and was recognized by stem directed conformation sensitive broadly neutralizing antibodies CR6261, F10 and F16v3 with affinity comparable to the full-length HA ectodomain. These results clearly suggest that this mutant protein is properly folded in its native pre-fusion conformation and thus can be an excellent candidate for eliciting stem directed broadly neutralizing antibodies. All these stabilized versions of stem derived immunogens will be tested for immunogenicity and cross-protection with different viral challenges. Chapter 6 describes the development of a method for mapping antibody epitopes (especially conformational epitopes) down to the residue level. Using a panel of single cysteine mutants, displayed on the yeast cell surface, this bypasses the need for laborious and time consuming protein purifications steps used in conventional methods for epitope mapping. We made a panel of single cysteine mutants, covering the entire surface of the antigen (CcdB, a bacterial toxin protein), displayed each mutant individually as well as in a pool, representing all mutants together on the yeast cell surface, and covalently labeled the cysteine with biotin-PEG2-maleimide to mask the area. The effect on antibody binding was monitored to identify the residues and relative positions important for antibody interactions with the displayed antigen by flow cytometry. By using this method we were able to map the conformational as well as linear epitopes of a panel of monoclonal antibodies down to the residue level with ease, and also identify the regions on the antigen which contribute to the antigen city during immunization in different animals. Since, this method is quite easy, rapid and gives in-depth information about antigenic epitopes, it can be useful in rational design of epitomes specific vaccines and other antibody therapeutics. It can easily be extended to other display systems and is a general approach to probe macromolecular interfaces.

La influenza es una enfermedad altamente infectocontagiosa de la cual se tienen registros históricos descriptivos desde la época griega y de certeza etiológica tan solo hace casi ocho décadas atras. Su agente causal es el virus del influenza de los que se conoce exiten tres grandes tipos: A B y C. El tipo A tiene la propiedad de circular por diversos reservorios biológicos, tales como el hombre, los cerdos y las aves, entre otros. Representa además una elevada variabilidad genética lo que le permite continuos cambios o derivas antigénicas menores responsables de la influenza humanas epidémicas y a veces reordenamientos amplios con cambios antigénicos mayores los que originan la temida influenza pandémica. En un mundo cada vez mas globalizado, con una población superior a los seis mil millones de personas, marcado por grandes inequidades sociales y con cambios climáticos evidentes, los virus de la influenza serán un riesgo permanente para la seguridad de la humanidad. La clínica de los diversos subtipos virales pueden balancearce desde las formas inaparentes hasta las formas graves de gripe o influenza, dependiendo de la virulencia del subtipo viral infectante y del huesped. Aunque nuestra tecnología diagnóstica y de inmunización ha avanzado sorprendentemente, la preparación y disposición de los nuevos kits diagnósticos suelen tardar al principio y en el caso de las vacunas estas no están disponibles para el un nuevo subtipo viral pandémico en el momento que se las necesita. El desarrollo de antivirales contra la gripe no es notorio, contándose aprobados para uso humano los amadantanes como la amantadina y la rimantadina y los inhibidores de la neuraminidasa como oseltamivir y sanamivir principalmente. Claramente los más vulnerables en este contexto son los países en vías de desarrollo y en especial aquellos mas pobres, hecho que nos debería llamar a una profunda reflexión. / Influenza is a highly contagious disease. There are some historical descriptions of this condition by ancient Greek physicians, and the etiological agents have been known only for the last eight decades. The causative agent is the influenza virus, which has three main types: A, B, and C. Type A is capable of circulating within many different biological reservoirs, including humans, swine, and birds. It also has high genetic variability, which allows it to have minor antigenic drifts or mutations which are responsible of epidemics in humans. Sometimes changes are quite marked, leading to pandemics. In a globalized world, with more than 6 billion inhabitants, with many social inequities and evident climate changes, influenza viruses are a permanent risk for mankind. Clinical features for the different viral subtypes may vary from subtle infections to full blown and severe, life-threatening forms. Event with the great advances in diagnostics and immunization, the manufacture and distribution of new diagnostic kits may take some time, and new vaccines are not always readily available. Specific therapies against influenza are not well developed. There are two groups of drugs, the so called adamantane derivatives, such as amantadine and rimantadine, and the neuraminidase inhibitors, such as oseltamivir and zanamivir. Most vulnerable areas for the new flu pandemics include developing countries, particularly the poorest ones, so that the greatest effort must be made for helping these areas.

Avian influenza H5N1 is one kind of important bird flu. Unfortunately, this virus has swiftly evolved and become highly pathogenic to humans and poultry, resulting in 100% of death in infected poultry and over 60% of mortality among infected human population. Moreover, the virus tends to reassort with other influenza viruses, such as the current swine flu H1N1, to establish themselves in environments and further this epidemic all over the world. The World Health Organization (WHO) has in fact warned that highly pathogenic avian influenza H5N1 poses a graver risk of a global human pandemic than at any time since the Hong Kong outbreak (H3N2) in the 1960s. / Finally, avian influenza is an inter-disciplinary issue across virology, medical geography, and spatial epidemiology. How to quantify and integrate knowledge from different disciplines remains a challenge in fully understanding the disease. We propose a method to formally integrate genetic analysis that identifies the evolution of the H5N1 virus in space and time, epidemiological analysis that determines socio-environmental factors associated with H5N1 occurrence and statistical analysis that identifies outbreak dusters. Our integrated results show a significant advance in findings over reports in, for instance, Gilbert et al. (2008) and we believe our findings are more precise and informative in representing the occurrence and the space-time dynamics of H5N1 spread. Overall, unlike traditional influenza studies, our work sets up a solid foundation for the inter-disciplinary study of this and other spatial infectious diseases. / First, we apply multifractal detrended fluctuation analysis to determine the temporal scaling behavior of outbreaks in Asia, Europe, Africa, and the whole of the world between December 2003 to March 2009. Long-range correlation and multifractality, two important properties characterizing the scaling behavior of complex dynamics, are first detected in the outbreak time series. In addition, this study identifies different temporal scaling behaviors of outbreaks of these continents 8,nd specific seasonal patterns in Asia. These findings confirm our perspective that avian-influenza outbreak behaviors are self-similar over time and are spatially heterogeneous. / One key to preventing such a calamity is to obtain a thorough understanding of the mechanisms of avian influenza transmission and its spatio-temporal patterns of dispersal. The issues at stake are outbreaks' spatial and temporal patterns, the interrelationship of these with the evolution of influenza viruses in such a way that geography is understood as a dimension of the disease's virology, and the human and avian behaviors and socio-ecological environments associated with H5Nl spread. This thesis sets out to study these problems in detail and propose solutions. / Second, we conduct a spatial analysis for global trends and local clusters of H5N1 outbreaks at multiple geographical scales. Currently, the local K function used in a point pattern analysis searches outbreak clusters, assuming the disease is spatially homogeneous. The thesis proposes a much more efficient method to measure the degree of clusters accurately. The modified function works by weighting outbreaks through distances, counting the number of the weighted outbreaks for each lattice point no matter whether the disease emerges in a grid. This weighted local K function extends cluster analysis from a point pattern to lattice data. Spatial representation in these terms then seeks to explore local patterns of H5N1 over a continuous space. / Third, we study a set of socio-environmental factors, which are plausibly associated with the occurrence of H5N1. Spatial epidemiological models are built for predicting the disease at both continental and national levels, covering Indonesia, China, and the whole of East-Southeast Asia. We evaluate the statistical models using 1,000 bootstrap replicates, showing a consistently high rate of prediction, assessed by statistics: AUC, Kappa Index, and pseudo R square. / Ge, Erjia. / Advisers: Yee Leung; Tung Fung. / Source: Dissertation Abstracts International, Volume: 73-06, Section: A, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 169-197). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Darbo tikslas. Įvertinti Lietuvos sveikatos priežiūros įstaigų darbuotojų požiūrį į vakcinaciją. Metodika. Kad įvertinti Lietuvos sveikatos priežiūros įstaigų darbuotojų požiūrį į vakcinaciją, buvo atliktas paplitimo tyrimas. Buvo apklausta 1693 respondentai, dirbantys asmens sveikatos priežiūros įstaigose. Duomenys apdoroti SPSS (13) kompiuterine programa. Analizei taikytas Pearson‘o χ2 testas ir statistiniam išvadų tikrinimui pasirinktas reikšmingumo lygmuo 0,05. Rezultatai. Anketinė apklausa parodė, kad Lietuvos sveikatos priežiūros įstaigų darbuotojai teigiamai žiūri į vakcinaciją. Nors skiepijasi 54,8% bendrosios praktikos gydytojų, 55,9% terapeutų ir kitų specialistų bei 51,6% slaugytojų, visgi jie nurodė, kad vakcina žymiai efektyvesnė ir mažiau kainuoja nei kitos medicininės paslaugos – 67,0% bendrosios praktikos gydytojų, 69,1% terapeutų ir kitų specialistų bei 60,3% slaugytojų ir kad skiepai duoda žymiai daugiau naudos nei žalos (84,9% bendrosios praktikos gydytojų, 87,5% terapeutų ir kitų specialistų bei 71,7% slaugytojų). Iš dažniausiai nurodytų nesiskiepijimo priežasčių, dauguma respondentų nurodė, kad nesiskiepija nuo gripo dėl to, kad saugosi nuo gripo stiprindami sveikatą (19,3% bendrosios praktikos gydytojų, 18,9% terapeutų ir kitų specialistų bei 15,8% slaugytojų), niekada gripu neserga (20,0% bendrosios praktikos gydytojų, 17,0% terapeutų ir kitų specialistų bei 15,2% slaugytojų), įgyjamas natūralus imunitetas (22,2% bendrosios praktikos gydytojų, 8,0%... [toliau žr. visą tekstą] / Objective: To determine the Lithuanian health care institutions workers opinion about vaccination. Methods: In order to determine the Lithuanian health care institutions workers opinion about vaccination spread research was performed. Were polled 1693 respondents working in health care institutions. The data was analysed using SPSS computer program. There was used Pearson Chi-Square test and to check statistical reliability was choose the difference statistically insignificant 0,05. Results: Questionnaire date shows positive Lithuanian health care institutions workers opinion about vaccination. Even though only 54,8% of common practice physicians, 55,9% therapeutists and other specialists and 51,6% nurses were vaccinated themselves but they indicated that vaccination is more effective and less expensive than other medical service – 67,0% of common practice physicians, 69,1% therapeutists and other specialists and 60,3% nurses and that vaccination is more useful than harmful (84,9% of common practice physicians, 87,5% therapeutists and other specialists and 71,7% nurses). The major reason why the respondents are against vaccination, especially against influenza virus, is that they strengthen their health to protect themselves against virus (19,3% of common practice physicians, 18,9% therapeutists and other specialists and 15,8% nurses), never have flu (20,0% of common practice physicians, 17,0% therapeutists and other specialists and 15,2% nurses), and acquire natural immunity... [to full text]

Made available in DSpace on 2015-02-27T17:39:04Z (GMT). No. of bitstreams: 2 44.pdf: 1010144 bytes, checksum: 1cbcb330900b0f5162dfcade1c16d471 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Existem inúmeras doenças que podem ser consideradas de risco para a medicina veterinária e para saúde pública entre elas se destaca a Influenza A. O agente etiológico desta enfermidade é um vírus pertencente a família Orthomyxoviridae, gênero Influenzavirus A. Vários animais, inclusive os cães, estão inseridos na cadeia epidemiologia desta enfermidade. O sucesso epidemiológico do vírus influenza deve-se ao surgimento de novas variantes antigênicas, devido à elevada frequência de rearranjo genético e as consequentes modificações antigênicas nas glicoproteínas de superfície viral. Os freqüentes rearranjos genéticos, a ocorrência recente de surto de influenza canina e os resultados de estudos envolvendo o vírus influenza em cães vêem despertando a atenção das autoridades sanitárias de diversos países, devido à capacidade adaptativa desse vírus e o estreito contato do homem com os cães. Em virtude da inexistência de dados que evidencie a circulação do vírus influenza em cães no Brasil, o presente trabalho teve como objetivo a detecção de anticorpos contra aos subtipos H3N8, H2N2 e H1N1 do vírus influenza A em cães oriundos da zona oeste da cidade do Rio de Janeiro. Do total de 304 soros analisados pela prova de HI, 70,4% apresentaram reatividade com títulos entre 10 e ≤80 para o subtipo H3, 30,6% para o subtipo H2 e 48,% positivos para o subtipo H1. Os resultados obtidos tornam evidente a circulação desses subtipos entre os caninos, ressaltando a necessidade de uma maior vigilância sanitária, do estabelecimento de rotina diagnóstica e epidemiológica da influenza canina, alertando as autoridades sanitárias responsáveis sobre o risco de possíveis surtos de influenza entre os caninos e os possíveis problemas que possam advir para os criadores, proprietários e para saúde pública. / Existem inúmeras doenças que podem ser consideradas de risco para a medicina veterinária e para saúde pública entre elas se destaca a Influenza A. O agente etiológico desta enfermidade é um vírus pertencente a família Orthomyxoviridae, gênero Influenzavirus A. Vários animais, inclusive os cães, estão inseridos na cadeia epidemiologia desta enfermidade. O sucesso epidemiológico do vírus influenza deve-se ao surgimento de novas variantes antigênicas, devido à elevada frequência de rearranjo genético e as consequentes modificações antigênicas nas glicoproteínas de superfície viral. Os freqüentes rearranjos genéticos, a ocorrência recente de surto de influenza canina e os resultados de estudos envolvendo o vírus influenza em cães vêem despertando a atenção das autoridades sanitárias de diversos países, devido à capacidade adaptativa desse vírus e o estreito contato do homem com os cães. Em virtude da inexistência de dados que evidencie a circulação do vírus influenza em cães no Brasil, o presente trabalho teve como objetivo a detecção de anticorpos contra aos subtipos H3N8, H2N2 e H1N1 do vírus influenza A em cães oriundos da zona oeste da cidade do Rio de Janeiro. Do total de 304 soros analisados pela prova de HI, 70,4% apresentaram reatividade com títulos entre 10 e ≤80 para o subtipo H3, 30,6% para o subtipo H2 e 48,% positivos para o subtipo H1. Os resultados obtidos tornam evidente a circulação desses subtipos entre os caninos, ressaltando a necessidade de uma maior vigilância sanitária, do estabelecimento de rotina diagnóstica e epidemiológica da influenza canina, alertando as autoridades sanitárias responsáveis sobre o risco de possíveis surtos de influenza entre os caninos e os possíveis problemas que possam advir para os criadores, proprietários e para saúde pública.

A pandemic subtype of influenza A sometimes replaces (e.g., in 1918, 1957, 1968) but sometimes coexists (e.g., in 1977) with the previous seasonal subtype. This research aims to determine a condition for replacement or coexistence of influenza subtypes. We formulate a hybrid model for the dynamics of influenza A epidemics taking into account cross-immunity of influenza strains depending on the most recent seasonal infection. A combination of theoretical and numerical analyses shows that for very strong cross-immunity between seasonal and pandemic subtypes, the pandemic cannot invade, whereas for strong and weak cross-immunity there is coexistence, and for intermediate levels of cross-immunity the pandemic may replace the seasonal subtype. Cross-immunity between seasonal strains is also a key factor of our model because it has a major influence on the final size of seasonal epidemics, and on the distribution of susceptibility in the population. To determine this cross-immunity, we design a novel statistical method, which uses a theoretical model and clinical data on attack rates and vaccine efficacy among school children for two seasons after the 1968 A/H3N2 pandemic. This model incorporates the distribution of susceptibility and the dependence of cross-immunity on the antigenic distance of drifted strains. We find that the cross-immunity between an influenza strain and the mutant that causes the next epidemic is 88%. Our method also gives an estimated value 2.15 for the basic reproduction number of the 1968 pandemic influenza. Our hybrid model agrees qualitatively with the observed subtype replacement or coexistence in 1957, 1968 and 1977. However, our model with the homogeneous mixing assumption significantly over estimates the pandemic attack rate. Thus, we modify the model to incorporate heterogeneity in the contact rate of individuals. Using the determined values of cross-immunity and the basic reproduction number, this modification lowers the pandemic attack rate slightly, but it is still higher than the observed attack rates. / Graduate

La grippe est une maladie respiratoire aigüe, due à une infection par des virus influenza et qui représente un problème important de santé publique. Une meilleure compréhension des interactions entre le virus influenza et son hôte nous permettra de mieux comprendre la physiopathologie de l'infection grippale, et donc, à terme, de mieux se protéger contre la maladie. La morbidité et la mortalité, causées par les infections grippales sévères, sont associées à une dérégulation de la réponse immunitaire, au niveau pulmonaire. Cette inflammation délétère serait à l'origine de dommages collatéraux du poumon, entrainant une diminution de la capacité respiratoire du patient. Bien que les mécanismes impliqués ne soient pas totalement élucidés, de récents travaux mettent en évidence un rôle central des cellules endothéliales dans la dérégulation de la réponse de l'hôte face à l'infection grippale. Lors d'une agression de l'endothélium, le processus physiologique de l'hémostase (activation plaquettaire, coagulation et fibrinolyse) s'active afin de permettre la cicatrisation de la plaie et de maintenir l'intégrité des vaisseaux sanguins. Dans de nombreuses maladies inflammatoires, la seule dérégulation de l'hémostase est directement liée à une réponse inflammatoire délétère. Lors de ma thèse, nous avons émis l'hypothèse que l'hémostase pouvait être à l'origine de la dérégulation inflammatoire durant les infections grippales. Nos données montrent le rôle de deux facteurs fortement impliqués dans l'hémostase : le récepteur activé par la thrombine, PAR-1 (Protease Activated Receptor J) ainsi que le plasminogène, dans l'inflammation délétère des poumons et dans la pathogénicité des virus influenza. Outre le rôle de l'hémostase, nous avons également pu mettre en évidence que le virus influenza incorpore des protéines cellulaires dans l'enveloppe virale, lui permettant d'échapper au système immunitaire, ce qui pourrait aussi contribuer à la dérégulation de la réponse de l'hôte. L'ensemble des résultats obtenus ont permis de mieux comprendre les mécanismes à l'origine d'une réponse immunitaire dérégulée dans les infections grippales et de proposer de nouvelles cibles thérapeutiques pour lutter contre la maladie / Influenza is an acute respiratory disease caused by infection with influenza virus and is a major public health problem. A better understanding of the interaction between influenza virus and host allow us to better understand the pathophysiology of influenza infection, and thus, ultimately, to better protect themselves against the disease. Morbidity and mortality caused by severe influenza infections are associated with dysregulation of the immune response in the lung. This deleterious inflammation is the cause of lung collateral damage, causing a decrease in the patient's breathing capacity. Although the mechanisms involved are not fully understood, recent studies point to a central role of endothelial cells in the deregulation of the host response to influenza infection. During endothelium aggression, the physiological process of hemostasis (platelet activation, coagulation and fibrinolysis) is activated in order to allow wound healing and to maintain the integrity of blood vessels. In many inflammatory diseases, the only dysregulation of hemostasis is directly linked to a deleterious inflammatory response. During my thesis, we hypothesized that hemostasis could be the cause of the inflammatory dysregulation during influenza infections. Our data show the role of two factors strongly involved in hemostasis: the thrombin activated receptor, PAR-1 (protease activated receptor 1) and plasminogen, in the deleterious lung inflammation and in the pathogenicity of influenza virus. Besides the role of hemostasis, we have also been able to show that the influenza virus incorporates cellular proteins in the viral envelope, allowing it to evade the immune system, which could also contribute to the deregulation of the host response. All the results obtained allowed to better understand the mechanisms involved in immune response dysregulation during influenza infection and suggest new therapeutic targets to fight against the disease

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Science), Vaal University of Technology. / Influenza-A virus is a single stranded negative sense RNA virus that is a member of a Orthomyxoviridae group. The virus is diverse and consists of 16 haemagglutinin (H) and 9 neuraminidase (N) glycoproteins subtypes that form a serotype. Avian influenza virus (AIV) has been detected in more than 100 bird species from 26 different families, although Anseriformes and Charadriiformes are considered the natural hosts of the virus. A 12-month study was conducted at the African Pride Irene Country Club lodge in Pretoria where the prevalence of AIV was monitored in a community of wild birds. The African Pride Irene Country Club lodge houses a population of wild bird species such as Egyptian geese (Alopochen aegytptiaca), Yellow-billed duck (Anas undulata), Red knobbed coot (Fulica cristata), African sacred ibis (Threskiornis aethiopicus) and Hadeda ibis (Bostrycha hagedash). A total of 3674 faecal samples were collected over a period of 12 months and screened for AIV group using the Matrix gene (M-gene) real time reverse-transcriptase PCR (rRT-PCR). Positive samples were submitted for virus isolation in embryonated chicken eggs. In addition, the RNAs were screened using H5 and H7 subtype specific rRT-PCR and a conventional universal RCR assay that targets the HA gene was also used. Polymerase Chain Reaction (PCR) products were requenced using Sanger sequencing and the viral isolates were subjected to Next Generation sequencing (NGS). Twenty percent of the samples tested positive for the AIV group and four virus subtypes were identified. One virus isolate was identified through NGS as H3N6; two through conventional PCR and Sanger sequencing as H9Nx and H6Nx. Of the twenty percent samples that tested positive for AIV 98% were identified as H7Nx by subtype specific through rRT-PCR. The highest frequency of AIV positive samples was detected between the months of January and February 2017 (20%), with smaller peaks detected in february and March 2016 (0.3%). Lower peaks were also detected between the months July and November 2016 (0.1%), respectively. A high prevalence of AIV was detected in the late summer months with a frequency of 65% positive, although a low prevalence was also detected in the autumn (0.6%) winter (0.6%) and spring 0.08%). Thus, the study provides a valuable insight into the seasonal prevalence of AIV in a heterogeneous wild duck population in Gauteng Province.

This purpose of this quantitative study was to examine health beliefs among Caribbean-born university students regarding acceptance or rejection of influenza vaccination among populations at institutions of higher education. In addition, acculturation was addressed as a factor affecting cultural health beliefs. A survey was completed by 98 students enrolled at Florida International University during the spring 2018 semester. Linear regression was used to analyze whether cultural health beliefs and acculturation were predictive of beliefs about influenza vaccination and beliefs about perceived barriers to influenza vaccination. The study findings showed cultural health beliefs of the students were statistically significant predictors of their beliefs about influenza vaccination and perceived barriers to influenza vaccination. Also, the levels of acculturation were a statistically significant predictor of students' cultural health beliefs and beliefs about perceived barriers to influenza vaccination. After 5+ years of acculturation in the United States, the students surveyed still held cultural beliefs and perceived barriers to influenza vaccination that contributed to their lack of acceptance of the vaccination. The information gained from this study gives credence to the need for designing health interventions and health messages on influenza vaccination that are culture specific for a college-age population if influenza vaccination acceptance is to be promoted.