Es wird angenommen, dass das Influenzavirus-Glykoprotein Hämagglutinin (HA) für seine Funktion sowohl bei der Virusfreisetzung als auch bei der Fusion von viraler und zellulärer Membran mit Cholesterin- und Sphingolipidreichen Domänen, sogenannten Membran-Rafts, assoziiert sein muss. Aus diesem Grund sollte in dieser Arbeit die Membran-Raft-Affinität von HA in lebenden Zellen mittels FLIM-FRET gemessen werden. Dabei wurde mit Hilfe der Fluroreszenz-Lebenszeit-Messung (FLIM) der Förster-Resonanz-Energie-Transfer (FRET) von fluoreszenzmarkiertem HA auf einen etablierten Raft-Marker bestimmt. Diese Messungen zeigten, dass beide Proteine in gemeinsamen Klustern in der Plasmamembran vorkommen. Durch Cholesterinentzug und durch den Einsatz von Cytochalasin D, welches die Mikrofilamente zerstört, konnte diese Klusterbildung reduziert werden. Demnach tragen sowohl die Membran-Rafts als auch das Aktinnetzwerk zu dieser Klusterbildung bei. Mittels FLIM-FRET konnte zusätzlich bestätigt werden, dass die Signale für die Detergenslöslichkeit von HA in Triton-Extraktionsexperimenten, die Palmitylierung und die stark hydrophoben Aminosäuren zu Beginn der Transmembrandomäne (TMD), auch im lebenden System eine wichtige Rolle spielen. Zusätzlich konnten biochemische Experimente zeigen, dass die hydrophoben Aminosäuren zu Beginn der HA-TMD den intrazellulären Transport, nach der Trimerbildung, entscheidend verzögern. Diese Verzögerung ist vermutlich auf einer erschwerten Integration dieser Proteine in die Membran-Rafts begründet. Die virale Fusion mit der Wirtszellmembran wird durch eine pH5-Behandlung vermittelte Konformationsänderung von HA ausgelöst. FLIM-FRET-Messungen zeigten für die pH5-Konformation von HA eine verglichen mit der pH7-Konformation verringerte Klusterbildung mit dem Raft-Marker. Somit ist offensichtlich, dass die Membranfusion-vermittelnde HA-Konformation eine verringerte Raft-Affinität besitzt. Diese verringerte Raft-Affinität könnte eine wichtige Rolle bei der Störung der Lipide an der Fusionsstelle spielen und somit die Bildung und/oder Vergrößerung der Fusionspore erleichtern. / It has been supposed that the hemagglutinin (HA) of influenza virus is recruited to cholesterol- and sphingolipid-enriched domains, also named membrane-rafts, to accomplish its function in virus budding and membrane fusion. This study aimed at verifying the affinity of HA for membrane-rafts in living cells using fluorescence-lifetime imaging microscopy to measure Förster’s resonance energy transfer (FLIM-FRET). FLIM-FRET revealed strong clustering between a fluorescence-tagged HA-protein and a well-established raft-marker in CHO cells. Clustering was significantly reduced when rafts were disintegrated by cholesterol depletion and when microfilaments were disrupted with cytochalasin D. Thus, membrane-rafts as well as the actin meshwork contribute synergistically to clustering. Clustering was also reduced by the removal of the known signals for the association of HA with detergent-resistant-membranes, the palmitoylation and the first amino acids in the transmembrane region (TMR). Since these mutations are obviously important for the raft-association of HA their function during the transport through the ER and the Golgi-complex was studied. These investigations showed that the exchange of the first three amino acids of the HA-TMR led to a decelerated transport after trimer-formation of the protein, probably due to retarded integration of these proteins into membrane-raft domains. Mediating viral fusion with the host cell membrane requires an irreversible conformational change of HA. FLIM-FRET studies of this low pH conformation unveiled that the clustering with the raft-marker is decisively reduced compared to the pre-fusion conformation of the protein. It might be assumed that the fusion-mediating conformation of HA reduces the proteins affinity for membrane-rafts. Therefore it is likely that this reduced affinity for rafts after the conformational change is relevant to cause perturbation of lipids at the fusion site and thereby facilitating the formation and/or enlargement of the fusion pore.

Seasonal influenza, or the flu, impacts over 3 million people each year. Within the health sector, nosocomial infection and absenteeism are frequently associated with the flu. The Centers for Disease Control and Prevention (CDC) recommend flu vaccination for all eligible individuals, especially health care workers (HCWs). Interventions associated with increased HCW vaccination include educational programs and occupational health campaigns to address misconceptions regarding vaccine safety and efficacy. This project evaluated the impact of a voluntary, web-based education module to encourage registered nurse (RN) vaccination. The logic and health belief models served as the theoretical frameworks. In a nonequivalent group design, an educational program addressing evidence-based barriers to vaccination was delivered at 1 acute-care hospital and was not delivered at a comparison hospital within the health system. A total of 192 surveys (116 at intervention facility) were returned over 3 weeks. Statistically significant differences (x2 = 7.210, p = 0.007) were found for RNs who accepted influenza vaccination after education when compared to the RNs not receiving education. The 15% higher vaccination rate for RNs receiving education (91.1% vs. 76.1%) translates into more than 100 additional vaccinated RNs if applied across both hospitals. This project found that a simple but tailored web-based educational program is effective in converting RNs to vaccination acceptance. Increased vaccination produces societal change by reducing nosocomial and community influenza transmission. Reduced influenza infection improves community health as well as patient safety. Future work should address community-wide HCW education initiatives and evaluate their impact on quality and financial indicators at the hospital and community levels.

Seasonal influenza is associated with signiï¬?cant morbidity and mortality among older adults, aged 65 and older. Since vaccination is the single most effective preventive measure against seasonal influenza, clinicians and senior citizen center administrators need a better understanding of the perceptions of older adults concerning the reason for poor influenza vaccine uptake. The purpose of this study was to identify perceived factors that may be associated with poor influenza vaccination uptake among older adults. The health belief model (HBM) guided the study. The research questions examined perceptions predicting the odds of influenza vaccination uptake among older adults. This quantitative cross-sectional study consisted of administration of a newly developed 33-item questionnaire to a convenience sample of 147 older adult participants. A 2-week reliability test-retest on 50 participants indicated the instrument had moderate internal consistency (α -?¥ 0.7). Paired-sample t tests were not significant (p > .05), indicating that participants provided reliable responses across time. Ordinal regression analysis indicated that all HBM constructs were significantly associated (susceptibility, barriers, benefits, cues to action, and self-efficacy p = .000; severity p = .002) with frequency of influenza disease and recency of influenza vaccine uptake within 1 year. The social change implications from this study may help to improve vaccination uptake among older adults by providing senior public health decision makers and direct care clinicians with informed knowledge on perceptions and barriers that may play a role in influenza vaccination decision-making among older adults.

Although wild birds are natural hosts of avian influenza viruses (AIVs), these viruses can be highly contagious to poultry and a zoonotic threat to humans. The propensity of AIV for genetic variation through genetic shift and drift allows virus to evade vaccine mediated humoral immunity. An alternative approach to current vaccine development is induction of CD8+ T cells which responds to more conserved epitopes than humoral immunity and targets a broader spectrum of viruses. Since the memory CD8+ T lymphocyte responses in chickens to individual AIV proteins have not been defined, the modulation of responses of the memory CD8+ T lymphocytes to H5N9 AIV hemagglutinin (HA) and nucleocapsid (NP) proteins over a time course were evaluated. CD8+ T lymphocyte responses induced by intramuscular inoculation of chickens with AIV HA and NP expressing cDNA plasmids or a non-replicating human adenovirus vector were identified through ex vivo stimulation with virus infected, major histocompatibility complex (MHC) matched antigen presenting cells (APCs). The IFN? production by activated lymphocytes was evaluated by macrophage production of nitric oxide and ELISA. MHC-I restricted memory T lymphocyte responses were determined at 10 days and 3, 5, 7 and 9 weeks post-inoculation (p.i). The use of non-professional APCs and APC driven proliferation of cells with CD8+ phenotype correlated with the activation of CD8+ T lymphocytes. The responses specific to nucleocapsid protein (NP) were consistently greater than those to the hemagglutinin (HA) at 5 weeks when the CD8+ T cell responses were maximum. By 8 to 9 weeks p.i., responses to either protein were undetectable. The T lymphocytes also responded to stimulation with a heterologous H7N2 AIV infected APCs. Administration of booster dose induced secondary effector cell mediated immune responses which had greater magnitudes than primary effector responses at 10 days p.i. Flow cytometric analysis (FACS) of the T lymphocytes demonstrated that memory CD8+ T lymphocytes of chickens can be distinguished from naive lymphocytes by their higher expression of CD44 and CD45 surface antigens. CD45 expression of memory lymphocytes further increases upon ex vivo stimulation with APCs expressing AIV. This is the first characterization of avian memory responses following both primary and secondary expression of any individual viral protein.

Computational evolutionary analyses, particularly phylogenetics and ancestral reconstruction, have been extensively exploited under different algorithms and evolutionary models to better understand genome evolution from both small- and large-scale perspectives in order to assign genotypes based on assortment, resolve species relationships and gene annotation issues, further understand gene gain/loss within individual gene families, measure functional divergence among homologs, and infer ancestral character states. These evolutionary studies provide us with insights into biologically relevant issues including paleoenvironments inferred from resurrected proteins, developmental physiology associated with functional divergence of duplicated genes, viral epidemics and modes of transmission in attempt to better prepare, prevent and control diseases, evolution of lineage-specific pathogenicity, and attempts to create a synthetic ancient organism that would benefit the field of synthetic biology. Our work also provides us with greater insights into the accuracies and limitations of ancestral sequence reconstruction methods. In total, our work highlights the diverse questions that evolutionary studies attempt to address and the different biological levels that can be studied to answer these questions.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2006. / Härtill 5 uppsatser.

Orientador: Hélio José Montassier / Banca: Liana Brentano / Banca: Manoel Victor Franco Lemos / Resumo: A técnica de imunocaptura associada com a reação de transcrição reversa e reação em cadeia da polimerase (IC-RT-PCR) executadas tanto pelos procedimentos convencional como em tempo real foram testadas para a detecção rápida do gene da glicoproteína de Matriz (M) do vírus de influenza aviária (AIV) em amostras de líquido cório-alantóide (LCA) e em suabes traqueais e cloacais. O presente trabalho teve como objetivo desenvolver e otimizar a técnica de IC-RT-PCR para o diagnóstico do vírus da Influenza aviária. Os resultados obtidos foram comparados com um sistema empregando "beads" magnéticas em microplacas (AMBION), que é o método padrão de extração de RNA usado no laboratório de referência para diagnóstico de influenza aviária, o National Veterinary Services Laboratory - Ames, EUA (USDA), acrescido ainda de outros métodos de extração tradicionalmente usados nos laboratórios de referência para AIV, como os procedimentos com o uso do solvente orgânico Trizol® (Invitrogen) e com um sistema robotizado e que utiliza "beads" magnéticas (MagNA Pure - ROCHE). A técnica de IC-RT-PCR em tempo real neste estudo detectou a estirpe H2N2 do AIV, sem que nenhum outro dos RNA-vírus heterólogos testados fossem detectados (vírus das doença de Gumboro, de Newcastle e da bronquite infecciosa aviária). Os limites de detecção do IC-RTPCR foram iguais aos obtidos na técnica de extração com o kit da AMBION e menores do que aqueles que foram observados para os métodos de extração com Trizol® (Invitrogen) e com o MagNA Pure. O IC-RT-PCR demonstrou ser um sistema de diagnóstico capaz de conciliar simplicidade operacional e um menor custo com sensibilidade e especificidade analíticas iguais às do procedimento padrão atualmente adotado, podendo ser inclusive por laboratórios dotados de uma infra-estrutura mais simples / Abstract: The polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR), including real-time RT-PCR have been used for the rapid detection of Matrix glycoprotein gene (M gene) Avian influenza virus (AIV). Despite the availability of various RNA extraction methods for using in RT-PCR, isolation and detection of viral RNA are still difficult due to the unstable nature of viral RNA molecules and the presence of PCR inhibitory substances. In this study, a simple method using immune-capture (IC) to recover viral RNA from H2 AIV samples was developed and compared to one standard and two others reference methods used for viral RNA extraction, such as Ambion MagMAXTM kit and Trizol® (Invitrogen) and Magnapure kit (Roche), respectively, with subsequent analysis by real-time RT-PCR. The real-time IC-RT-PCR developed in was able to detect specifically H2N2 AIV strain, without detecting non-related avian RNA-virus pathogens, such as Newcastle disease virus, avian infectious bronchitis virus and Gumboro disease virus. Comparable detection limits were found for IC and the standard RNA extraction method using Ambion MagMAXTM kit, either for the detection of AIV in allantoic fluid suspension or in seeded tracheal and cloacal swab samples by conventional or real time RT-PCR techniques. These methods were less sensitive than Trizol® (Invitrogen) and Magnapure kit (Roche) procedures. Thus, IC was rapid and as sensitive and specific as current standard AIV RNA extraction method for real time or conventional RT-PCR, besides it conciliated simplicity and lower cost and can be applied simultaneously for direct detection of AIV in a large number of samples, including less-equipped laboratories / Mestre

Influenza illness occures masively in human population for centuries. Influenza is an acute disorder of viral origin. Currently influenza can be found all over the world. The viruses of influenza are circulating continously in all continents not only in the human population, but in animals too. Every year the population is affected by the seasonal epidemic of different magnitude. Influenza illness pose an important medical, social and economical problem. Next to the seasonal outbreaks occuring every year the influenza pandemic can appear after several tens of years. The pandemic of influenza would mean spread all over the world and it is caused by the new variant of the influenza virus. In past the influenza pandemics caused mortality of millions of people all over the world, high economical losses and social collapse of the states in the history. That´s why the particular states prepare their national pandemic plans. The early preparedness for possible influenza pandemic elaborated in the national pandemic plans and the measures following from the plans constitute a tool for reduction of extent, impact and outcomes of pandemic. The quality of preparedness can influence the economical functions of states, medical impacts of illness and final number of the victims. The aim of the dissertation is to compare the proposed measures responding to the influenza pandemic arising from the national pandemic plans of the Czech and Slovak Republic, to ascertain the state of preparedness to the influenza pandemic following from the pandemic plan of the Czech Republic and the situation in awareness of the civil population about the influenza pandemic in the Czech Republic. The theoretical part of the dissertation is focused on the epidemiological characteristic of the influenza illness. In the sphere of interest the issue of influenza pandemics is explained in terms of their history and the next field of the dissertation is focused on the pandemic plans. The research part of the dissertation is focused on the solution to the given research questions. Two research questions were specified to accomplish the defined aim: What are the differences between the approaches and measures in the pandemic plans of the Czech and Slovak Republic? What is the knowledge and awareness of civil population about the influenza pandemic in the Czech Republic? Regarding the issue the methodology the first research question included analysis of the content of the pandemic plans of the Czech and Slovak Republic and performance of the Checklist method in order to obtain an overview on approach, procedures and standard of preparedness in the territory of both states and possibilities their reciprocal comparison. Under the second research question the awareness and knowledge of the civil population about the pandemic influenza in the Czech Republic was investigated. For acquiring the data of researching study there was used The method of quantitative research was used to acquire the data for the research survey. The form of twenty questions was created. The first part of the questionnaire was focused on the general knowledge about influenza and the second part on the influenza pandemic and related measures. The respondents chose from the answers defined in advance. There wasn´t used the random sampling of the respondents. Fifty respondents from the non-representative sample filled out the form for acquiring the basic knowledge and finding out theoretical information about the influenza pandemic. Subsequently the statistical analysis was made from the obtained data using the methods of descriptive and mathematic statistic. It follows from the analysing results that the knowledge about the influenza illness and influenza pandemic exists among the civil population which means the laic public, but they are the lower extent and do not correspond to the normal frequency distribution.

As doenças respiratórias são muito comuns na produção intensiva de suínos, já em javalis são escassas informações sobre prevalência, etiologia e apresentação clínico-patológica destas enfermidades. No entanto, a presença de patógenos respiratórios comuns entre javalis selvagens e confinados e suínos domésticos já foi relatada. Este trabalho descreve as principais lesões macroscópicas e histológicas de pneumonias de javalis e os agentes comumente envolvidos. Foram examinados pulmões de javalis, ao abate, provenientes de criatórios comerciais e a principal lesão macroscópica foi consolidação crânio-ventral dos lobos craniais e médios e lesões crônicas cursando com hiperplasia linfóide na histologia. O principal agente bacteriano detectado foi o Mycoplasma hyopneumoniae (58,6%). Outros patógenos bacterianos detectados foram Actinobacillus pleuropneumoniae (48,8%), Haemophilus parasuis (49,6%), Mycoplasma hyorhinis (41,3%), Pasteurella multocida (9,1%) e Streptococcus suis (9,1%). Na segunda parte do trabalho, a pesquisa de patógenos virais foi direcionada para o Vírus da influenza suína (VIS) com objetivo de estudar o envolvimento em pneumonias de javalis de criatórios e a relação com agentes bacterianos encontrados. O vírus pandêmico A/H1N1/2009 foi detectado em 18,3% (11/60) e sua identidade foi confirmada por sequenciamento. A carga viral para H1N1 clássico variou de 4,58 a 6275 cópias/μL e para o H1N1 pandêmico, de 4,65 a 3863 cópias/μL. Nenhuma amostra apresentou título viral após a inoculação em ovos embrionados. As lesões histológicas principais foram broncopneumonia crônica difusa e pneumonia intersticial mononuclear leve, além de hiperplasia linfóide. As amostras positivas por RT-PCR para o VIS para o pH1N1 foram testadas por IHQ, sendo todas negativas para influenza A, mas todas eram positivas para M. hyopneumoniae. Quando testadas por bacteriologia, 18,2% das amostras foram positivas para P. multocida. O estudo mostrou que as pneumonias em javalis de criatório apresentaram lesões e patógenos associados similares aos encontrados em suínos domésticos ao abate. Este é o primeiro relato da infecção pelo vírus pH1N1 em javalis no Brasil. / Respiratory diseases are very common in swine intensive production, although in wild-boars the knowledge of the prevalence, etiology and clinic-pathological presentation of these diseases are very limited. However, the presence of common respiratory pathogens among wild-boar, captive wild-boar and domestic pigs has been reported. This paper describes the main macroscopic and histologic pneumonic lesions of captive wild-boars and pathogens commonly involved. Captive wild-boar lungs at slaughter were examined and the main macroscopic lesion observed was cranio-ventral consolidation of cranial and middle lobes and chronic lesions associated with lymphoid hyperplasia by histology. The main bacterial pathogen detected was Mycoplasma hyopneumoniae (58.6%). Other bacterial pathogens detected were Actinobacillus pleuropneumoniae (48.8%), Haemophilus parasuis (49.6%), Mycoplasma hyorhinis (41.3%), Pasteurella multocida (9.1%) and Streptococcus suis (9.1%). In the second part of this work, the survey of viral pathogens was directed to swine influenza virus (SIV) in order to study the involvement in captive wild-boar pneumonias and the relation with bacterial pathogens. The A/H1N1/2009 pandemic virus was detected in 18.3% (11/60) and its identity was confirmed by sequencing. The classical H1N1 viral load ranged from 4.58 to 6275 copies/uL and the pandemic H1N1, from 4.65 to 3863 copies/uL. No samples had viral titers after inoculation in embryonated eggs. The main histological lesions were chronic diffuse bronchopneumonia and interstitial mononuclear pneumonia as well as mild lymphoid hyperplasia. Samples positive to pH1N1 were assayed by IHC for SIV, all with negative results, and to M. hyopneumoniae, all were positive. When assayed by bacteriology, 18.2% of samples were positive to P. multocida. This study showed that pneumonia in captive wild-boar had similar lesions and associated pathogens were similar to those found in domestic pigs at slaughter. This is the first report of pH1N1 virus infection in captive wild-boars in Brazil.

Influenza is a highly contagious viral disease with epidemic and pandemic spread of the character, which annually affects 10% of the world's population, in the course of a pandemic even 40-50%. The originator of the disease is a virus that is present as a type A, type B or type C. Source of infection is the man to transfer air or contaminated objects. Susceptibility is widespread. The disease predominantly affects the respiratory tract, has a sudden beginning and manifests itself primarily with fever, chills, muscle pain, joint pain and headache. The most common complication is inflammation of the lungs. In the diagnosis are rapid tests for the detection of the Antigen. Treatment is symptomatic, antivirals are available the type of neuraminidase inhibitors M2, which easily gives rise to a resistance of influenza virus. The basis for the prevention of influenza is annual vaccination, which dramatically reduces the risk of hospitalization and death, especially among the elderly and the chronically ill. Coverage in our population is low, and that despite the fact that the flu is the cause of the deaths of thousands of people around the world. A year in the Czech Republic will die according to qualified estimates the flu one to two thousand people. Due to the possible prevention of influenza, it is important to disseminate information on the possibilities of prevention by vaccination and educate the general public about the seriousness of this disease. Diploma thesis discusses influenza disease as such and on the issue of vaccination against influenza among the elderly in connection with the adoption of the NAP to increase vaccination coverage against the flu. The theoretical part summarises the commonly known knowledge on the causative agent of the disease, the way of transmission, symptoms, diagnosis, complications, treatment, and prevention by vaccination.