Eine Influenza A Virus Infektion induziert die Expression zahlreicher Gene, einschließlich der TypI Interferone, die eine erste Abwehrlinie gegen virale Infektionen bilden. Hierbei ist IFNb das wichtigste Zytokin. IFNb wird durch einen multimeren Komplex, das Enhanceosom kontrolliert, das Bindungsstellen für die Transkriptionsfaktoren AP-1, NF-kB und IRF-3 in seiner Promotorsequenz besitzt. In früheren Arbeiten konnten wir zeigen, dass die Influenza Virus-induzierte AP-1 abhängige Genexpression über den JNK/SAPK-Signalweg erfolgt (Ehrhardt, 1999; Ludwig et al., 2001). Unter den, an DNA Elemente bindenden AP-1 Faktoren waren solche, die aufgrund von Phosphorylierung durch die JNKs reguliert werden, wie beispielsweise ATF-2. Weiterhin korrelierte die Induktion der AP-1 abhängigen Genexpression mit der starken Aktivierung von JNK und seiner upstream Regulatoren in permissiven Zellen. Die Virusmengen transfizierter und infizierter Zellen, in denen JNK inhibiert wurde, waren höher im Vergleich zu Virusmengen der Kontrollzellen. Demzufolge kann die Virus-induzierte Aktivierung von JNK und AP-1 nicht der Virusreplikation dienen, sondern gehört vielmehr zu einer antiviralen Immunantwort. Daten aus einem Virus-freien, auf Plasmiden basierenden vRNA Replikations-System deuten darauf hin, dass die JNK Aktivierung aus der Akkumulation viraler RNA resultiert. Entsprechend bewirkte die Infektion von Zellen mit einem Virus, dem das virale NS1 Protein fehlt, welches RNA binden und somit "wegfangen" kann, eine gesteigerte JNK Aktivität im Vergleich zu den Kontroll-Infektionen. Damit konnte das NS1 Protein als erstes virales Protein identifiziert werden, das der Virus- und dsRNA-induzierten Aktivierung des JNK/SAPK-Signalweges entgegen wirkt. Der Transkriptionsfaktor IRF-3 wird spezifisch infolge einer viralen Infektion aktiviert und ist daher ein potenter Kandidat, die schnelle und starke antivirale Genexpression zu regulieren. Infolge einer Influenza Virus Infektion wird IRF-3 phosphoryliert, wandert in den Kern und bindet dort an Promotoren, die die antivirale Genexpression steuern. Bislang sind die IRF-3 Kinase und zelluläre Signalwege, die eine IRF-3 Phosphorylierunge induzieren, unbekannt. Um in unserem Labor Signalmediatoren, die upstream von IRF-3 liegen, zu suchen, wurde ein IRF-3 responsives Promotor-Reportergen-Plasmid, aus dem IFNb Promotor stammend, konstruiert. Die kleine Rho-GTPase Rac1 wurde als erster nicht an RNA bindender, zellulärer Mediator identifiziert, der in die Influenza Virus-induzierte IRF-3 abhängige Genexpression involviert ist. Die Inaktivierung der Rho-GTPasen durch das spezifische Inhibitor Toxin B oder dominant negatives Rac1 resultierten in der Inhibierung der Virus- und dsRNA-induzierten IRF-3 Phosphorylierung und DNA Bindung, sowie der IRF-3 abhängigen Promotoraktivität, beispielsweise des IFNb Promotors. Damit konnten zwei wichtige Komponenten der Virus-induzierten Immunantwort identifiziert und charakterisiert werden. / Infection of cells with Influenza A virus induces the expression of a variety of genes, including the type I interferons which are a first line of defense against viral infections. IFNb, the most important cytokine, is controlled by a higher order complex, the enhanceosom, which contains binding sites for the transcription factors AP-1, NF-kB and IRF-3. We could show that the Influenza Virus induced AP-1 dependent gene expression occurs via the JNK/SAPK pathway (Ehrhardt, 1999; Ludwig et al., 2001). Among the AP-1 factors which were identified to bind their cognate DNA element during viral infection are those, that are regulated via phosphorylation by JNKs, such as ATF-2. Accordingly, the induction of AP-1 dependent gene expression correlates with a strong activation of JNK and its upstream activators MKK4 and 7 in permissive cells. Virus yields from transfected and infected cells in which JNK signaling was inhibited by different approaches were higher compared to the levels from control cells. Therefore we conclude that virus-induced activation of JNK and AP-1 is not exploited by the virus to support its replication but rather is required for the innate antiviral immune response. Data obtained with a virus-free plasmid-based vRNA replication system indicated that JNK activation is a cause of viral RNA accumulation during infection. This was supported by the observation, that infection of cells with a virus lacking viral NS1 protein, which is known to bind and to sequester RNA from cellular signaling intermediates, caused a strongly enhanced JNK activity compared to control infections. Furthermore, the NS1 protein was identified as the first viral protein that antagonizes virus- and dsRNA-induced activation of the stress response signaling pathway mediated through Jun N-terminal kinase. IRF-3 is specificially activated in response to viral infection and is therefore the most potent candidate to regulate the fast and strong antiviral gene expression. After an Influenza virus infection IRF-3 becomes phosphorylated and migrates to the nucleus where it binds to antiviral gene promoters. However, the IRF-3 kinase and the cellular signaling pathways leading to IRF-3 phosphorylation are unknown. To investigate signaling mediators upstream of IRF-3, we have constructed an IRF-3 responsive promoter-reporter gene plasmid derived from the IFNb promoter. The small Rho-GTPase Rac1 was identified as the first non-RNA binding cellular mediator involved in the Influenza virus-induced IRF-3 dependent gene expression. Inactivation of these Rho GTPases by the specific inhibitor toxin B or dominant negative Rac1 resulted in the inhibition of virus- and dsRNA-induced IRF-3 phosphorylation and DNA binding as well as of IRF-3 dependent promoter activity, e.g. of the IFNb promoter. Thus two important components of virus-mediated immune response were identified and characterised.

Influenza B viruses are a significant cause of disease and influenza B antigens are present in all human vaccines. Achieving suitable yields of seed viruses is often difficult for vaccine manufacturers. With influenza A viruses increases in yields have been achieved by the preparation of reassortants between a high-yielding donor strain and an epidemic strain. However, reassortment of influenza B viruses for the preparation of seeds has not been usually undertaken due to the lack suitable donor strains. Such an approach, which formed the basis of this thesis, could improve vaccine yields, lower costs and introduce a further element of predictability to vaccine manufacture. Potential donor strains were prepared from B/Lee/40 (B/Lee) by two approaches involving the selection of stable cold- and high- temperature mutants. Initial passaging was undertaken in specific-pathogen-free (SPF) chicken embryo kidney (CEK) cultures and later passage in SPF embryonated chicken eggs. Both approaches were successful, although a smaller number of viable progeny could be isolated from plaques obtained at 38„aC. Potential donor strains, isolated by selection at either 25 or 38„aC and plaque-purified in SPF CEK cultures, were tested for haemagglutinin and infectious titre, in comparison with the original parental strain by three methods, and for differences in antigenicity by cross-haemagglutination-inhibition tests. Potential donor strains selected at temperatures of 25„aC (C25) and 38„aC (H38) produced haemagglutination titres of 320 units/50ƒÝL and infectivities of 8.57 and 8.39 50% egg infectious doses, respectively, when grown in eggs at the permissive temperature (34„aC). Reassorting experiments using the B/Lee-derived potential donor strains C25 and H38 and the epidemic strain, B/Johannesburg/5/99 (B/Johannesburg), showed that the preparation of reassortant progeny with both epidemic strain HA and NA was difficult. Only 1/24 of the resulting reassortants possessed both the HA and NA of the epidemic strain. None of the reassortant progeny produced in reassorting experiments using C25 and H38 and the epidemic strain B/Panama/45/90 (B/Panama) possessed the desired 6:2 gene constellation (i.e. genes for the two surface antigens of the epidemic strain and the remainder from the donor strain). The infectious titre of selected progeny from the reassortment experiments were determined by three methods and compared with their respective epidemic parents. Yields of several influenza B epidemic strains and potential donor strains were measured after growth in Madin-Darby canine kidney (MDCK) cells prepared in serum-containing (SC) and animal- and human-derived protein-free (AHPF) media. Optimal multiplicities of infection were determined for B/Panama, B/Johannesburg and C25 in MDCK cultures grown in SC medium. A series of experiments were then undertaken to determine the maximum virus yields in MDCK cells grown in SC medium, followed by a further experiment using C25, B/Panama, B/Johannesburg, and selected reassortants after preparation in AHPF medium. Cell culture yields from 5/6 viruses grown in MDCK cells prepared in AHPF medium were higher than in cells prepared in SC medium and approached those obtained in eggs.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

This dissertation examines issues related to avian influenza (AI) disease. This is done via three essays that individually examine: (1) the impacts of climate change on the probability and expected numbers of AI outbreaks and associated economic loss; (2) the effects that media coverage of AI outbreaks has on meat demand in the United States, and (3) the potential effectiveness of AI mitigation strategies on poultry production and welfare under a simulated AI outbreak in United States. The climate change and spread of AI outbreaks study finds that the probability and expected number of AI outbreaks increases as climate change proceeds. Particularly, past climate change has contributed to the current spread of AI disease by 11% and the future climate change will increase this spread by another 12%. Moreover, the underreporting probability of AI outbreaks is also examined and results show that the underreporting probability is much higher in countries with lower gross domestic production level, larger export of poultry products and more numbers of AI confirmed human deaths. Therefore, disease prevention and control plans should focus on these economically poor and climatically changed regions. AI outbreak information has significant effects on meat demand in the United States. In particular, impacts of overseas AI human deaths on meat demand equal 0.02% for beef, -0.005% for pork, and -0.01% for chicken for sample when there was no AI occurred in the United States, while it has smaller impacts on meat expenditure when using the whole sample. In addition, human deaths due to AI disease will increase beef demand and decrease that for pork and chicken. However, AI media coverage in short-run has insignificant effect on meat demand, which suggests that consumers are more cautious when cases occur within the United States as opposed to international cases. In the study on the effects and welfare implications of AI mitigation strategies, results find that vaccination strategy is welfare decreasing under most cases of demand shocks but is desirable in some regions when both domestic and excess demand decrease. Under the assumption of one AI outbreak in the United States, the associated mitigation costs because of past climate change are relatively small.

Influenza virus is a RNA virus that exists as different types and subtypes. Influenza A virus strains are known to cause disease in several bird and mammalian species. Wild birds are believed to constitute the natural reservoir for influenza A virus. In humans, influenza A virus causes yearly seasonal influenza epidemics of respiratory disease resulting in high morbidity and severe economic consequences. Due to the virus’ ability to change its antigenic properties by mutation, yearly vaccination is required for protection from the disease. There are many different subtypes of influenza virus which are characterized according to two surface structures - the hemagglutinin and neuraminidase proteins - , for example; H5N1. These subtypes have the ability to recombine, and thereby creating new variant combinations. If a subtype that the living population of humans has not encountered before starts to spread among humans, it can result in a pandemic. Pandemic outbreaks have occurred at irregular intervals throughout history and have had a devastating impact on mankind. For example the Spanish influenza pandemic of 1918 is thought to have killed more than 50 million people. Influenza A virus is also an important cause of disease in poultry where virus strains of some subtypes may change into forms that are highly pathogenic. These virus strains may transmit directly to man and multiple other species. This has been the case in the ongoing outbreak that started in South East Asia in 2003. All known subtypes of influenza A virus have been isolated from wild birds living in aquatic environments, mainly dabbling ducks. These species are considered to be the reservoir for influenza A virus. The virus causes sub clinical gastrointestinal infection in ducks. High amounts of virus are excreted in the feces and spread via the fecal-oral route through water where it can persist for a prolonged time. There are still many unknowns about the ecology of influenza virus in the wild bird reservoir. This thesis includes five articles where data are presented that add new knowledge on this subject. We add proof that wild ducks are indeed the host for most influenza A virus subtypes by presenting data from a meta-analysis on all published screening data from wild birds and by presenting data from a four year screening of migratory ducks that were caught and sampled at Ottenby Bird Observatory. Our investigations have shown that the prevalence of influenza virus in the wild duck population of western Eurasia shows temporal differences in comparison to the results found in studies in North America. The prevalence in western Eurasian ducks is high during the period August to December and also rises in the spring. These findings are of importance for the understanding of how influenza virus is perpetuated in nature. During the course of the study only low pathogenic subtypes were isolated. Of concern is the high frequency of isolation of virus strains of the H5 and H7 subtypes that are prone to change into highly pathogenic variants in poultry. Many of the strains isolated in our study are similar to the ones that have caused influenza outbreaks in poultry in Europe during the last seven years. This indicates that wild bird surveillance for influenza A virus can be of major value as a sentinel system to prevent outbreaks in domestic poultry. Studies on Black-headed Gulls (Larus ridibundus) revealed a previously unknown subtype, H16. This finding widened the spectra of known influenza A virus subtypes in nature. Influenza A virus was also isolated in samples from Guillemots (Uria aalge) in the Baltic Sea. This was the first time influenza A virus was isolated from this species in Europe. The isolated virus strains contained a mix of genes, some of which must have been derived from influenza A virus strains present in the North American bird population. This finding proves that limited exchanges between the virus strains present on the American and the Eurasian continents exist, which is of concern for evaluating the risk of spread of highly pathogenic virus strains by wild birds to the Americas. / Influensavirus är RNA virus och indelas i olika typer och subtyper. Influensa A virus orsakar sjukdom hos ett flertal fågel- och däggdjursarter. Vilda fåglar anses utgöra den viktigaste reservoaren för influensa A virus. Hos människa orsakar influensa A virus årliga epidemier av luftvägssjukdom med hög sjuklighet och stora ekonomiska konsekvenser för samhället. Eftersom frekventa mutationer orsakar ändringar i virusets ytstrukturer krävs årlig vaccination med nytt anpassat vaccin för att ge skydd mot sjukdom. Det finns många olika subtyper av influensa A virus. Dessa karaktäriseras med två av virusets ytstrukturer; hemagglutinin och neuraminidas, vilket till exempel skrivs H5N1. Virus av olika subtyper kan rekombinera och på så sätt skapa nya varianter. Om en subtyp som tidigare ej cirkulerat bland världens befolkning orsakar ett utbrott kan detta leda till en världsomfattande epidemi, en så kallad pandemi. Pandemier har drabbat mänskligheten med viss regelbundenhet genom historien och haft förödande konsekvenser. Till exempel orsakade pandemin ”Spanska sjukan” under åren 1918-1920 mer än 50 miljoner dödsfall. Influensa A virus orsakar också förödande utbrott i fjäderfäbesättningar. Virus av vissa subtyper kan mutera till högpatogena varianter och orsaka så kallad högpatogen aviär influensa. Dessa högpatogena varianter kan även överföras till och orsaka sjukdom hos människa och andra djur vilket varit fallet under det pågående utbrott av H5N1 som startade i sydöstra Asien 2003. Alla kända subtyper av influensa A virus har isolerats i material från vilda fåglar vilka lever i vattenmiljö, framförallt från änder. Dessa arter anses därför utgöra influensavirusets reservoar i naturen. Hos änder orsakar viruset framförallt en subklinisk infektion i gastrointestinalkanalen och sprids genom faekal-oral överföring via vatten i vilket viruset kan förbli aktivt en längre tid. Det finns fortfarande många obesvarade frågor angående influensa A virus ekologi bland vilda fåglar. I denna avhandling presenteras fem artiklar som tillför ny kunskap inom detta område. I avhandlingen styrks bevisen för att vilda änder utgör virusets reservoar i naturen dels genom en metaanalys av samtliga publicerade data rörande fynd av influensa A virus hos vilda fåglar, dels med hjälp av data från fyra års provtagning från flyttande vilda änder vid Ottenby fågelstation. Resultaten påvisar temporala skillnader i influensvirusets prevalens i den västeuroasiatiska andpopulationen jämfört med den nordamerikanska. Prevalensen i den västeuroasiatiska andpopulationen är hög under perioden augusti till december och i viss mån även under våren. Dessa fynd talar för att influensavirus kontinuerligt cirkulerar i andpopulationen. Under studien av förekomsten av influensa A virus hos änder isolerades enbart olika lågpatogena subtyper. Subtyperna H5 och H7 var vanligt förekommande. Dessa subtyper är benägna att utvecklas till högpatogena varianter om de sprids till fjäderfäbesättningar med svåra konsekvenser som följd. Genom studier av virus släktskap visas att de virus vi isolerat från vilda änder är snarlika de som orsakat utbrott bland fjäderfä i Europa under de senaste sju åren. Detta styrker värdet av att övervaka förekomsten av influensavirus hos vilda fåglar för att på så sätt förhindra utbrott av sjukdom bland fjäderfä. Undersökning av prover från skrattmås (Larus ridibundus) ledde fram till upptäckten av en helt ny subtyp av influensavirus; H16. Därmed utvidgades spektret av kända subtyper i naturen. Influensa A virus isolerades från sillgrisslor (Uria aalge) i Östersjön vilket inte tidigare gjorts hos denna art i Europa. Dessa virus innehöll gener från både nordamerikanska och euroasiatiska fågelpopulationers virus. Det visar att det finns ett utbyte av virus mellan fågelpopulationerna på de skilda kontinenterna. / On the day of the defence data the status on article IV was Submitted and the title was "Multi-year surveillance of influenza virus type A in migratory waterfowl in northern Europe".

Natural killer (NK) cells are lymphocytes of the innate immune system that play a major role in the destruction of both tumours and virally-infected cells. The cytotoxicity of NK cells is tightly controlled by signals received through activating and inhibitory receptors. NK cells express a variety of inhibitory receptors such as Ly49 receptors. Ly49 receptors bind to class I MHC molecules that expressed on normal cells. Using Ly49-deficient (NKCKD) mice we show that Ly49-KD NK cells successfully recognize and kill influenza virus-infected cells and that NKCKD mice exhibit better survival than wild-type mice. Moreover, influenza virus infection has a propensity to upregulate cell surface expression of MHC-I on murine lung epithelial cells in vivo. Significantly, we demonstrate increased lung damage of WT-mice versus NKCKD mice after influenza virus infection as determined by histological analyses. This data indicated that absence of Ly49 inhibitory NK receptors greatly enhances survival of infected mice.

Public health planning for influenza is based on morbidity and mortality estimates derived from statistical models. Lower than anticipated 2009 H1N1 pandemic death estimates have raised questions about the method. Examining the statistical method is important for future policy and program development. We compared the main methods of estimating influenza burden through a systematic literature review and by comparing statistical estimates of influenza-attributable burden at the Ottawa Hospital (TOH) to clinical estimates validated through chart review. We identified heterogeneity in methods used to estimate influenza-attributable mortality in the literature which resulted in within-season estimate variation by study. We found statistical estimates of influenza burden at TOH to be 4-8 times greater than clinically validated data. We also found no significant association between the outcomes examined and epidemic periods at TOH. The findings of this study suggest discordance between model estimates by model approach and between model estimates and validated findings. Examining reasons for these discordances should be pursued.

No description available.

Linkages between influenza prevalence and climate (e.g. precipitation, temperatures, El Nino Southern Oscillation ENSO) have been suspected, but definitive evidence remains elusive. This analysis investigated a climatic relationship between influenza mortality (measured by multiple caused pneumonia and influenza deaths) and influenza morbidity (measured by isolates tested for influenza). Influenza-climate linkages were analyzed at multiple spatial scales (e.g. local analysis, and regional analysis) and multiple temporal scales (e.g. annualized mortality counts, and mortality counts based on cumulative percentiles). Influenza mortality and morbidity were found to have significant correlations to seasonal temperatures, precipitation, and ENSO. Influenza-climate associations varied spatially and temporally, and underscore the importance of considering geographic scale in investigative analyses of disease. Evidence for an influenza-climate relationship provides a greater understanding of the enviro-climatic factors that can contribute to an influenza epidemic, and provides an impetus for further studies that incorporate climatic factors in influenza risk modeling.