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".

No description available.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 178-187). Also available in print.

Two posttranslational modifications, ISGylation and phosphorylation, impact the replication of influenza A virus, a human pathogen responsible for high mortality pandemics. The ubiquitin-like ISG15 protein is induced by type 1 interferon (IFN) and is conjugated to many cellular proteins by three enzymes that are also induced by IFN. Experiments using ISG15-knockout (ISG15-/-) mice established that ISG15 and/or its conjugation inhibits the replication of influenza A virus, but inhibition was not detected in mouse embryo fibroblasts in tissue culture. The present study is focused on the effect of ISG15 and/or its conjugation on the replication of influenza A virus in human cells in tissue culture. IFN-induced antiviral activity against influenza A virus in human cells was significantly alleviated by blocking ISG15 conjugation using small interfering RNAs (siRNAs) against ISG15 conjugating enzymes. IFN-induced antiviral activity against influenza A virus gene expression and replication was reduced 10-20-fold by suppressing ISG15 conjugation. Unconjugated ISG15 does not contribute to this antiviral activity. Consequently human tissue culture cells can be used to delineate how ISG15 conjugation inhibits influenza A virus replication. SiRNA knockdowns were also used to demonstrate that other IFN-induced proteins, specifically p56, MxA and phospholipid scramblase 1, also inhibit influenza A virus gene expression in human cells. The research on phosphorylation focused on the viral NS1A protein, a multifunctional virulence factor. Although threonine phosphorylation of the NS1A protein was discovered 30 years ago, the sites of phosphorylation and its function had not been identified. A recombinant influenza A virus encoding an epitope-tagged NS1A protein was generated, enabling the purification of NS1A protein from infected cell extracts. Mass spectrometry identified phosphorylation at T49 and T215. A recombinant virus in which phosphorylation at 215 was abolished by replacing T with A is attenuated, and an apparently aberrant NS1A protein is produced. Attenuation did not occur when T was changed to E to mimic a constitutively phosphorylated state, or surprisingly when T was changed to P to mimic avian NS1A proteins. These results suggest that T215 phosphorylation in human viruses and P215 in avian viruses can support analogous functions. / text

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

Continually reported human cases of highly pathogenic avian influenza (HPAI) H5N1 virus infection create heightened threat to public health, due to the disease severity and high lethality. Acute respiratory syndrome (ARDS) has been found to be the most severe form of acute lung injury caused by H5N1 virus infection. Studies have highlighted that the unusually high virulence of H5N1 virus infection is associated with the cytokine dysregulation and enhanced viral replication in the host. In reference to the past experience during Spanish 1918 influenza pandemic and SARS, it is crucial that a novel therapeutic target is explored and employed in time for the effective control of emerging diseases. The pandemic potential of H5N1 influenza virus urges well preparedness not only in terms of containment measures, but also in the treatment aspect of the severe human H5N1 disease. To date, therapeutic approaches are limited to the use of vaccine, antiviral drugs and corticosteroids. It has been suggested that commercially available antiviral drugs are prone to induce resistance mutations; and are effective in the protection against influenza virus infection only if administered during the early course of disease development. Moreover, vaccine development does not grant a promising therapeutic strategy at the time of a pandemic as it takes time for the development and distribution of safe and reliable vaccine. In attempts to search for a novel adjunctive therapy in addition to currently available agents, indirubin-3’-oxime (IDO) and indirubin derivative, E804 have been tested to show the effect in cytokine suppression and antiviral activity against H5N1 influenza virus infection in vitro. These compounds have been extracted and purified from a natural herb called Isatis tinctoria which is frequently used for herbal remedy in treating respiratory symptoms in traditional Chinese medicine. In this study, it was demonstrated that IDO and E804 treatment in H5N1 influenza virus infected human alveolar epithelial cells effectively inhibit the proinflammatory cytokine induction and viral replication. This physiologically relevant in vitro alveolar epithelial cell model and the efficacy of IDO and E804 provide new insights to the development of new treatment option for severe human H5N1 disease. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Influenza virus has long been a major threat to public health worldwide. The virus can be highly deadly because of antigenic shift. Since the H5N1 outbreak in Hong Kong in 1997, avian flu is regarded as the next pandemic threat. For combating the disease, it is essential to investigate more on the influenza virus, in particular H5N1. Nucleoprotein (NP) is a major component of the ribonucleoprotein complex (RNP) in the influenza virus. NP exhibits both structural and functional roles for influenza virus assembly and propagation and is involved in mediating the transcription-replication process. The NP of the virus binds the RNA genome and acts as a key adapter between the virus and the host cell. It therefore plays important roles and represents an attractive drug target. Recently, the X-ray structure of H5N1 NP was solved to a resolution of 3.3 Å , which provides valuable clues on how NP carries out its functions. However, the N-terminal 1-20 residues were not resolved in the H5N1 NP crystal structure. This N-terminal region is thought to contain a nuclear localization signal (NLS), a cellular splicing factor BAT1/UAP56 binding site, and a nuclear export signal. It has been suggested that the N-terminal NLS binds to importin (a cytosolic protein) for the nuclear import of NP. In the present study, the solution structure of H5N1 NP N-terminal peptide (NP20) in membrane mimetic solvent condition was determined using Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopies. The CD results show that NP20 adopted an α-helical conformation. The NMR data indicate that NP20 formed a single α-helix spanning from residues Gly5 to Gly16. Surface electrostatic potentials further showed that the NP20 peptide is amphipathic in nature, which may be important for its binding with importin. NMR titration experiments have been carried out between NP20 and importin. Addition of importin into the solution of NP20 peptide caused significant broadening of the NMR signals of NP20 and progressive changes of the chemical shifts of NOE cross-peaks at increasing importin concentration confirm that NP20 could bind with importin. Therefore, the present study supports that NP20 region is the binding site of importin mediating the import of NP into the host cell nucleus. In conclusion, the knowledge gained from this study provides a better understanding on the structure of NP20 and its interaction with the host importin protein, and may serve as a template for the development of novel antiviral drug targeting NP with improved therapeutic index. / published_or_final_version / Chemistry / Master / Master of Philosophy