The influenza A viral hemagglutinin (HA) protein binds to sialic acid (SA) groups of cellular surface glycoproteins to achieve viral attachment and entry. The SA binding specificity of HA is one of the major determinants for controlling viral tropism and host specificity. Fibronectin (FN) is a ubiquitinious glycoprotein secreted on cell surface, either circulating in plasma, or as one of the best characterized components of the extra cellular matrix. With its binding properties towards different types of molecules and pathogens, it has been utilized by different bacterial and viral pathogens for binding, entry, propagation and pathogenesis. The binding affinity and region of plasma FN to influenza A viral glycoprotein was identified in early 1980s. Evidence also suggests the binding is SA associated. FN associates with different viral pathogens. However, evidence of FN direct involvement in influenza A pathogenesis remains unknown. The objective of this thesis is to test the involvement of cellular FN in influenza A viral infection. To perform the study, FN siRNA and anti-FN antibody were applied. This study demonstrated possible involvement of FN in the replication of human H1N1 and highly pathogenic avian H5N1 viruses. It also discovered that FN is very important for the replication of H1N1 virus, but not H5N1 virus. Interestingly, the result suggested that FN does not affect the initial virus-host binding, but it has an effect on post-attachment events. Key amino acid positions controlling the SA binding specificity of seasonal human or avian influenza A viruses have been identified in the HA. In this thesis, reverse genetics and mutagenic work identified that viruses with a α2,3-linked SA (SA α2,3) binding preference were not inhibited by anti-FN antibody, while viruses with a α2,6-linked SA (SA α2,6) specificity were severely inhibited. This surprising finding of SA binding preference related FN involvement in post-attachment event led to the further investigation on the structural involvement of FN and viral entry pathway analysis. The 9th and 10th of type III repeating units of FN form the cell-binding domain of the protein for cell attachment. From site specific antibody inhibitory studies, the cell binding region of FN near the synergy adhesion site(SAS) and Arg-Gly-Asp-Ser(RGDS) cell adhesion signal was identified to be important for the replication of viruses that have a α2,6 SA binding preference, but it was also found to be independent of α5β1 integrin receptor. After attaching to a host cell, the virus was internalized in an endosome via clathrin- or caveolin- mediated endocytosis. By application of pathway inhibitors, the FN association with viral entry pathway was evaluated. Though this study failed to identify a single specific FN mediated viral entry pathway, this pathway study indicated the possibility of FN various involvement in influenza viral entry. The study indeed indicated that viruses have difference SA binding preferences are different in their choices in viral entry pathways. This thesis did not only introduce cellular FN as a novel host factor, but also identified possible target and brought new light in the control of influenza A viral infection. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

During the 1997 Hong Kong ‘bird flu’ incident, three subtypes of influenza viruses, including H5N1, H9N2 and H6N1, were co-circulated at the live-poultry markets. Genetic analyses revealed that all these viruses shared the same internal gene complex and might have been all involved in generation of the HK/97-like H5N1 virus. Subsequent epidemiological and genetic studies found that both H6N1 and H9N2 viruses became established and prevalent in minor poultry in the region. However, the genesis pathway for each of these viruses has not been defined. It is also unclear about these three subtypes further interact with each other and evolve in the field, along with the emerging reassortant variants. To address these questions, H6 subtype of avian influenza viruses isolated from terrestrial minor poultry from 2000 to 2005, and from 2006 to 2007 in our influenza surveillance in southern china has been genetically and antigenically analyzed in this study. Genetic and phylogenetic analyses of representative strains indicated that all H6N1 isolates from 2000 to 2007 had W312-like hemagglutinin and neuraminidase genes. These H6N1 viruses have become established in the minor poultry, mainly in quail and chukar, in this region. However, phylogenetic analyses revealed that the internal genes of the H6N1 virus lineage were derived from multiple origins with different evolutionary pathways. Evolution analyses of different gene segments of H6N1 viruses revealed imbalance dynamic evolutionary rates between surface genes and internal genes, which suggests that this virus lineage was more likely a descendant of the HK/97-like H5N1, rather than its precursor virus. Similar to what have been observed in the H5N1 and H9N2 virus lineages, the internal gene complex of the H6N1 viruses was found to undergo extensive reassortment. Many novel internal gene segments of H6N1 viruses were first recognized in the reassortant H9N2 virus particles, suggesting that the gene flow is likely from H9N2 to H6N1. The co-circulation of different virus lineages in southern China has greatly increased the genetic diversity of influenza viruses in this region. Analyses of the dynamics of different H6N1 reassortant variants also showed that some of them became persistent, but others were transient in the field. The increasingly diversified H6N1 and other subtypes of viruses will naturally increase the opportunity of interspecies transmission and dissemination, and may pose renewal threat for public health. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

The outbreaks of human influenza caused by highly pathogenic avian influenza H5N1 virus have attracted a lot of attention and public concern. Effective and universal vaccines may be the best means for prevention and control of the influenza. Taking into account that viral clearance and recovery from influenza A virus infection have been demonstrated to be correlated to specific cytoxic T lymphocyte (CTL) instead of neutralizing antibodies, it is important to develop effective vaccines which are capable of inducing not only neutralizing antibody but also CTL responses. Furthermore, T cell epitopes are usually more conserved than neutralizing epitopes. However, rare information concerning human T cell epitopes specific to H5N1 virus has been reported so far. This study was designed to test our hypothesis that novel and potent human CTL and Th epitopes specific to NP protein of H5N1 virus may be identified in vaccinated and/or infected HLA-A2/DR1 transgenic mice (SURE/L1), while protective epitopes may be further defined from the identified T cell epitopes in the mice challenged with lethal dose of the virus. We used SURE/L1 mouse model because it contains HLA-A2 (*0201) and -DR1 (*0701), both are the second highest frequency of HLA class I and II in Chinese. Since the NP gene is relatively conserved among different clades or strains of H5N1 virus, we selected viral protein NP as the target. Furthermore, we screened the T cell epitopes in splenocytes not only from vaccinated mice but also from survived mice infected with gradually increased dose of H5N1 virus, because the T cell epitopes identified in both vaccinated and infected mice or in infected mice alone might have higher potential to be protective epitopes. In this study, a novel HLA-DR1 (class II) restricted T cell epitope NP368-382, NPII-7, was identified in both vaccinated and infected mice. Two doses of NPII-7 peptide boosting in the mice induced very strong Th1 and CTL responses but no NP specific antibody responses. The vaccination of additional 2 doses of NPII-7 also provided partial protection against lethal challenge of H5N1 virus in the mice, whereas NP DNA vaccination alone did not show any protective effect. The protective effect may be attributed to the strong Th1 and CTL responses induced by the NPII-7 vaccination, because both NP DNA and NPII-7 vaccinations could not induce neutralizing antibody response. Notably, a HLA class II restricted peptide, NPII-7, may induce not only Th1 responses but also more strong HLA class I restricted T cell (CTL) responses. It may probably due to that the HLA-DR1 restricted T cell epitope (NENMEAMDSNTLELR) contained the full sequence of a reported HLA-A2 restricted CTL epitope (AMDSNTLEL), named NP-17 in this study. Although it needs to be further defined whether this novel epitope is really a HLA-DR1 restricted T cell epitope, or it shares the activity of HLA-A2 restricted T cell epitope, or it is just an alternate HLA-A2 restricted T cell epitope, this study has identified a novel T cell epitope and proved that it is a protective T cell epitope. / published_or_final_version / Microbiology / Master / Master of Philosophy

Although several risk factors for severe influenza infection have been identified in previous studies, many patients having multiple risk factors only developed mild symptoms while many healthy young patients developed severe complications when infected with A(H1N1)pdm09. Thus, there are still undiscovered factors that affect the progression and severity of influenza. The early innate immune response may be critical in determining the disease progression. Non-neutralizing antibodies existed in the early stage of infection may contribute to the outcome of the disease. In this study, the association of disease severity with the titre and avidity of non-neutralizing antibodies in early stage of influenza infection was investigated. It has been shown that the titre of non-neutralizing antibody was higher in more severe patients in the early stage of infection. Higher antibody avidity was also found to be associated with more severe disease independently. These findings tend to support the view that antigenic drift leads to an excessive production of pro-inflammatory non-neutralizing antibodies in the patients and associated with severe outcome. Since patients with more severe disease tend to have a delayed clearance of the virus and allow more transmission, the antigenically shifted or drifted influenza virus may gradually become predominant in human population. This idea suggested that the predominance of influenza virus with NA-H275Y mutation in 2007-2008 was contributed by the co-existing, fitness restoring secondary adaptive mutation in HA. NA-H275Y was identified in previous studies to be the mutation encoding for the influenza virus to resist against oseltamivir but would also change the property of NA as a result of compromised viral fitness. Therefore, influenza virus carrying NA-H275Y is unlikely to emerge and spread in human population. However, NA-H275Y mutated strains of influenza virus emerged and spread globally in the influenza season of 2007-2008 and quickly become the predominant strain in 2008. Previous study found NA-R222Q and NA-V234M were the mutations responsible for restoring the viral fitness in oseltamivir resistant clinical isolates. Still, this cannot fully explain the predominance of the resistant strains over the susceptible strains. Therefore secondary adaptive mutation in HA was believed to be present and cause antigenic change to the resistant strains of influenza. In this study, mutual information analysis and HA structural analysis were conducted to screen out HA-A189T and HA-Y94H to be the candidates co-exist with NA-H275Y and possibly critical for antigenic changes. This study further suggested that HA-Y94H mutation leads to a change of antigenic property of the virus by examining the antigenicity and growth kinetics of the recombinant viruses carrying the selected HA mutations. HA-94 may be critical for determining both the receptor binding property and antigenic property of the virus. Review on the evolution of seasonal influenza viruses from 2005 to 2008 suggested that the emergence of HA-Y94H mutation may enhance the presence of NA-H275Y and helps the viruses carrying NA-H275Y to spread and dominate over the oseltamivir susceptible strains during 2007 and 2008. / published_or_final_version / Microbiology / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 91-106) Also available in print.

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

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

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