Introduction With the emergence of the H5N1 virus in humans that was of entirely avian origin, a better understanding of potential receptors of the influenza A virus is needed. It is widely accepted that terminally sialylated glycoconjugates on the surface of the red blood cells are receptors to which the influenza A virus binds to and causes the agglutination of red blood cells. By isolating glycans found on red blood cells, perhaps it is possible to find potential receptors that influenza A virus has preferential binding to. There has been a general shift of using turkey red blood cells rather than chicken red blood cells for the haemagglutination of viruses over the past few decades. The influenza virus’s loss in ability to agglutinate chicken red blood cells but keep the ability to agglutinate turkey red blood cells a puzzling mystery. By comparing the differences, perhaps it is possible to elucidate structures influenza viruses prefer to bind to. Methods Mass spectrometricanalysis of purified glycans will allow us to narrow down the structures of the most abundant glycans found on the erythrocyte surface. Lectin staining with flow cytometry is used to identify the receptor specificity of the influenza viruses. Haemagglutination assay in conjunction with glycan binding array data from the CFG will allow us to pinpoint the possible structures that give the viruses the ability to bind using treatments with sialidases. Results The mass spectrometric data was good and the basic glycan structures were elucidated according to their mass to charge ratio. The proportion of the different glycans for each of the erythrocyte type was clearly shown. The lectin staining gave more accurate results have selecting a single cell population and it was clear that turkey red blood cells had more α2,3 and α2,6 linked glycans than the chicken red blood cells. The haemagglutination assay aided the identification of differentiating theα2,3 linked and α2,6 linked liking viruses. The glycan array binding data was obtained but some results were absent. Conclusion It is certain that turkey red blood cells are better than chicken red blood cells for use in the haemagglutination assays as they present some glycans that are present in human bronchial epithelials. The most abundant sialylated glycan are the α2,6 linked sialylated glycans. The most abundant glycans on chicken red blood cells are either absent or in low abundance on the HBEs. Though the turkey red blood cells have some glycans that are present on HBEs, the glycan profile is very different. To agglutinate red blood cells, viruses are likely to bind to the bisecting glycans and shorter antennae glycans. In methodology: for lectin staining experiments, one must take caution in the lectin used as it may affect results. Sialidase S treated RBCs are a good method to distinguish α2,3 linked glycans liking viruses. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Neuraminidase (NA) inhibitors are one of two classes of antiviral compounds available for the control of influenza infections. The H275Y NA mutation confers resistance to the NA inhibitor oseltamivir carboxylate among the N1 influenza subtype and has been identified from resistant variants with distinct epidemiological outcomes in human. Specifically, dominance of oseltamivir-resistant variant of the A/Brisbane/59/2007-like viruses was reported during the 2008 to 2009 influenza season, until it was replaced by the 2009 pandemic H1N1 virus [A(H1N1)pdm09]. Since the emergence of the 2009 pandemic, the fitness and transmission potential of oseltamivir-resistant A(H1N1)pdm09 variants carrying the H275Y mutation has been a concern. This project aims to systematically evaluate the fitness of viruses carrying the H275Y mutation for A(H1N1)pdm09 and seasonal H1N1 viruses. A panel of recombinant viruses with their NA gene derived from the A(H1N1)pdm09 A/California/04/09 (CA04), seasonal H1N1 A/New Caledonia/20/1999 (NewCal) or A/Brisbane/59/2007 (Brisbane) was generated in the genetic background of CA04. The H275Y mutation in all three viruses led to a reduced affinity for 3’-sialylactose (3’SL) or 6’-sialylactose (6’SL). Similarly, lowered enzyme activity was observed across H275Y-carrying viruses in 3’SL and 6’SL, with the exception of RG-CA04NA-H275Y at catalyzing 3’SL. Differential 3’SL and 6’SL substrate usage was observed between the NA of seasonal H1N1 and A(H1N1)pdm09 viruses. Reduced infectivity was also observed for recombinant CA04 viruses carrying the H275Y mutation with decreased infectivity in mucin-secreting primary human airway epithelial cells when compared to their oseltamivir-sensitive counterparts. In the ferret model, the pathogenicity of RG-CA04NA-H275Y and RG-CA04BrisbaneNA-H275Y viruses was attenuated albeit the transmissibility was minimally affected when compared to RG-CA04 wild-type virus. In parallel, recombinant seasonal H1N1 viruses encoding the surface glycoproteins of NewCal and Brisbane were tested in ferrets. Results indicated that NewCal and Brisbane viruses carrying the H275Y mutation displayed comparable transmission efficiencies to the wild-type NewCal virus via direct-contact and respiratory-droplet settings. These results suggest that the H275Y NA mutation only leads to a minor reduction in viral fitness, with its transmission potential being minimally affected in the naïve ferret model. / published_or_final_version / Public Health / Master / Master of Philosophy

The seasonal Influenza A viruses are respiratory pathogens causing epidemics annually with mild illnesses, while sporadically, novel influenza viruses emerge and trigger pandemics associated with more widespread and sometimes severe disease. The biological basis for severity of influenza disease remains unclear though it is recognized that the interplay between the influenza viruses and the host immune responses both contribute to viral pathogenesis. As macrophages are key sentinels of the innate immune response and play a crucial role in being the “first responders” as well as contributing to shaping the subsequent (pathogen‐specific) adaptive immune response, the objective of this research was to bring insights on the outcomes of the interactions of influenza viruses with the macrophages. The occurrence of Antibody‐Dependent Enhancement (ADE) of Influenza infection in macrophages was investigated. ADE occurs when non‐neutralized virus‐antibody complexes find alternative entry routes into host cells, mainly through the Fc‐receptor pathway and has been demonstrated predominantly in macrophages. Addition of human serum from some individuals to influenza A virus (either H5 pseudoparticles or pandemic (H1N1) virus) led to enhanced infection of murine macrophage‐like cells as illustrated by a two to five fold increase in detection of influenza M‐gene copies. Immunofluorescence microscopy indicated that serum‐mediated pandemic (H1N1) infection led to an increase in the number of infected cells than in controls. As the fold change in viral gene copies paralleled the fold increase of infected cells I concluded that ADE infection provide pandemic (H1N1) virus with increased opportunity to infect cells rather than simply increase the viral load per cell. In order to strengthen our results, and make them more physiologically relevant, experiments were then performed with human primary cells with clinical sera. However, ADE was not demonstrated in primary human macrophages, suggesting that ADE may be cell type or host specific. The second research question investigated was whether the different state of human primary macrophage differentiation or activation in vitro determined the susceptibility to influenza infection. Recently, work by others has shown a diverse range of macrophage phenotypes that arise by differences in macrophage differentiation and activation. In addition to the classical activation pathway (caMΦ), new mechanisms of activation, designated as alternative activation (aaMΦ), have been reported. Classically and alternatively activated macrophages display different phenotypes and properties, such as molecule expression patterns, cytokine secretion, and gene signatures. This study constitutes the first systematic comparison of Influenza A virus infection of these different subsets of human primary monocyte‐derived macrophages. When assessed for their permissiveness to different influenza A viruses, aaMΦΦshowed greater susceptibility to influenza A infection than caMΦ. This work also documents the receptor patterns and the gene expression profile of these macrophages in response to influenza virus infection in vitro. The results point to differences in susceptibility of the classically and alternatively activated human macrophages to pandemic H1N1 and other influenza A viruses and reveal intrinsic differences between these macrophage subtypes. Further investigations are needed to define the cellular and molecular determinants that define susceptibility of different macrophage subsets to influenza A infection. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

Hepatitis B virus covalently closed circular DNA (HBV cccDNA) forms a mini-chromosome structure inside infected hepatocyte nuclei and plays an important role in chronic hepatitis B infection. Methylation of cccDNA-bound histone and the associations of heterochromatin HP1 complex related proteins with cccDNA were investigated in this thesis using transient transfection study system and chromatin immunoprecipitation assay. Di- and tri-methylation of histone H3 lysine 4 residue (H3K4), which plays an activating role in eukaryotic transcription, were found to associate with cccDNA in a way in parallel to the level of HBV replication in our system. On the other hand, tri-methylation of H3K9, which plays an inhibitory role in eukaryotic transcription, was found to associate with cccDNA during decline of HBV replication. During the decline of HBV replication, cccDNA was associated with histone methyltransferases SUV39H1 and SUV420H1 and histone demethylase PLU1. The dynamic of the association of heterochromatin protein 1 (HP1) to cccDNA was similar to that of SUV39H1. The association of cccDNA with five HP1 complex-related proteins (three DNA methyltransferases Dnmt3a, Dnmt3b and Dnmt1 and two methylated DNA binding proteins MBD1 and MeCP2) was studied, and their associations could be roughly divided into two stages. From 72 hours to 96 hours post-transfection, there was an increased association of cccDNA with Dnmt3a, Dnmt3b and MBD1, which was in parallel to the increased association of HP1 and SUV39H1with -cccDNA. From 96 hours to 120 hours after transfection, an increased association of Dnmt1 and MeCP2 with cccDNA was detected, which was correlated to that of SUV420H1. At the time when HBV replication was declining at 120 hours post-transfection, a highest association of SUV39H1, SUV420H1, HP1 and all 5 HP1 complex-related proteins with cccDNA was found. In conclusion, methylation of cccDNA-bound histone was associated with HBV replication. Activating H3K4 methylation was found to correlate with increase in HBV replication, while inhibitory H3K9 methylation correlated with decrease in HBV replication. The association of HP1 was in parallel to that of SUV39H1, indicating that HP1-SUV39H1 complex might be involved, and thereby recruiting other proteins for transcription suppression. Recruitment of DNA methyltransferases and methylated DNA binding proteins to cccDNA provided further evidence that methylation of cccDNA plays a role in transcription suppression. This study identified the associations of methylated histone and other related proteins with cccDNA and their correlations with viral replication. These results enhance our knowledge in HBV replication cycles and transcription regulation. It may show a novel area in development of antiviral drugs such as histone methyltransferase modulators. / published_or_final_version / Medicine / Master / Master of Philosophy

Mammals have complicated antiviral innate immunity to combat viral infection and this poses a strong selection pressure on the viruses. As a result, many viruses have evolved different strategies to disrupt the function of hosts’ antiviral innate immunity. Herpes simplex virus type 1 (HSV-1) is one of the examples. HSV-1 is a common and important human pathogen. HSV-1 infection induces type I interferons (IFNs) which restrict viral replication potently. To ensure persistent infection and successful replication, HSV-1 encodes several IFN-suppressing proteins. One example is Us11. Interaction between Us11 and various cellular proteins, such as PKR, RIG-I and PACT, were shown by other studies. However, exactly how Us11 suppresses IFN function remains to be elucidated. In this study, I discovered that Us11 specifically inhibits PACT induced activation of RIG-I. In HSV-1 infected cells, PACT and Us11 associate with each other tightly and this interaction prevents the interaction of PACT with RIG-I. It was also found that RNA binding domains on both PACT and Us11 are important for the association. In infection experiments, the increased production of IFN- during the infection of PACT-competent cells with Us11-deficient HSV-1 recombinant virus was not observed in infected PACT-compromised cells, suggesting the requirement of PACT for Us11 suppression of IFN production. To conclude, this study provides an explanation for Us11 antagonism of IFN production. My findings suggest that PACT is a novel target of HSV-1 IFN-antagonizing protein Us11. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Influenza A virus infection is a major cause of human morbidity and mortality. T cell immunity is believed to play critical roles for host defenses against influenza A infection. Once intracellular influenza A infection is established, viral clearance is mainly dependent on virus-specific CD8+ T cells. CD4+ T cells are important for adaptive immunity to natural influenza A infection or vaccination by providing help to B cells for antibody production and also providing help to CD8+ T cells for the generation of cytotoxicity. In addition, virusspecific CD4+ and CD8+ T cells are rich sources of effector cytokines, such as IFN-and TNF-, which can promote the function of antigen presenting cells and have direct antiviral activity. Cross-subtype reactive CD4+ and CD8+ memory T cells also affect the clearance of virus infection even in those who lack virus-specific antibodies. Therefore, the aim of our study is to assess the influenza virus-specific T cell responses and define their possible protective role in pandemic H1N1 virus and seasonal influenza infection in human. First we determined whether healthy adults have the cross-reactivity of memory CD4+ and CD8+ T cells against pandemic virus. In April of 2009, 7 pandemic H1N1 infected patients and 17 their healthy contacts who had no pandemic influenza infection were recruited in this study. By using intracellular IFN-staining and flow cytometry, we examined their pandemic H1N1 virus and seasonal influenza H1N1-specific CD4+ and CD8+ T cell responses. Healthy contacts did have measurable but low frequencies of cross-reactive influenza-specific CD4+ and CD8+ T cells, though the frequencies of these T cells specific to pandemic H1N1 virus were slightly lower than that specific to seasonal H1N1 virus. Furthermore, when compared the pandemic H1N1-specific T cell responses between healthy contacts and patients with pandemic H1N1 infection, we can found that the healthy contacts have higher pandemic H1N1 specific-T cell responses than patients, suggesting these pre-existing pandemic H1N1 specific-T cells may have protection from pandemic influenza virus infection. In addition, we conducted a prospective T cell immunity and influenza surveillance study in a cohort of more than 200 healthy volunteers before the influenza season and investigated whether the pre-existing T cell immunity is related to the protection from influenza infection in the next coming influenza season. Using intracellular IFN-staining assay, we examined their pre-existing seasonal influenza H1N1, H3N2, seasonal influenza B virus-specific CD4+ and CD8+ T cell responses. Due to the small number of cases of influenza infection in the coming influenza season, the results only showed a trend that the subjects who have higher frequency of influenza virus strain-specific T cells may have lower chance to suffer from same strain of influenza infection, which to some extent, reflect the pre-exist memory T cells have association with the protection in the coming influenza season. In conclusion, T cells play an important role in defensing against influenza infection. The higher influenza virus specific-T cells response activity in healthy adults may have a protection against influenza virus infection. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

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Analysis of the proteins of capripoxvirus (KS-1) revealed a 32kd protein that is one of the major structural proteins of the virus and is localised in the virus envelope. Monospecific serum prepared against the 32kd envelope protein neutralised the virus indicating that this protein contains neutralising epitopes. Lymphocyte proliferation studies, using the 32kd protein and peripheral blood mononuclear cells from capripoxvirus (KS-i) vaccinated sheep, showed that this protein strongly induced cellmediated immune responses. The 32kd protein is capripoxvirus specific and induced antibodies in early stages of capripoxvirus infections. Immunoblot analysis of antibody responses against this protein has provided a basis for the differential diagnosis of capripoxvirus and orf virus infections. The 32kd protein bound to the surface of cultured lamb testis cells. The binding of the 32kd protein was completely inhibited by prior incubation of cells with purified capripoxvirus (KS-1) but not by bovine serum albumin. Trypsin treatment of capripoxvirus (KS-1) degraded the majority of the 32kd protein with a minimal effect on a few other virus proteins. Trypsin removed an external 10kd fragment from the 32kd protein, leaving a 22kd fragment associated with the virus. In addition, the trypsin treatment reduced the virus infectivity by at least ten fold, suggesting that the cell surface binding domain of the 32kd protein is located within the external 10kd fragment. The monospecific serum to the 32kd protein had no effect of the infectivity titre of the trypsin treated virus further supporting the concept that the external 10kd fragment of the 32kd protein is involved in binding of the virus particle to the cell surface. A degenerate oligonucleotide probe, based on an internal amino acid sequence obtained from V8 protease cleavage products of the 32kd protein, was used to identify the gene encoding the 32kd protein. The gene encoding the 32kd protein was identified within the 2.8kb HindI1l Q1 fragment of the capripoxvirus (KS-1) genome. The nucleotide sequence analysis of the Hindu Q1 fragment revealed five open reading frames (Q11L, Q12R, Q13L, Q14R and Q15L), one of these open reading frames, Q13L, is capable of encoding a 30.6kd protein and contains the complete internal amino acid sequence obtained from the V8 protease cleavage products of the 32kd protein, indicating that the Q13L encodes the 32kd envelope protein of capripoxvirus (KS-1). The deduced amino acid sequence of the Q13L shows a 34.1% identity and 61.3% similarity with that of H3L open reading frame of vaccinia virus.

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