Influenza B virus is a major human pathogen causing highly contagious respiratory disease. It accounts for approximately ~30% of influenza virus infection per year. The effector domain of the NS1 protein of influenza B virus (NS1B protein), encompassing the carboxy terminal two thirds of the protein, suppresses interferon-β (IFN-β) synthesis in virus-infected cells by unknown mechanism(s). The induced IFN-β mediates innate immunity. To elucidate the mechanism by which the NS1B effector domain suppresses the production of IFN-β, we identified cellular proteins that interact with the NS1B effector domain. Two approaches were used. The approach that succeeded employed the transfection into cells of plasmids expressing the NS1B effector domain containing two affinity tags. After double affinity purification, co-purified cellular proteins were identified by mass spectrometry. We identified Brd2 as a cellular protein that interacts with the NS1B protein. We established that Brd2 specifically binds to the NS1B effector domain in vitro, in vivo, and in virus-infected cells. Serial mutagenesis experiments showed the phenylalanine at position 171 (F171) of the NS1B protein is essential for Brd2 binding. To determine the function of the interaction of Brd2 with the NS1B protein, we generated a recombinant virus encoding an NS1B protein in which F at position 171 was replaced by an alanine. The F171A mutant virus was attenuated, and unlike the wild-type virus, induced the synthesis of IFN-β mRNA. IRF3, a key transcription factor for transcription of the IFN-β gene, was activated in mutant virusinfected cells, but not in wild-type virus-infected cells. Transfection assays implicated the activation of the TBK1 kinase as the step in IRF3 activation that is induced in mutant virus-infected cells. We interpreted these results as showing that Brd2 binding to the NS1B protein is required for suppressing IRF3 activation and IFN-β induction. Attempts at further confirmation by depletion of endogenous Brd2 using RNA interference were not successful because of inefficient knock-down efficiency and nonspecific IFN-β induction. A further complication is that another bromodomain protein, Brd4, interacts with the NS1B protein and could compensate for depletion of Brd2. / text

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.

No description available.

Lam, Ka Han. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 77-82). / Abstracts in English and Chinese. / Acknowledgement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Content --- p.vii / List of Abbreviations and Symbols --- p.xi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Severity of influenza --- p.1 / Chapter 1.2 --- Introduction of influenza viruses --- p.3 / Chapter 1.2.1 --- Virion and genome structure --- p.4 / Chapter 1.2.2 --- The replication cycle of influenza viruses --- p.5 / Chapter 1.3 --- Influenza virus NP --- p.8 / Chapter 1.3.1 --- The importance of NP in RNP structure maintenance --- p.9 / Chapter 1.3.2 --- NP self oligomerization --- p.10 / Chapter 1.3.3 --- NP-RNA interaction --- p.12 / Chapter 1.3.4 --- NP and other interacting partners --- p.13 / Chapter 1.4 --- Aim of the project --- p.16 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Biological materials --- p.18 / Chapter 2.2 --- Construction of NP mutants --- p.19 / Chapter 2.3 --- Luciferase assay --- p.22 / Chapter 2.4 --- Western blot --- p.23 / Chapter 2.5 --- Protein expression and purification --- p.23 / Chapter 2.6 --- Circular dichroism spectroscopy --- p.24 / Chapter 2.7 --- Static Light scattering --- p.24 / Chapter 2.8 --- Surface plasmon resonance --- p.25 / Chapter 2.9 --- Co-immunoprecipitation (co-IP) --- p.26 / Chapter Chapter 3 --- Identification of residues crucial for NPB oligomerization and ribonucleoprotein activity / Chapter 3.1 --- Introduction --- p.27 / Chapter 3.2 --- Result --- p.31 / Chapter 3.2.1 --- NPB mutants showed deficiency in overall transcription and replication activity --- p.31 / Chapter 3.2.2 --- Expression and purification of NP mutants with low RNP activity --- p.37 / Chapter 3.2.2.1 --- Expression of MBP-tagged NP variants --- p.37 / Chapter 3.2.2.2 --- Purification of MBP-tagged NP variants --- p.38 / Chapter 3.2.3 --- Secondary structures of NP variants were comparable t o wild type NP --- p.41 / Chapter 3.2.4 --- NP variants with low RNP activity were abnormal in oligomerization in vitro --- p.42 / Chapter 3.2.5 --- NP variants with low RNP activity were impaired in homo-oligomer formation in vivo --- p.45 / Chapter 3.2.6 --- Discussion --- p.47 / Chapter Chapter 4 --- Identification of residues crucial for NP 一 RNA interaction and ribonucleoprotein activity / Chapter 4.1 --- Introduction --- p.56 / Chapter 4.2 --- Result --- p.58 / Chapter 4.2.1 --- NPB mutants showed deficiency in overall transcription and replication activity --- p.58 / Chapter 4.2.2 --- Expression and purification of NP variants with low RNP activity --- p.62 / Chapter 4.2.3 --- Secondary structures of NP variants were comparable t o wild type NP --- p.63 / Chapter 4.2.4 --- NP variants with low RNP activity were abnormal in RNA binding --- p.64 / Chapter 4.3 --- Discussion --- p.68 / Chapter Chapter 5 --- Conclusion and future prospect --- p.73 / Copyright --- p.76 / References --- p.77

The interferon (IFN) system forms an essential part of the innate immune response, up-regulating hundreds of IFN-stimulated genes (ISGs) in response to viral infection. A key protein in this response is the human myxovirus resistance protein MxA, an IFN-induced GTPase with broad-spectrum antiviral activity, capable of inhibiting many RNA and DNA viruses. One of the most studied antiviral effects of MxA is the inhibition of influenza A virus replication, yet the molecular mechanism of antiviral activity is still unknown. Influenza A viruses are inhibited by MxA at two distinct stages of viral replication; during viral entry and following primary transcription of viral mRNAs. The antiviral effects of MxA during viral entry are highly dependent on IFN, however activity exerted after primary transcription can occur in the absence of IFN. This study provides evidence that MxA exerts its antiviral activity at these two stages of viral replication through distinct mechanisms, and outlines a potential model of MxA antiviral activity following primary transcription. A potential third antiviral mechanism of MxA is proposed based on the findings that MxA is able to regulate cellular lipid metabolism, thereby potentially affecting virion composition. Mutational analysis of MxA highlights the significance of GTPase activity to the antiviral effects of MxA, while also demonstrating that natural single nucleotide polymorphisms in MxA have the potential to severely impair or prevent antiviral activity. Finally, this thesis shows for the first time that MxA exhibits antiviral activity against influenza B viruses. Overall this thesis provides new information illustrating how MxA provides potent antiviral activity against influenza viruses. Such information is vitally important as understanding the molecular basis of how proteins such as MxA function against many human pathogens is fundamentally important in our efforts to create better long-term treatment options for all viral diseases.

Introduction: Epidemiological studies have indicated that 5-38% of influenza like illnesses (ILI) develop into severe disease due to, among others, factors such as; underlying chronic diseases, age, pregnancy, and viral mutations. There are suggestions that dual or multiple virus infections may affect disease severity. This study investigated the association between co-infection between influenza A viruses and other respiratory viruses and disease severity. Methodology: Datum for samples from North West England tested between January 2007 and June 2012 was analysed for patterns of co-infection between influenza A viruses and ten respiratory viruses. Risk of hospitalization to a general ward ICU or death in single versus mixed infections was assessed using multiple logistic regression models. Results: One or more viruses were identified in 37.8% (11,715/30,975) of samples, of which 10.4% (1,214) were mixed infections and 89.6% (10,501) were single infections. Among patients with influenza A(H1N1)pdm09, co-infections occurred in 4.7% (137⁄2,879) vs. 6.5% (59⁄902) in those with seasonal influenza A virus infection. In general, patients with mixed respiratory virus infections had a higher risk of admission to a general ward (OR: 1.43, 95% CI: 1.2 – 1.7, p = <0.0001) than those with a single infection. Co-infection between seasonal influenza A viruses and influenza B virus was associated with a significant increase in the risk of admission to ICU/ death (OR: 22.0, 95% CI: 2.21 – 219.8 p = 0.008). RSV/seasonal influenza A viruses co-infection also associated with increased risk but this was not statistically significant. For the pandemic influenza A(H1N1)pdm09 virus, RSV and AdV co-infection increased risk of hospitalization to a general ward, whereas Flu B increased risk of admission to ICU/ death, but none of these were statistically significant. Considering only single infections, RSV and hPIV1-3 increased risk of admission to a general ward (OR: 1.49, 95% CI: 1.28 – 1.73, p = <0.0001 and OR: 1.34, 95% CI: 1.003 – 1.8, p = 0.05) and admission to ICU/ death (OR: 1.5, 95% CI: 1.20 – 2.0, p = <0.0001 and OR: 1.60, 95% CI: 1.02 – 2.40, p = 0.04). Conclusion: Co-infection is a significant predictor of disease outcome; there is insufficient public health data on this subject as not all samples sent for investigation of respiratory virus infection are tested for all respiratory viruses. Integration of testing for respiratory viruses’ co-infections into routine clinical practice and R&D on integrated drugs and vaccines for influenza A&B, RSV, and AdV, and development of multi-target diagnostic tests is encouraged.