published_or_final_version / Microbiology / Master / Master of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

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

Like the majority of host cell mRNAs, the mRNAs of influenza virus are capped and polyadenylated. The NS1 protein of influenza has been implicated as a translational activator for both influenza and reporter gene mRNAs. Data is presented showing that influenza A virus infection resulted in an increased ratio of cap-dependent to cap-independent translation. This ratio increase was largely due to an increase in cap-dependent translation. These experiments employed a bicistronic reporter construct measuring cap-dependent and cap-independent translation in a single sample. Expression of NS1 alone resulted in a small, but reproducible increase in the ratio of cap-dependent to cap-independent translation. Additionally, with use of an NS1 deleted mutant influenza A virus (delNS1) it is shown that infection without NS1 expression produced less of a translation ratio increase compared to wild-type virus infection. Furthermore, expression of NS1 rescued a more wild-type ratio increase in delNS1 infected Vero cells. These results implicate NS1 as playing a role in increasing the ratio of cap-dependent to cap-independent translation in influenza A virus infected cells. Additionally, eIF4E-binding protein-1 (4E-BP1), a member of the protein family that inhibits cap-dependent translation through their inhibition of the cap-binding protein, eukaryotic initiation factor 4E (eIF4E), is shown to be inactivated throughout the majority of the influenza A virus infection process.

Dendritic cells (DC) are a heterogeneous population of hematopoietic cells that play a versatile role in orchestrating immune responses against an array of invading pathogens, including influenza virus. These cells reside in lymphoid organs as well as in non-lymphoid tissues such as mucosal surfaces of respiratory and gastro-intestinal system. Recent investigations have suggested that in the steady state, dendritic cells are derived mainly from bone marrow precursor cells without a monocytic intermediate whereas during inflammation or infection, monocytes readily differentiate to generate monocyte derived dendritic cells (MoDC). The ability of virus infected monocytes to differentiate into MoDC was investigated and the results demonstrated that in vitro infection of monocytes with influenza virus impaired their development into MoDC. It was also observed that influenza infection of monocytes, pre-treated with GM-CSF and IL-4 for DC differentiation, was minimally-productive and non-cytopathic. In spite of successful viral genome transcription, viral protein synthesis was restricted at an early stage. However, despite of the limited replication, influenza virus infected monocytes failed to develop the distinctive DC- like morphology when cultured with GM-CSF and IL- 4 as compared to their mock infected counterparts. Infected cells, after 4 days in culture, expressed reduced amounts of CD11c, CD172a (myeloid marker), CD1w2 (CD1b) and CCR5. Influenza virus infected monocytes also retained substantial non-specific esterase activity, a characteristic for monocytes and macrophages. Antigen presentation capability of infected cells was also affected as indicated by decreased endocytosis. Production of IL-12, a pro-inflammatory cytokine and IL-10, a reciprocal inhibitory cytokine, was coordinately modified in influenza virus infected monocytes in order to arrest their differentiation into DCs. At least limited viral replication was necessary to impede the differentiation process completely. However, viral NS1 protein activity, as evidenced with a mutant influenza virus, was not essential for this inhibition. This identified a new strategy by influenza virus to interfere with DC differentiation and evade a virus specific immune response.

Seasonal influenza epidemics are a major public health concern, causing three to five million cases of severe illness and about 250,000 to 500,000 deaths worldwide. Given the unpredictability of these epidemics, hospitals and health authorities are often left unprepared to handle the sudden surge in demand. Hence early detection of disease activity is fundamental to reduce the burden on the healthcare system, to provide the most effective care for infected patients and to optimize the timing of control efforts. Early detection requires reliable forecasting methods that make efficient use of surveillance data. We developed a dynamic Bayesian estimator to predict weekly hospitalizations due to influenza related illnesses in the state of Texas. The prediction of peak hospitalizations using our model is accurate both in terms of number of hospitalizations and the time at which the peak occurs. For 1-to 8 week predictions, the predicted number of hospitalizations was within 8% of actual value and the predicted time of occurrence was within a week of actual peak. / text

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.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / Influenza A virus (IAV) is a negative sense, single-stranded, segmented RNA virus with eight gene segments. It is an important respiratory pathogen which causes annual epidemics and occasional pandemics worldwide in humans and leads to considerable economic problems for the livestock industry. To control and prevent this significant disease, understanding the pathogenesis of IAVs is critical. Although some molecular mechanisms regarding virulence have been determined, IAV pathogenesis is not completely understood and is difficult to predict. The eight viral gene segments of IAV were thought to encode for 10 viral proteins. Since 2001, eight additional viral proteins have been identified, including PB1-F2, PB1-N40, PA-X, NS3, PA-N155, PA-N182, M42, and PB2-S1. However, the functions of these novel proteins in influenza virus replication as well as pathogenesis have not been fully elucidated. Although PB1-F2 protein is an important virulence factor of IAV, the effects of this protein on viral pathogenicity of swine influenza virus (SIV) remain unclear. In Chapter 2, we investigated the contribution of the PB1-F2 protein to viral pathogenicity of a virulent triple-reassortant (TR) H1N1 SIV in different hosts, pigs and mice. Our data indicate that PB1-F2 expression in virulent TR H1N1 SIV modulates virus replication and pathogenicity in the natural host, pigs, but not in mice. In addition, single amino acid (aa) substitution at position 66 (N/S) in the PB1-F2 has a critical role in virulence in mice but no effect was found in pigs. A novel IAV protein, PA-X consists of the N-terminal 191aa of PA protein and a unique C-terminal 41 (truncated form) or 61 (full-length form) aa residues encoded by +1 ribosomal frameshifting. Although several studies have demonstrated the PA-X protein as an important immune modulator and virulence factor, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on viral pathogenicity and host response remains unclear. In Chapter 3, we showed that expression of either truncated or full-length PA-X protein in 2009 human pandemic H1N1 (pH1N1) viruses suppresses host antiviral response by host shutoff activity which promotes viral growth and virulence in mice when compared to loss of PA-X expression. Furthermore, full-length PA-X expression displayed stronger impact on viral pathogenicity and host immune response compared to truncated PA-X expression. Taken together, our results provide new insights into the impact of PB1-F2 and PA-X proteins on virus replication, pathogenicity and modulation of host immune responses. This knowledge is important for better understanding of IAV pathogenesis.