Influenza is a viral infection that affects global health and economy with its endemic and sometimes pandemic spread. Rapid detection of Influenza viruses enables antiviral use and can bring financial savings. It is also essential for the global surveillance of prevalent Influenza strains. RT-PCR is considered the most specific and sensitive method for detection of Influenza, but Influenza mutates at a high rate and it is therefore crucial that RT-PCR methods are updated regularly. In 2014, Cepheid released their Xpert Flu/RSV XC assay, which can detect Influenza A and B and RSV by multiplex RT-PCR in approximately one hour. The aim of this study was to evaluate this assay at Laboratoriemedicin Västernorrland by using the laboratory’s previous PCR assay for detection of Influenza viruses as reference method. Real-time RT-PCR was used to compare Xpert Flu/RSV XC to the reference method. A dilution series was performed to estimate the methods’ PCR efficiencies and precision was calculated from quadruplicates of a positive control sample. Clinical specimens (n=42) were used to evaluate the diagnostic sensitivity and specificity of Xpert Flu/RSV XC. Objective statistical analysis of PCR data was performed and discussed. The Xpert Flu/RSV XC was equivalent to the reference method and demonstrated high diagnostic sensitivity and specificity. Estimated PCR efficiencies were however low. With the introduction of Xpert Flu/RSV XC to the laboratory follows many potential benefits, primarily in form of a simplified pre analytical procedure and a shortened analysis time. The Xpert Flu/RSV XC assay enables fast diagnosis of Influenza infection.

This work was aimed to investigate the basic viral infection protocols mainly focusing on Nicotiana benthamiana hairy root cultures and wild-type tobacco mosaic virus (TMV). The application of transgenic virus containing the gene for green fluorescent protein (GFP) for foreign protein production in plant tissue cultures and whole plants was also studied. The effect on viral accumulation of the form of plant tissue culture used, such as hairy roots, shooty teratomas and suspended cells, was investigated. Viral infection was shown to have no effect on culture growth and morphology. Hairy root cultures are a superior host for viral propagation and production in vitro. The maximum specific rate of viral accumulation occurred mainly during the root growth phase. The average maximum virus concentration in the hairy roots was 0.82 ?? 0.14 mg g-1 dry weight and virus protein represented a maximum of approximately 6% of total soluble protein in the root biomass. Proportional scale-up of TMVinfected hairy roots in shake flasks and bioreactors can be achieved without changing the average virus concentration accumulated in the hairy roots. The level of viral accumulation was much lower in N. benthamiana hairy roots infected with transgenic virus containing GFP (TMVGFPC3) compared with TMV and low levels or no GFP was detected. Viral accumulation and GFP production in whole plants was studied using different generations of transgenic TMV-GFPC3 virus. Hybrid viruses with the foreign gene GFPC3 deleted may have been formed in successive TMV-GFPC3 generations, resulting in the loss of GFP production and enhanced viral infectivity. In vitro generated RNA transcript and first generation TMV-GFPC3 were found to be more suitable for infection than the second generation TMV-GFPC3. However, the accumulation of GFP and virus concentration did not occur at the same ratio. Provided a more genetically stable transgenic viral vector is used for infection, transient viral infection of hairy roots can be a potential alternative system for foreign protein production than plants grown in the field as the containment or safety issues can be addressed.

Human cytomegalovirus (HCMV) is a highly prevalent opportunistic infection and a major pathogen in immune-compromised patients. The virus exhibits a wide cell tropism and is able to lytically infect virtually any cell type, with detectable gene expression and release of new virions, but not the neutrophil. This cell is the first immune cell to engage most pathogens, engulfing and killing them before undergoing apoptosis and clearance by macrophages. However certain viruses and bacteria are able to evade host defences and use the neutrophil as a “Trojan horse” for replication and dissemination. In this context, enhanced neutrophil survival may promote infection. This work describes a profound neutrophil survival phenotype elicited by contact with live or UV-inactivated HCMV, in the absence of lytic viral gene expression. The effect does not involve signalling through candidate Toll-like receptors, but is dependent on activation of the ERK MAPK and NFκB signalling pathways, and is viral strain-dependent, restricted to clinical strains of the virus. Furthermore, HCMV triggers the secretion of a bioactive secretome that induces a similar paracrine anti-apoptotic effect in fresh neutrophils, and stimulates monocyte chemotaxis and differentiation to a phenotype that is permissive for HCMV infection. This “transferrable” effect is not due to residual virus or the presence of well-known neutrophil survival factors such as IL-6 or IL-8, but is mediated by a heat-stable protein or lipid, secreted late in culture. These results are supported by data in neutrophils isolated from patients with CMV viraemia and pneumonitis which show increased survival ex vivo, and will be further investigated using plasma membrane profiling by amino-oxybiotinylation and tandem mass tag mass spectrometry. This technique, used for the first time here in a primary cell type, allows quantitative proteomics to be performed for the first time in the neutrophil. This work demonstrates that the technique provides a comprehensive readout of all neutrophil plasma membrane proteins in a sample, with high plasma membrane purity and minimal neutrophil activation and necrosis, validated by flow cytometry. Furthermore, this has been applied to generate plasma membrane profiles for the resting, inflammatory and apoptotic neutrophil, revealing a number of neutrophil cell surface molecules not found by previous membrane proteomic methods. This technique has the potential to analyse the effect of HCMV and other pathogens on the expression profile of the neutrophil surface membrane and to examine how neutrophil signalling and function is modulated. These data shed light on the role of neutrophil apoptosis as a potential promoter of HCMV infection, and have the potential to increase our understanding of both the neutrophil’s response to pathogen invasion and to generate future approaches to combating HCMV dissemination and pathogenesis.

In marine environments, eukaryotic marine microalgae coexist with the viruses that infect them. Marine microalgae are the main primary producers in the oceans and are at the base of the marine food web. Viruses play important roles in top-down control of algae populations, cycling of organic matter, and as evolutionary drivers of their hosts. Algae must adapt in response to the strong selection pressure that viruses impose for resistance to infection. In addition to biotic selection pressures such as viral infections, algae must also adapt to their abiotic environment. Global climate change is affecting temperature, salinity, pH, light and nutrient concentrations in the oceans, particularly in surface waters, where microalgae live. Currently, little is known about how consistent the effects of viruses on their hosts are, whether the cost of host resistance varies across environments, and whether there is a trade-off between maintaining resistance to viruses and adapting to other environmental changes. The marine picoeukaryote Ostreococcus tauri is abundant in Mediterranean lagoons, where it experiences large fluctuations in environmental conditions and co-occurs with lytic viruses (Ostreococcus tauri viruses – OtVs). Viral infection causes lysis of susceptible (S) cells, however a small proportion of cells are resistant (R) and avoid lysis. Some resistant O. tauri populations can coexist with infectious viruses, and it has been proposed that these viruses are produced by a minority of susceptible cells within a mainly resistant population. These populations are referred to as resistant producers (RP). Virus production in RP populations is unstable and eventually they shift to R populations. I used O. tauri and one of its viruses, OtV5, as a model system to investigate whether cells that are susceptible or resistant to virus infection adapt to environmental change differently and whether there is a cost of being resistant. For the first time, I evolved susceptible and resistant hosts of a marine alga separately under a range of environments and directly compared their plastic and evolved responses. I showed that resistant populations of O. tauri maintained their resistance for more than 200 generations in the absence of viruses across all environments, indicating that the resistance mechanism is difficult to reverse. Furthermore, I did not detect a cost of being resistant, as measured by population growth rate and competitive ability. Virus production in RP populations stopped in all environments and all populations became R. In addition, I found that virus production in RP O. tauri populations can fluctuate before completely ceasing, and that phosphate affected the length of time it took for virus production to stop. These results, combined with mathematical modelling of O. tauri infection dynamics, provide support for the prediction that RP populations consist of a mixed population of susceptible and resistant cells. By examining multiple environments and resistance types, we can better understand first, how microalgae populations adapt to environmental change and second, the ecological and evolutionary consequences of maintaining resistance to viruses in common marine picoeukaryotes.

abstract: Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by progressive autoimmune destruction of insulin-producing pancreatic β-cells. Genetic, immunological and environmental factors contribute to T1D development. The focus of this dissertation is to track the humoral immune response in T1D by profiling autoantibodies (AAbs) and anti-viral antibodies using an innovative protein array platform called Nucleic Acid Programmable Protein Array (NAPPA). AAbs provide value in identifying individuals at risk, stratifying patients with different clinical courses, improving our understanding of autoimmune destructions, identifying antigens for cellular immune response and providing candidates for prevention trials in T1D. A two-stage serological AAb screening against 6,000 human proteins was performed. A dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) was validated with 36% sensitivity at 98% specificity by an orthogonal immunoassay. This is the first systematic screening for novel AAbs against large number of human proteins by protein arrays in T1D. A more comprehensive search for novel AAbs was performed using a knowledge-based approach by ELISA and a screening-based approach against 10,000 human proteins by NAPPA. Six AAbs were identified and validated with sensitivities ranged from 16% to 27% at 95% specificity. These two studies enriched the T1D “autoantigenome” and provided insights into T1D pathophysiology in an unprecedented breadth and width. The rapid rise of T1D incidence suggests the potential involvement of environmental factors including viral infections. Sero-reactivity to 646 viral antigens was assessed in new-onset T1D patients. Antibody positive rate of EBV was significantly higher in cases than controls that suggested a potential role of EBV in T1D development. A high density-NAPPA platform was demonstrated with high reproducibility and sensitivity in profiling anti-viral antibodies. This dissertation shows the power of a protein-array based immunoproteomics approach to characterize humoral immunoprofile against human and viral proteomes. The identification of novel T1D-specific AAbs and T1D-associated viruses will help to connect the nodes in T1D etiology and provide better understanding of T1D pathophysiology. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2015

Early growth response (Egr) gene 2 and 3 are genes encoding transcription factors important for maintaining immune homeostasis. Here we define a fundamental role of Egr2 and 3 to control T cell proliferation and differentiation of effector T cells. Egr2 and Egr3 deficiency in T cells resulted in impaired T cell proliferation, but hyper-activation and excessive differentiation of T cells in response to viral infection, while, conversely, sustained Egr2 expression enhanced proliferation, but severely impaired effector differentiation in to T helper (Th) subsets, such as, Th1 and Th17 subtypes. T-bet is important for differentiation of effector T cells in response to pathogen and in particular it is a master regulator for modulating the T helper 1 lineage specific differentiation programme. Although T-bet has been extensively studied in T cells, the regulation of T-bet function is less well known. We have now discovered that Egr2 and 3 are potent inhibitors for Tbet function in CD4 and CD8 effector T cells. Together with Egr2 and 3, T-bet is induced in naïve T cells by antigen stimulation, but the expression was reciprocally regulated by IFNγ, which inhibited Egr2 and 3, but promoted Tbet expression. The expression of Egr2 and 3 in CD4 T cells under TH2 and TH17 condition was essential to suppress TH1 differentiation in vitro. In response to viral infection, sustained Egr2 expression in T cells profoundly inhibited differentiation of effector cells, while Egr2 and 3 deficient T cells produced excessive levels of IFNγ. We found that both Egr2 and 3 can directly interact with the Tbox domain of T-bet, block its DNA binding and inhibit T-bet mediated production of IFNγ. Thus, Egr2 and 3 are antagonists for T-bet function in effector T cells and essential for the control of T cell differentiation and immune pathology.

Hosts have two main strategies for coping with infections: resistance and tolerance. Resistance is aimed at preventing or eliminating parasites, whereas the goal of tolerance is to maintain performance regardless of parasite burden. The balance between resistance and tolerance within a host may mediate host competence, or the propensity of a host to infect other hosts or vectors. Hosts with high tolerance and low resistance to an infection, for instance, may be highly competent and possess the greatest potential to act as superspreaders. These superspreading hosts will contribute disproportionately to transmission, thus posing the greatest risk to other hosts within a population or community. Understanding the drivers of heterogeneity in host competence therefore has broad implications for the management of infectious diseases in nature. Host tolerance is typically quantified as the slope of the relationship between host performance and parasite burden. The majority of host tolerance studies have been conducted at the level of genotypes, populations and species. Individual hosts often exhibit variation in competence, with some individuals contributing more or less to transmission than the population/species average. Despite the clear importance of understanding tolerance at the individual-level, such studies are rare and may be particularly challenging in field contexts due to the need for repeated performance-burden measurements. I used the house sparrow (HOSP) – West Nile virus (WNV) system to investigate differences among two alternative approaches to estimating individual tolerance: the scope and position methods. The scope method estimates tolerance traditionally as the slope of multiple performance-burden measurements over time within an individual; alternatively, the position method required only one measurement for each individual, thus characterizing tolerance via among-individual variation in host defense. We found strong relationships between scope and position estimates of individual tolerance, suggesting that the position method may be an appropriate proxy to use in field studies. I also compared tolerance estimates derived from different metrics of performance. There were weak correlations among these estimates of tolerance, implying that tolerance estimated by measuring a single trait may not be indicative of tolerance at the level of the whole individual or their contribution to disease processes. Understanding the physiological mediators of host competence may help to pinpoint at-risk and risky individuals (or genotypes, populations and species) within natural communities, thus facilitating the development of more targeted disease management strategies. Cytokines and glucocorticoids have been identified as potent mediators of host defense. Pro-inflammatory cytokines may act to promote resistance, whereas anti-inflammatory cytokines and glucocorticoids tend to mediate host tolerance. I investigated the dynamics of pro-inflammatory cytokine IFN-γ, anti-inflammatory cytokine IL-10, and the major avian glucocorticoid, corticosterone (CORT), following WNV exposure in HOSP. I then assessed the influence of these three mediators on resistance and tolerance to WNV infection. I found unusual dynamics for the three mediators across the infection period: IFN-γ expression was not induced by WNV exposure, IL-10 expression was dampened by WNV exposure, and CORT levels were higher in unexposed individuals. Despite the unique response of HOSP to WNV exposure seen here, we did find that constitutive expression of IFN-γ and IL-10 mediate resistance and tolerance to WNV, respectively. Unexpectedly, we also found evidence for protective (pro-resistance) effects of CORT, which contrasts with previous evidence for the role of CORT in mediating WNV infections. Combined, the results of this study suggest that hosts with constitutively high IL-10 and low IFN-γ expression may have high potential to act as superspreaders of disease, thus becoming critical targets in designing WNV-control strategies in passerines. The methods by which we quantify host tolerance may greatly affect the conclusions we are able to draw from such studies. To date, a variety of definitions and techniques have been used to study tolerance in animals. In chapter three, I briefly summarize past plant and animal tolerance research, highlighting discrepancies among researchers in their motivations, definitions and techniques for studying tolerance. For instance, I discuss biases in the literature regarding the use of range versus point tolerance, vigor, and laboratory versus field studies. In particular, I expound upon the nature of the performance metrics used in the majority of tolerance estimations in the literature, and discuss the ecological implications of these metrics. To conclude, I offer suggestions for overcoming the challenges associated with studying tolerance and encourage a unified way forward in the field, emphasizing the selection of system-specific and ecologically relevant tolerance metrics. My thesis research has employed physiological and behavioral methods in an ecological context to better understand the heterogeneities that exist in host competence. By combining empirical data in the HOSP-WNV system with conceptual and methodological strategies for assessing host defenses, this research has broadened our knowledge of host responses in the WNV system in a manner that may be applicable to understanding and managing disease dynamics in diverse natural communities.

Transcriptional regulation of CD8+ T cell differentiation during acute and chronic viral infections is an intricate web made up of many of transcription factors. While several transcription factors have been elucidated in this process, there are still many more that remain elusive. In this work, we look into the role of two transcription factors, IRF4 and Runx2, and their role in CD8+ T cell terminal effector cells and memory precursor cells during acute LCMV-Armstrong infection. We found that IRF4 expression was regulated by TCR signal strength during infection, and that IRF4 expression levels directly correlated with the magnitude of the effector cell response. IRF4 was also shown to regulate T-bet and Eomes, two transcription factors critical for CD8+ T cell differentiation into effector and memory cells. From these results, we were interested in the potential role of IRF4 during chronic LCMV-clone 13 infection, where ratios of T-bet and Eomes are critical for viral clearance. We found that haplodeficiency of IRF4 in the T cell compartment lead to an increase in the ratio of Eomes to T-bet in T cells, which in turn affected the proportion of Eomeshi versus T-bethi cells and resulted in a loss in ability to clear viral infection. Irf4+/-Eomes+/- compound heterozygous mice were generated to test if decreasing Eomes expression would rescue the Irf4+/- phenotype. Irf4+/-Eomes+/- mice were phenotypically similar to WT mice in terms of Eomes to T-bet ratios, and were able to clear viral infection, demonstrating a critical role of IRF4 in regulating T-bet and Eomes during chronic viral infection. Next we looked into the role of Runx2 during acute LCMV-Armstrong infection and found that Runx2-deficient pathogen-specific CD8+ T cells had a defect in the total number of memory precursor cells compared to WT controls. We further showed that Runx2 was inversely correlated with TCR signal strength, and that Runx2 expression was repressed by IRF4. From these work, we have introduced two more transcription factors that are critical for CD8+ T cells differentiation during acute and chronic viral infection. Given the sheer number of transcription factors known to regulate these processes, having a full understanding of the transcriptional network will allow us to find the best targets for therapeutic intervention for treatments ranging from vaccine development and autoimmunity to cancer immunotherapy and treatment of chronic viral infections.

Nosocomial virus infections cause significant morbidity and mortality. Besides influenza viruses, the disease burden of parainfluenza virus type 3 (PIV-3) is comparatively high among hospitalized patients and severe disease courses can occur. PIV-3 showed the highest rates of nosocomial infections of a panel of respiratory viruses. Therefore, a retrospective observational study was conducted among patients with either PIV-3 or influenza viruses, which served as reference pathogen. The aim was to compare the seasonal dynamics and clinical characteristics of nosocomial infections with these highly transmittable viruses. Nosocomial infection occurred in 15.8% (n = 177) of all influenza cases, mainly in the first half of a season. About 24.3% (n = 104) of the PIV-3 cases were nosocomial and occurred mainly in the second half of a season. Both nosocomial rates of influenza and nosocomial rates of PIV-3 varied between the seasons. Community acquired and nosocomial cases differed in underlying medical conditions and immunosuppression. Knowledge of the baseline rates of nosocomial infections could contribute to the implementation of appropriate infection control measures.

A dengue é doença causada pelo vírus dengue (DENV), que acomete cerca de 390 milhões de pessoas no mundo, representando uma ameaça à saúde pública mundial. Até o momento não se dispõe de tratamento específico ou de vacinas para a maioria da população. Um dos maiores obstáculos para o desenvolvimento de vacinas ou para a compreensão da biologia do vírus é a falta de modelos animais que mimetizem a doença vista em humanos. Neste trabalho, o principal objetivo foi a busca de modelos alternativos de infecção com um isolado clínico (JHA1) de DENV sorotipo 2 (DENV2) capaz de infectar camundongos imonocompetentes após administração pela via intracraniana. Foram testadas duas vias alternativas de inoculação viral (intraperitoneal e intravenosa) em camundongos adultos imunocompetentes, além da via intracraniana em camundongos C57BL/6. Por fim, tentativas de mapeamento de mutações relacionadas à neurovirulência do JHA1 em camundongos imunocompetentes. Os resultados obtidos contribuem para a caracterização do JHA1 e para a busca de modelos experimentais alternativos de DENV. / Dengue fever is a disease caused by the dengue virus (DENV), which affects around 390 million people worldwide, posing a threat to global public health. To date, there is no specific treatment or vaccination for the majority of the population. One of the major obstacles to vaccine development or understanding the biology of the virus is the lack of animal models that mimic the disease seen in humans. In this work, the main objective was to search for alternative models of infection with a clinical isolate (JHA1) of DENV serotype 2 (DENV2) capable of infecting immunocompetent mice after administration by the intracranial route. Two alternative routes of viral inoculation (intraperitoneal and intravenous) were tested in immunocompetent adult mice, in addition to the intracranial route in C57BL/6 mice. Finally, attempts to map mutations related to JHA1 neurovirulence in immunocompetent mice. The results obtained contribute to the characterization of JHA1 and to the search for alternative experimental models of DENV.