Le virus de la fièvre hémorragique de Crimée-Congo (VFHCC) est un virus transmis par des tiques, appartenant au genre Nairovirus de la famille des Bunyaviridae. Il est réparti sur plus d’une trentaine de pays de plusieurs continents : Europe, Asie et Afrique ; avec une mortalité moyenne estimée de 30%. Le VFHCC peut causer à l’homme une maladie hémorragique très sévère et parmi divers symptômes, il peut induire une inflammation aigüe et des lésions hépatiques. Les phagocytes mononucléaires, les hépatocytes et les cellules endothéliales ont été décrits comme étant des cellules cibles lors d’études cliniques et post-mortem ainsi que lors d’études en modèle murin. Nous avons analysé, lors d’étude in vitro, la réponse cellulaire de cellules présentatrices de l’antigène (CPAs) et d’hépatocytes humains. Afin de mieux comprendre la pathogenèse du VFHCC, nous avons comparé la réponse de ces cellules avec celle du virus Dugbe (DUGV), un nairovirus génétiquement le plus proche de VFHCC considéré comme faiblement pathogénique. Finalement, afin améliorer la détection de DUGV in vitro et lors d’études épidémiologiques de terrain, nous avons développé un test moléculaire en temps réel pour détecter et quantifier DUGV.Nous avons observé que le VFHCC induisait une réponse inflammatoire chez les CPAs, en revanche, DUGV induisait une réponse plus forte, ce qui suggère que VFHCC induirait une inhibition sélective de certains médiateurs de la réponse pro-inflammatoire. Lors de l’infection in vitro des hépatocytes par le VFHCC, nous avons observé que le virus induisait du stress du RE, l’activation de l’IL-8 un médiateur pro-inflammatoire, et la modulation des deux voies pro-apoptotiques. Quand nous avons comparé cette réponse à celle induite par DUGV nous avons trouvé que la différence majeure était l’absence d’apoptose. Ces différences pourraient, en partie, expliquer le rôle du foie dans la pathogenèse induite par le VFHCC. / Crimean-Congo hemorrhagic fever virus (CCHFV) is a widely distributed tick-borne member of the Nairovirus genus (Bunyaviridae) inducing an average mortality rate of 30% in humans. CCHFV induces a severe hemorrhagic disease in infected patients that includes, among other bleeding symptoms, acute inflammation and liver lesions. The mononuclear phagocytes, the hepatocytes and the endothelial cells were described to be the main target cells in both human clinical studies and animal model in vivo studies.We analysed the in vitro cellular response of host antigen presenting cells (APC) and hepatocytes. Then, to better elucidate the pathogenesis of CCHFV, we compared the response of these cells after infection with Dugbe virus (DUGV), a mild pathogenic virus genetically close to CCHFV. In order to improve DUGV detection in vitro and in field studies, we also developed a molecular real-time quantitative tool to detect and quantify DUGV.We found that CCHFV induced an inflammatory response in both APCs tested; however DUGV induced a higher cytokine/chemokine response in these target cells than CCHFV. Our results suggest that CCHFV was able to selectively inhibit the activation of some inflammatory mediators in the in vitro infection and that CCHFV/DUGV cellular response differences could be relevant in pathogenesis. On the other hand, when we in vitro infected hepatocytes with CCHFV, we observed that it was able to induce ER-stress, activate IL-8 secretion and modulate both mitochondrial and death receptor pathways of apoptosis. When we compared this cellular response with that induced by DUGV, we found that the most striking difference was the absence of apoptosis. These differences could, in part, explain the role of the liver in the pathogenesis induced by CCHFV.

Infections with dengue viruses (DENV) constitute both a global problem as well as locally in Florida. DENV comprise four distinct serotypes of single-stranded RNA viruses and belong to the family Flaviviridae. DENV are among the most medically important arboviruses in the world and cases may currently exceed 400 million per annum. Additionally, dengue established its first recorded endemic transmission cycle in the state of Florida in over a half century, first within the Florida Keys during 2009-10 followed by an unrelated outbreak in Martin County in 2013. The clinical profile of DENV infections ranges from a mild febrile illness to severe illness including hemorrhaging and/or shock, occasionally leading to death. Asymptomatic and mild cases also play a role in maintaining transmission cycles. The early diagnosis and management of patients at the clinical level have both proven to be major obstacles in the control of DENV and are important at both the individual and community levels. Individually, the proper management of patients that will progress to severe illness demands that they are identified in order to receive supportive treatment and mitigate associated morbidity and mortality. At the community level, early diagnosis may reduce transmission by limiting the possibility of vector contact with viremic individuals. Early diagnosis is dependent on the detection of viral markers, while a number of host factors may inform prognosis. The microsphere-based immunoassay (MIA) is capable of detecting up to 100 different targets in a single sample and therefore would be useful as a single assay for determining both. This study attempted to develop a diagnostic and prognostic MIA using the DENV NS1 glycoprotein and 5 host markers as targets. For the purposes of DENV NS1 detection in MIA, a set of monoclonal antibodies (mAbs) were subjected to immunoprecipitation and/or Western blot analysis in order to determine immunoreactivity. Two mAbs, 3A5.4 and 3D1.4, were chosen for use in MIA as a capture antibody and a detection antibody, respectively, and the results compared to a commercially available DENV NS1 ELISA. The 5 markers chosen for MIA trials included GM-CSF, IFN-γ, IP-10, IL-10, and MCP-1 and were selected from a panel of 27 markers screened initially in two in vitro models of DENV infection in addition to serum samples. The two cell lines investigated were HPMEC ST1.6R and u937 as both are thought to play important roles in models of DENV pathogenesis. The results of the DENV NS1 detection MIA were initially promising but were ultimately unsuccessful. When measuring host markers in the MIA, results pointed towards certain profiles that may be of future use. IL-10 was found to be elevated in a statistically significant manner in DENV qRT-PCR+ samples (p=0.035) when compared to negative sera. MCP-1 elevation was found to be of borderline significance (p=0.058). Other potential markers based on the results reported here include IP-10, IL-6, IL-8, VEGF, and RANTES. The ultimate goal of measuring host markers is to gain the ability to differentiate patients that will progress to severe illness from those that will recover. In conclusion, despite the failure of the MIA to detect DENV NS1 in human sera, our results in determining host markers and developments leading to successful DENV NS1 detection ELISAs elsewhere lead us to believe that this approach remains promising. Major drawbacks of this study included the lack of samples from patients that experienced severe DENV illness as a comparative group in addition to small sample sizes. Future goals should include determining the reasons for the failure of the MIA in detecting DENV NS1, selecting a panel of appropriate markers to differentiate non-severe from severe cases of DENV prior to progression, and optimizing the inclusion of these markers to an appropriate number.

Doctor of Philosophy / Division of Biology / Kristin Michel / The biting midge, Culicoides sonorensis, vectors orbiviruses of economic importance, such as epizootic hemorrhagic disease virus (EHDV). Due to the limitations in available molecular tools, critical Culicoides-orbivirus interactions underlying vector competence remain unclear. To provide a foundation for the study of midge-EHDV interactions, RNA interference (RNAi) was developed as a reverse genetic tool, and EHDV-2 infection dynamics were determined within C. sonorensis. To develop RNAi, exogenous double-stranded RNA (dsRNA) was injected into C. sonorensis adults specific to the C. sonorensis inhibitor of apoptosis protein 1 (CsIAP1) ortholog (dsCsIAP1). A significant decrease in CsIAP1 transcripts was observed in whole midges, with highest reduction in the midgut. In addition, dsCsIAP1-injected midges had increased mortality, a loss of midgut tissue integrity, and increased caspase activity. The longevity and midgut phenotypes were partially reversed by the co-injection of dsRNA specific to the C. sonorensis initiator caspase Dronc ortholog and CsIAP1. These results demonstrated that RNAi can be achieved in the midge midgut through injection of target dsRNAs into the hemolymph. Furthermore, the time course of EHDV-2 infection within C. sonorensis was characterized. EHDV-2 infection was observed in the midgut and secondary tissues, including the salivary glands, by 5 days post-feeding (dpf). These data are consistent with dissemination of EHDV-2 to secondary susceptible tissues throughout the midge via the hemolymph and indicate that virus transmission by C. sonorensis may occur as early as 5 dpf. This work provides a foundation for the future study of Culicoides-orbivirus interactions, including the antiviral role of RNAi at the midgut barrier.

Hereditary hemorrhagic telangiectasia (HHT), also known as Osler-Weber-Rendu syndrome, is a rare genetic blood disorder that leads to abnormal bleeding due to absent capillaries and multiple abnormal blood vessels known as arteriovenous malformations. A feature of HHT is high-output heart failure due to multiple arteriovenous malformations. High-output heart failure can lead to recurrent epistaxis Kiesselbach area syndrome (REKAS), further exacerbating heart failure through increased blood loss and resultant anemia. We report a patient with HHT who presented with high-output heart failure contributing to REKAS. In patients with REKAS, we propose if anemia is present, REKAS can be avoided by correcting the anemia by increasing the hemoglobin level to greater than 9 to 10 g/dL. This decreases hyperdynamic circulation and reduces pressure in the blood vessels of the nose.

Cerebral hemorrhages are a type of stroke causing bleeding in the brain from a ruptured brain vessel or an aneurysm. In the emergency department (ED), advanced practice nurses (APNs') play a pivotal role in identifying cerebral hemorrhages. Many APNs' lack the skills necessary to assess, diagnose, and manage this type of stroke. A delay in diagnosis of cerebral hemorrhages carries a high risk of morbidity and mortality and can lead to lifelong disability or death. The framework of this project is the Patricia Benner theory of novice-to-expert, which is a 5-step approach designed to strengthen nursing skills with the goal of developing nurses into experts in the neuroscience field. This project addressed whether a validated educational program for APNs' in the ED will increase knowledge to improve the assessment, diagnosis, and management of the cerebral hemorrhage stroke patient. A panel of experts were provided a comprehensive educational plan with multiple case scenarios. A 5-point Likert questionnaire was administered and a statistical analysis was performed. Results indicated the panel strongly agreed that the educational initiative increased APNs' knowledge and fostered a culture of change for APNs' in the ED. Potential implications of this educational module will be considered in satellite organizations impacting APNs' growth and improving patient care throughout the health system. The project determined that the significance of social change included increased APNs' knowledge and advanced professional growth, lowers mortality rates, and promotes an evidence-based quality improvement project.

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Trauma är den ledande dödsorsaken i Sverige för människor mellan 15 och 44 år och en stor andel dör till följd av blödning som uppkommer vid skadetillfället. Blödning fortsätter också vara den ledande orsaken till traumarelaterad död som kunde ha varit förebyggbar både civilt och militärt. Traumaomhändertagandet är komplext, ofta tidskritiskt och ambulanspersonalen är ofta de som först får vårda dessa patienter ute på skadeplats och därav blir ambulanspersonalens första bedömning och omhändertagande av stor betydelse. Vätskebehandling för kritiskt skadade traumapatienter i hemorragisk chock eller hotande hemorragisk chock är ett omdiskuterat ämne och Sverige saknar nationella riktlinjerna för omhändertagande och behandling prehospitalt vid hemorragisk chock och trauma och de regionala riktlinjer som finns skiljer sig ibland till viss del åt mellan länen. Syftet med studien var att undersöka effekten av ambulanssjuksköterskans vätskebehandling för patienter i hemorragisk chock vid trauma. Som metod har en litteraturöversikt genomförts där totalt 15 studier inkluderats som är publicerade mellan 2009 och 2019. I resultatet framkom två huvudteman – vätskebehandlingens effekt på mortalitet och vätskebehandlingens effekt på koagulation. Samtliga fyra studier som undersökt hur koagulationsförmågan påverkas av kristalloid hyperton och/eller isoton vätskebehandling hos patienter med eller med risk för hemorragisk chock utsatta för trauma har kommit fram till att den försämras desto mer vätska patienten får. Resultatet visade oklar evidens gällande vätskebehandlingens effekt för mortaliteten på patienter i hemorragisk chock vid trauma. Däremot påvisade ingen av studierna att mortaliteten minskade. Slutsats var att majoriteten av artiklarna talar för att stora mängder vätska prehospitalt minskar eller inte gör någon skillnad för överlevnaden för kritiskt skadade traumapatienter vid hemorragisk chock. Många faktorer spelar in och det är svårt att dra några slutsatser utifrån resultatet och mer forskning behövs inom området. / Trauma is the leading cause of death in Sweden for people between the age of 15 and 44 years and a large proportion of people die because of bleeding that occurs at the time of the injury. Bleeding also continues to be the leading cause of trauma-related death that could have been preventable both in a civilian and military setting. Trauma care is complex, often time-critical, and the ambulance nurses are often the first to care for these patients on the scene and therefore the first assessment and care of these patients is of great importance. Fluid resuscitation for critically injured trauma patients in hemorrhagic shock or threatening hemorrhagic shock is a debated topic and Sweden lacks national guidelines for trauma care and treatment prehospital. The regional guidelines sometimes for some manner differ between the counties in Sweden. The aim of this study was to determine the impact of fluid resuscitation for patients with hemorrhagic shock after trauma. As a method, a literature review was carried out, which included a total of 15 studies published between 2009 and 2019. The result revealed two main themes - the impact of fluid resuscitation on mortality and the impact of fluid resuscitation on coagulation. All four studies that examined how coagulation ability is affected by crystalloid hypertonic and/or isotonic fluid resuscitation in patients at risk of hemorrhagic shock after trauma, the severity seems to be dependent on the amount of fluid infused, the more fluid the more severe coagulation abnormalities. The result showed unclear evidence of the effect of fluid resuscitation in mortality for trauma patients in hemorrhagic shock. However, none of the studies showed it decreased in mortality. In conclusion, the majority of articles show that large amount of fluid given in prehospital care have no impact or did have a negative impact on survival of critically injured trauma patients in hemorrhagic shock. Many factors come into play and it is difficult to draw any conclusions based on the results and more research are needed.

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