KRATEK POVZETEK PROJEKTA
Virusne hemoragične mrzlice (VHM) so sistemske vročinske bolezni, ki jih pogosto spremljajo krvavitve. Povzročajo jih zoonotski virusi, katerih rezervoar so glodavci in/ali členonožci, zaradi česar so bolezni endemične v različnih regijah. Za VHM so značilne motnje strjevanja krvi, žilna prepustnost, poškodbe endotelija in krvavitve. Klinična slika je od blage do življenjsko ogrožajoče. Teža bolezni je odvisna od virusnega povzročitelja, imunskih dejavnikov gostitelja in okolijskih pogojev. Zaradi globalizacije, podnebnih sprememb in migracij so se nekatere VHM razširile izven endemičnih območij, kar predstavlja globalno grožnjo javnem zdravju. Okužbe z ortohantavirusi spadajo med VHM in povzročajo dva sindroma: hemoragično mrzlico z renalnim sindromom (HMRS) v Evropi in Aziji in hantavirusni srčno-pljučni sindrom v Amerikah. Čeprav se sindroma obravnavata kot ločeni bolezni, jima je skupna okužba endotelijskih celic kapilar različnih organov, kar sproži sistemsko vnetje. V Evropi HMRS povzročata PUUV in DOBV, pri čemer okužbe s PUUV običajno povzročijo blažjo obliko bolezni. Okužbe z DOBV so redkejše, vendar povzročajo hujšo obliko bolezni z višjo smrtnostjo. Bolniki okuženi z DOBV imajo pogosto akutno odpoved ledvic, motnje vida in hudo trombocitopenijo. Zemljepisno lega Slovenije omogoča soobstoj obeh virusov na istem endemičnem območju, kar omogoča raziskovanje vpliva genetskih linij ortohantavirusov na izid HMRS, ki še ni razjasnjen. Ortohantavirusi ne povzročajo neposredne citopatologije ali apoptoze endotelijskih celic, zato je povečana prepustnost endotelija verjetno posledica imunskih in vnetnih odzivov gostitelja. Mehanizmi, ki vodijo v motnje hemostaze, še niso popolnoma pojasnjeni. Patogeni ortohantavirusi lahko sprožijo nenadzorovane hemostatske procese, ki povzročijo razširjeno intravaskularno koagulacijo (DIK) in povečajo tveganje za tromboze ter krvavitve. Genetski dejavniki gostitelja, vključno z vrstami HLA in polimorfizmi genov za citokine, dodatno vplivajo na težo bolezni. Klinične slike so zelo različne, raziskave pa pogosto prinašajo nasprotujoče si rezultate zaradi. Razumevanje razmerja med virusnimi dejavniki, imunskim odzivom in endotelijsko disfunkcijo je ključno za razvoj učinkovitih terapij za HMRS. Trenutno zdravljenje temelji na podporni terapiji, razvoj ciljnih terapij pa ovirajo pomanjkanje ustreznih živalskih modelov in omejeno poznavanje patogeneze ortohantavirusov. Cilj predlaganega projekta je opredelitev krvavitev pri HMRS. Na podlagi retrospektivnih kliničnih in laboratorijskih podatkov bomo postavili klinično lestvico za kvantifikacijo teže HFRS, ki jo bomo validirali na prospektivnih podatkih. Z amplikonskim pristopom bomo pridobili celotne genome vseh treh segmentov DOBV in PUUV, ter na primerjavi sekvenc določili genetski linije, ki so povezane s hujšim potekom HMRS. S postavitvijo ko-kultur v pogojih in vitro bomo določili izražanje genov ter citokine, kemokine in rastne faktorje, ki sodelujejo pri aktivaciji endotelija in motnjah hemostaze. S povezovanjem kliničnih, imunoloških in viroloških dejavnikov želimo izboljšati razumevanje porušenja hemostaze in krvavitev pri HMRS. Določitev ključnih dejavnikov ne bo izboljšalo samo zdravljenja HFRS, ampak bo pripomoglo tudi k razvoju protivirusnih zdravili za zdravljenje drugih VHM s podobnimi molekularnimi mehanizmi.
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SUMMARY
Viral hemorrhagic fevers (VHF) are systemic febrile illnesses that are often accompanied by bleeding. They are caused by zoonotic viruses whose reservoir is rodents and/or arthropods, making these diseases endemic in different regions. VHF is characterized by coagulopathy, vascular dysregulation, endothelial damage and hemorrhagic manifestations. The clinical presentation ranges from mild to life-threatening. The main pathophysiological features of VHF include increased levels of proinflammatory cytokines, endothelial dysfunction and coagulopathy. The severity of the disease depends on the viral pathogen, host immune factors and environmental conditions. Due to globalization, climate change and migration, some VHFs have spread beyond endemic areas and pose a global threat. Orthohantavirus infections are classified as VHFs and cause two syndromes: Hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. Although these syndromes are considered separate diseases, they share the common feature of infecting endothelial cells in capillaries of different organs and causing systemic inflammation. In Europe, HFRS is mainly caused by the Puumala virus (PUUV) and the Dobrava virus (DOBV). PUUV infections generally lead to a milder course of the disease, while DOBV infections occur less frequently but are associated with a more severe clinical outcome and a higher mortality rate. Patients infected with DOBV often develop acute renal failure, visual impairment and severe thrombocytopenia. The geographical location of Slovenia allows both viruses to coexist within the same endemic area and thus provides a unique opportunity to study the impact of the genetic lineage of orthohantaviruses on the course of HFRS, which remains unclear. Hantaviruses do not cause direct cytopathology or apoptosis in endothelial cells, suggesting that the increased endothelial permeability is primarily caused by host immune and inflammatory responses. However, the mechanisms leading to hemostatic dysregulation are not yet fully understood. Pathogenic hantaviruses can trigger uncontrolled hemostatic processes that lead to disseminated intravascular coagulation (DIC) and increase the risk of thrombotic and hemorrhagic complications. Genetic host factors, including HLA types and cytokine gene polymorphisms, further influence the severity of the disease. The clinical presentation is highly variable and research results are often contradictory. Understanding the interplay between viral factors, the immune response and endothelial dysfunction is crucial for the development of effective HFRS therapies. Current treatment relies on supportive care, while the development of targeted therapies is hampered by the lack of suitable animal models and limited knowledge of orthohantavirus pathogenesis. The aim of the proposed project is to characterize the hemorrhagic manifestations in HFRS. Based on retrospective clinical and laboratory data, we will create a clinical scoring system to quantify the severity of HFRS, which will be validated on prospective data. Using an amplicon-based NGS approach, we will obtain the complete genomes of all three segments of DOBV and PUUV and identify genetic lineages associated with severe HFRS outcomes. By establishing co-cultures under in vitro conditions, we will determine the expression of genes, cytokines, chemokines and growth factors involved in endothelial activation and hemostatic dysregulation. By integrating clinical, immunologic and virological factors, we aim to improve the understanding of hemostatic dysfunction and bleeding in HFRS. The identification of key factors will not only improve the treatment of HFRS, but also contribute to the development of antiviral therapies for other life-threatening VHFs with similar molecular mechanisms.