HAEMOSTASIS AND VIRUS INFECTIONS - EPIDEMIOLOGY, PATHOGENESIS & PREVENTION - Marco Goeijenbier The studies reported in this thesis were performed at the department of Viroscience of the Erasmus Medical Center, Rotterdam, the Netherlands. Financial support for reproduction of this thesis generously funded by ISBN: Cover design: Stefanie van den Herik | uitgeverij BOXpress Printed & Lay Out: Proefschriftmaken.nl || Uitgeverij BOXPress Haemostasis and Virus infections – Epidemiology, Pathogenesis & Prevention – Hemostase en virus infecties -Epidemiologie, Pathogenese & Preventie- PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus prof.dr. H.A.P. Pols en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op vrijdag 12 juni 2015 om 9.30 uur door Marco Goeijenbier geboren te Voorburg Promotiecommissie Promotoren: Prof.dr. E.C.M. van Gorp Prof.dr. A.D.M.E. Osterhaus Overige leden: Prof.dr. M. P.G. Koopmans Prof.dr. J.C.M. Meijers Prof.dr. T. Kuiken Co-promotoren: Dr. B.E.E. Martina Dr. J.F.P. Wagenaar Table of Contents CHAPTER 1 INTRODUCTION: “General introduction and outline of this thesis” Taken in part from: J. Med. Vir. 2012, 84(10):1680-96 Neth. J. Med. 2014, 72(9):442-8 CHAPTER 2 EPIDEMIOLOGY: “Viral haemorrhagic fever in the Netherlands and the republic of Suriname” 2.1 A viral haemorrhagic fever case in the Netherlands and review of the literature Taken in part from: Crit. Rev. Microbiol. 2013, 39(1):26-42 Neth. J. Med. 2011, 69(6):285-9 2.2 The hanta hunting study: underdiagnosis of Puumala hantavirus infections in symptomatic non-travelling leptospirosis-suspected patients in the Netherlands Euro Surveill. 2014, 14;19(32) 2.3 Emerging viruses in the Republic of Suriname: retrospective and prospective study into chikungunya and hanta virus circulation, 2008-2012 and 2014. Submitted CHAPTER 3 PATHOGENESIS: “Clotting and haemorrhage in infectious diseases” 3.1 Effect of Puumala hantavirus infection on Human Umbilical Vein Endothelial Cell hemostatic function: Platelet interactions, increased Tissue Factor expression and fibrinolysis regulator release Front. Microbiol. 2015 6:220 3.2 A comparative proteomic analysis of Human Umbilical Vein Endothelial Cells after infection with Puumala hantavirus and Leptospira interrogans, causative agents of rodent-borne haemorrhagic fevers Submitted 3.3 Markers of endothelial cell activation and immune activation are increased in patients with severe leptospirosis and associated with disease severity Submitted 3.4 Activation of coagulation and tissue fibrin deposition in experimental influenza in ferrets. BMC Microbiol. 2014 30;14:134 CHAPTER 4 ECOLOGY & PREVENTION: “Newly identified risk factors and tools of prevention” 4.1 The association between hantavirus infection and selenium deficiency in mainland China Viruses 2015 7(1):333-51 4.2 Safety and immunogenicity of an MVA-based influenza A/H5N1 vaccine. A randomized phase I/IIa clinical trial. Lancet Infect Dis. 2014 14(12):1196-207 CHAPTER 5 SUMMARIZING DISCUSSION 5.1 Summarizing discussion and directions for future studies Taken in part from Neth. J. Med. 2015 accepted manuscript 5.2 Nederlandse samenvatting en discussie voor niet ingewijden 5.3 References About the Author Dankwoord CHAPTER 1 GENERAL INTRODUCTION AND OUTLINE OF THE THESIS Taken in part from: Viral infections and mechanisms of thrombosis and bleeding M. Goeijenbier1 & M. van Wissen2, C. van de Weg1, E. Jong2, V.E.A. Gerdes2, J.C.M. Meijers3, D.P.M. Brandjes2, E.C.M. van Gorp1 J. Med. Vir. 2012, 84(10):1680-96 Ebola Virus Disease; a review on epidemiology, symptoms, treatment and pathogenesis M. Goeijenbier1, J.J.A. van Kampen1, C.B.E.M. Reusken 1 , M.P.G. Koopmans1, E.C.M van Gorp1 1. Erasmus MC, Rotterdam, the Netherlands, Department of Virology 2. Slotervaart Hospital, Amsterdam, the Netherlands, Department of Internal medicine 3. Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, the Netherlands Neth. J. Med. 2014, 72(9):442-8 Virus infections and mechanisms of thrombosis and bleeding Viral infections of any kind can influence the haemostatic balance. This means that during, or shortly after infection with certain viruses the ability of blood to clot in humans can be drastically changed which can lead to both thrombotic as well as haemorrhagic complications. Direct or indirect activation of the endothelium by viruses (or other pathogens) may result in alterations in the coagulation- and the fibrinolytic systems (1). Normally, coagulation is a balance between procoagulant and (natural) anticoagulant mechanisms and actually it seems that a well regulated activation of coagulation is part of the host’s defence against infectious agents (2). However, the inflammation caused by certain viral infections may lead to such alterations in coagulation resulting in an imbalance between the pro- and anticoagulant state. The clinical picture of altered coagulation in several viral infections manifests itself in mild or even severe bleeding (haemorrhage), thrombosis, or both. In severe cases this may even lead to disseminated intravascular coagulation with the formation of micro vascular thrombi in various organs (3). Disseminated intravascular coagulation contributes to multiple organ failure and is associated with high mortality in both bacterial and nonbacterial diseases (3;4). It is not yet clear why some viruses cause haemorrhage (e.g. the viral haemorrhagic fevers like hantavirus and Ebola), others are associated with thrombosis (e.g. cytomegalovirus and influenza) and others show both (e.g. varicella zoster virus) (5-8). In addition, the bleeding complications caused by viral haemorrhagic fever pathogens vary in severity, such as more minor bleeding complications in dengue and more severe bleeding seen in patients infected with Crimeon Congo Haemorrhagic fever virus or one of the Old-World hantaviruses. Both bleeding and thrombotic complications during or shortly after viral infection are associated with a worse outcome of disease, while for most of the viral infections no targeted therapy is available and only supportive care can be provided. Therefore, a better understanding is needed of the pathogenesis of bleeding and thrombotic complications due to viral infections. Principles of haemostasis, coagulation, and fibrinolysis: general aspects One of the most important functions of the endothelial cell layer is the maintenance of haemostasis, a physiologic mechanism that maintains blood in a fluid state within the vasculature and responds with coagulation at time of injury to prevent exsanguination (9). For this to work properly, a perfect balance between pro- and anti-coagulant factors is necessary. The formation of a blood clot is a well-regulated process comprising three elements: 1) primary haemostasis, 2) secondary haemostasis/coagulation, and 3) fibrinolysis (Figure 1) (10). The onset of clotting arises when the vasculature is damaged and blood is exposed to platelet activating- and pro-coagulant factors. The first response, primary haemostasis, is the adherence and aggregation of activated platelets to the side of injury, due to Von Willebrand factor (VWF). Von Willebrand factor is a very sensitive marker of endothelial cell activation and is a large glycoprotein synthesized in the vascular endothelium. Endothelial cells store VWF in the Weibel-Palade bodies in the form of unusually large vWF multimers. The function of vWF comprises adhesion of platelets to exposed subendothelium, forming the basis for the haemostatic plug. At the side of injury activated platelets provide cell-surface phospholipids to further facilitate secondary haemostasis, the formation of fibrin strands, which stabilise the platelet plug. Coagulation results from a series of linked coagulation protease-zymogen reactions, ultimately resulting in the formation of fibrin. Tissue factor is the main initiator of the coagulation cascade, which is localised in the subendothelium, but also on non-circulating leukocytes and possibly on platelets. Thrombin generation is induced by the assembly of the tissue factor-factor VIIa complex. Thrombin is able to convert fibrinogen into (insoluble) fibrin. Coagulation is regulated by different inhibitory mechanisms. A first mechanism is made up of the circulating inhibitors of blood coagulation: antithrombin and heparin cofactor II (both inhibitors of thrombin), and tissue factor pathway inhibitor. Two other circulating inhibitors of blood coagulation are protein C and protein S (the latter of which is a cofactor for the proper functioning of activated protein C). A second inhibitory mechanism consists of the endothelium- bound modulators heparin sulphate and thrombomodulin, which facilitate the inhibitory activity of antithrombin and the activation of protein C, respectively. The third element, the fibrinolytic system, is necessary to degrade the formed fibrin strands. This system is initiated by tissue plasminogen activators and urokinase after their synthesis by, and release from, endothelial cells. These activators initiate the conversion of plasminogen to plasmin, which hydrolyses polymerised fibrin strands into soluble fibrin degradation products, thus degrading the fibrin clot. The activity of the fibrinolytic system is, among other things, regulated by plasminogen activator inhibitor type I, which may greatly increase during acute phase reactions. Thrombin-activatable fibrinolysis inhibitor is also an inhibitor