Ebola fever pathogenesis

1. Ebola fever pathogenesis State Research Center of Virology and Biotechnology “Vector”

2. For studying the key moments of the Ebola-fever pathogenesis we use a method of comparison pathophysiological reactions at animals with various sensitivity to the same virus.

3. Gradient of animal susceptibility to EBO Non-susceptible animals I Rabbits inoculated with wild –type EBO Weakly susceptible animals II Guinea pigs inoculated with wild –type EBO Susceptible animals III Guinea pigs inoculated with adEBO Because our comparison involves 3 host-virus combinations running the gamut from inapparent processes (rabbits) to nonfatal febrile infection (wild-type EBO in guinea pigs) to lethal infection (adEBO in guinea pigs), we term this comparison a "susceptibility gradient” method for investigating the complex of pathological processes that occur on EBO challenge.

4. I n v e s t i g a t i n g p a r a m e t e r s Coagulation Virological Biochemical Hematological Immunological Obtaining of database ANALYSIS

5. The “susceptibility gradient” method requires the comparing of a large number of various pathophysiological parameters, which must be presented in an easy comparable form. • In connection with this we elaborated the way of effective data visualization on the base of Excel macroses. • The data are presented in a form of graphic tables allowing demonstration of investigated results in one flat.

6. Vertical rows in tables reflect the susceptibility level. Horizontal rows show pathophysiological parameters. We used different colours to point the range of parameters changing above and below the initial level.

7. Such way of data presentation permited to see important moments in parameter dynamics in susceptible and insusceptible to EBO animals. • We used the plus/minus system for the assessment of general tends of observed pathophysiological effects.

10. Data presented shows the counterdirected dynamics of immunological and the complex of hematological, biochemical, and coagulation values in susceptible and non-susceptible to EBO animals.

11. Average estimation of effects with respect to the groups of investigating parameters III Hm II Bh Cg I Im Hm – hematological parameters Bh – biochemical parameters Cg – coagulation parameters Im – immunological parameters

12. Thus, the activation of immune system appears to be essential for resistance to EBO, and the degree of suppression of immunity determines the level of animal susceptibility to EBO.

13. Coagulation cascade in Ebola fever However, the syndrome and the catastrophic result of infection is coagulation disorders. Initiating stimulus providing the starting of coagulation cascade Activation of coagulating factors Consumption of coagulating factors Consumption of platelets Formation of fibrinogen clots Activation of fibrinolitic system (maybe, superactivation) Total bleedings and hemorrhage

14. Most of the modern conceptions of filoviral hemorrhagic fever pathogenesis try in either case to explain the mechanism of uncontrolled coagulation cascade that develops in the infected organism. • The initiating stimulus for coagulation reactions may be cytokines, which enter the blood in an avalanche from destroyed cells of the mononuclear phagocyte system as a result of filovirus reproduction in them (Feldmann et al, 1996, J. Virol).

15. The factor feeding the coagulation cascade also may be the cytotoxic influence of autoantibodies to an adventitial protein, which are generated because of the molecular mimicry of Ebola virus GP site to this protein (Tilson et al, 1996, Clin. Immunol. Immunopathol.). liver • We also observed the autoimmune agression and cytotoxic action of immune complexes in guinea pigs infected with adapted EBO. Blood vesel

16. It is known that the primary target of EBO is macrophages. Perhaps, the imbalance of immunomodulating properties of macrophages leads to the decrease of its and neutrophil phagocytic activity, in part, to immune complexes that provides the cytotoxic action of the last.

17. So, we presume that initiating stimulus for coagulation cascade is the cytotoxic action of immune complexes.

18. EBO EBO intervention leads to the blastogenesis development (blastic wave)in lymphoid organs What antibody? As a consequence there is an appearance in blood of juvenile forms of lymphocytes, which transform into plasmocytes. Day 3 Day 9 Appearance of plasmocytes is a sign of antibody production.

19. Taking into account the appearance of such single prolymphocytes in guinea pigs inoculated with inactivated EBO, and the high titers of antibodies to EBO in these animals we think that specific antibodies to EBO were also generated in adEBO-infected animals but totally formed immune complexes and settled in tissues. • Prolymphocytes in blood of adEBO infected guinea pigs is a result of blastogenic reaction in lymphoid organs. • Such prolymphocytes were also observed in blood of guinea pigs inoculated with inactivated EBO and wild-type EBO.

20. __________experiment 1 __________experiment 2 • In guinea pigs infected with adEBO the elimination of circulating immune complexes from blood was observed (in 2 independent experiments). • We think this elimination is provided by adsorbtion of immune complexes in tissues. Wild-type EBO adEBO

21. So, the analysis data presented here and a great mass of non-presented here data allowed noting the principle moments in pathophysiological processes after EBO challenge, which distinguished susceptible and insusceptible animals. • This differences helped us to make a scheme of Ebola fever pathogenesis in order to understand the main stages of Ebola infection.

22. We think there are several parallel waysof event developmentin the organism from the momentof EBO intervention: • reproduction of EBO in macrophages, endotheliocytes, hepatocytes; • suppression of neutrophil functions; • cell apoptosis; • activation of antioxidant systems.

23. Ebola virus Reproduction in macrophages Suppressive factor settling on neutrophils (maybe sGP) Reproduction in liver Reproduction in endotheliocytesand fibroblasts Apoptosis of cells Activation of antioxidation systems (decrease of malonic dialdehyde amount on day 3) and following exhaustion of them

24. EBO reproduction in the macrophages on the one hand, and the suppression of neutrophils on the other hand leads to the breach of neutrophil-macrophage interaction. • The breach of neutrophil-macrophage interaction in case of EBO may cause the non-adequate presentation of EBO antigens to T lymphocytes. • As a result, the following activation of B lymphocytes leads to a producing both of specific antibodies with the ability to form immune complexes capable to settle in tissues, and of autoantibodies with a potential to destroy cells. • These last events are seen to be the central link in our scheme.

25. B Ebola virus Reproduction in macrophages Suppressive factor settling on neutrophils (maybe sGP) Breach of macrophage functions Breach of phagocytic mechanisms of neutrophils M Breach of neutrophil-macrophage interaction N Generalized destruction of macrophages Non-adequate presentation of Ebola virus antigens to T lymphocytes T Reproduction in liver Cytokines release into blood (TNF-alpha etc) Decrease of lymphocyte amount in blood and lymph nodes on day 5-7 Activation of B lymphocytes (single mitoses in lymphoid cells in blood on day 3) Reproduction in endotheliocytesand fibroblasts Producing of antibodies capable to form immune complexes and to settle in tissues Producing of autoantibodies with great potential to destroy cells Apoptosis of cells Activation of antioxidation systems (decrease of malonic dialdehyde amount on day 3) and following exhaustion of them

26. The settling of immune complexes in different tissues and cells resulting in damage to these tissues and breach of functions is the next important point of our scheme. • Immune complexes adsorption in liver and kidneys accelerates the development of hepatorenal syndrome caused by EBO reproduction in these organs. • The settling of immune complexes on immune cells leads to lymphocyte destruction, apoptosis and decrease of their amount in blood and lymph nodes, to acceleration of macrophage death, to suppression of neutrophil functions.

27. B Ebola virus Settling of immune complexes on different tissues and cells : Reproduction in macrophages Suppressive factor settling on neutrophils (maybe sGP) Breach of macrophage functions Breach of phagocytic mechanisms of neutrophils M Breach of neutrophil-macrophage interaction N Generalized destruction of macrophages Non-adequate presentation of Ebola virus antigens to T lymphocytes T on macrophages on neutrophils Reproduction in liver Cytokines release into blood (TNF-alpha etc) Activation of B lymphocytes (single mitoses in lymphoid cells in blood on day 3) Necroses in liver and breach of its functions (raise of ALT and AST activities on day 5-9) Reproduction in endotheliocytesand fibroblasts Producing of antibodies capable to form immune complexes and to settle in tissues Producing of autoantibodies with great potential to destroy cells on lymphocytes on liver cells Signs of hepatorenal syndrome on kidney cells on platelets Damage of tissues and cells (endotheliocytes first of all) Apoptosis of cells Activation of antioxidation systems (decrease of malonic dialdehyde amount on day 3) and following exhaustion of them Breach of kidney functions (rise of beta-lipoproteids urea and amount on day 7-9 ) Release into blood of toxic products Strengthening of lipid peroxidation (raise of malonic dialdehyde amount on day 5-6) Decrease of lymphocyte amount in blood and lymph nodes on day 5-7

28. Appearance of juvenile granulocytes and thrombocytes in guinea pigs infected with adEBO evidenced on activation of granulocytic and megakariocytic hemopoiesis in bone marrow with the export of immature cells into blood. • This activation of bone marrow cells might be caused by different cytokines and proliferative factors, which appeared during the infection course.

29. Immunological disorders Export of eosinophils on day 9 Exportt of juvenile granulocytes on day 5-9 Export of juvenile lymphocytes on day 3, 9 B Ebola virus Settling of immune complexes on different tissues and cells : Сoagulation disorders Reproduction in macrophages Suppressive factor settling on neutrophils (maybe sGP) Export of juvenile thrombocytes on day 5-9 Breach of macrophage functions Breach of phagocytic mechanisms of neutrophils Breach of neutrophil-macrophage interaction M N Generalized destruction of macrophages Non-adequate presentation of Ebola virus antigens to T lymphocytes T on macrophages on neutrophils Reproduction in liver Cytokines release into blood (TNF-alpha etc) Decrease of lymphocyte amount in blood and lymph nodes on day 5-7 Activation of B lymphocytes (single mitoses in lymphoid cells in blood on day 3) Necroses in liver and breach of its functions (raise of ALT and AST activities on day 5-9) Reproduction in endotheliocytesand fibroblasts Producing of antibodies capable to form immune complexes and to settle in tissues Producing of autoantibodies with great potential to destroy cells on lymphocytes on liver cells Imbalance of cytokine amount in blood Signs of hepatorenal syndrome on kidney cells on platelets Damage of tissues and cells (endotheliocytes first of all) Apoptosis of cells Activationof antioxidation systems (decrease of malonic dialdehyde amount on day 3) and following exhaustion of them Breach of kidney functions (rise of beta-lipoproteids urea and amount on day 7-9 ) Release into blood of toxic products Strengthening of lipid peroxidation (raise of malonic dialdehyde amount on day 5-6)

30. The attachment of immune complexes to thrombocytes intensifies their death and, along with the consumption of platelets in coagulation reactions, accelerates the development of lethal coagulopathy. • We think that lethal coagulation disorders are due to the total combination of several pathogenic mechanisms, which simultaneously start the coagulation cascade raising it to an uncontrolled level. • Such pathogenic mechanisms may be the increased lipid peroxidation; the release into the blood of toxic products from cells that have been destroyed by immune complexes, and the increased level of cytokines.

31. Immunological disorders Export of eosinophils on day 9 Exportt of juvenile granulocytes on day 5-9 Export of juvenile lymphocytes on day 3, 9 B Ebola virus Settling of immune complexes on different tissues and cells : Сoagulation disorders Reproduction in macrophages Suppressive factor settling on neutrophils (maybe sGP) Export of juvenile thrombocytes on day 5-9 Breach of macrophage functions Breach of phagocytic mechanisms of neutrophils M Breach of neutrophil-macrophage interaction N Generalized destruction of macrophages Non-adequate presentation of Ebola virus antigens to T lymphocytes T on macrophages on neutrophils Reproduction in liver Cytokines release into blood (TNF-alpha etc) Decrease of lymphocyte amount in blood and lymph nodes on day 5-7 Activation of B lymphocytes (single mitoses in lymphoid cells in blood on day 3) Necroses in liver and breach of its functions (raise of ALT and AST activities on day 5-9) Reproduction in endotheliocytesand fibroblasts Producing of antibodies capable to form immune complexes and to settle in tissues Producing of autoantibodies with great potential to destroy cells on lymphocytes on liver cells Imbalance of cytokine amount in blood Signs of hepatorenal syndrome on kidney cells on platelets Damage of tissues and cells (endotheliocytes first of all) Apoptosis of cells Activation of antioxidation systems (decrease of malonic dialdehyde amount on day 3) and following exhaustion of them Breach of kidney functions (rise of beta-lipoproteids urea and amount on day 7-9 ) Release into blood of toxic products Strengthening of lipid peroxidation (raise of malonic dialdehyde amount on day 5-6) Initiation of coagulation cascade Activation of fibrinolitic system (appearing of paracoagulation products in plasma on day 5-9) Consumption of blood platelets on day 7-9 Development of lethal coagulation disorders (decrease of prothrombin index, bleeding, sporadic hemorrhages in internal organs) Scheme of Ebola fever development

32. Most investigators use as a rule only susceptible animals. But the susceptibility gradient makes it possible to identify the principal differences in immune reactions that differentiate susceptible animals from resistant ones, and to assess the interaction of different components, both immune and non-immune, in host defense.