Ebola Hemorrhagic Fever

Introduction:
The Ebola virus induces a lethal viral hemorrhagic fever in humans and primates, and is characterized by its active immune suppression, and a systemic inflammatory response, thereby impairing the vascular, coagulation, and immune systems, leading to multiorgan failure and ultimately death.2 In 1967, laboratory personnel of a primate facility in Marburg, Germany were infected with a hemorrhagic virus, which was subsequently isolated into animal models via inoculation from patient blood samples, where electron microscopy revealed a novel negative-strand RNA viral strain. This outbreak of the Marburg virus (MARV), a sister species of Ebola, caused a case-fatality rate as high as 22%.3 Similar cases of hemorrhagic fever were documented in 1976 when endemic outbreaks occurred near simultaneously in southern Sudan, and northern Zaire, now the Democratic Republic of the Congo (DRC). The World Health Organization conducted an epidemiological study of the outbreak, and recorded substantially higher fatality rates, ranging from 53% to 88% for the various regional studies.4, 5 The causative pathogen was named the Ebola virus after a small river in northwestern DRC near which the outbreaks occurred. Years later, the isolated viruses from the incident were examined by oligonucleotide mapping of the virion RNA, and distinguished two genetically distinct subtypes of the virus, Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV).6 The ZEBOV has empirically been the most virulent and fatal of the strains, causing up to a 90% mortality rate. Since then, several other members of the species have been indentified, Cte d'Ivoire ebolavirus (CIEBOV), Bundibugyo ebolavirus, and a fifth stain not known to be infectious toward humans, Reston ebolavirus (REBOV).7 All recent cases have been endemic to Central Africa, caused largely by the reemergence of ZEBOV in regions, of Gabon, Republic of Congo, and DRC, and of SEBOV in areas of south Sudan and Uganda.2 Aside from the natural causation, there are periodic incidents of research personnel being inadvertently infected, the latest being in March, 2009 when a German scientist pricked herself with an infected needle.8 The high mortality rate and 1|Page

subsequent safety concerns of the Ebola virus have catapulted this diseased to an important public health treat and a bioterrorist pathogen of the highest national security priority, a Category A Pathogen by both the Centers for Disease Control and Prevention and the National Institutes of Helath.9 Thus, the Ebola virus has been under intense scrutiny, to identify protein drug targets, and to develop treatments for the disease.

Structure and Genome:

The Ebola virus and Marburg virus fall into the family Filoviridae, characteristic of filamentous particles. The genomes of all filoviruses are composed of non-segmented, negative sense single stranded RNA that is approximately 19-kb long. From the 3 end, the Ebola virus contains seven genes, encoding for eight proteins; nucleoprotein (NP), virion structure protiens (VP35, VP40), glycoprotein (GP), VP30, VP24, and RNA dependent RNA polymerase (L).10 The L protein coupled with the VP35 form the RNA polymerase, with the L protein providing the mechanic functionality of the active complex. The viral glycoprotein plays an important its pathophysiology, as it has been implicated in increasing vascular permeability.11 The GP is post-translationally cleaved by furin to yield a GP1 and GP2 subunit. The GP1 forms transmembrane trimeric spikes across in the viral envelope, which cause the virus to bind to endothelial cells lining the interior surface of blood vessels. GP2 has been implicated in mediated fusion of viral and host membranes through clathrin and caveolae membrane pathways.1 The secreted form of the GP also plays a central role in invading the immune system by interfering with the activation of neutrophils. Ebola virus particles are 80 nm in diameter, but vary in length, ranging up to 14,000 nm.12

Pathogenesis:
The Ebola virus is first contracted through host mucosal linings, or by abrasions in the epithelium that allow fluid transfer of infected materials.13 Exposure and infection is also possible in a laboratory setting though needle punctures and blood. The virus has a proclivity to replicate in monocytes, macrophages, and dendritic

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cells, which expose it to the lymphatic system, and to the liver and spleen through the blood. The infected macrophages and monocytes release tissue factor and cause coagulation irregularities, characteristic of Ebola Hemorrhagic Fever.14 The viral replication in infected cells overwhelms protein synthesis and begins secreting massive quantities of dimeric GP. This sGP binds to neutrophils, and plays a pivotal role in preventing an early immune response to the viral invasion. Then the virus begins to invade endothelial cells and violently replicates by taking over the host proteomic machinery causing cell death, which causes a loss of vascular integrity. This uncontrolled and explosive viral replication throughout the body cause widespread and severe focal necrosis. The impaired immune system recognizes this cell damage and releases high concentration of proinflammatory mediators such as TNF-, Interleukins (IL-2, IL-6, IL-8, and IL-10), etc., in the late stages of the disease, causing hemorrhage and shock, leading to multi-organ failure.15

Drug Targets:
There are several important proteins that are central to Ebola virus host invasion and pathogenesis. The glycoproteins are crucial to viral homing and endocytosis, as well as early interference with the immune system. This has made it an attractive target for vaccines and entry inhibitors, and has led to the development of several working vaccines in primates.1 The trimeric GP is found on the viral membrane and is involved in host attachment. Lee et. al crystalized the structure the trimeric GP bound to an antibody isolated from a human survivor, leading to the possibility of an antibody based treatment. (Fig.1)

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Fig. 1 The ZEBOV Glycoprotein a) Schematic domain of the ZEBOV glycoprotein in its genome. b) The molecular surface of the trimeric GP viewed from its side (left) and from the top (right) c) A view of the chalice and cradle that hold the GP trimer.1

Another viable target is the L-protein involved in the RNA-dependent-RNA polymerase. (Fig. 2) This protein is an ideal target for antiviral interventions not only because its suppression should lead to a nearly complete loss of all RNA synthesis, but also because of the absence of similar proteins in mammalian cells.16 Through the use of small interfering RNA (siRNA), it is possible to interfere with the replication of the viral genome. Geisbert et. al used lipid encapsulated siRNA targeting ZEBOV L polymerase, as well as two other viral proteins involved in inhibiting type 1 interferon response. They treated a group of macaques with anti-ZEBOV siRNAs at 2mg/kg through bolus intravenous infusion, and after seven treatments, they completed protected the rhesus monkeys against from hemorrhagic fever. (Information in the L-polymerase does not include structure.)

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Fig. 2 A diagram of the Ebola virion with the relative positions of all its proteins.

An entirely different approach to drug targeting is to identify and suppress proteins that are actively involved in propagating the viral genome and protein machinery. Using siRNA gene-targeting technology, it is possible to elucidate which gene products play critical roles in Ebola virus infection.17 Kolokoltsov et al. demonstrated via siRNA screening, that phosphatidylinositol-3-kinase and calcium/calmodulin kinase related networks were important for ZEBOV infection. LY294002 (CAS number: 154447-36-6, InvivoGen) was used to target phosphatidylinositol-3-kinase (PI3K) and has shown to have no appreciable side effects in previous studies. 2-(4morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, also known as LY294002 (C19H17NO3) was shown to act in vivo as a highly selective inhibitor of (PI3). (Fig. 3) When used at a concentration of 50 M, it specifically abolished PI3 kinase activity (IC50=0.43 g/ml; 1.40 M) but did not inhibit other lipid and protein

kinases such as PI4 kinase, PKC, MAP kinase or c-Src (1).18 Fig. 3 The molecular structure of LY294002 (C19H17NO3) 307.343 g/mol LY294002 was originally patented to prevent aberrant tumor-

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associated angiogenesis. The other pathway identified in the study was the calcium/calmodulin kinase, which was inhibited by KN-93 (CAS Number: 139298-40-1, Sigma-Aldrich), by preventing CAMK2 from associating with calmodulin, a necessary step in the kinase process. N-[2-[N-(4-Chlorocinnamyl)-Nmethylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4methoxybenzenesulfonamide phosphate salt, aslo known as KN-93 (C26H29ClN2O4S H3PO4) inhibited CaM kinase II activation by Fig. 4 The molecular structure of KN-93 (C26H29ClN2O4S H3PO4) 599.03 g/mol pretreatment (IC50 ~1 M).

Conclusion:
Since the Ebola virus is such a dangerous and virulent strain, research on it is restricted largely to Biosafety Level 4 (BSL-4) facilities. Thus, drug development on the virus is slow and expensive, and consequently there is no approved treatment of the Ebola Hemorrhagic Fever. However, new importance has been given to the disease, in light of its possible use as a bioterrorist weapon, and several new vaccines and drugs are showing promising results.

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References
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