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Examen écrit Université de Genève Faculté de Médecine Ebola, an update Emilie BRANCHE Directeur de thèse: Prof. Francesco NEGRO Co-directrice de thèse: Dr. Sophie CLÉMENT Examinateurs: Prof. Laurent KAISER Dr. Glauciá PARANHOS-BACCALA 2015

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Examen écrit

Université de Genève

Faculté de Médecine

Ebola, an update

Emilie BRANCHE

Directeur de thèse: Prof. Francesco NEGRO

Co-directrice de thèse: Dr. Sophie CLÉMENT

Examinateurs: Prof. Laurent KAISER

Dr. Glauciá PARANHOS-BACCALA

2015

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Abstract

The current outbreak of Ebola principally affecting Guinea, Sierra Leone and Liberia has

caused at least 24 000 infections and over 9 000 deaths as of March 2015. The "imported"

cases in Spain, United Kingdom and United States of America lead to an important surge of

media, medical and research interest. This review summarizes the current knowledge about

Ebola virus including the epidemiology, the pathogenesis as well as the treatments both

currently available and in development. We will see that Ebola pathogenesis is definitely

complex and that both coagulation and immune response play a crucial role in its etiology.

I - Introduction

1- Classification

Ebola virus belongs to the Ebolavirus genus in the Filoviridae family in the Mononegavirales

order. Filoviridae family includes Marburgvirus with Marburgvirus, Cuevavirus with Lloviu

cuevavirus and Ebolavirus. Since the original description in 1976 of the Zaire Ebolavirus , four

species have emerged, Bundibugyo, Cote d'Ivoire or Tai Forest, Reston and Sudan ebolavirus

[1] (Figure 1).

Figure 1 : Taxonomy of the Filoviridae family adapted from Kuhn JH et al, 2010 [2]

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All these species are pathogenic for humans, except Reston ebolavirus, which is pathogenic

for non-human primates. These viruses have been sequenced and their molecular evolution

described [3]. The current circulating Ebolavirus (EBOV) shares 97% homology with Zaire

Ebolavirus [4]. This review will mainly focus on EBOV.

2 - Epidemiology

The first Ebola cases appeared in 1976 in Sudan [5] and in Zaire [6]. In southern Sudan, 284

cases appeared causing 151 deaths (53% of death) in 6 months (June to November). In

northern Zaire (near the Ebola river giving the name to this virus), 318 cases were described

leading to 280 deaths (88% of death) in 2 months (September and October).

Since these original cases, approximately 20 additional outbreaks occurred between 1976

and 2013, causing nearly 2500 deaths in the Democratic Republic of Congo, Sudan, Gabon,

Ivory Coast, Uganda and Zaire [7, 8]. Since December 2013, a new outbreak is occurring in

several African countries (Mali, Senegal, Nigeria, Guinea, Sierra Leone and Liberia). One case

has been described in United Kingdom, another in Spain and 4 cases in the United States of

America. This outbreak caused 24 282 infections so far with a mortality rate of 40% [9]

(Figure 2 and Figure S1).

Figure 2 : 8 March 2015: situation of EBOV outbreak.

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a - Reservoir

Although the primary animal host for EBOV is still unclear, fruit bats seem to be its reservoir.

After several outbreaks in Gabon and Zaire between 2001 and 2005 that devastated local

gorilla and chimpanzee populations, a team of researchers captured 1030 animals including

bats, birds and small terrestrial vertebrates close to infected gorilla and chimpanzee

carcasses. They could detect immunoglobulin G (IgG) specific for EBOV virus in sera from

three different bat species, Hypsignathus monstrosus, Epomops franqueti and Myonycteris

torquata. Moreover, viral genome was detected in the same bat populations in organs

known to be the principal targets of EBOV, namely the liver and the spleen [10]. However,

many questions regarding the mechanism by which bats are infected and transmit the

viruses remain unanswered. Importantly, the transmission is not only due to direct contact

between human or non-human primates and living or dead bats. Indeed the majority of new

infections during outbreaks were due to human-human contacts through blood, secretions

or body fluids including sweat, saliva and tears [11].

b - Clinical characteristics

During the early stages, EBOV infection triggers several symptoms such as fever, severe

headache, muscle pain, intense weakness, fatigue, diarrhea, vomiting and abdominal

(stomach) pain. During the intermediate or advanced stages, inflammatory factors-induced

vasodilatation results in both internal and external hemorrhages (bleeding or bruising). In

addition to coagulation system disorders, the infection of kidney and liver leads to organ

dysfunctions. Body injury and viral spread in blood circulation and organs lead to a vicious

downward spiral. If viral spread cannot be controlled, patients may succumb to organ failure

or secondary bacterial infection. Hemorrhage observed during EBOV disease is due to

disseminated intravascular coagulation (DIC) development. This pathology is characterized

by a widespread activation of clotting cascade resulting in blood clots formation in blood

vessels, impairing the tissue blood flow and leading to ischemia and organ damages. In

addition, this blood clot formation exhausts the coagulation factors, preventing normal

coagulation and leading to hemorrhages. The mechanisms leading to DIC will be further

detailed below (see chapter III.2). Furthermore, there is a weak inflammatory response

coupled with a significant lymphoid cell apoptosis leading to lymphopenia, which seems to

be a marker of poor prognosis. These symptoms may appear anywhere from 2 to 21 days

after EBOV exposure, but the average is between 8-10 days. In most cases, the cause of

death is mainly due to organ failure (such as liver or spleen) rather than to hemorrhagic

fever. A major complication for the EBOV diagnostic is that symptoms observed during the

early stages of infection are non-specific and difficult to distinguish from other endemic

diseases, such as Lassa fever, malaria, cholera or typhoid fever.

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II - The virus

1 - Morphology and genome organization

Viral particles have a filamentous morphology. The origin of the name of Filoviridae family

comes from the Latin word filum referring to this particular morphology (Figure 3a). EBOV is

an enveloped, single strand, non-segmented, negative sense RNA virus. The 19kb viral

genome contains seven genes separated by regulatory regions composed of the 3'

nontranslated region (NTR), highly conserved transcription stop and start signals and the

5'NTR. The conserved transcription stop and start signal either overlap or are separated by

intergenic regions (IGR) [12]. Even if the viral genome contains only seven genes, more

proteins are produced through cotranscriptional editing of the GP (glycoprotein) gene [13].

Encoded proteins are nucleoprotein (NP), polymerase cofactor VP35, matrix protein VP40,

glycoprotein (GP), transcriptional activator VP30, second matrix protein VP24 and RNA-

dependent-RNA polymerase (L) proteins. In addition, through RNA editing EBOV is able to

express two truncated secreted proteins, glycoprotein (sGP) and small glycoprotein (ssGP)

[13] (Figure 3b). The viral RNA is encapsidated by NP and associated to VP35, VP30 and L to

form the ribonucleoprotein (RNP) complex. The RNP is surrounded by a matrix structure,

containing the matrix proteins VP40 and VP24, and finally by a host cell-derived membrane

in which the surface glycoprotein GP is embedded [14]. GP self-associates as a trimer, linked

by a single disulfide bond to form spikes at the virion surface [15] (Figure 3c). In addition to

their structural function, these proteins play several roles notably in immune system evasion

as described below (see the chapter V)

Figure 3 : EBOV morphology observed by electron microscopy (a) (CDC, 2005) and schematic organization of genome (b) [14] and virion (c) [16]

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2 - EBOV protein functions

a - RNP complex

NP, VP35, VP30 and L proteins play a fundamental role in RNP complex formation and in viral

transcription and replication.

NP is a 739 amino-acids (aa) protein encoded by the first gene located at the 3' region of the

genome. It plays a central role in virus replication, NP together with VP24 and VP35 are

necessary and sufficient for the formation of nucleocapsids that are morphologically

indistinguishable from those from EBOV infected cells [17].

VP35 protein is composed of 321 aa (35kDa). In addition to its role in nucleocapsid formation

by creating a link between L and N, VP35 is also a cofactor of the RNA-dependent RNA

polymerase complex. It plays an important role in antiviral and IFN response inhibition

detailed later (see chapter V.2).

VP30 (288aa, 32kDa) interacts also with NP in the RNP complex. VP30 is a transciptional

activator. VP30 can switch from a phosphorylated inactive state to an active state, through

dephosphorylation by the cellular protein phosphatase 1 (PP1). This regulation maintains the

balance between transcription and replication, as VP30 activity is required for the

transcription initiation [18]. When VP30 is active, transcription and protein synthesis occur,

while when it is inactive, viral replication takes place.

The L gene encodes a large protein of 2212 aa (252kDa), highly conserved across Ebola

species. This RNA-dependent-RNA polymerase (similar to the other polymerases of negative

single stranded RNA virus) is responsible for the viral transcription as well as for the RNA

replication. Moreover, it regulates the GP editing leading to the generation of sGP and ssGP.

b - Matrix proteins: VP40 and VP24

VP40 is composed of 326 aa (35kDa) and is the most conserved and the most abundant

protein in the virion. It is not clear whether the majority of VP40 in the cytoplasm or

premembrane zone is monomeric [19-22] or dimeric [23] or both [24]. This VP40 form was

found to be critical for both the transport of the nucleocapsid to the cell surface and for its

incorporation into virions [23]. Nevertheless, this monomeric or dimeric conformation can

be switched into either octameric or hexameric structures that have distinct functions.

Octamer formation is critically dependent on RNA binding [25], as no octamer can be

observed in the absence of RNA [26], suggesting that they may play an important role in

EBOV transcription and replication [20]. Hexamers are believed to be induced by the VP40

binding to plasma membrane [19, 27] and may be implicated in the initiation of virus

assembly, binding and budding via their interaction with the cytoplasmic tails of viral GP

and/or the RNP complex [28, 29]. Hexameric VP40 induces host cell membrane curvature

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needed for viral egress [24, 30]. This matrix protein plays a central role in the formation of

the filamentous structure of EBOV virions [23, 29]. However, how VP40 induces the

formation of the particular filamentous morphology of the particle is mostly unknown. In

addition, a soluble secreted form of VP40 was observed during EBOV infection in vitro and

was also found in the serum of virus-infected animals albeit in low amounts [31]. The role of

this soluble form of VP40 as well as the mechanism by which it is released are unknown.

Nevertheless, the early appearance of anti-VP40 antibodies in EBOV infected patients could

be explained by the presence of this secreted VP40 [32, 33]. These observations suggest that

soluble VP40 may play a role in EBOV pathogenicity.

VP24, composed of 251 aa (28kDa), plays a structural role of matrix but has also a function

during EBOV life cycle. In contrast to what has been reported in previous studies, Watt A et

al (2014) demonstrated that VP24 has only a very modest influence on genome replication

and transcription. Nevertheless, it plays an important role in particle infectivity due to its

function in nucleocapsid assembly and more specifically in RNA incorporation into viral

particles [34]. Like VP35, VP24 interferes with IFN response (see below in chapter V.2).

c - GP

The GP gene of EBOV contains an editing site allowing the translation of three differents

proteins (Figure 4). The first isoform is a structural protein, translated into a glycoprotein

precursor (GP0) further cleaved by a cellular proprotein convertase furin [35]. This produces

a surface subunit GP1 and a transmembrane subunit GP2 that are able to form a

heterotrimer. GP plays a role in virion attachment and fusion but this process remains poorly

understood. GP1 contains an excessively O-linked glycosylated mucin-like region (MLR) at C-

terminal, a heavily N-linked glycosylated glycan cap domain (GCD) and a receptor binding

domain (RBD) which mediate the binding to a variety of host cell surface factors including T-

cell immunoglobulin and mucin domain 1 (Tim-1) [36]. MLR is required for neither the viral

entry nor the cellular tropism [15], as MLR-deleted GP is able to mediate viral attachment

and entry, but it may influence the EBOV capacity to escape the immune system [37]. GP2

with the fusion peptide is required for the virus-host membrane fusion. In addition to this

transmembrane GP form, several soluble GPs have been described. A trimeric soluble GP,

called shed GP, is produced by the release of virion-attached GP byTNF-α-converting enzyme

(TACE) through a cleavage site proximal to the transmembrane anchor. Moreover, GP gene

encodes two non-structural forms of GP that are soluble and secreted in important quantity

by infected cells. The soluble GP (sGP) is homodimeric whereas the small soluble GP (ssGP) is

monomeric. During EBOV infection, the ratio between sGP and GP transcripts is

approximately 75% / 20% and ssGP represents 5% of GP transcripts [38]. These secreted GPs

are easily detectable in the blood of infected patients [39] and play several roles in both

cytoxicity induced by EBOV and immune evasion as detailed later (see chapter III.3 and

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V.1.b). In addition, a study demonstrated that sGP can substitute GP1 to form sGP-GP2

complex, suggesting a role for sGP as a structural protein [40].

Figure 4 : Processing of EBOV glycoproteins from Cook et al, 2013 [41]

3 - Viral life cycle

EBOV life cycle is similar to life cycles of other viruses with negative single strand RNA

(Figure 5). After GP binding to attachment factors (including DC-SIGN, L-SIGN) [42] and entry

receptors, such as Tim-1 [43, 44], whole virions are internalized via macropinocytosis and

trafficked to the endosomal compartment [45, 46]. GP1 is then cleaved by the endosomal

cysteine proteases cathepsin B (CatB) and L (CatL) that remove the hyper-glycosylated

region, which exposes the RBD in order to bind the Niemann-Pick C1 (NPC1) cholesterol

transporter. GP1-NPC1 interaction leads to conformation change of trimeric GPs and allows

the insertion of three fusion peptides located at the N-terminal region of GP2 in endosomal

membrane. This step is essential for the fusion process, allowing viral genome release into

the cytoplasm [47, 48]. The released viral RNA is then first transcribed. Due to the presence

of transcription stop and start signal in the regulatory region between each gene, the

negative-strand RNA genome is transcribed by the L polymerase into seven monocistronic

mRNAs. These mRNAs are capped and polyadenylated. It is believed that for EBOV, such as

for all negative RNA viruses, the polymerase accesses to the viral genes via a single

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polymerase binding site at the 3' end. Once bound the viral polymerase progresses along the

RNA template by stopping and reinitiating at each gene junction and transcribes genes in a

sequential and gradient manner. Accordingly the first gene, NP, is transcribed at the highest

level whereas the last gene, L is transcribed at the lowest level. Then, replication likely

begins when enough NP is present to encapsidate neo-synthetized antigenome and

genomes. GP-encoding mRNAs transit to the endoplasmic reticulum (ER) where GP is

synthesized and form trimers. After the addition of N and O-linked glycans in the ER and

Golgi apparatus, GPs are delivered to the plasma membrane by secretory vesicles. NP, VP35

and VP30 proteins associate with viral RNA to form RNP complex, and with matrix proteins

(VP40 and VP24) and GP proteins. Eventually, viral particles bud at the cell surface and are

released.

Figure 5 : EBOV life cycle, from White JM 2012 A new player in the puzzle of filovirus entry [49]

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III - Pathogenesis

1 - Target cells and tissues

The detailed pathogenesis of the disease is not well understood. Nevertheless, it has been

found that EBOV has a broad cell tropism, infecting a wide range of cell types. In situ

hybridization and electron microscopy analyses of tissues from patients with fatal disease or

from experimentally infected non-human primates showed that monocytes, macrophages,

dendritic cells (DCs), endothelial cells, fibroblasts and several types of epithelial cells such as

hepatocytes and adrenal cortical cells support EBOV replication [50-54]. Temporal in vivo

studies in non-human primates experimentally infected with EBOV determined that

monocytes, macrophages, DCs but also natural killer (NK) cells are the first and favorite

targets of the virus, whereas all others cells cited above are infected much later during the

course of the disease, proximal to death [51, 52, 55]. Monocytes, macrophages, and DCs

appear to play a major role in the dissemination of the virus. Immunohistochemical studies

have shown that the virus disseminates from lymph nodes via lymphatic and vascular

systems to several organs including liver, spleen, lung, kidney, pancreas, large and small

intestines and skin amongst others [50, 54]. Nevertheless, the most prominent damages are

observed in liver and spleen. In these organs, cell necrosis and apoptosis were detected. The

same was observed in lymph nodes leading to the lymphoid depletion detailed below see

chapter IV.2). In the liver, hepatocytes and Kupffer cells are infected, leading to hepatic

dysfunction directly resulting from viral damages or circulatory impairment. EBOV infection

leads to coagulopathy through damages to both liver, which is the production site of clotting

factors, as well as certain coagulation inhibitors, [56] and endothelial cells, which provide

tissue factor (TF also known as thromboplastin), tissue factor pathway inhibitor (TFPI) and

receptor for protein C activation [50, 57]. These organ disorders contribute more to the

patient death than the hemorrhagic fever.

2 - Coagulation anomalies and vascular endothelium impact

Coagulopathy has been observed during EBOV infection and might have several causes

including activation of cytokine secretion, platelet aggregation and consumption, activation

of the coagulation cascade, deficiency of coagulation factors due to both liver and

endothelium damages. Indeed, it has been described that pro-inflammatory cytokines such

as IL-6 are increased in human and non-human primates infected by EBOV [58, 59]. IL-6 is

known to trigger the coagulation cascade. Accordingly, the transcriptional targets of IL-6

including several proteins that either increase the transcription of pro-coagulant proteins

like TF or decrease the transcription of anticoagulant proteins such as antithrombin [60].

Moreover, EBOV infected monocytes and macrophages induce an increase of TF protein

level in macaques circulation [61]. EBOV infection also causes hepatic necrosis and apoptosis

leading to an impairment of the synthesis of critical coagulation factor including protein C,

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protein S and fibrinogen [62, 63]. Deregulation of this coagulation pathway leads to

disseminated intravascular coagulation (DIC) which is observed during infection and likely

contributes to hemorrhage symptoms and multi-organ failure [6, 61, 64]. In addition to these

problems in the coagulation pathway, the widespread injury to endothelial cells via a direct

cytotoxic effect of GP (detailed later in chapter III.3) is observed in EBOV infection and is

another mechanism triggering DIC. These cells have several properties, one of these being

the capacity to regulate the process of coagulation and fibrinolysis and to modulate the

fibrin deposition. At steady state, endothelial cell surface is thought to be essentially

anticoagulant or non-thrombogenic. The control of coagulation is exerted by endothelial

cells at different critical steps of the clotting cascade. Briefly, endothelial cells are the main

source of TFPI, which blocks TF, the principal initiator of the coagulation cascade [65, 66]. TF

is a transmembrane glycoprotein receptor expressed in response to injury at the surface of a

variety of cells, including platelets, monocytes, macrophages, fibroblasts, and endothelial

cells [67]. In addition, endothelial cells express a large amount of heparan sulfate and related

glycosaminoglycans to neutralized clotting enzymes such as factor Xa and thrombin [65].

Eventually, endothelial cells play a critical role in the protein C anticoagulant pathway by

deregulating its expression [68]. EBOV infection leads to impairment of the endothelial

barrier integrity and to an increased endothelial permeability [51]. In addition, several

factors secreted by both infected monocytes and macrophages can exert changes in the

vascular endothelium in a variety of ways. This includes either an indirect induction of

endothelial cell activation, by infecting and activating leukocytes and triggering the synthesis

and local production of pro-inflammatory soluble factors, or a direct induction of changes in

endothelial cell expression of cytokines, chemokines and cell adhesion molecules in the

absence of immune mediators (as a direct result of virus infection, mechanism detailed in

chapter III.3). Mediators released from EBOV-activated endothelial cells can modulate

vascular tone, thrombosis, and/or inflammation including nitric oxide (NO), prostacyclin,

interferons (IFNs), interleukin (IL)-1, IL-6, and chemokines such as IL-8, IL-6, IL-7 [61]. All

these endothelial cells impairments are implicated in DIC syndrome and hemorrhage

development. However, as previously mentioned, the hemorrhage observed during EBOV

infection is insufficient to cause the death, as the massive loss of blood is atypical and, when

is present, is largely restricted to the gastrointestinal tract. Nevertheless, it seems that pro-

inflammatory cytokines secreted by monocytes, macrophages or DCs and both apoptosis

and necrosis observed in several organs including liver and spleen induced by EBOV infection

might participate to malfunction of both vascular system and coagulation, leading to general

failure of several organs, septic shock and death.

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3 - Direct toxicity

In vitro studies have shown that GP has direct cytotoxic properties on endothelial cells via

morphology changes leading to cell rounding and detachment [69, 70]. Indeed, studies

identified a reduction at the cell surface of the expression of adhesion molecules such as

integrins or immune molecules (including major histocompatibility complex class I [MHC]

and the epidermal growth factor receptor) induced by GP expression. This is believed to

contribute to the cell rounding and consequent loss of cell adhesion observed in infected

cells [70-74]. This finding suggests that GP and more particularly the MLR of GP plays an

important role in endothelial cell toxicity and could be responsible for both endothelial

integrity disruption and increased endothelial permeability, triggering hemorrhage

development during the disease [69]. The mechanism by which GP has this toxic effect has

been shown to be dependent on GTPase dynamin. Through its interaction with dynamin, GP

disrupts the normal intracellular trafficking of the cell surface proteins essential for cell

attachment and immune signaling [70]. Nevertheless, the importance of GP cytotoxicity in

viral pathogenesis is however controversial. Indeed, direct damages to the endothelial cells

by virus replication have been observed only in animal models at terminal stages of the

disease [51]. A study demonstrated that moderate expression of GP (similar to the amount

observed in EBOV infected cells during the early stages of infection) did not result in

morphological changes and was not cytotoxic, suggesting that cell rounding and

downregulation of the surface markers are late events in EBOV infection, whereas

production and massive release of virus particles occur at early steps [75].

It has been described that EBOV-infected cells release proteolytic endosomal enzymes, such

as the cathepsin proteases implicated in extracellular matrix degradation and disease

progression [76, 77]. The secretion of cathepsins by EBOV-infected cells suggests that these

molecules may be implicated in direct cytotoxicity induced by EBOV and contribute to the

vascular endothelium destruction because these proteases in vitro catalyze the degradation

of extracellular matrix and induce cell rounding and detachment in vitro.

A recent study showed that GP increases the NK cell toxicity. In fact, mouse macrophages

infected with VSV particles containing EBOV-GP instead of their glycoprotein (VSVΔG/EBOV-

GP) particles causes an increase in NK cell cytotoxicity through a decrease of MHC-I

expression [55].

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IV - Immune response during EBOV infection

Several studies have shown that EBOV infection was associated with aberrant innate

immune responses and with global suppression of adaptive immunity (Figure 6).

1 - Innate response

After the epithelial barrier, innate immunity is defined as the first line of defense against

pathogenic microbial exposure. Innate immune responses are not specific to a particular

pathogen in contrast to the adaptive immune responses. Innate immune responses involve

several pathways in order to distinguish self from non-self. The recognition of non-self leads

to the activation of several cells, such as monocytes, macrophages, granulocytes, DCs,

natural killer (NK) cells but also to the complement activation (soluble factors) followed by

the cytokines production such as interferon (IFN). The interferon system represents a major

innate defense against infections by viruses and other pathogens. Three classes of IFNs have

been described. Type I IFNs, comprising IFN-α and IFN-β, are produced by many cell types.

Type II IFNs, with IFN-γ, are generated by activated T cells and NK cells. Type III IFNs,

including IFN λ1–3, are incompletely characterized, but are believed to mediate an antiviral

response as well. The IFN response begins with the recognition of diverse pathogen-

associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Viruses

contain several PAMPs recognized by specific PRRs. Double strand RNA (dsRNA), single

strand RNA, CpG-DNA, 5'-triphosphate RNA or single strand DNA which are recognized by

Toll-like receptors (TLR)-3 , TLR 7/8, TLR9, retinoic acid-inducible gene I (RIG-I) or single

stranded DNA melanoma differentiation associated gene 5 (MDA-5). EBOV, being a negative

strand RNA, induces IFN signaling through TLR-3, TLR-7/8 and RIG-I. The receptors can be

localized in the cytoplasm, like RIG-I and MDA-5, or in membranes, like TLRs. Receptor

activation leads to IFN production via IFN regulatory factor (IRF)-3 and IRF-7. This secreted

IFN binds to its receptor composed of two subunits, IFN α receptor 1 (IFNAR1) and IFNAR2 at

the cell surface in order to activate JAK-STAT pathway. This signaling pathway leads to the

phosphorylation and subsequent dimerization of the transcription factor STAT, allowing its

shuttle into the nucleus to induce transcription of interferon-stimulated genes (ISG)[78]. IFN

response leads to an antiviral state but we will see later that several EBOV proteins can

interfere at several levels with the JAK/STAT pathway (see chapter V.2). Altogether, these

mechanisms are often sufficient to counter invading viruses. In addition, when they fail to do

so, they favor the generation of host mediated humoral and cellular immune responses that

limit and in most cases eliminate the invading pathogen. Several viruses, however, such as

EBOV, have developed a variety of mechanisms to escape the innate immune system

(detailed below). EBOV infection targets antigen-presenting cells (APC) during the early

stages of infection. Since these cells play a critical role in immune responses, their infection

by EBOV has dramatic consequences, notably by preventing their maturation. Indeed, in

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vitro studies demonstrated that EBOV-infected DCs do not express the DC

maturation/activation markers such as CD80, CD86, CD40, CD83 and MHC of class I and II

needed to CD4+ and CD8+ T-cell co-simulation and activation [79, 80]. In addition, EBOV

infection prevents cytokine and chemokine production implicated in inflammation

regulation and immune response such as IFN-α, IFN-β, tumor necrosis factor (TNF) -α, IL-1β,

IL-10, IL-6, IL-2, IL-8, IL-12 [79, 81]. During viral infection, NK cells quickly respond by

triggering exocytosis of perforin and granzymes and secretion of IFN-γ, respectively

mediating the destruction of infected cells or the macrophage activation. NK cells activation

requires several signaling molecules including IL-12 (for the cytokines production), IFN-α and

IFN-β (for the development of cytotoxic effector function) secreted by mature/activated

DCs. Since EBOV infection prevents DC maturation/activation, NK cells activation is

decreased [82] , which further favors virus replication. Therefore, a proper activation of NK

cells could be critical for the protection of EBOV infection [83].

In such a dysregulated immune response context, it has been observed that despite a high

viral load and necrotic lesions in fatal EBOV cases, only a minimal inflammation is observed

in infected organs and tissues [50], probably due to a weak immune system activation. Yet,

and in contrast to the negative impact of EBOV infection on DCs and NK cells, the infection of

monocytes and macrophages by EBOV leads to an important secretion of pro-inflammatory

cytokines such as IL-1β, IL-6, IL-8, IL-15, IL-16, TNF-α but not IFN-α and chemokines such as

macrophage inflammatory protein (MIP)- 1α, MIP-β [84-86]. All these disturbances in

immune cell activation and pro- and anti-inflammatory cytokines production contribute to

facilitate the uncontrolled viral replication observed during EBOV infection. Indeed, it has

been shown that the early innate response correlates with the survival of EBOV-infected

patients. Therefore, the rapid initiation of innate response may limit EBOV infection and

could be a critical condition to host survival [32].

2 - Adaptive response

Adaptive response is the third line of defense, after epithelial barrier and innate response. It

is triggered after a few days of infection and is more powerful than innate immunity in

combating the infection. In contrast with innate system, adaptive system develops a specific

response to the antigen and allows establishing an immune memory. Briefly, after DC

maturation and activation by pathogen detection, DCs migrate to lymph nodes to present

antigens on their surface via MHC-I or II and express co-stimulatory factors (CD80, CD84,

CD40) in order to activate T lymphocytes (CD4+ and CD8+). When pathogen-specific T cells

are activated, they proliferate, leave the lymph node and migrate to infected tissues. CD8+ T

cells directly kill the infected cells through their cytotoxic activity and CD4+ T cells (Th1)

activate macrophages via both TCR-MHC-II interaction and cytokine production. Another

CD4+ T cells (Th2) population remains in the lymph node and stimulates the proliferation

and differentiation of pathogen-specific B cells through both MHC-II presentation and

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cytokine production in order to promote the antigen-specific antibody production or

proliferation of memory B cells.

We have seen that EBOV replicates efficiently in DCs without cytokine and chemokine

production and without inducing their maturation/activation. This lack of DC activation most

likely results into poor immune responses by NK as seen before but also into weak T and B

cell activation. In addition, fatal cases of EBOV infection are associated with a lack of

detectable adaptive immunity. It has been observed that EBOV infection induces a

substantial lymphopenia due to CD4+ and CD8+ T cell depletion and necrosis observed at

least in spleen, thymus and lymph nodes of non survivors compared to survivors; the same

was observed in experimentally infected non-human primates [50, 87, 88], and the different

mechanisms implicated in this phenomenon will be described below (paragraph

"lymphopenia"). Nevertheless, despite significant lymphocyte apoptosis, it has been

demonstrated that a functional and specific, albeit insufficient, adaptive immune response is

present in lethal EBOV infection [89], occurring even in the presence of incompletely

activated DCs. There is an increased percentage of CD4+ and CD8+ T cells expressing high

levels of CD44, a T-cell activation and maturation marker, close to the end of lethal EBOV

infection. CD8+ T cells play an important role in EBOV infection. Indeed, in lethal mice model

of EBOV, the IFN-γ production by CD8+ T cells in response to EBOV infection was observed at

the end of the disease [89]. In addition, this important source of IFN-γ could explain the

macrophage and monocyte activation observed during EBOV infection. Moreover, transfer

of EBOV-specific CD8+ T cells from mice infected with EBOV during 7 days protects naive

mice from EBOV challenge. [89]. Together, these data support the hypothesis that functional

adaptive immune responses are present, at the end of the disease in lethal EBOV-infected

mice but is insufficient in part due to massive lymphocyte apoptosis.

Concerning B cells, a clinical study performed during the 1996 outbreak in Gabon described

humoral immune responses in EBOV infected patients, as antibodies directed against GP

have been found in surviving patients [90]. In addition, important levels of IgG and IgM,

specific to NP, VP40 and VP35, have been found by ELISA in all survivors early in disease or

during early convalescence. In contrast, no viral antigen-specific IgG have been found in fatal

cases and only weak IgM levels have been detected in one-third of fatal cases [33]. These

results suggest that a prompt and vigorous humoral response may help survivors to limit and

finally control viral dissemination. Furthermore, it has been observed that this

immunoglobulin deficiency is not associated with a decrease of B cells. The mechanism by

which EBOV impacts on immunoglobulin levels therefore remains poorly understood [33,

91]. Nevertheless, the impact of EBOV infection on T cell activation and proliferation could

alter B cell activation.

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Lymphopenia

The mechanism by which EBOV induces a lymphopenia is not fully understood, likely in part

because a direct mechanism cannot be involved since EBOV does not infect lymphocytes.

As discussed above, EBOV readily infects and replicates in DCs, interfering with their

activation/maturation and therefore with their ability to initiate the adaptive immune

response and the associated lymphocytes expansion [61, 80].

We have seen before that the release of NO, which is a physiological vasodilator and anti-

platelet factor, was increased by the endothelial cells activated by EBOV [61, 92]. In vivo

study demonstrated that blood levels of NO were much higher in fatal cases (increasing with

disease severity), and extremely elevated levels could have negatively affected vascular tone

and contributed to virus-induced shock [93]. In addition of its role in endothelial barrier, NO

could also play a role in lymphopenia. Briefly, it has been shown that NO promotes apoptotic

pathways in numerous cell types including lymphocytes through the indirect activation of

caspases [94], moreover NO inhibits T and B cell proliferation via the downregulation of

MHC-II, co-stimulation molecules and/or cytokines (such as IL-12) [95].

The death receptor pathway activation could be implicated in lymphocyte apoptosis

observed during EBOV infection. EBOV infection could induce both intrinsic (mitochondrial

mediated pathway) and extrinsic (death receptor pathway) cell death cascades as crosstalk

occurs between these two pathways. In the intrinsic pathway, intracellular stress factors

(such as oxidative stress or DNA injury) via Bax and Bak proteins cause depolarization of the

mitochondrial membrane, thereby inducing the release of cytochrome C and activating the

caspase cascade beginning with caspase-9. Bcl-2 protein is known to inhibit apoptosis via its

interaction with Bax and Bak proteins [96]. In fatal EBOV cases, a decrease of Bcl-2 mRNA

level has been observed in PBMC during the disease, whereas a strong increase has been

detected in survivors at the time of T-cell activation [33]. The extrinsic pathway is initiated

by ligand-receptor interaction at the cell surface including either Fas Ligand with Fas or TRAIL

with TRAIL receptors (such as DR4 and DR5). Such interactions lead to caspase (caspase 8

and then caspases 3 and 7) activation via the adaptor protein Fas-associated death domain

(FADD) and finally induce DNA degradation and cell death [97]. With regards to the extrinsic

pathway, EBOV infection increases TRAIL expression in cultured monocyte-like cells, and

some EBOV-infected monkeys exhibit an increase of soluble Fas in their sera [86].

Furthermore, TRAIL and Fas mRNA expressions are increased in the PBMC of infected

monkeys [52].

Another explanation for lymphopenia induced by EBOV implicates GP. Indeed GP contains a

domain with a significant homology with the "immunosuppressive peptide" found in

glycoproteins of various oncogenic retroviruses known to often induce immunosuppression

[98]. This will be detailed below (see chapter V.1.c). In addition, it was postulated that sGP

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could play a role in lymphocyte apoptosis by interacting with circulating lymphocytes, as it

was detected in large amounts in the blood [99]. Nevertheless, an in vivo study showed that

sGP was not able to induce T cell apoptosis neither by itself nor by death receptor co-

stimulation; further studies are required to investigate the ability of sGP to induce apoptosis

via the intrinsic pathway [100].

Altogether, immune system dysfunctions (weak innate and adaptive immune system

activation or lymphopenia) contribute to the uncontrolled spread and growth of the virus.

This suggests that a strong immune response may result in protection against EBOV

infection, which may guide the design of new therapeutic strategies to control lethal EBOV

disease.

Figure 6 : Model of EBOV pathogenesis in primates. Adapted to Bray M 2005 [101]

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V - Immune response evasion by EBOV viral proteins

Several mechanisms by which EBOV escapes immune system have been suggested (Figure

7).

Figure 7 : Potential mechanisms by which various EBOV proteins evade host innate and acquired immune systems. Adapted from Ansari AA 2014 [102]

1 - GP implications

a - Glycosylation and MLR

EBOV infection elicits only low level of neutralizing antibodies against GP in humans and

other animals [14]. As mentioned above, the heavy glycosylation of GP is implicated in the

immune system escape. These glycans located in the MLR sequence promote the generation

of antibodies against the more variable GP1 domain, which are not able to confer a strong

protection [103]. In mice, removal of the MLR of GP1 can lead to the production of more

efficient antibodies directed against the conserved glycoprotein core structure, confirming

the impact of this MLR domain in masking neutralizing epitopes [104]. Moreover, O- and N-

glycosylations impedes the recognition of GP by neutralizing antibodies through steric

shielding [41, 74, 105-107]. In addition, MLR is necessary and sufficient to decrease the

expression of cell surface proteins such as MHC-I and several members of the integrin family.

This domain blocks the access to MHC-I needed for CD8+ T cell stimulation [69, 105]. An

additional mechanism by which glycosylations play an important function in the immune

escape involves N-linked GP2 glycosylation. Indeed, mutation of one of the two N-linked GP2

glycosylation sites prevents the interaction between GP1 and GP2 required for GP

localization at the plasma membrane and is implicated in antigenicity and immunogenicity of

EBOV GP. All these results suggest that it might be possible to enhance immunity by specific

modifications in the GP glycosylation [37].

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In addition, we have seen that EBOV infection leads to DC maturation defects and

consequently to a failure of efficient T cell activation [79, 80]. In vitro studies using EBOV-

virus like particles (VLP) containing VP40 demonstrated that VLPs, contrary to EBOV

infection, have the capacity to activate DCs. MLR is the domain required for DC activation via

a recognition of MLR by toll like receptor (TLR)-4 and NF-kappaB and MAPK signaling

pathway activation [108, 109]. Indeed, VLPs with wild-type GP but not with MLR-deleted GP

can activate TLR-4-dependent responses. In EBOV infection context, these results suggest

that MLR plays a major role in the abnormal DC activation observed during the disease.

b - Role of sGP and shed GP

Secreted GPs, sGP and shed GP, have been shown to be important in immune evasion.

Because sGP shares 295 amino acids with GP and is the predominant transcript for the GP

gene, it has been postulated that sGP probably competes with virion-attached GP. Indeed,

the majority of antibodies from EBOV-surviving patients and monkeys are directed against

sGP rather than against GP1/2 [110, 111]. It is possible that the majority of antibodies

binding sGP are non-neutralizing, but it is likely that the weak amount of neutralizing

antibody production is absorbed by the much more abundant sGP. Indeed, it has been

demonstrated that sGP serves as a decoy for neutralizing antibodies [112]. In addition to its

role in adaptive response evasion, sGP has been reported to bind to neutrophils through the

Fcγ receptor thereby inhibiting early neutrophil activation [113]. Concerning the shed GP, in

a guinea pig model of EBOV infection, this secreted GP is present in significant amounts in

the blood of infected animals. Shed GP inhibits the neutralizing activity of EBOV antibodies,

and the increase of shed GP in infected animals observed between days 6 and 9 post

infection correlates with the course of disease and the lethal outcome at day 9 [114]. All

these findings suggest that secreted GPs may play an important role in the pathogenesis.

c - Immunosuppressive domain in GP

Several retroviruses including Avian reticuloendotheliosis virus (ARV) and Feline leukemia

virus (FeLV), have a particular peptide in their envelope protein named p15E or

"immunosuppressive peptide", that has immunosuppressive properties. For example, this

peptide inhibits the T cell activation normally induced by concanavalin stimulation [115], the

proliferation of murine cytotoxic T cells [116] and macrophage recruitment to the

inflammatory site in mice [117]. Amino acid sequence comparison has uncovered a high

homology between this "immunosuppressive peptide" and 160 residues at the C-terminal

part of EBOV-GP which could explain the immunosuppressive effect mediated by EBOV-GP

[98]. More recently, a study identified a 17-mer peptide in this region as the

immunosuppressive domain of EBOV-GP. This peptide induces a significant decline of CD4+

and CD8+ T cells. In addition, this peptide induces a decrease of IL-2 receptor at the T cell

surface, but also inhibits IFN-γ, IL-2 and IL-10 expression leading to an inhibition of T cell

proliferation and activation [118]. The mechanism by which immunosuppressive peptide

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acts on CD4+ and CD8+ cells is unknown but it has been hypothesized that it inactivates

these cells by directly contacting them or indirectly through its previously described effect

on APCs.

2 - IFN pathway inhibition by VP35 and VP24

EBOV uses several mechanisms in order to inhibit IFN production (Figure 8). It has been

shown that VP35 is responsible for the absence of IFN-α production and prevents the

activation of IFN-stimulated response element (ISRE)-containing promoters when either

transfected dsRNA or viral infection is used as the activating stimulus [119]. A more detailed

study of the mechanism by which VP35 influences the host IFN response showed that it

inhibits the IFN synthesis at several levels. VP35 can bind viral dsRNA and inhibit the

recognition by helicase RIG-I implicated in the IFN pathway and then the IFN-α and -β

production [120]. The ability of VP35 to block IFN production was also correlated with its

ability to inhibit the phosphorylation of IRF-3 through interaction with kinases including IκB

kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK-1) [121], and thus inhibiting its nuclear

translocation and activation [122]. A SUMOylation of IRF-7 induced by VP35 was recently

described as an additional mechanism of repression of the transcription of IFN genes [123].

Co-immunoprecipitation experiments demonstrated that VP35 interacts with PIAS1 (protein

inhibitor of activated STAT-1) and Ubc9, two proteins involved in the small ubiquitin-like

modifier (SUMO) conjugation cascade [124, 125]. Besides that, Feng et al have

demonstrated that VP35 protein is a RNA binding protein with a stronger affinity for dsRNA

than PKR. Consequently, VP35 competes with PKR for EBOV dsRNA binding and prevents the

phosphorylation of translation initiation factor eIF-2 (eIF-2) by PKR required to stop protein

synthesis and thus viral replication [126].

In addition to VP35, VP24 is another important player in the counteraction of IFN pathway

by EBOV. VP24 inhibits the IFN pathway by preventing the nuclear accumulation of STAT-1

[127]. Actually, VP24 binds to karyopherin-α, a nuclear transporter, with very high affinity to

compete with STAT-1 and inhibit its nuclear transport [128, 129]. In addition to the JAK-STAT

pathway, the p38 mitogen-activated protein (MAP) kinase pathway is also critical for the IFN

response [130]. Engagement of the IFN receptor by IFN activates a cascade of MAP kinases,

leading to the phosphorylation of the alpha isoform of p38 (p38-α) [131]. Phosphorylated

p38-α then triggers the phosphorylation of downstream transcription factors that participate

in IFN responses. It is well established that p38 is essential for gene transcription via ISRE or

GAS elements [130-132]. It has been observed that VP24 inhibits the p38 MAP kinase

pathway by preventing the phosphorylation of p38-α [133]. The dual action of these two

viral proteins, VP35 and VP24, may thus contribute to a potent inhibition of the IFN pathway,

permitting an efficient virus replication and dissemination in the host.

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Figure 8 : EBOV proteins interfering with interferon signaling

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VI - Diagnosis and treatments Although EBOV is considered to be a significant public health problem, no licensed drug or

vaccine is currently available [134-136]. The most effective measure for controlling disease

propagation is the isolation of patients and strict barrier nursing procedures to protect

healthcare workers. Meanwhile, symptomatic and supportive care is the treatment of

choice. Nevertheless, owing to the advances of basic EBOV research, several promising drugs

and vaccine candidates [137] are under development.

1 - Diagnosis methods

As written above, the clinical symptoms in the early stages of EBOV infection are very similar

to others viral diseases such as flu and other respiratory infections, common enteritis or

other infections frequently occurring in African including malaria and Lassa fever. Therefore,

especially in the early stages, virological testing is very important for the diagnosis.

Specific EBOV-antibodies detection by ELISA and immunofluorescence has been developed

but as mentioned above, EBOV antibodies are produced only in small quantity, especially in

fatal cases.

The inoculation of a cell culture with patient sera or other body fluids or tissue extracts is the

classical method to isolate and amplify EBOV. Then, EBOV is detected by PCR or

immunofluorescence using viral-specific primers or antibodies respectively. Antigen blood

tests are based on the detection of virus proteins using specific antibodies and are hardly

influenced by virus variability. The high viremia in EBOV patients often facilitates antigen

detection, although the tests are clinically less sensitive than PCR [138, 139]. As EBOV has a

specific filamentous morphology, the direct detection of EBOV by electron microscopy in

organ section and serum is possible but high virus concentrations are needed [140]. A major

disadvantage of these diagnosis methods is the time required to isolate the virus (days to

week) and the need of biosafety level 3 or 4 facilities. The detection by electronic

microscopy is not routinely used because of its high cost. Therefore, the most used method

is based on nucleic acid tests, as it requires 24-48h to obtain results in a very sensitive

fashion. Very recently a new test that provides results within 15 minutes has been

developed, the ReEBOVTM Antigen Rapid Test. This test, which is based on the detection of

the VP40 protein rather than nucleic acids, is able to correctly identify about 92% of EBOV

infected patients and to exclude 85% of those not infected with the virus. In addition to its

rapidity, the antigen test is easy to perform and does not require electricity, which therefore

would favor its use in lower health care facilities or mobile units [141].

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2 - Treatments and Vaccines

Currently, the majority of treatments used aim at treating symptoms induced by EBOV. For

example, as EBOV inhibits IFN signaling, exogenous INF-α or INF-β have been used and could

delay the occurrence of viremia or increase survival time, but they cannot rescue non-

human primates from lethal infection [142, 143]. As EBOV infection indirectly impairs the

coagulation pathway by provoking the depletion of clotting factors through aberrant and

excessive coagulation, the recombinant nematode anticoagulant protein c2 (rNAPc2) and

the recombinant human activated protein C (rhAPC), originally used for anticoagulation

purposes, have been tested and gave promising results in infected monkey [63, 144].

rNAPc2, which has shown 33% efficacy in non-human primates [144], is in Phase II trial for

thrombosis prevention. Nevertheless no human trial is planned for EBOV treatment [145].

Several treatments targeting a specific step of viral life cycle including entry, RNA synthesis

and translation have been developed.

a - Candidates to block the viral entry

In order to block the virus entry, researchers purified patients-derived polyclonal or

monoclonal antibodies specifically targeting the main neutralizing epitopes on EBOV-GP. The

antibody KZ52, derived from a survivor of the Kikwit EBOV outbreak in 1995, displays a

potent neutralizing activity and has been shown to protect guinea pigs [146] but not non-

human primates [147]. During the past years, researchers have developed three generations

of antibody cocktail formulations. The first one was based on the combination of two

human-mouse chimeric antibodies, ch133 and ch226, which presented strong neutralizing

activity against EBOV in vitro. Unfortunately, trials in non-human primates challenged with

EBOV were not convincing [148]. A second generation of anti-EBOV antibody cocktail

formulas, ZMAb and MB-003 consisting of three different neutralizing antibodies derived

from EBOV GP, have been tested in non-human primates. ZMAb, containing mAbs 1H3, 2G4

and 4G7, showed 100% protection in Cynomolgus macaques [149]. The MB-003 cocktail,

including antibodies of c13C6, h-13F6, and c6D8, showed 67% protection in macaques [150].

It seems that human trial has started so far for this treatment. This technology may be

insufficiently robust to promote the production of neutralizing antibodies to fight the

current EBOV outbreak. A recent study has established a better optimized antibody

combination derived Zmab and MB-003, named Zmapp and containing c13C6, 2G4 and 4G7.

This new mAbs combination demonstrates a successful protection in non-human primates

[151]. Phase I safety and efficacy trials have been initiated in January 2015, but the

conclusions are not yet available [145].

In addition to the neutralizing antibodies, other treatments have been developed to block

viral entry. Since the first C-terminal heptad repeat (CHR)-peptide-based HIV entry inhibitor

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discovered in 1992 [152], this potential treatment strategy has been applied against many

enveloped viruses, including EBOV [153, 154]. Briefly, as the CHR domain of GP2 plays a role

during the fusion step in the endosomes, exogenous CHR could be able to compete with viral

CHR and prevent the viral fusion. This treatment showed inhibition activity against three

EBOV species, including Zaire, Sudan and Reston Ebolavirus [153]. Other therapeutic

candidates have been described to prevent the fusion step including Cat L/B inhibitor [155]

and NPC binding compounds [156].

b - Candidates to block viral RNA synthesis and/or translation

Others drugs targeting RNA synthesis and translation have been developed. Nucleot(s)ides

analogues including Ribavirin, Favipiravir and Brincidofovir have been tested. Ribavirin could

not limit the replication of EBOV and failed to protect animals from lethal challenge [157,

158]. Interestingly, Favipiravir showed efficient antiviral activity in mouse models for EBOV

infections [159]. Clinical efficacy trials began in Guinea in December 2014, however more

data are required in order to draw a conclusion [145]. Brincidofovir (CMX001), showed

potent anti-EBOV activity in vitro, and has been used to treat EBOV patients but its

mechanism of action is unclear. However, a new phase II clinical trials of Brincidofovir has

been launched to test its potential safety and antiviral activity in EBOV infected patients

[160]. A new clinical efficacy trial began in Liberia in January 2015, but due to the lack of

patients this trial has been stopped. In addition, to date no precise results are available

because this drug is often combined with other drug therapies [145]. Finally, BCX-4430,

another nucleoside analogue, interferes with the function of RNA polymerase of EBOV, and

confers protection to EBOV-challenged rodent animals [161]. BCX-4430 is in phase I safety

trial and efficacy trials will begin providing that the safety results from Phase I will be

satisfactory [145].

Others strategies using small interfering RNAs (siRNAs) have been developed. Especially,

siRNAs specifically directed against the RNA sequences of RNP complex, VP24, and VP35

were tested [162]. For instance AVI-6002, a mixture of iRNA targeting mRNA sequences of

VP24 and VP35 protected five of eight rhesus monkeys from EBOV challenge [163]. For this

drug, the phase I safety is completed but there are no human trial planned at this time [145].

c - Vaccines

Several vaccine candidates have been tested on rodent and non-human primates [164]. The

first trials were done with inactivated viruses but this method was quickly abandoned. A lot

of viral vectors have been used to produced anti-EBOV vaccines including Venezuelan equine

encephalitis virus [165], adenovirus [166], virus Parainfluenza [167] or Vesicular stomatitis

virus (VSV) [168].

Attenuated recombinant VSV vaccine expressing EBOV GP protects non-human primates

from EBOV infection. Interestingly, it has been used to successfully treat a scientist infected

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by EBOV [169]. Clinical trials are in progress in several countries including United States,

Canada, Germany, Gabon and Switzerland. Concerning the last one, clinical trials are

performed in Geneva and began in September 2014. Initial data obtained were very

promising but the development of unexpected mild to moderate joint pain 10 to 15 days

after injection had lead to the suspension of this trial. In January 2015, the trial resumed

using a lower dose and final results are expected soon.

The appearance of reverse genetic tools for EBOV allowed the opening of a new way in the

design of vaccine vectors. For example, it has been shown that EBOV recombinant carrying

mutations in the domain of the VP35 involved in the suppression of IFN production loses its

virulence in a guinea pig model [170]. It also effectively protects guinea pigs during EBOV

infection. However, this method is unsafe since the recombinant EBOV could mutate and

therby regain its pathogenic potential in the vaccinated patient. VLPs expressing

immunogenic proteins such as the NP, GP and VP40 EBOV were also tested [171], but this

approach is expensive and difficult to implement.

VII - Conclusion

This review summarizes the major knowledge on EBOV accumulated during almost 40 years.

Unfortunately, the entry receptors, virus life cycle, immune response and evasion during

infection are not fully understood. As of today, there are no vaccines or efficient treatment

available. However, this virus has caused a lot of deaths since 1976. But the interest for the

research even if it seems to correlate with death cases (Figure S2), was scanty for many

years probably due to the fact that outbreaks spread only in Africa, and thus far away from

Western countries. Interestingly, two cases of Marburg virus (a virus close to EBOV and with

similar symptoms) have been detected in 2008 in the Netherlands [172] and the United

States [173]. These cases alerted the international community on the risk of emergent viral

diseases and have had a positive impact on the number of publications related to EBOV

(Figure S2). In addition, the ongoing outbreak, has caused a huge increase of publications on

EBOV (Figure S3). After almost 40 years and thousands of deaths, EBOV finally begins to

receive some attention from researchers, and more precisely from the organizations that

fund basic research and the pharmacological companies.

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Supplementary information:

Figure S1 : Worldwide geographic distribution of filovirus hemorrhagic fever cases, 1967–2014. From Martines et all (2015) [50]

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Figure S2 : Ebola fatal cases and scientific publications on Ebola, 1976- 2014 [174]

Figure S3 : Ebola fatal cases and scientific publications on Ebola, March 2014 - October 2014 [174]