This document provides a summary of the current Ebola virus outbreak as of March 2015. It discusses the epidemiology of Ebola virus, including the four known species and the current West African outbreak that has caused over 24,000 infections and 9,000 deaths. It also summarizes the classification, morphology, and genome organization of Ebola virus, describing the seven genes and proteins encoded including nucleoprotein, polymerase cofactor, glycoprotein, and RNA-dependent RNA polymerase. The functions of these proteins in viral transcription, replication, and evasion of the immune response are examined.

1. 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

2. 2
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]

3. 3
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.

4. 4
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.

5. 5
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]

6. 6
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

7. 7
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

8. 8
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

9. 9
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]

10. 10
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,

11. 11
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.

12. 12
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].

13. 13
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

14. 14
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

15. 15
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.

16. 16
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

17. 17
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]

18. 18
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].

19. 19
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

20. 20
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.

21. 21
Figure 8 : EBOV proteins interfering with interferon signaling

22. 22
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].

23. 23
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

24. 24
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

25. 25
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.

26. 26
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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]