Ebola and Marburg haemorrhagic fever viruses: major scientific

advances, but a relatively minor public health threat for Africa

E. M. Leroy1,2, J-P Gonzalez1 and S. Baize3

1) Centre International de Recherches Medicales de Franceville, Franceville, Gabon, 2) MIVEGEC (IRD 224, CNRS 5290, Universites Montpellier 1), Institut

de Recherche pour le Developpement, Montpellier, France and 3) Unite de Biologie des Infections Virales Emergentes, Institut Pasteur, IFR128-Biosciences

Gerland-Lyon Sud, Lyon, France

Abstract

Ebola and Marburg viruses are the only members of the Filoviridae family (order Mononegavirales), a group of viruses characterized by

a linear, non-segmented, single-strand negative RNA genome. They are among the most virulent pathogens for humans and great

apes, causing acute haemorrhagic fever and death within a matter of days. Since their discovery 50 years ago, filoviruses have caused

only a few outbreaks, with 2317 clinical cases and 1671 confirmed deaths, which is negligible compared with the devastation caused by

malnutrition and other infectious diseases prevalent in Africa (malaria, cholera, AIDS, dengue, tuberculosis …). Yet considerable human

and financial resourses have been devoted to research on these viruses during the past two decades, partly because of their potential

use as bioweapons. As a result, our understanding of the ecology, host interactions, and control of these viruses has improved consid-

erably.

Keywords: Ebola, immunity, Marburg, outbreak, reservoir, vaccine

Clin Microbiol Infect

Corresponding author: E. M. Leroy, Centre International de Re-

cherches Medicales de Franceville, Franceville, Gabon

E-mail: eric.leroy@ird.fr

Introduction

Ebola (EBOV) and Marburg (MARV) are the only members

of the Filoviridae family and are among the most virulent

pathogens for humans and great apes. The most virulent spe-

cies of these viruses induce acute haemorrhagic fever and

death within a few days in up to 90% of symptomatic individ-

uals [1,2]. The explosive disease course, high case fatality

rate, and lack of specific treatments or vaccines make EBOV

and MARV a major public health issue for Africa, and a cate-

gory A biothreat [3,4]. In the field, clinical specimens must

be handled according to WHO guidelines on viral haemor-

rhagic fever agents in Africa [5], and analyzed in biosafety

level 4 laboratories.

Research on filoviruses has been closely tied to outbreak

activity. Some advances in the biology and pathogenesis of

MARV and EBOV were made just after the discovery of these

agents, in 1967 and 1976, respectively. The two viruses were

then largely neglected until EBOV re-emerged in 1995 in Kik-

wit, Democratic Republic of Congo (DRC), where it caused a

large outbreak that received considerable media attention

worldwide. The extraordinarily high lethality, multifocal haem-

orrhaging, and specificity to African ecosystems enhanced the

fascination of Ebola for the international community, which

saw this disease as a potential threat in the post-Cold War

context [6]. This partly explains why research on the biology,

epidemiology, ecology and pathophysiology of these viruses

has advanced markedly over the past 15 years, leading

recently to the development of potential antiviral drugs and

candidate vaccines. Yet the overall disease burden of filovirus-

es over the past 45 years is minimal compared with major

infectious diseases such as AIDS, malaria, cholera, dengue and

tuberculosis [7,8]. This paper reviews research advances in

Ebola and Marburg ecology, virus–host interactions and dis-

ease control during the past 15 years.

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Filoviruses as Prototypes of EmergingPathogens

An emerging pathogen is defined as either a truly novel or a

hitherto unknown agent, or a pathogen whose incidence has

increased considerably in susceptible populations. EBOV and

MARV fulfil one criterion of this definition, in that they were

identified relatively recently. The first reported cases of

human filovirus infection occurred in 1967 in Marburg, Ger-

many, and in Belgrade, Serbia (former Yugoslavia), when lab-

oratory technicians were infected while manipulating African

green monkeys (Cercopithecus aethiops) imported from

Uganda [9–12]. An unknown infectious agent was recovered

from the victims’ blood and organs and was named ‘Marburg

virus’ after the outbreak location. EBOV first emerged in the

form of two near-simultaneous outbreaks in 1976 in Sudan,

near the border with DRC, and in DRC, near the border

with Sudan [13,14]. An unknown causative agent was isolated

from patients in the DRC outbreak and was named for the

river Ebola, which flows past Yambuku, the outbreak epicen-

tre (Fig. 1).

The morphology of MARV and EBOV is unique among

viruses (Fig. 2a,b). Using electron microscopy, they often

appear as long filamentous particles about 14 000 nm long

and 80 nm wide, whereas other particles assume the shape

of the number 6 or a hairpin [15–17]. MARV and EBOV are

the only members of the Filoviridae family. This family belongs

to the order Mononegavirales, which also includes Rhabdoviri-

dae, Paramyxoviridae and Bornaviridae, a group of viruses

characterized by a linear, non-segmented, single negative-

stranded RNA genome [18–23].

These viruses cause an explosive disease with multiple

haemorrhaging occurring within a few days and a high fatality

rate (up to 90%), which was rarely observed in viral diseases

[24–29]. Generally, after an incubation period ranging from 2

to 21 days (mean 4–9 days), non-specific symptoms such as

fever, headache, nausea and muscle pain occur abruptly.

There are rapidly followed by gastrointestinal problems

(stomach pain, vomiting, diarrhoea), respiratory disorders

(throat and chest pain, cough) and neurological manifesta-

tions (prostration, confusion, delirium), indicating systemic

viral dissemination and multi-organ failure. Haemorrhagic

manifestations vary in severity and location, and often include

skin rash, conjunctival injection, nosebleeds, melaena, hae-

matemesis and bleeding at venepuncture sites. In fatal cases

the symptoms and haemorrhaging rapidly become severe,

and death generally ensues in a context of tachypnoea,

coma and shock. The virus disappears rapidly from the

bloodstream after the resolution of systemic symptoms in

survivors, and prolonged convalescence is frequent, with epi-

sodic non-specific symptoms such as asthenia, myalgia and

fever. More specific signs, associated with transient persis-

tence of the virus in immunologically protected sites such as

the eyes and testicles have been reported during the early

stages of recovery. The virus has been isolated for up to

2 months in semen [30–32]. Biological parameters are not

specific for filovirus infection and most data come from

experimentally infected animals [33,34]. They include mild

and early leucopenia, associated with lymphopenia, thrombo-

cytopenia (< 100 000 platelets/mm3), hyperproteinaemia, and

marked transaminase elevation. The prothrombin time is

always prolonged and fibrin split products are often

detected, indicating disseminated intravascular coagulation

[35,36].

(a)

(b)

Republic

of Congo

2001–02

2003

2005

Democratic

Republic of Congo

1976

1995

2007

2008

Sudan

1976

1979

2004

Uganda

2000

Ivory Coast 1995

Uganda

2007

Gabon

1994

1996

1996–97

2001–2002

Kenya

1980

1987

South Africa

1975

Democratic

Republic of Congo

1998–2000

Angola

2005

Uganda

2007

2008

CIEBOV

BEBOV

SEBOV

ZEBOV

Marburg

Ebola

FIG. 1. Filovirus outbreaks in Africa. Reported outbreaks or isolated

cases of haemorrhagic fever caused by Marburg virus (MARV) (a)

and Ebola virus (EBOV) (b) are indicated with the corresponding

year and colour-coded according to virus species.

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Filoviruses: a Questionable Public HealthBurden for Africa

Since their discovery 45 years ago, filoviruses have been

responsible for only a few outbreaks, resulting in 2317 clini-

cal cases and 1671 confirmed deaths (reviewed in Fig. 1 and

Table 1, [37]). Hence, the disease burden of filovirus infec-

tion in Africa is extremely small compared with other infec-

tious diseases and malnutrition.

As said previously, the first MARV outbreak occurred in

Europe and was associated with 31 cases, including 25 pri-

mary cases (i.e. infected directly by animals) with seven

deaths [9–11]. Since then, only sporadic cases have been

reported, in South Africa in 1975 (three cases, one death)

and in Kenya in 1980 (two cases, one death) and 1987 (one

fatal case) [31,38,39]. MARV subsequently caused two large

outbreaks, in 1998–2000 in DRC and in 2004–2005 in

Angola. The longest MARV outbreak hit the Durba area

(eastern DRC) between 1998 and 2000, affecting 154 people

and killing 83% of victims [40]. Most patients worked in

underground gold mines. The Durba outbreak was charac-

terized by the circulation of multiple viral strains, pointing to

multiple independent introductions from the unknown

(a) (b)

(c)

VP40

VP24

NPVP30RNA

VP35LMembrane

GP1,2

trimer

(d)

FIG. 2. 10Ultrastructure and structure of filovirus virons. Particles of various forms are visualized by negative staining by uracyl acetate (a, b). Part

of a rod-shaped particle showing the tubular nucleocapsid and the surface spikes (c). Schematic representation of a filovirion indicating protein

composition, the tubular nucleocapsid and the surface spikes (d). (From Kuhn JH 9. Filoviruses: a compendium of 40 years of epidemiological, clini-

cal, and laboratory studies. In: Calisher CH editor. Springer, Wien, NewYork; 2008).

TABLE 1. List of filovirus outbreaks with the corresponding

location, number of clinical cases and fatality rate

Virus Date Location CasesFatality(%)

Marburg virus 1967 Germany – Yugoslavia 31 231975 South Africa 3 331980 Kenya 2 501987 Kenya 1 100

1998–2000 DRC 154 832005 Angola 252 902007 Uganda 4 252007 Uganda 1 02008 Uganda 1 100

Ebola virusZaire

1976 DRC 318 881977 DRC 1 1001994 Gabon 49 651995 DRC 315 881996 Gabon 37 57

1996–1997 Gabon 60 752001–2002 Gabon and

Republic of Congo123 79

2003 begin Republic of Congo 143 902003 end Republic of Congo 35 83

2005 Republic of Congo 11 752007 DRC 264 712008 DRC 32 47

Ebola virusSudan

1976 Sudan 286 531979 Sudan 34 652000 Uganda 425 532004 Sudan 17 42

Ebola virusCote d’Ivoire

1994 Ivory Coast 1 0

Ebola virusBundibugyo

2007 Uganda 102 42

DRC, Democratic Republic of Congo.

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natural reservoir [29,40,41]. The largest recorded MARV

outbreak occurred in Uige province of northern Angola in

2004–2005 [42]. It was the first recorded outbreak in West

Africa, causing 252 cases and 227 deaths (fatality rate 90%).

Complete genomic characterization showed that the culprit

strain was closely related to previous East African isolates

[42]. Finally, small MARV outbreaks occurred in western

Uganda in 2007 and 2008 among workers in lead and gold

mines of the Kitaka Cave near Ibanda village [43,44]. Two of

the six victims died.

Ebola virus first emerged in the form of two near-simulta-

neous outbreaks in 1976 caused by two different species,

namely Sudan Ebola virus (SEBOV) in Sudan and Zaire Ebola

virus (ZEBOV) in DRC [13,14]. In Sudan, the outbreak was

centred in the Nzara and Maridi and was responsible for 284

cases. The mortality rate was 53%, characteristic of SEBOV

infection. The epicentre of the DRC outbreak was in Yamb-

uku, about 800 km from Maridi. This outbreak resulted in

318 cases with a mortality rate of 89%, characteristic of

ZEBOV infection. Later there was an unconfirmed lethal case

involving a 9-year-old girl living in Tandala, DRC, [45], fol-

lowed by another SEBOV outbreak in 1979, again in Nzara

and Maridi, with 34 cases and 22 deaths [46]. After a 15-year

period in which no further cases were recorded, EBOV

re-emerged in 1994 for a 3-year period. This new phase was

marked by the identification of a new species, Cote d’Ivoire

Ebola virus (CIEBOV), and by four ZEBOV outbreaks. The

only case caused by CIEBOV occurred in 1994, when an eth-

nologist sickened a few days after autopsying a chimpanzee

found dead in the Tai national park in Ivory Coast [47–49].

A large outbreak then occurred in 1995, in and around the

town of Kikwit, south of DRC, with 315 cases and a mortal-

ity rate of 81% [50]. Despite the deployment of more

sophisticated scientific and medical resources than those

available in 1976, this outbreak was as large as the 1976 out-

break, probably because it affected a town with several hun-

dred thousand inhabitants. Three other outbreaks, all as the

result of ZEBOV, struck northeast Gabon between 1994 and

1997 [51–54]. In total, these outbreaks involved 140 cases

and caused 75 deaths. The first occurred in gold-digger

camps located in the heart of the forest. The second out-

break hit the village of Mayibout, located south of Mekouka,

among children who had carried and butchered a chimpan-

zee carcass found in the forest. The third outbreak occurred

in 1996–1997, with 60 cases and 45 deaths over a 6-month

period. Fifteen cases and 11 deaths were recorded in Libre-

ville, the capital of Gabon, and a South African nurse was

infected by a Gabonese physician who had travelled to

Johannesburg. The period 2000–2008 was marked by

repeated ZEBOV outbreaks, SEBOV resurgence, and the dis-

covery of a new species of Ebola virus, Bundibugyo Ebola

virus (BEBOV). Between 2001 and 2005, Gabon and Republic

of Congo (RC) were hit by five ZEBOV outbreaks [55–58].

The first outbreak occurred within the cross-border area

between northeast Gabon and northwest RC, and resulted

in 143 cases and 128 deaths. RC was again affected twice in

2003 (respectively 143 and 35 cases, and 128 and 29 deaths),

and then in 2005 at Etoumbi, where only 11 cases (nine fatal-

ities) were reported. Two outbreaks of Sudan Ebola virus

also occurred during this period. One hit Uganda in 2000,

comprised three foci and was the largest of all recorded

EBOV outbreaks, causing 173 deaths among its 425 victims

[59,60]. A second SEBOV outbreak occurred in Sudan in

2004 in the town of Yambio, located near Nzara and Maridi.

There were 17 cases and seven deaths [61–63]. The latest

species to be discovered, Bundibugyo Ebola virus (BEBOV),

was discovered in 2007 in Uganda, where it was responsible

for a large outbreak [64] with 116 cases and 30 deaths (fatal-

ity rate 26%).

These four EBOV species are all pathogenic for humans

and arose in sub-Saharan Africa. An additional species, Res-

ton Ebola virus (REBOV), was isolated in 1989 from Asian

cynomolgus monkeys (Macaca fascicularis) imported from the

Philippines and housed in a quarantine facility in Reston, Vir-

ginia, USA. The monkeys developed a haemorrhagic disease

associated with high lethality, and subsequent outbreaks

among cynomolgus monkeys were reported in animal facili-

ties in Texas, USA, Italy and the Philippines [39,65–68].

Although no humans fell sick during these episodes, several

monkey handlers in the United States and in the Philippines

were shown to have seroconverted [69,70]. Recently, REB-

OV was also isolated from domestic Philippino swine with a

severe respiratory syndrome and co-infected by porcine

reproductive and respiratory syndrome virus [71].

Filoviral Genes and Proteins

The MARV and EBOV genomes are about 19 000 nucleo-

tides long and are transcribed into eight major subgenomic

mRNAs, which encode seven structural proteins: 3¢ leader,

nucleoprotein (NP), virion protein 35 (VP35), VP40, glyco-

protein (GP), VP30, VP24, and RNA-dependent RNA poly-

merase (L)-5¢ trailer; and one non-structural protein (sGP),

shown in Fig. 2. The central core of the virion is composed

of the ribonucleoprotein complex, which consists of the

genomic RNA molecule encapsulated by NP linked to the

inner matrix proteins VP30 and VP35 and the RNA-depen-

dent RNA polymerase (Fig. 2d). This complex is involved in

transcription and replication [72–74]. The matrix proteins

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VP40 and VP24 are linked to the ribonucleoprotein complex

at the inner surface of the lipid bilayer of the viral envelope,

which is derived from the host cell. VP40 and VP24 are

involved in viral nucleocapsid formation, viral budding or

assembly, and host range determination [72,75–82]. The viral

envelope contains only the glycoproteins, organized as tri-

meric spikes (Fig. 2c,d) consisting of two fragments (extracel-

lular protein, GP1, and membrane-anchored protein, GP2)

which result from furin-like enzyme cleavage of precursor

polyproteins and remain linked by a disulphide bond [74,83–

85]. GP binds preferentially to endothelial and monocytic

cells via GP1, mediating target cell entry, inducing endothelial

cell disruption and cytotoxicity in blood vessels in vitro, and

causing immunosuppression in vitro via a retrovirus-like pep-

tide [74,86–94]. EBOV differs from MARV and from other

members of the Mononegavirales in that infected cells

secrete large amounts of a non-structural glycoprotein

(sGP), the primary product of the GP gene, which is

expressed from non-edited mRNA species [93,95,96]. The

role of sGP is poorly understood. Finally, the matrix proteins

VP35 and VP24 seem to play a key role in the pathogenicity

of EBOV and MARV, by inhibiting antiviral responses and

particularly type 1 interferon (IFN) synthesis [97–102].

Filoviruses Genomics

The Filoviridae family comprises two genera, Marburgvirus and

Ebolavirus, and belongs to the order Mononegavirales, a

group of viruses characterized by a genome consisting of a

linear, non-segmented, single negative strand of RNA [18–

23]. Molecular evolutionary analyses of the glycoprotein

genes suggests that EBOV and MARV diverged several thou-

sand years ago [103]. The genus Marburgvirus consists of a

single species, Marburgvirus, comprising five viruses differing

from one another by up to 21% at the nucleotide level, while

there are five known species of Ebolavirus that have different

geographic locations, case fatality rates, and nucleotide

sequence divergences of about 32–41% [16].

Our knowledge of filovirus distribution and diversity may

be limited because EBOV and MARV have only been identi-

fied and characterized in sick or dead humans and animals

[40,42,43,53–56,64,68,71,104–107]. Numerous studies have

attempted to determine the regions of EBOV and MARV cir-

culation. Remotely sensed data and niche ecologic modelling

suggest that filoviruses may circulate within a much wider

region than their outbreak areas [108–111]. Using a sensitive

and specific ELISA method, an overall ZEBOV-specific IgG se-

roprevalence of 15.3% was found in Gabon, the highest ever

reported [112]. The seroprevalence was significantly higher

in forested areas (19.4%) than in grassland (12.4%), savannah

(10.5%) and areas rich in lakes (2.7%). These results are con-

sistent with previous reports of seroprevalence rates ranging

from 1.8% to 21.3% throughout central Africa using poorly

specific immunofluorescence antibody testing 2methods [113–

117]. More convincingly, two recent small serosurveys, based

on the same ELISA as the one we used, have shown high

antibody prevalence in some forested regions of Central

Africa. Indeed, a 9.3% (15/161) prevalence of ZEBOV-specific

IgG was found in unaffected villages surrounding Kikwit a few

weeks after the 1995 outbreak [118], and a 13.2% (25/190)

seroprevalence was found in the Aka Pygmy population in

the Central African Republic, where no ZEBOV outbreaks

have been reported [119]. Such high seroprevalence rates

indicate a high level of ZEBOV circulation in the tropical rain

forest environment and suggest that human rural populations

are highly exposed to the virus, with mild or asymptomatic

infection by outbreak strains [120] or less pathogenic strains,

or simple exposure to inert viral particles.

Filoviruses in Wildlife

Filovirus haemorrhagic fevers are typical zoonotic diseases

transmitted accidentally by direct contact with live or dead

animals. The role of wildlife species in the human epidemiol-

ogy of ZEBOV is only partly understood (Fig. 3). In addition

to its high pathogenicity for humans, ZEBOV has caused

massive outbreaks among gorillas and chimpanzees, killing

thousands of animals during the last decade in parts of Ga-

bon and RC [56,121–125]. Another study conducted in the

Tai forest of Ivory Coast showed that the disappearance of

11 members (26%) of a group of 43 chimpanzees during

November 1994 may have been the result of CIEBOV [126].

Such massive outbreaks in great apes could have major impli-

cations for animal conservation. The identification of multiple

strains during the 2001 Gabon/RC outbreak and the recent

identification of two phylogenetically divergent lineages sug-

gest independent introductions into great ape and human

populations following multiple viral spill-overs from a reser-

voir host [56,107,127]. In this ‘multi-emergence’ hypothesis,

Ebola outbreaks would occur episodically in certain ecologi-

cal conditions caused by habitat disturbances or climatic phe-

nomena [108,109]. Although the idea of multi-emergence

makes no reference to a particular timescale, this theory also

implicitly assumes that ZEBOV was present in Equatorial

Africa long before the first documented outbreak in 1976, as

supported by various serosurveys [128–130].

Although many field and laboratory studies have been

conducted since the first recorded outbreak in an attempt

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to identify the reservoir species of MARV and EBOV, conclu-

sive evidence that bats are natural hosts for filoviruses was

obtained only recently [14,45,131–138]. Antibodies and

nucleotide sequences specific for ZEBOV [127,139] were

detected in the liver and spleen of three fruit bat species in

Gabon and RC (Hypsignathus monstrosus, Epomops franquetti

and Myonycteris torquata), and antibodies and nucleotide

sequences specific for MARV [140,141] were found in a fruit

bat species in Gabon (Rousettus aegyptiacus) and in two insec-

tivorous bat species in DRC (Rhinolophus eloquens and Mini-

opterus inflatus). More recently, MARV was isolated from

cave-dwelling Rousettus aegyptiacus bats in Uganda [43].

Finally, a recent study showed that the 2007 Luebo outbreak

in DRC was linked to massive fruit bat migration, strongly

suggesting that humans could be infected directly by bats

(Fig. 3) [142].

Pathogenesis of Ebola Virus Infection andImmune Responses

Antigen-presenting cells such as dendritic cells and macro-

phages are major EBOV targets [143,144]. Indeed, by their

presence in mucosal tissues and skin, these cells are infected

early and serve as the substrate for the first replicative viral

cycles. Because of their extensive distribution among the dif-

ferent organs and tissues, and their mobility, dendritic cells

and macrophages are probably responsible for viral dissemi-

nation and systemic infection [145]. Intense viral replication

then occurs in secondary lymphoid organs and liver, and the

virus subsequently spreads to hepatocytes, endothelial cells,

fibroblasts and some epithelial cells [146,147]. The pathogen-

esis of filovirus infection is reviewed in Fig. 4.

However, despite widespread viral replication, the

observed damage to endothelia, liver and other organs is not

sufficiently severe to cause terminal shock and death. Endo-

thelial infection is infrequent, occurring in the terminal stages

with no clear evidence of cytopathic effects [36]. Similarly,

although multifocal hepatic necrosis may be linked to viral

replication in liver, the observed hepatocellular lesions can-

not directly lead to death [146,147]. A pathogenic role of

the host response is therefore suspected.

Extensive infection of monocytes and macrophages leads to

aberrant release of inflammatory mediators and chemokines

such as interleukin-1b (IL-1b), tumour necrosis factor-a (TNF-

a), IL-6, IL-15, IL-16, IL-1 receptor antagonist, soluble TNF

receptor, IL-10, NO), IL-8, growth regulated oncogene-a,

CCL3, CCL4, CXCL10, monocyte chemotactic protein-1, and

eotaxin [144,148–151]. This ‘cytokine storm’, particularly

prominent in the terminal stages of the disease in humans and

non-human primates, has dramatic consequences. It is proba-

ble that TNF-a, NO), and other vasoactive compounds play a

crucial role in the associated vascular leakage, by increasing

endothelial permeability, reducing vascular tone, and altering

endothelial cell functions [144,152–154]. In addition, some of

these mediators may induce the expression of adhesion mole-

cules at the surface of endothelial cells, so allowing neutroph-

ils and monocytes to invade sites of infection where they may

cause bystander tissue damage [155]. Infected macrophages

are involved in the coagulopathy and in the induction of dis-

seminated intravascular coagulation by their abundant expres-

sion of tissue factor [35,156,157]. Other consequences of the

massive antigen-presenting cell infection are lymphoid deple-

tion in the spleen, lymph nodes and thymus, and extensive

apoptosis of T lymphocytes and natural killer cells, as

observed in blood and tissues of human and non-human pri-

mate fatalities [145,150,154,158–161]. T-cell death cannot be

the result of a direct viral cytopathic effect, as lymphocytes

are not infected by EBOV, but would rather result from inter-

actions with infected antigen-presenting cells and soluble medi-

Reservoirs of ZEBOV & MARV:

fruit bats species

Intermediary susceptible species:

virus amplification in great apes and duikers

Outbreak

Suspected

Direct

contact

FIG. 3. 11Model of the natural cycle of filovirus. The diagram shows animal-to-human transmissions leading to outbreak appearance. Several Zaire

Ebola virus (ZEBOV) outbreaks appeared when hunters handled the infected carcasses of chimpanzee, gorilla and duiker. Several ZEBOV and

Marburg virus (MARV) outbreaks were suspected to be associated with exposure to fruit bats.

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ators [36,150,158]. A viral protein with superantigen activity

might also be involved [162]. Although T-cell depletion proba-

bly induces profound immunosuppression, the consequences

for disease progression are unclear [163,164]. Fatal Ebola

haemorrhagic fever is also associated with defective humoral

responses, with absent specific IgG and barely detectable IgM

[158,165]. This impairment of adaptive immunity may result

from extensive infection of dendritic cells. Indeed, EBOV-

infected dendritic cells produce only a limited panel of cyto-

kines without full activation and maturation, which may impair

their ability to trigger adaptive immunity [143,145,166,167].

Some viral factors are also able to counteract innate

immunity and thereby impede the control of viral replication.

Indeed, VP35 blocks IFN-a/b synthesis by preventing the acti-

vation of interferon regulatory factors 3 and 7

[97,99,101,102,168]. VP35 also interferes with the activation

of dsRNA-dependent protein kinase [169], and, like VP30

and VP40 [170], is a suppressor of RNA silencing [171].

VP24 impairs the nuclear accumulation of tyrosine-phosphor-

ylated signal transducer and activator of transcription 1 and

thereby inhibits IFN-a/b and IFN-c signalling [172,173]. Inhibi-

tion of type I IFN responses seems to be crucial for EBOV

virulence, as viruses that have lost this property by VP35

mutation are attenuated in vitro and in vivo [174,175].

In striking contrast with fatal outcome, effective control of

EBOV infection is associated with balanced immune

responses. A transient and well-regulated inflammatory

response is observed early in the disease not only in patients

that survive, but also in asymptomatic subjects [120,149].

This response may help to control the infection and to

induce specific immunity. An early and robust humoral

response is strongly correlated with survival in symptomatic

patients [158,165]. In asymptomatic EBOV-infected subjects,

antibodies are detected about 3 weeks after infection and in

moderate amounts [120]. In survivors and asymptomatic sub-

jects, high antibody titres persist for years [176]. Although

FIG. 4. A model of pathogenesis of filovirus infection, based on findings with Zaire Ebola virus. Dendritic cells and macrophages are early targets

of filoviruses. The virus spreads from the initial site of infection to secondary lymphoid organs and liver where intense replication takes place.

Then, other cells are infected such as hepatocytes, endothelial cells, fibroblasts and some epithelial cells. The inhibition of type I interferon (IFN)

production by viral proteins leads to relentless viral replication in most organs. The extensive infection of antigen-presenting cells (APC) leads to

altered inflammatory response and uncontrolled release of mediators. This ‘cytokine storm’ contributes to the pathogenesis by attracting inflam-

matory cells towards infected tissues, inducing coagulopathy and increasing endothelial permeability and vascular leakage. In addition, infected

APC and/or soluble factors are responsible for the defective adaptive immunity and the massive apoptosis of T lymphocytes. Multifocal necrosis

of hepatocytes, liver failure, destruction of secondary lymphoid organs and other tissue damage may result from direct cytopathic effects but also

from the host response. Together, these events lead to multi-organ failure, impairment of the vascular system, terminal shock and death.

COLOR

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antibodies probably participate in the control of infection,

their precise role is unclear. Importantly, control of EBOV

infection is also characterized by a lack of T-cell apoptosis

and by the circulation of activated T cells (probably including

cytotoxic T lymphocytes), coinciding with a fall in viraemia

and the disappearance of symptoms [158]. A similar

response also occurs in asymptomatic individuals [177], sug-

gesting that T-cell responses are crucial for controlling the

infection. The factors underlying the radically different out-

comes of EBOV infection are unclear, but may include the

route of infection, the size of the inoculum, the cell types

that are initially infected, previously acquired immunity [112],

heterologous immunity, and MHC status [178]. However, it

is unlikely that outcomes were related to different virus

strains, as no difference was found in the coding sequences

of viruses isolated from survivors and fatalities and different

outcomes can be observed in patients infected with the same

viral source [51,105].

Treatment and Prevention

Although several advances in the treatment and prevention

of EBOV infection have recently been described, there is no

licensed vaccine, and the only way to limit outbreaks is to

isolate patients. The development of therapeutic measures is

hampered not only by the need to manipulate EBOV in bio-

safety level 4 facilities, but also by the limited number of

people affected since the discovery of filoviruses. However,

the increasing frequency of EBOV outbreaks in humans and

great apes [56,122–124] and the recent emergence of new

EBOV species [47,64], give evidence for the intense circula-

tion of EBOV in the endemic area.

The main challenge is to develop a vaccine conferring

cross-protection against heterologous EBOV species, includ-

ing emerging strains, ideally after a single dose. Several vac-

cine candidates have shown good efficacy in non-human

primates over the last 10 years. The use of live viral vectors

or virus-like particles to produce EBOV GP is a promising

approach, providing sterilizing immunity after a single dose

[179–184]. Recent studies have demonstrated the feasibility

of a multivalent vaccine able not only to protect against the

filovirus species used to formulate the candidate

[180,185,186] but also to cross-protect against an emerging

species [187]. However, several concerns must be dealt with

before human use, such as the safety of the viral vector, pos-

sible pre-existing immunity to the vector, and formulation of

a single-dose cross-protective vaccine. Results obtained with

candidate vaccines confirm the major role of T-cell

responses, and of CD8+ T cells in particular, in the control

of filovirus infections, as well as the presence of cross-react-

ing immunogenic epitopes on the viral GP.

A vesicular stomatitis virus-based candidate also has thera-

peutic potential, conferring substantial post-exposure protec-

tion when administered very rapidly to non-human primates

[188,189]. Passive immunization with convalescent plasma or

monoclonal antibodies seems to be ineffective [190–192],

tending to confirm that humoral responses are not primarily

responsible for the control of EBOV infection. Similarly,

recombinant type I IFN administration fails to protect non-

human primates [193]. One promising approach is to manip-

ulate the coagulation system by inhibiting the tissue factor

pathway with a factor VIIa/tissue factor inhibitor [156] or by

activating the natural anticoagulant protein C pathway with

recombinant activated protein C [194]. However, the sur-

vival benefit is limited. Another approach is to target viral

replication with small interfering RNA or antisense oligonu-

cleotides [195–198], although the latter is limited by the

need to use virus species-specific sequences, which are not

known in the early stages of an outbreak.

Conclusions

Substantial advances in our understanding of the ecology of

EBOV and MARV, as well as host–virus interactions and dis-

ease control, have been made during the past two decades.

This period was marked by the first recorded MARV out-

break in West Africa, resurgence of Ebola Sudan in Sudan

and Uganda, the discovery of two new species (Cote d’Ivoire

and Bundibugyo), and escalation of ZEBOV outbreaks in the

border region of Gabon and the Republic of Congo. Bats

have been identified as a major filovirus reservoir, and many

human outbreaks have been shown to have arisen through

the handling of infected chimpanzee and gorilla carcasses.

The 2007 ZEBOV outbreak was linked to fruit bats, although

the precise mechanism of transmission to humans was not

identified. MARV and ZEBOV induce profound suppression

of adaptive immunity, characterized by massive B-lymphocyte

and T-lymphocyte apoptosis largely mediated by the TRAIL

(TNF-related apoptosis-inducing ligand) and Fas pathways. A

recent study suggests that superantigenic activity may be

involved and numerous studies have shown that VP35 and

VP24 play an essential role in ZEBOV suppression of IFN-a/

b production.

Transparency Declaration

Conflicts of interest: nothing to declare.

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