C Elegans

2003 Nature PublishingGroup
PERSPECTIVES
The total number of infected individuals was
284, with 151 deaths
1,2
. By theend of August, a
second epidemic started in the equatorial rain
forest of northern Zaire, now Democratic
Republic of the Congo (FIG. 1). The presumed
index casecameto theYambuku Mission hos-
pital for treatment of acute malaria. However,
it remainsunclear whether thisindividual was
the source of the epidemic or became infected
in the hospital. Most subsequent cases had
contact with patients, but for more than 25%
of cases the only known risk factor that was
elucidated wasreceipt of injectionsat the hos-
pital. Although transmission was focused in
theoutpatient clinicsof thehospital, therewas
subsequent dissemination in the surrounding
villagesto people who were caring for sick rel-
atives or attending childbirth, or through
other forms of close contact. The case fatality
rate in this outbreak was 88% (318 cases, of
which 280 died), much higher than with the
Sudan outbreak (53%)
2,3
.Virus was isolated
from patients of both outbreaks and named
Ebola virus (EBOV) after a small river in
northwestern Zaire. For several years, a direct
link between the two EBOV epidemics was
assumed. Later, it wasdiscovered that the out-
breaks were caused by two distinct species of
filoviruses, today known as Sudan ebolavirus
(SEBOV) and Zaireebolavirus(ZEBOV).
Notably, the first filovirusto be discovered
was Marburg virus (MARV), which was iso-
lated during an outbreak of haemorrhagic
fever in Europe in 1967 (REF. 4). Until the
recent outbreak in the northern part of the
Democratic Republic of the Congo, there
have been only a few isolated cases of MARV
haemorrhagic fever in Africa. This outbreak
hasbeen the largest so far with more than 100
cases, high mortality and several distinct
introductions.
Emergence/re-emergence of Ebola viruses.
EBOV surprised everyone when it emerged
in the United States in 1989. The virus was
detected in Cynomolgus monkeys (Macaca
fascicularis), which were imported from the
Philippines into a primate facility in Reston,
Virginia
5
. Despite initial arguments, no clear
link to Africa could be established, so the pre-
sumption prevails that this new virus, which
isa distinct speciesknown asReston ebolavirus
(REBOV), could be of Asian origin. The 1989
monkey outbreak spread through affected
roomsby droplet contact with adjacent cages
or to distant cages and to different rooms by
larger dropletsand/or small-particle aerosols.
The potential airborne route of transmission
and the presumptive lower human patho-
genicity (no disease was found in infected
workers) remain interesting features that are
associated with theREBOV species.
Since the first description of EBOV, the
virus has re-emerged several times in central
Africa, which isconsidered to be endemic for
EBOV (FIG. 1). In 1992 and 1994, two episodes
of mortality were recorded among a troop of
chimpanzees in the Tai National Park in the
western Ivory Coast. Several of the dead ani-
mals showed signs of haemorrhages, and a
necropsy was carried out on one of the ani-
malsin the field.A 34-year-old woman devel-
oped fever, headache and myalgia eight days
after carrying out thenecropsy. Fivedayslater,
she developed a syndrome that wassimilar to
that described for surviving EBOV-infected
patients.A distinct EBOV species, Ivory Coast
ebolavirus(ICEBOV), was confirmed as the
causeof thisdisease
6
(FIG. 1).
EBOV becamea real threat to public health
when it re-emerged in Kikwit, Democratic
Republic of the Congo, in 1995. This out-
break received much attention from the
media and made EBOV one of the most
feared infectious agents worldwide. Subse-
quent outbreaks in Gabon and Uganda, as
Ebola virus, being highly pathogenic for
humans and non-human primates and the
subject of former weapons programmes,
is now one of the most feared pathogens
worldwide. In addition, the lack of pre- and
post-exposure interventions makes the
development of rapid diagnostics, new
antiviral agents and protective vaccines a
priority for many nations. Further insight into
the ecology, immunology and pathogenesis
of Ebola virus will promote the delivery of
these urgently required tools.
Ebola virus (EBOV) was discovered in 1976,
but remained largely unknown until 1995,
when it re-emerged in Kikwit, Democratic
Republic of the Congo (formerly Zaire). In
this article, we describe the discovery of the
virus and the events that have led to recent
attemptsat vaccinedevelopment.
Discovery of Ebola virus
First appearance. The story begins in 1976 in
Central Africa. In June and July, the first cases
of a haemorrhagic disease were reported from
Nzara, a small town in the Western Equatorial
Province of southern Sudan, bordering the
African rain forest (FIG. 1). Theoutbreak started
with the infection of individualsfrom a single
cotton factory (index cases) and quickly spread
to the close relatives of these individuals. The
epidemic was augmented by the exportation
of casesto neighbouring areas, and high levels
of transmission occurred in the hospital of
Maridi a teaching centrefor student nurses.
The outbreak lasted until November, during
which time approximately 15 generations
of person-to-person transmission occurred.
NATURE REVI EWS | I MMUNOLOGY VOLUME 3 | AUGUST 2003 | 6 7 7
Ebola virus: from discovery to vaccine
Heinz Feldmann, Steven Jones, Hans-Dieter Klenk
and Hans-Joachim Schnittler
TI ME L I NE
6 7 8 | AUGUST 2003 | VOLUME 3 www.nature.com/reviews/immunol
P E R S P E C T I V E S
infectious in tissue culture, indicating that
proteolytic cleavage is not a requirement for
infectivity
10
. The infectious clone system will
be instrumental for future studies on protein
function, genome transcription and replica-
tion, pathogenesis and the development of
therapeutic and prophylactic interventions.
reduce the cytotoxicity of transmembrane
GP
9,13
. A slightly different, and independently
established, system was used to study the role
of proteolytic cleavage for the activation of
transmembrane GP
10,14
. A recombinant virus
that expresses non-cleaved GP molecules on
the surface was isolated and shown to be
well as the present outbreak in Congo-
Brazaville, which hasaffected many great apes
that have a role in transmission to humans
7
,
keep this image alive and also emphasize the
importance of EBOV infections for local and
international public health. Since the tragic
eventsof 11 September 2001, another dimen-
sion was added bioterrorism. Active
weapon-development programmes to pro-
duce large quantities of filoviruses (MARV
and EBOV) were carried out in the past.
Today, EBOV is not only feared as one of the
most pathogenic human agents, but also asan
agent that poses a potential bioterrorism
threat
8
.
Molecular characterization
Originally MARV and EBOV were classified
in the family Rhabdoviridae, but in 1982, the
family Filoviridaewas created on the basis of
the unique morphological, morphogenic,
physiochemical and biological features (FIG. 2
and BOX 1). The molecular characterization of
EBOV began at the end of the 1980s. Despite
all of the attention on EBOV, surprisingly, the
first complete genome sequence of a filovirus
was published for MARV. Today, full-length
sequences are available for two MARV iso-
lates, the Mayinga strain of ZEBOV, as well as
the Philippine and Pennsylvania strains of
REBOV (FIG. 2).
A scientific breakthrough in the field was
made with the development of the infectious
clone for EBOV
9,10
(FIG. 2). This success has to
be attributed to the knowledge that was
gained from work done with artificial mini-
genome systems
11,12
. The infectiousclone sys-
tem was used to create an editing site mutant
that led to the overexpression of transmem-
brane glycoprotein (GP) and the loss of
expression of solubleglycoprotein (sGP)
9
. The
recombinant virushad an increased cytopath-
ogenic effect, but reduced virus production,
indicating that RNA editing is required to
Disc overy of
Marb urg virus
the first filovirus
Classific ation of
Eb ola virus into a
d istinc t family
Filoviridae
Disc overy of Sudan
ebolavirus (SEBOV)
and Zaire ebolavirus
(ZEBOV)
Emergenc e of
Reston ebolavirus
(REBOV)
First c omp lete genome
seq uenc e of a filovirus (Lake
Victoria marburgvirus, strain
Musoke)
Emergenc e of Ivory
Coast ebolavirus
(ICEBOV)
First c omp lete
genome seq uenc e
of ZEBOV
Estab lishing of the small rod ent
mod el for ZEBOV imp ortant
for the d evelop ment of antiviral
agents and vac c ines
Protec tion of non-
human p rimates
against a lethal
c hallenge with ZEBOV
Develop ment of the
ZEBOV minigenome-
b ased reverse- genetic s
system
Infec tious c lone
for ZEBOV
Re- emergenc e of ZEBOV
in Kikwit, Democ ratic
Rep ub lic of the Congo
this outb reak mad e Eb ola
virus known world wid e
1967 1976 1982 1989 1992 1993 1994 1995 1998 1999 2000 2001
T imeline | Key events
Gabon, 1994
Unknown cases
Gabon, 1996
50 cases / 50% lethality
Gabon, 1996/1997
Gabon, Congo 2001/2002
Congo, 2003
Democratic Republic
of the Congo, 1976
Democratic Republic
of the Congo, 1995
Ivory Coast, 1994
1 case / survivor
Durba, Uganda, 2000/2001
Sudan, 1976
Sudan, 1979
Figure 1 | Geographical locations of Ebola-virus outbreaks. O utbreaks of Ebola virus (EBO V)
were reported from one western and several central African countries. Case numbers and lethalityare
indicated. The stars indicate countries where EBO V-specific antibodies have been reported in parts of the
population. The reports are mainlybased on antibodydetection byindirect immunofluorescence assay
(IFA) (data are adapted from REF. 91).
cells
31,32
. However, the direct binding of sGP
to neutrophilshasbeen challenged by others
33
.
Relatively high levelsof sGP, thelarger cleavage
fragment GP1 and soluble GP1GP2 (BOX 1)
are released into the medium of filovirus-
infected cells, and sGP has been detected in
patient serum. These findings led to the
hypothesisthat solubleglycoproteins,especially
sGP, might effectively bind antibodies that
might otherwise be protective
34,35
. It has also
been hypothesized that these soluble glyco-
proteins might function as mediators in the
activation of mononuclear phagocytic cells
and endothelial cells
21,36
(FIG. 3). In addition,
filovirus transmembrane GP has a carboxy-
terminal region that resembles a putative
immunosuppressivedomain that isalso found
in glycoproteins encoded by retroviruses
37,38
,
but it is not known whether this domain
actually contributesto immune suppression.
Concepts in pathogenesis
Although important scientific achievements
have been made in the past, our knowledge of
the pathogenic mechanismsof EBOV and the
host immune response is still limited. EBOV
is known to cause the most severe form of
haemorrhagic disease in both humans and
non-human primates, and shows the highest
case-fatality rates for viral haemorrhagic
fevers (reported up to 88%). The disease is
caused by marked replication of virus
together with immune and vascular dysregu-
lation. In this way, it is as much an immune
syndrome asa virus-induced vascular disease.
Aswith someother viral haemorrhagic fevers,
infection with EBOV is associated with fluid-
distribution problems, hypotension, coagula-
tion disorders and bleeding, which finally
result in the sudden onset of severe shock.
EBOV haemorrhagic fever can, therefore, be
compared to a syndrome that is provoked by
systemic treatment with cytokinesor induced
by an endotoxin
15
.
Impaired immuneresponse. In humans and
monkeys, there is marked disruption of the
parafollicular regionsin the spleen and lymph
nodes, and replication of filovirusesin mono-
nuclear phagocytic cells(cytolytic in vitro) has
been shown
1621
. Widespread presence of
bystander apoptosis of lymphocytes during
infection with EBOV has been noted in non-
human primateshowever, there is, asyet, little
information on the role of apoptosis, in gen-
eral, in the pathogenesis of EBOV, other than
an observed concomitant lossof CD8
+
T cells
and plasma cellsfrom theimmunesystem
18
.
There is also evidence that filoviruses
interfere with innate immunity, which nor-
mally has an important role in the control of
virus replication, allowing the adaptive
immune system to clear the virus. It has been
shown i n i nfected humans that there are
clear differences in the expression of cyto-
kines between fatal cases and patients who
survive
2225
. In particular, thepresenceof inter-
leukin-1(IL-1) and elevated concentrations
of IL-6in the plasma during the symptomatic
phase have been indicated asmarkersof non-
fatal infection, whereas the release of IL-10
and high levelsof neopterin and IL-1 receptor A
(IL-1RA) in the plasma, as soon as a few days
after the onset of disease, isindicative of a fatal
outcome (FIG. 3). Studies with ZEBOV have
shown that the virus blocks double-stranded
RNA (dsRNA)- and virus-mediated induction
of interferon (IFN)-responsive promoters
and the IFN- promoter. In particular, viral
protein 35 (VP35) was shown to function as
an inhibitor of the type I IFN response and
might, therefore, be an important feature in
the pathogenicity of EBOV
26,27
. The impor-
tance of the type I IFN response can also be
shown in the mouse model
28
. Although the
mouse-adapted ZEBOV strain seems to have
been selected during serial passage for itsabil-
ity to block the production of type I IFNs,
knockout micethat lack an effectivetypeI IFN
response are susceptible to lethal infection
by several non-adapted EBOV and MARV
strains
29
. Recently, it was also shown that
infected monocyte-derived dendritic cellswere
impaired in thesecretion of pro-inflammatory
cytokines, the production of co-stimulatory
moleculesand thestimulation of T cells
30
.
For EBOV, it has been reported that sGP
interacts with the host immune response by
binding to neutrophils through CD16b
the neutrophi l-speci fi c form of the Fc-
receptor III (FcRIII) and, subsequently,
i nhi bi ti ng the early activati on of these
Box 1 | Filoviruses
Filoviruses constitute a separate family in the order Mononegavirales. The family consists of
the genera Marburgvirus(MARV) and Ebolavirus(EBOV); the genus Ebolavirusis further
subdivided into four distinct species: Ivory Coast ebolavirus(ICEBOV), Reston ebolavirus
(REBOV), Sudan ebolavirus(SEBOV) and Zaireebolavirus(ZEBOV). Filoviruses consist of
a single, negative-stranded, linear RNA genome that is non-infectious and does not contain
a poly(A) tail. After entry into the cytoplasm of host cells, it is transcribed to generate
polyadenylated subgenomic messenger RNA species. The genome has the following
characteristic gene order: 3 leader, nucleoprotein (NP), virion protein 35 (VP35), VP40,
glycoprotein (GP), VP30, VP24, polymerase (L) protein and 5 trailer (FIG. 2). Transcription
and translation leads to the synthesis of seven structural polypeptides with presumed
identical functions for all of the different filoviruses. Four proteins are associated with the
virus genomic RNA in the ribonucleoprotein complex: NP, VP30, VP35 and the L protein.
NP, VP35 and L are essential and sufficient for the transcription, as well as the replication,
of MARV, whereas EBOV-specific transcription also depends on the presence of VP30. The
three remaining structural proteins are associated with the membrane. GP is a type I
transmembrane protein that forms the spikes on the virus particle. It is synthesized as a
precursor (preGP) that is post-translationally cleaved by furin or a furin-like endoprotease
into the disulphide-linked fragments GP1 and GP2. The homotrimeric GP1GP2
functions in receptor binding and fusion and is the target for the neutralizing host immune
response. Of the two membrane-associated non-glycosylated proteins (VP24 and VP40),
VP40 functions as the matrix protein. The structure and function of VP24 has not yet been
studied. A non-structural, soluble glycoprotein (sGP) is expressed as the primary product
(non-edited mRNA) of the GPgene by EBOV, but not by MARV
36
.
Ultrastructural studies indicate an association of virus particles with coated pits for the
initiation of infection, indicating that filoviruses enter cells by receptor-mediated endocytosis.
GP mediates receptor binding and subsequent fusion. The asialoglycoprotein receptor, folate
receptor-, integrins (especially the 1 group), DC-SIGN (dendritic-cell specific intercellular
adhesion molecule-grabbing nonintegrin) and L-SIGN (liver/lymph node-SIGN) have all been
described as potential attachment factors to promote infection with filoviruses
40,8386
. Uncoating
is presumed to occur in a manner analogous to that of other negative sense RNA viruses.
Transcription and genome replication take place in the cytoplasm and follow, in general, the
models of Paramyxoviridaeand Rhabdoviridae. Transcription starts at the conserved start site
and polyadenylation occurs at a series of uridine residues in the stop site. The 5-terminal non-
coding sequences favour hairpin-like structures for all viral mRNA. Replication involves the
synthesis of a full-length positive-stranded copy. During infection, nucleocapsids accumulate
intracellularly and form intracytoplasmic inclusion bodies. Virions are released by budding
through the plasma membrane
36
.
For further reading on the history, epidemiology, molecular biology and pathogenesis of
filoviruses, we refer the reader to several review articles and books (REFS2,36,8790).
Determinants of cytotoxicity. Infection with
filovirus leads to a moderate cytopathic effect
in susceptible tissue culture and host target
cells. However, the mechanism of cell destruc-
tion is unknown. It is possible that either the
marked production and accumulation of
virusproteinsor the maturation of viruspar-
ticlesat the plasma membrane are involved in
this process. Alternatively, a virus protein
might havespecific cytotoxic potential. Several
groups have shown cell alteration after the
expression of EBOV-encoded GP
13,39,40
. In one
study, it wasreported that a serine-threonine-
rich mucin-like domain of GP1 mediated
cytotoxicity in human embryonic kidney
293T cells and endothelial cells
13
. A second
study showed the detachment of 293T cells
after the expression of EBOV-encoded, but
not MARV-encoded, GP. Cell detachment, in
thiscase, occurred without cell death. Thiswas
largely attributed to a domain in the extracel-
lular region of GP2 and seemed to involve a
phosphorylation-dependent signal cascade
39
.
The ectodomain of GP and its anchorage to
the cell membrane are required for GP-
induced morphological changes
40
. Using a
reverse geneticssystem, it wasalso shown that
the cytotoxicity of EBOV dependson the level
of expression of GP. Overexpression of GP
leadsto the early detachment and cytotoxicity
of infected cells
9
. These data show that the
expression of GP, which iscontrolled by RNA
editing a mechanism that is not required
for virusreplication isevolutionarily linked
to theneed to control cytotoxicity.
Impaired endothelial function. The distur-
bance of the bloodtissue barrier, which is
controlled mainly by endothelial cells, is
another important factor in pathogenesis. The
endothelium seemsto be affected in two ways:
directly by infection with filoviruses, leading
to activation and eventual cytopathogenic
replication, and indirectly by a mediator-
induced inflammatory response (FIG. 3). Such
mediators seem to originate from filovirus-
activated cellsof the mononuclear phagocytic
system, which are the main target cells of the
virus
15,1921,23,41
. Increased levels of cytokines,
such as tumour-necrosis factor (TNF) and
IFN-, were detected in the serum of infected
individuals
23
. In particular, increased levels
of IFN- were associated with fatal cases
22,23
.
In vitro, it was shown that the virus-induced
cytokine release led to the activation of the
endothelium, as shown by a breakdown of
the endothelial barrier function
19
(FIG. 3).
Although the molecular mechanisms are
largely unknown, experimental data have
provided evidence for changes in the protein
organization of the endothelial-cell junctions,
NP
3 5
35 40 sGP/GP 30 24 L
35
L
NP
T7
30
NP
VP30
VP35 VP40/VP24
GP1, 2
L
Transcription/translation
RNP
Virus
Full length Ebola
virus genome
Nucleus
a
c
d
b
Figure 2 | Filovirus particles. a | Surface structure of a particle. Purified M arburg virus (M ARV)
particles were spotted on poly-L lysine-coated glass coverslips, air-dried and analysed by atomic
force microsopy. The image shows a filovirus particle of about 900 nm in length and 80 nm in
diameter (Image courtesy of H. Schillers, Department of Physiology, University of M uenster, G ermany
with permission from Schattauer from REF. 92 (2003)). b| Schematic illustration of a particle. Four
proteins are involved in the formation of the ribonucleoprotein complex (RNP): polymerase or large (L)
protein, nucleoprotein (NP), virion structural protein 30 (VP30) and VP35. The glycoprotein (G P) is a
type I transmembrane protein and is anchored with the carboxy-terminal part in the virion membrane.
Homotrimers of G P form the spikes on the surface of the virion. VP40 and VP24 are membrane-
associated proteins. c | A schematic illustration of the Ebola virus (EBO V) genome. The genome
consists of a single, negative-stranded, linear RNA molecule. d| G eneration of EBO V particles from
cloned complementary DNA. The system is based on the co-transfection of six different plasmids:
five expression plasmids under the control of the chicken -actin promoter, encoding the four virus
proteins that are required for transcription and replication (NP, VP30, VP35 and L) of the EBO V
genome and the bacteriophage T7 polymerase, and a plasmid under the transcriptional control of
the T7 promoter for transcribing the full-length EBO V genome (for details, see REF. 10).
Clinical and laboratory data also indicate
disturbances in haemostasis during infec-
ti on wi th fi lovi ruses. Although thrombo-
cytopaenia is observed in severe infections
of humans and non-human primates, stud-
ies on the role of disseminated intravascular
coagulati on (DI C), consumpti on coagu-
lopathy, and platelet and endotheli al dys-
functions are either missing, controversial
and/or i ncomplete
44
(FIG. 3). Duri ng i nfec-
tion with filoviruses, DIC can be regularly
observed in non-human primates and has
been descri bed i n humans, but i s less
i mportant i n the mouse model of EBOV
i nfecti on
28,45
. In the non-human pri mate
model, the endothelium remains relatively
i ntact morphologi cally, even at the end
stages of the disease, but, an increase in per-
meability was observed. Recent preliminary
in vivo data (T. Gei sbert et al., personal
communication) indicated that the coagu-
lati on abnormali ti es mi ght be tri ggered
by the release of cytoki nes from i nfected
mononuclear phagocytoti c cells, as di s-
cussed earlier, rather than destruction of the
endothelium itself.
in particular the cadherincatenin complex
of the vascular endothelium
15
. This might
explain the imbalance of fluid between the
intravascular and extravascular tissue spaces
that is observed in patients. In vitro, the
increased permeability could be blocked by
antibodiesthat neutralize TNF
19
, indicating a
crucial role for this pro-inflammatory
cytokine in virus-induced shock. It has been
shown by others that both TNF and IFN-,
can alter the endotheli al barri er functi on
i n tissue-culture models and in vivo
19,42
.
Therefore, infection with filovirusmight lead
to an uncontrolled release of cytokines, simi-
lar to that observed in lipopolysaccharide
(LPS)-induced shock during infection with
Gram-negative bacteria. Interestingly, recently
reported preliminary data indicated that there
are differences between filovirus- and LPS-
induced cytokine release (U. Strher et al.,
personal communication).
Present data indicate that the activation of
endothelial and mononuclear phagocytotic
cells could be triggered either by infection
with virus or by the binding of soluble viral
or cellular factors
19,21
(FIG. 3). Several soluble
glycoproteins are expressed and secreted or
released by alternative mechanisms during
infection with filoviruses(BOX 1). Comparison
of the different strains of filovirus and virus
variantsraisesquestionsthat concern therela-
tive roles of these proteins in pathogenesis.
MARV, which causes comparable disease in
primatesand humans, releasesGP1 in similar
amounts to EBOV, but does not express sGP
due to a different organization of its gene
encoding GP
36
. Similarly, a ZEBOV variant
that expresses only small amounts of sGP is
highly pathogenic in animal models
43
, whereas
the less pathogenic strain REBOV produces
high levelsof sGP
34
. Thisarguesagainst a gen-
eral role of sGP in the pathogenesis of
filovirusesand might point to a potential role
of secreted GP1 and/or thesolubleectodomain
of GP1GP2. Thismight highlight the impor-
tance of cleavage of GP in pathogenesis, asthe
production of solubleGP1 dependson theeffi-
cacy of proteolytic cleavage
14,36
. Interestingly,
proteolytic cleavageisnot required for infectiv-
ity of the virus
10
. However, all of this does not
exclude a role for sGP as a biologically active
protein in infection.
Virus entry
M onocyte/macrophage
Release of cytokines
and chemokines
?
Cytotoxic T cell
IL-1RA, IL-10
Fatal infection
IFN-, TNF
Soluble viral glycoproteins
released from infected cells
(sGP, GP1 and soluble GP1GP2)
Vascular leakage
Coagulation disorders
Dysregulation of the vascular tone
M onocytes/macrophages
become activated and
produce infectious particles
Immune response
IL-1, IL-6
Non-fatal infection
+
b
a
Figure 3 | Pathogenesis model. The concept of the pathogenesis of filovirus haemorrhagic fever is illustrated. Viruses that enter the body through
lymphatic and/or blood vessels get direct access to sessile monocytes/macrophages that become activated independently of virus replication.
a | Infected cells might extravasate into tissues to infect other cells, such as hepatocytes, and induce focal necrosis. b| The production of cytokines by
monocytes/macrophages can either promote or inhibit the immune response. Pro-inflammatory cytokines, such as interferon- (IFN-; additionally
produced by cytotoxic T cells
22
) and tumour-necrosis factor (TNF), can induce the activation of endothelial cells and increase vascular leakage. Direct
infection of endothelial cells occurs, but probably has a minor role in the pathogenesis. The role of soluble glycoproteins is not based on experimental data
(see also REFS36,92).
vaccines requires industrial support and this
di d not seem to be feasi ble, knowi ng that
there would not be a market for the vaccine.
Thi s vi ew has changed wi th the exi sti ng
threat from bioterrorism. Aside from better
and more rapid diagnostics and antivirals,
the development of a protective vacci ne
against List A agents, of which EBOV is one,
today ranks at the top of the priority list for
most countries. If a vaccine were available,
present poli ci es would probably i nclude
the vacci nati on of all at-ri sk medi cal per-
sonnel such as fi rst-responders, other
public health workers, laboratory workers
and military personnel.
Attempts to develop a vaccine for EBOV
began shortly after the viruswasdiscovered in
1976. Preparations of formalin-fixed or heat-
inactivated virions were used in the early
attempts to develop a vaccine against EBOV,
using guinea pigs and non-human primates
asthe animal models. Unfortunately, the level
of protection that was achieved was inconsis-
tent. Guinea pigs were partially protected
70
and in one study, four out of five baboons
survived challenge with EBOV after vaccina-
tion with an inactivated EBOV vaccine
71
.
However, other studies indicated that inacti-
vated EBOV did not induce sufficient immu-
nity to protect hamadryl baboons reliably
against a lethal challenge
72
.
Recently, investigators have mainly con-
centrated on the use of subunit vaccines that
are based on a si ngle or combi nati ons of
virus-encoded structural proteins to induce
protective i mmuni ty agai nst challenge
wi th EBOV. GP, nucleoprotei n (NP), and
the vi rus structural protei ns VP24, VP30,
VP35 and VP40 have been tested as vaccine
candidates using naked DNA, adenovirus,
vaccinia virus or Venezuelan equine enceph-
ali ti s vi rus (VEEV) repli cons as delivery
mechanisms
7378
(TABLE 1). Only EBOV GP
is exposed on the cell surface of the virion
and so seems to be the only target for neu-
trali zi ng anti body. It i s not surpri si ng,
therefore, that i mmuni zati on wi th VEEV
replicons that express the other virus pro-
teins resulted in low endpoint titres (<1:20)
in the 80% plaque reduction neutralization
test (PRNT
80
). Speci fi c, non-neutrali zi ng
anti bodi es were produced i n response to
i mmuni zati on wi th VP24, VP30, VP35,
VP40 and NP, but i t i s not clear what role
these anti bodi es have i n protecti on. The
EBOV GPVEEV replicon resulted in anti-
body titres of between 10 and 320 using the
PRNT
50
, and protected 8 out of 10 gui nea
pigs and 18 out of 20 mice against challenge,
and the EBOV NPVEEV vector protected
all 20 mice that were immunized
75
.
Animal models.Non-human primatesare the
preferred animal model for human filovirus
infection, as these animals are fatally infected
with human virulent, non-adapted strains
of EBOV and MARV. Numerous species of
non-human primate have been used, such as
baboons, African green monkeys, and Rhesus
and Cynomolgusmacaques, and the pathol-
ogy of infected non-human primates is simi-
lar to that seen in humans
6064
. ZEBOV has
been adapted for infection of both guinea
pigs
65,66
and mice
28
by serial passage in each
species. The pathogenesis of these adapted
strains is similar to that in non-human pri-
matesand humans. Notably, in all cases, there
is a conserved tropism for mononuclear
phagocytic cells, pathological changes to the
liver and spleen, high levels of viraemia and
high mortality. The mouse-adapted strain
seems to have reduced virulence in non-
human primates, but is virulent in guinea
pigs
67
. Lymphocyte apoptosis was not
reported to be a main feature of ZEBOV
infection in mice or guinea pigs
28,66
, but was a
consistent feature of disease in humans
22
and
non-human primates
18
. Coagulopathy in
particular, haemorrhageand fibrin deposition
is not a main feature of disease caused by
either of the rodent-adapted strains
6668
. This
pathological feature is not consistently
observed in primates and the occurrence in
humans has not been well investigated
because of the difficulty in obtaining sam-
ples
69
. For vaccine research, the guinea pig isa
poor immunological model with a restricted
selection of specific reagents. However, mice
provide an excellent model system with a
wide range of reagents, as well as transgenic
and mutant strains. The differencesin pathol-
ogy between model systems must be consid-
ered when planning vaccine studies, asshould
the observation that it isapparently harder to
protect non-human primates than either
species of rodent
69
. Phase III clinical trials in
humans will be difficult to conduct for any
EBOV vaccine, because of the sporadic nature
of the outbreaksand the potential difficulty in
obtaining ethical approvals. In this case,
licensing authorities will require that efficacy
and safety have been shown in at least two
animal species, and the most logical choices
would bemiceand non-human primates.
Vaccinedevelopment. There has been a long-
standing discussion about the requirement
for vaccinesfor EBOV and other rarely occur-
ring viruses that induce haemorrhagic fever.
However, in the past, the rare appearance of
EBOV haemorrhagic fever and the remote
locationsof the outbreaksdid not favour vac-
cine development. Development of effective
Immunotherapy and vaccines
Immune response and immunotherapy.
Despite being clearly immunosuppressive,
there is good evidence for protective immu-
ni ty duri ng EBOV haemorrhagi c fever.
However, the mechanisms of recovery from
infection are not well understood. In con-
trast to survivors and asymptomatic cases
that show IgM and IgG antibody responses
to virus antigens, fatal infections usually end
with high viraemia and little evidence of a
humoral i mmune response
22,24,46
. If hi gh-
quali ty neutrali zi ng anti bodi es could be
given to patients at the time of disease onset,
then this might limit the severity of disease,
even i f i t fai ls to prevent i t completely.
Neutralizing antibodies specific for GP were
shown to have protective and therapeuti c
properties in rodent models
4749
. An attempt
to overcome the lack of serum available from
convalescent patients was made by Russian
investigators, who developed hyper-immune
horse serum
50
; thi s was shown to protect
baboons from experimental infection with
EBOV
51
. The treatment was also seen to
be effective in guinea pigs, but failed to pro-
tect Cynomolgus monkeys from challenge
wi th EBOV
52
. Furthermore, horse anti -
bodi es are not the best choi ce for a donor
antisera, because horses produce a subclass
of i mmunoglobuli n (IgG
T
) that i s hi ghly
immunogenic in humans
53
. Antiserum from
other ani mals for example, sheep and
goats i s tolerated better by the human
and pri mate i mmune systems because i t
does not contai n IgG
T
54
and, therefore,
mi ght be a better source for therapeuti c
polyclonal antisera.
Protection in humans by convalescence
sera has been anecdotally reported and con-
valescent plasma wastransfused into patients
during the EBOV outbreak in Kikwit
55
.
However, the efficacy of the treatment has
been questioned because of the reduced
severity of the disease at thispoint of the out-
break, even in non-treated patients. An evalu-
ation of the efficacy of passive immunization
in controlled clinical trials is missing at pre-
sent. As with most RNA viruses, EBOV can
rapidly mutate to produce antibody-escape
mutants
56
. This indicates that antibody ther-
apy might require hyperimmune polyclonal
serum or a panel of monoclonal antibodiesof
different epitope specificitiesto be successful.
Despite the protective properties of antibod-
ies, there hasbeen some debate about the role
of antibodies in enhancing EBOV infection
and potentially exacerbating the disease
5759
.
If this holds true for the infected host, strate-
gies for vaccine development might have to
be revisited.
The use of human adenovirusesasvaccine
vectors is problematic because pre-existing
immunity in the human population might
eventually limit the efficacy of this approach.
However, a study by Sullivan and colleagues
76
shows the first successful protection of non-
human primates against challenge with
EBOV, although thishasbeen achieved previ-
ously for MARV using a GP-based alphavirus-
replicon vaccine
81
. The study givesusinsights
into the mechanisms and indicates that
appropriate live vaccine vectors or replica-
tion-deficient vectors might ultimately pro-
vide a successful human vaccine. If further
enhancements in vaccine strategies are to be
possible, the full spectrum of assays that are
available to immunologistsin the twenty-first
century must be used in future vaccine stud-
ies. Particularly important would be the use of
MHC classI tetramersto determineresponses
of CTLsto EBOV-encoded antigens, aswell as
the use of intracellular flow cytometry to
determine cytokine responses to immu-
nization and detailed study of the immuno-
pathological responses to EBOV infection in
unimmunized animals.
Finally, the recent development of EBOV-
like particles
82
might provide an interesting
delivery system for a protective host immune
response that seems to depend on both the
cell-mediated effector mechanisms and the
humoral immune response
69,78
. Virus-like
particles, which are generated by the expres-
sion of virus membrane proteins, might
overcome the safety limitations that are asso-
ciated with the use of attenuated filovirus
particlesor live vaccine vectors, aswell aspre-
existing immunity to vectors such as vaccinia
or adenovirus.
Concluding remarks
The first cases of EBOV were reported from
Sudan and Zaire in 1976, but the virus has
only received real attention from scientists
since 1995. Until recently, vaccine develop-
ment was not considered to be a priority and
despite several promising results, vaccine
development is still in the experimental
stages. The process of moving an experi-
mental vaccine candidate into clinical trials
i s time-consuming and expensive. It has
become clear that the rodent models (mouse
and guinea pig), although important for the
development of antivirals, therapeutics and
vaccines, are not necessarily predictive for the
efficacy of the same agents in non-human
primates, which are the gold standard for pre-
dicting efficacy in humans.At present, there is
no convincing evidence for any successful
strategy in post-exposure prophylaxis. So,
therapeutic antibodies might be the most
Replicon immunization resultsin the pro-
duction of virus antigen by host cells, leading
to antigen presentation by dendritic cells in
the context of both MHC class I and class II
molecules. This should lead to the develop-
ment of both cytotoxic and helper T-cell
responses, in addition to the production
of antibody, but unfortunately these T-cell
responses were not measured. Consequently,
when these VEEV repliconEBOV GP/NP
vaccine constructs failed to protect non-
human primates from challenge with EBOV,
it was impossible to determine whether a
deficiency in antibody or cell-mediated
immunity was responsible
78
. Recombinant
vaccinia viruses that express EBOV GP, NP,
VP35, VP40 and VP24 (REFS79,80) failed to
protect guinea pigs, with the exception of the
GPvaccinia virusconstruct, which protected
three out of five guinea pigs, but failed to pro-
tect Cynomolgusmacaques
78
. Once again, the
antibody titres that were determined for the
vaccinia virusconstructsusing enzyme-linked
immunosorbent assay (ELISA) and PRNT
80
were low but, notably, T-cell proliferation and
cytotoxic T lymphocyte(CTL) responseswere
not measured, resulting in the lossof relevant,
and possibly crucial, immunological data.
There has been some success with DNA
vaccination using genesencoding both GP and
NP
73,74
. EBOV GP DNA vaccination of guinea
pigsresulted in variableprotection, depending
on the vaccine regimen, and premature sacri-
fice of the experimental animals makes inter-
pretation of thechallengeresultsproblematic
74
.
Immune serum from the guinea pigs did not
inhibit the growth of EBOV in vitro, and there
was no passive protection conferred to naive
animals after the transfer of serum. DNA
immunization of mice with both EBOV GP
and NP constructs resulted in some degree of
protection from challenge and the develop-
ment of CTL responses to both antigens. The
most successful strategy so far involvesusing a
DNA primefollowed by an adenovirusboost
76
.
Cynomolgusmonkeyswere immunized twice
with DNA encoding the GPs of ZEBOV,
SEBOV and ICEBOV, and ZEBOV NP fol-
lowed by a booster of adenovirus expressing
ZEBOV GP five months after priming. The
monkeys were challenged with six plaque-
forming unitsof ZEBOV and all four animals
survived after challenge.Antibody responses,
T-cell proliferation and CTL responses indi-
cated that antibody and T memory helper cells
are essential for the protection, and that cell-
mediated immunity, although possibly impor-
tant, isnot an absolute requirement. Thisisin
agreement with observations that passive
transfer of serum can be protective and might
give some support to the concept of antibody
therapy (discussed earlier).
Table 1 | Comparison of Ebola virus vaccine candidates
Vaccine Gene Animal Survivors/ References
system product model challenged
Vaccinia virus GP Guinea pig 3/5 79
Vaccinia virus GP Cynomolgus macaque 0/3 78
VEEV replicon VP24 M ouse 37/60* 77
VEEV replicon NP M ouse 20/20 75, 77
VEEV replicon GP Guinea pig 8/10 75
VEEV replicon GP M ouse 18/20 75
VEEV replicon GP Cynomolgus macaque 0/3 78
VEEV replicon NP Cynomolgus macaque 0/3 78
VEEV replicon GP and NP Cynomolgus macaque 0/3 78
DNA NP Guinea pig 5/8 74
DNA NP M ouse 7080%
73
DNA GP Guinea pig 13/15 74, 76
DNA GP M ouse 50100%
73
DNA sGP Guinea pig 8/11 74
DNA GP and NP Guinea pig 8/8 76
DNA and adenovirus GP and NP Cynomolgus macaque 4/4 76
*The total number represents the combined data for two mouse strains, one strain was better protected by
the vaccine than the other.
Survival was dependent on the dose of DNA administered. GP, glycoprotein;

NP, nucleoprotein; s, soluble; VEEV, Venezuelan equine encephalitis virus; VP, virion structural protein.
44. Chen, J. P. & Cosgriff, T. M . Hemorrhagic fever virus-
induced changes in hemostasis and vascular biology.
Blood Coagul. Fibrinolysis 11, 461483 (2000).
45. Fisher-Hoch, S. P. et al. Pathophysiologyof shock and
hemorrhage in a fulminating viral infection (Ebola).
J . Infect. Dis. 152, 887894 (1985).
46. K siazek, T. G. et al. Clinical virologyof Ebola hemorrhagic
fever (EHF): virus, virus antigen, and IgG and IgM
antibodyfindings among EHF patients in K ikwit,
Democratic Republic of the Congo, 1995. J . Infect. Dis.
179, S177S187 (1999).
47. M aruyama, T. et al. Recombinant human monoclonal
antibodies to Ebola virus. J . Infect. Dis. 179, S235S239
(1999).
48. Wilson, J. A. et al. Epitopes involved in antibody-
mediated protection from Ebola virus. Science 287,
16641666 (2000).
49. Parren, P. W. et al. Pre- and post-exposure prophylaxis of
Ebola virus infection in an animal model bypassive
transfer of a neutralizing human antibody. J . Virol. 76,
64086412 (2002).
50. Ktysnayanskii, V. P. et al. Preparation of hyperimmune horse
serum to Ebola virus. Vopr. Virusol. 39, 9192 (1994).
51. M ikhailov, V. V. et al. The evaluation in hamadryas
baboons of the possibilityfor the specific prevention of
Ebola fever. Vopr. Virusol. 39, 8284 (1994).
52. Jahrling, J. B. et al. Passive immunization of Ebola virus-
infected cynomologusmonkeyswith immunoglobulin from
hyperimmune horses. Arch. Virol. 11, 135140 (1996).
53. Fernandes, I., Takehara, H. A. & M ota, I. Isolation of IgGT
from hyperimmune horse anti-snake venom serum: its
protective ability. Toxicon. 29, 13731379 (1991).
54. Sjostrom, L. et al. A comparison of ovine and equine
antivenoms. Toxicon. 32, 427433 (1994).
55. M upapa, K . D. et al. Treatment of Ebola hemorrhagic
fever with blood transfusions from convalescent patients.
J . Infect. Dis. 179, S18S23 (1999).
56. Takada, A. et al. Identification of protective epitopes on
Ebola virus glycoprotein at the single amino acid level by
using recombinant vesicular stomatitis viruses. J . Virol.
77, 10691074 (2003).
57. Takada, A. et al. Infectivity-enhancing antibodies to Ebola
virus glycoprotein. J . Virol. 75, 23242330 (2001).
58. Geisbert, T. W. et al. Evidence against an important role
for infectivity-enhancing antibodies in Ebola virus
infections. Virology293, 1519 (2002).
59. Takada, A. et al. Antibody-dependent enhancement of
Ebola virus infection. J . Virol. 77, 75397544 (2003).
60. Ellis, D. S. et al. Ebola virus: a comparison, at
ultrastructural level, of the behaviour of the Sudan and
Zaire strains in monkeys. Br. J . Exp. Pathol. 59, 584593
(1978).
61. Davis, K . J. et al. Pathologyof experimental Ebola virus
infection in African green monkeys. Involvement of
fibroblastic reticular cells. Arch. Pathol. Lab. Med. 121,
805819 (1997).
62. Fisher-Hoch, S. P. & M cCormick, J. B. Experimental
filovirus infections. Curr. Top. Microbiol. Immunol. 235,
117143 (1999).
63. Ryabchikova, E. I., K olesnikova, L. V. & Netesov, S. V.
Animal pathologyof filoviral infections. Curr. Top.
Microbiol. Immunol. 235, 145173 (1999).
64. Zaki, S. R. & Goldsmith, C. S. Pathologic features of
filovirus infections in humans. Curr. Top. Microbiol.
Immunol. 235, 97116 (1999).
65. Bowen, E. T. et al. Ebola haemorrhagic fever:
experimental infection of monkeys. Trans. R. Soc. Trop.
Med. Hyg. 72, 188191 (1978).
66. Connolly, B. M . et al. Pathogenesis of experimental Ebola
virus infection in guinea pigs. J . Infect. Dis. 179,
S203S217 (1999).
67. Bray, M . et al. Haematological, biochemical and
coagulation changes in mice, guinea-pigs and monkeys
infected with a mouse-adapted variant of Ebola Zaire
virus. J . Comp. Pathol. 125, 243253 (2001).
68. Gibb, T. R. et al. Pathogenesis of experimental Ebola
Zaire virus infection in BALB/c mice. J . Comp. Pathol.
125, 233242 (2001).
69. Hart, M . K . Vaccine research efforts for filoviruses. Int. J .
Parasitol. 33, 583595 (2003).
70. Lupton, H. W. et al. Inactivated vaccine for Ebola virus
efficacious in guinea pig model. Lancet 2, 12941295
(1980).
71. M ikhailov, V. V. et al. An evaluation of the possibilityof
Ebola fever specific prophylaxis in baboons (Papio
hamadryas). Vopr. Virusol. 39, 8284 (1994).
72. Chepurnov, A. A. et al. Attempts to develop a vaccine
against Ebola fever. Vopr. Virusol. 40, 257260
(1995).
73. Vanderzanden, L. et al. DNA vaccines expressing either
the GP or NP genes of Ebola virus protect mice from
lethal challenge. Virology246, 134144 (1998).
15. Schnittler, H. J. & Feldmann, H. M olecular pathogenesis
of filovirus infections: role of macrophages and
endothelial cells. Curr. Top. Microbiol. Immunol. 235,
175204 (1999).
16. Baskerville, A. et al. Ultrastructural pathologyof
experimental Ebola haemorrhagic fever virus infection.
J . Pathol. 147, 199209 (1985).
17. Geisbert, T. W. et al. Association of Ebola-related Reston
virus particles and antigen with tissue lesions of monkeys
imported to the United States. J . Comp. Path. 106,
137152 (1992).
18. Geisbert, T. W. et al. Apoptosis induced in vitroand
in vivoduring infection byEbola and M arburg viruses.
Lab. Invest. 80, 171186 (2000).
19. Feldmann, H. et al. Filovirus-induced endothelial leakage
triggered byinfected monocytes/macrophages. J . Virol.
70, 22082214 (1996).
20. Ryabchikova, E., Kolesnikova, L. V. & Luchko, S. V. An
analysisof featuresof pathogenesisin two animal modelsof
Ebola virusinfection. J . Infect. Dis. 179, S199S202 (1999).
21. Strher, U. et al. Infection and activation of monocytes by
M arburg and Ebola viruses. J . Virol. 75, 1102511033
(2001).
22. Baize, S. et al. Defective humoral response and extensive
intravascular apoptosis are associated with fatal outcome
of Ebola virus-infected patients. Nature Med. 5, 423426
(1999).
23. Villinger, F. et al. M arkedlyelevated levels of interferon
(IFN)-, IFN-, interleukin (IL)-2, IL-10, and tumor necrosis
factor- associated with fatal Ebola virus infection.
J . Infect. Dis. 179, S188S191 (1999).
24. Leroy, E. M . et al. Human asymptomatic Ebola infection
and strong inflammatoryresponse. Lancet 355,
22102215 (2000).
25. Baize, S. et al. Inflammatoryresponsesin Ebola virus-
infected patients. Clin. Exp. Immunol. 128, 163168 (2002).
26. Harcourt, B. H., Sanchez, A. & O ffermann, M . K . Ebola
virus selectivelyinhibits responses to interferon, but not
to interleukin-1, in endothelial cells. J . Virol. 73,
34913496 (1999).
27. Basler, C. F. et al. The Ebola virus VP35 protein functions
as a type I IFN antagonist. Proc. Natl Acad. Sci. USA97,
1228912294 (2000).
28. Bray, M . et al. A mouse model for evaluation of
prophylaxis and therapyof Ebola hemorrhagic fever.
J . Infect. Dis. 178, 651661 (1998).
29. Bray, M . The role of the type I interferon response in the
resistance of mice to filovirus infection. J . Gen. Virol. 82,
13651373 (2001).
30. M ahanty, S. et al. Cutting edge: impairment of dendritic
cells and adaptive immunitybyEbola and Lassa viruses.
J . Immunol. 170, 27972801 (2003).
31. Yang, Z. et al. Distinct cellular interactions of secreted
and transmembrane Ebola virus glycoproteins. Science
279, 10341036 (1998).
32. K indzelskii, A. L. et al. Ebola virus secretoryglycoprotein
(sGP) diminishes FcRIIIB-to-CR3 proximityon
neutrophils. J . Immunol. 164, 953958 (2000).
33. M aruyama, T. et al. Ebola virus, neutrophils and antibody
specificity. Science 282, 845 (1998).
34. Sanchez, A. et al. The virion glycoprotein of Ebola viruses
are encoded in two reading frames and are expressed
through transcriptional editing. Proc. Natl Acad. Sci. USA
93, 36023607 (1996).
35. Volchkov, V. E. et al. Release of viral glycoproteins during
Ebola virus infection. Virology245, 110119 (1998).
36. Feldmann, H. et al. Biosynthesis and role of filoviral
glycoproteins. J . Gen. Virol. 82, 28392848 (2001).
37. Volchkov, V. E., Blinov, V. M . & Netesov, S. V. The
envelope glycoprotein of Ebola virus contains an
immunosuppressive-like domain similar to oncogenic
retroviruses. FEBS Lett. 305, 181184 (1992).
38. Ignatyev, G. M . Immune response to filovirus infections.
Curr. Top. Med. Microbiol. Immunol. 235, 205217 (1999).
39. Chan, S. Y., M a, M . C. & Goldsmith, M . A. Differential
induction of cellular detachment byenvelope
glycoproteins of M arburg and Ebola (Zaire) viruses.
J . Gen. Virol. 81, 21552159 (2000).
40. Takada, A. et al. Downregulation of 1 integrins byEbola
virus glycoprotein: implication for virus entry. Virology
278, 2026 (2000).
41. Gupta, M . et al. Passive transfer of antibodies protects
immunocompetent and immunodeficient mice against
lethal Ebola virus infection without complete inhibition of
viral replication. J . Virol. 75, 46494654 (2001).
42. Wong, R. K ., Baldwin, A. L. & Heimark, R. L. Cadherin-5
redistribution at sites of TNF- and IFN--induced
permeabilityin mesenteric venules. Am. J . Physiol. 276,
H736H748 (1999).
43. Volchkov, V. E. et al. M olecular characterization of guinea-
pig-adapted variantsof Ebola virus. Virology277,
147155 (2000).
promising short-term strategy. Despite some
evidence for antibody-mediated enhance-
ment of disease, therapeutic antibodies
should be carried into the next stage involving
the humanization of monoclonal antibodies,
the production of human monoclonal anti-
bodies and the evaluation of polyclonal and
reconvalescent sera. A major drawback in the
process of developing counter measures
against EBOV, other viral haemorrhagic
fevers and related diseases is the biocontain-
ment that is required for animal work with
these agents. Building new facilities is one
way to respond and the political support is
guaranteed in crisis situations. However,
aside from maintaining facilities and long-
term funding, the availability of well-trained
personnel becomesa crucial issue.
Heinz Feldmann isat theSpecial Pathogens
Program,National Microbiology Laboratory and
Department of Medical Microbiology,University of
Manitoba,Winnipeg,Canada.Steven Jonesisat the
Special PathogensProgram,National Microbiology
Laboratory and Department of Immunology,
University of Manitoba,Winnipeg,Canada.
Hans-Dieter Klenk isat theInstitutefor Virology,
Philipps-University of Marburg,Germany.
Hans-JoachimSchnittler isat theInstitutefor
Physiology,Medical Faculty
Carl-Gustav Carus,Technical University of
Dresden,Germany.
Correspondenceto H.F.
e-mail: heinz_feldmann@hc-sc.gc.ca
doi: 10.1038/nri1154
1. World Health O rganization. Ebola haemorrhagic fever in
Sudan, 1976. Bull. WHO56, 247270 (1978).
2. Pattyn, S. R. in Ebola Virus Haemorrhagic Fever 1st edn
1436 (Elsevier, Amsterdam, Holland, 1978).
3. World Health O rganization. Ebola haemorrhagic fever in
Zaire, 1976. Bull. WHO56, 271293 (1978).
4. Siegert, R. et al. Zur thiologie einer unbekannten von
affen ausgegangenen infektionskrankheit. Dtsch. Med.
Wochenschr. 92, 23412343 (1967).
5. Jahrling, P. B. et al. Preliminaryreport: isolation of Ebola
virus from monkeys imported to USA. Lancet 335,
502505 (1990).
6. LeGuenno, B. et al. Isolation and partial characterization of
a new strain of Ebola virus. Lancet345, 12711274 (1995).
7. Walsh, P. D. et al. Catastrophic ape decline in western
equatorial Africa. Nature 422, 611614 (2003).
8. Borio, L. T. et al. Hemorrhagic fever viruses as biological
weapons: medical and public health management. J AMA
287, 23912405 (2002).
9. Volchkov, V. E. et al. Recoveryof infectious Ebola virus
from cDNA: transcriptional RNA editing of the
glycoprotein gene controls viral cytotoxicity. Science291,
19651969 (2001).
10. Neumann, G. et al. Reverse genetics demonstrates that
proteolytic cleavage of the Ebola virus glycoprotein is not
essential for replication in cell culture. J . Virol. 76,
406410 (2002).
11. M hlberger, E. et al. Three of the four nucleocapsid
proteins of M arburg virus, NP, VP35, and L, are sufficient
to mediate replication and transcription of M arburg virus-
specific monocistronic minigenomes. J . Virol. 72,
87568764 (1998).
12. M hlberger, E. et al. Comparison of the transcription and
replication strategies of M arburg virus and Ebola virus by
using artificial replication systems. J . Virol. 73,
23332342 (1999).
13. Yang, Z. et al. Identification of the Ebola virus glycoprotein
as the main viral determinant of vascular cell cytotoxicity
and injury. Nature Med. 6, 886889 (2000).
14. Volchkov, V. E. et al. Processing of the Ebola virus
glycoprotein bythe proprotein convertase furin. Proc.
Natl Acad. Sci. USA95, 57625767 (1998).
92. Schnittler, H. J. & Feldmann, H. Viral hemorrhagic fever
a vascular disease? Thromb. Haemost. 89, 967972
(2003).
Acknowledgements
We wish to thank manycolleaguesin the field for their helpful dis-
cussionsand suggestions. In particular, we thank D. Dick and A.
Lacquement for critical review of the manuscript. O ur work on
filoviruses was supported by grants from the Deutsche
Forschungsgemeinschaft (DFG), the Canadian Institutesof Health
Research (CIHR), the K empkes-Stiftung, the European
Communityand Health Canada.
Online links
DATABASES
The following terms in this article are linked online to:
L o c u sL in k : http://www.ncbi.nlm.nih.gov/LocusLink/
IFN- | IL-1 | IL-6 | IL-10 | IL-1RA | TNF
FURTHER INFORMATION
T h e in te rn a tio n a l c o m m itte e o n ta x o n o m y o f viru se s:
http://www.ncbi.nlm.nih.gov/ICTV
S e le c te d a g e n t p ro g ra m m e : http://www.cdc.gov/od/sap
Access to this interactive links box is free online.
74. Xu, L. et al. Immunization for Ebola virus infection. Nature
Med. 4, 3742 (1998).
75. Pushko, P. et al. Recombinant RNA repliconsderived from
attenuated Venezuelan equine encephalitisvirusprotect
guinea pigsand mice from Ebola hemorrhagic fever virus.
Vaccine19, 142153 (2000).
76. Sullivan, N. J. et al. Development of a preventive vaccine
for Ebola virusinfection in primates. Nature408, 605609
(2000).
77. Wilson, J. A. et al. Vaccine potential of Ebola virus VP24,
VP30, VP35, and VP40 proteins. Virology286, 384390
(2001).
78. Geisbert, T. W. et al. Evaluation in nonhuman primates of
vaccines against Ebola virus. Emerg. Infect. Dis. 8,
503507 (2002).
79. Gilligan, K . J. et al. in Vaccines 97(eds Brown, F. et al.)
8792 (Cold Spring Harbor LaboratoryPress, Cold
Spring Harbor, New York, 1997).
80. Chepurnov, A. A. et al. Immunological properties of VP24
protein of Ebola virus expressed byrecombinant vaccinia
virus. Vopr. Virusol. 42, 115120 (1997).
81. Hevey, M . et al. M arburg virus vaccines based
upon alphavirus replicons protect guinea pigs and
nonhuman primates. Virology251, 2837 (1998).
82. Noda, T. et al. Ebola virus VP40 drives the formation of
virus-like filamentous particles along with GP. J . Virol. 76,
48554865 (2002).
83. Wool-Levis, R. J. & Bates, P. Characterization of Ebola
virusentrybyusing pseudotyped viruses: identification of
receptor-deficient cell lines. J . Virol. 72, 31553160
(1998).
84. Becker, S., Spiess, M . & Klenk, H. D. The asialoglycoprotein
receptor isa potential liver-specific receptor for M arburg
virus. J . Gen. Virol. 76, 393399 (1995).
85. Chan, S. Y. et al. Folate receptor- is a cofactor for
cellular entrybyM arburg and Ebola viruses. Cell 106,
117126 (2001).
86. Alvarez, C. P. et al. C-type lectins DC-SIGN and L-SIGN
mediate cellular entrybyEbola virus in cis and in trans.
J . Virol. 76, 68416844 (2002).
87. M artini, G. A. & Siegert, R. in Marburg Virus Disease
1st edn 1230 (Springer, New York, 1971).
88. K lenk, H. D. M arburg and Ebola viruses. Curr. Top.
Microbiol. Immunol. 235, 1230 (1999).
89. Peters, C. J. & LeDuc, J. W. An introduction to Ebola:
the virus and the disease. J . Infect. Dis. 179, S9S14
(1999).
90. Sanchez, A. et al. in Fields Virology4th edn (eds K nipe,
D. M . et al.) 12791304 (Lippincott-Williams & Wilkins,
Philadelphia, Pennsylvania, 2001).
91. Feldmann, H. et al. M arburg and Ebola viruses: past
advances and future needs. Nova Acta Leopoldina 313,
163178 (1999).

C Elegans

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

C Elegans

Uploaded by

Copyright:

Available Formats

2003 Nature PublishingGroup

Survival was dependent on the dose of DNA administered. GP, glycoprotein;

You might also like