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Ebolavirus
E Ebolavirus K S Brown and A Silaghi, University of Manitoba, Winnipeg, MB, Canada H Feldmann, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada ã 2008 Elsevier Ltd. All rights reserved.
History Ebola virus (EBOV) first emerged as the causative agent of two major outbreaks of viral hemorrhagic fever (VHF) occurring almost simultaneously along the Ebola River in Democratic Republic of Congo (DRC, formerly Zaire) and Sudan in 1976 (see Table 1). Over 500 cases were reported, with case fatality rates (CFRs) of 88% and 53%, respectively. It was later recognized that these two outbreaks were caused by two distinct species of EBOV (Zaire ebolavirus and Sudan ebolavirus). In 1989, a novel virus, Reston ebolavirus (REBOV), was isolated from naturally infected cynomolgus macaques (Macaca fascicularis) imported from the Philippines into the United States. All shipments except one were traced to a single supplier in the Philippines; however, the actual origin of the virus and mode of contamination for the facility have never been ascertained. While pathogenic for naturally and experimentally infected monkeys, limited data indicate that REBOV may not be pathogenic for humans as animal caretakers were infected without producing clinical symptoms. In 1994, the first case of Ebola hemorrhagic fever (EHF) occurred in western Africa in the Tai Forest Reserve in Coˆte d’Ivoire (Ivory Coast). An ecologist was infected by performing a necropsy on a dead chimpanzee whose troop had lost several members to infection with Coˆte d’Ivoire ebolavirus (CIEBOV). A single seroconversion was later documented, suggesting another nonfatal human case in nearby Liberia. Zaire ebolavirus (ZEBOV) reemerged in Kikwit, DRC, in 1995, causing a large EHF outbreak with 81% CFR. Sudan ebolavirus (SEBOV) reemerged in 2000–01 in the Gulu District in northern Uganda. There were over 425 cases (53% CFR), making it the largest EHF epidemic documented so far. Starting in 1994, an endemic focus of ZEBOV activity became obvious in the northern boarder region of Gabon and the Republic of Congo (RC) with multiple small EHF outbreaks over the past decade. Most infections there have been associated with the hunting and handling of animal
carcasses (mainly great apes). In addition, ZEBOV has virtually decimated the chimpanzee and gorilla populations in those areas. At least three laboratory exposures to EBOV have occurred; one in Russia (2004) was fatal.
Taxonomy and Classification Filoviruses are classified in the order Mononegavirales, a large group of enveloped viruses containing nonsegmented, negative-sense (NNS) RNA genomes. The family Filoviridae is separated into two distinct genera, Marburgvirus and Ebolavirus. The genus Ebolavirus is subdivided into four species – Zaire ebolavirus, Sudan ebolavirus, Coˆte d’Ivoire ebolavirus, and Reston ebolavirus. Filoviruses are classified as maximum containment (biosafety level 4 (BSL-4)) agents as well as category A pathogens based on their generally high mortality rate, person-to-person transmission, potential aerosol infectivity, and absence of vaccines and chemotherapy.
Biological and Physical Properties of Virion EBOV particles are pleomorphic, appearing as U-shaped, 6-shaped, circular forms, or as long filamentous, sometimes branched forms varying greatly in length (up to 14 000 nm), but have a uniform diameter of 80 nm (see Figures 1(a) and 1(b)). EBOV virions purified by ratezonal gradient centrifugation are bacilliform in outline and show an average length associated with peak infectivity of 970–1200 nm. Except for the differences in length, EBOVs seem to be very similar in morphology. Virions contain a helical ribonucleoprotein complex RNP or nucleocapsid roughly 50 nm in diameter bearing crossstriations with a periodicity of approximately 5 nm, and a dark, central axial space 20 nm in diameter running
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Ebolavirus
Table 1
Ebola hemorrhagic fever (EHF) outbreaks from 1976 to 2005
Ebola species
Year
Outbreak location (country)
Place of origin
Human cases (% case fatality rate)
Zaire ebolavirus
1976 1977 1994 1995 1996 1996
Yambuku (DRC) Tandala (DRC) Ogooue-Invindo province (Gabon) Kikwit (DRC) Mayibout (Gabon) Booue (Gabon); Johannesburg (South Africa) Ogooue-Invindo province (Gabon); Cuvette region (RC) Cuvette region (RC); Ogooue-Invindo province (Gabon) Mboma and Mbandza (RC) Etoumbi and Mbomo in Cuvette region (RC) Kampungu (DRC) Nzara, Maridi, Tembura, Juba (Sudan) Nzara, Yambio (Sudan) Gulu District in Mbarrara, Masindi (Uganda) Yambio Country (Sudan) Tai Forest (Ivory Coast) Liberia (Liberia) Reston, Virginia (also Pennsylvania and Texas) (USA) Siena (Italy) Alice, Texas (USA)
DRC DRC Gabon DRC Gabon Gabon
318 (88) 1 (100) 51 (60) 315 (79) 37 (57) 61 (74)
Gabon?a
124 (79)
RC?a
143 (90)
RC RC
35 (83) 12 (75)
DRC Sudan Sudan Uganda
Ongoing 284 (53) 34 (65) 425 (53)
Sudan Ivory Coast Liberia?a Philippinesb
17 (41) 1 (0) 1 (0) 4 (0)c
Philippines Philippines
0d 0d
2001–02 2002–03 2003 2005
Sudan ebolavirus
Coˆte d’Ivoire ebolavirus Reston ebolavirus
2007 1976 1979 2000–01 2004 1994 1995 1989 1992 1996
a
Place of origin unconfirmed. Reston virus has only been traced to a single monkey-breeding facility in the city of Calamba, the Philippines, which was depopulated in 1996 and is no longer in operation. c Mortality in monkeys was estimated at 82%. d Only monkeys were infected in these outbreaks; no reported human cases. DRC, Democratic Republic of the Congo; RC, Republic of the Congo. b
the length of the particle. The RNP complex is composed of the genomic RNA and the RNA-dependent RNA polymerase (L), nucleoprotein (NP), and virion proteins 35 and 30 (VP35 and VP30). A lipoprotein unit-membrane envelope derived from the host cell plasma membrane surrounds it. Spikes approximately 7–10 nm in length, spaced apart at 10 nm intervals, are visible on the virion surface and are formed by the viral glycoprotein (GP). Virus particles have a molecular weight of approximately 3–6 108 Da and a density in potassium tartrate of 1.14 g cm–3. Virus infectivity is quite stable at room temperature. Inactivation can be performed by ultraviolet (UV) light and g-irradiation, 1% formalin, bpropiolactone, and brief exposure to phenolic disinfectants and lipid solvents, like deoxycholate and ether.
Properties of Genome The EBOV genome consists of a molecule of linear, nonsegmented, negative-stranded RNA which is noninfectious,
not polyadenylated, and complementary to viral-specific messenger RNA. The genome amounts to c. 1.1% of the total virion. EBOV genomes are 19 kbp in length and fairly rich in adenosine and uridine residues. Genomes show a linear gene arrangement in the order 30 leader–NP–VP35–VP40–GP–VP30–VP24–L–50 trailer (see Figure 1(b)). All genes are flanked at their 30 and 50 ends by highly conserved transcriptional start (30 -CUnCnUnUAAUU-50 ) and termination signal sequences (30 UAAUUCUUUUU-5), respectively, all of which contain the pentamer 30 -UAAUU-50 . Most genes are separated by intergenic sequences variable in length and nucleotide composition. A feature of all EBOV genomes is the fact that some intergenic regions overlap by the conserved pentamer (UAAUU) sequence. ZEBOV and SEBOV show three such overlaps within the intergenic sequences of VP35/VP40, GP/VP30, and VP24/L, whereas REBOV shows only two between VP35/VP40 and VP24/L. Extragenic leader and trailer sequences are present at the 30 and 50 genome ends. These sequences are complementary at their very extremities, showing the potential to form
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(a) Spikes Nucleocapsid 80 nm
Envelope
NP
VP35
VP40
GP
Matrix
L
VP30 VP24
5⬘
3⬘ IR Leader
Overlap
Edit site
Overlap
Gene noncoding regions Extragenic and intergenic region (IR) sequences
Overlap
Trailer
Nucleoproteins Polymerase complex Membrane associated Nonstructural (sGP)
(b) Figure 1 Particle morphology. (a) Transmission electron microscopy of Ebola virus particle. Both graphs show images of Vero E6 cells infected with Zaire ebolavirus. (b) Ebola virus particle structure and genome organization. The upper part provides a scheme of the virus particle separated into four components. The inner core consists of the nucleocapsid which is a structure formed by the single-stranded, negative-sense RNA genome associated with the two nucleoproteins (NP, VP30) and the polymerase complex (L and VP35). The matrix is built by the viral matrix protein VP40 and a minor component VP24. The envelope is derived from the infected cell during assembly/budding. The spikes consist of a homotrimer of the glycoprotein (GP).
stem–loop structures. Phylogenetic analyses on the basis of the GP gene of the different Ebola species show a 37–41% difference in their amino acid and nucleotide sequences. Analysis within one species shows remarkable genetic stability between strains (the variation in nucleotide sequences has been shown to be <7% and even <2% among distinct ZEBOV strains), unexpected for an RNA virus, but highly indicative that these viruses have reached a high degree of fitness to fill their respective niches.
Properties of Viral Proteins Virions contain seven structural proteins with presumed identical functions for the different viruses (see Table 2). The electrophoretic mobility patterns of these proteins are characteristic for each species.
RNP Complex and Matrix Proteins Four proteins are associated with the viral RNP complex: NP, L, VP30, and VP35 (see Figure 1(b)). These proteins are involved in the transcription and replication of the genome. NP and VP30 represent the major and minor nucleoproteins, respectively. They interact strongly with the genomic RNA molecule, and are both phosphoproteins. VP30 is also a zinc-binding protein that behaves as a transcriptional activator. The L and VP35 proteins form the polymerase complex. The L protein, like other L proteins of NNS RNA viruses, represents the RNA-dependent RNA polymerase. Motifs linked to RNA (template) binding, phosphodiester bonding (catalytic site), and ribonucleotide triphosphate binding have all been described. VP35 appears to behave in a mode similar to that of the phosphoproteins found in other NNS RNA viruses, acting as a cofactor that affects the mode of RNA synthesis (transcription vs
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Table 2
Ebola virus proteins: functions and localization
Gene ordera
Ebola virus proteins
Protein function (localization)
1 2
Nucleoprotein (NP) Virion protein 35 (VP35)
3 4 (primary) 4 (secondary) 5
Virion protein 40 (VP40) Secreted glycoprotein (sGP) (nonstructural) Glycoprotein (GP) Virion protein 30 (VP30)
Major nucleoprotein, RNA genome encapsidation (component of RNP complex) Polymerase complex cofactor, type I interferon antagonist (component of RNP complex) Matrix protein, virion assembly, and budding (membrane associated) May have immunomodulatory role (secreted)
6
Virion protein 24 (VP24)
7
Polymerase (L)
a
Receptor binding and membrane fusion (membrane associated) Minor nucleoprotein, RNA binding, transcriptional activator (component of RNP complex) Minor matrix protein, virion assembly, type I interferon antagonist (membrane associated) RNA-dependent RNA polymerase, enzymatic portion of polymerase complex (component of RNP complex)
Gene order refers to 30 –50 gene arrangement as shown in Figure 1(b).
replication). VP35 is also known to be a type I interferon (IFN) antagonist; it blocks IFN-a/b expression by inhibiting IFN regulatory factor 3 (IRF-3). VP40 functions as the viral matrix protein and represents the most abundant protein in the virion. It plays a number of roles in viral infection related to assembly and budding of the viral particles. The production of VP40 by itself is sufficient to initiate the budding process for the production of viruslike particles (VLPs); however, the production of these particles is greatly enhanced by the addition of GP and NP. Recent studies have shown that EBOV hijacks the cellular protein machinery in order to mediate assembly and budding from the cellular membranes. These functions have been associated with overlapping late domain sequences (PTAP and PPEY) found in VP40. Late domains interact with cellular proteins such as Tsg101 and Nedd4 and affect a late step in the budding process. VP24 is thought to be a secondary (minor) matrix protein that, unlike VP40, is only incorporated into virions in small amounts. It has an affinity for the plasma membrane and perinuclear region of infected cells. The precise role of VP24 in replication is unclear; however, VP24 is a known type I IFN antagonist. Unlike VP35, VP24 blocks the translocation of phosphorylated STAT1 into the nucleus, subverting the antiviral response. More recently, VP24 has been associated with adaptation in rodent hosts. Glycoproteins The structural GP is a type I transmembrane protein inserted into the membrane as a trimer (see Figure 1(b)). It functions in viral entry, influences pathogenesis, and acts as the major viral antigen. The GP gene shows two open reading frames (ORFs) encoding for the precursors of GP (pre-GP) and a nonstructural secreted glycoprotein (pre-sGP), which is the primary product of this gene. Translation of pre-GP can only be achieved through
mRNA editing, where one adenosine residue is added at a seven-uridine-stretch template sequence, resulting in a frameshift of the primary ORF. GP is cytotoxic when expressed at higher levels, leading to the hypothesis that mRNA editing may be evolutionarily related to the control of overexpression of this protein. The N-terminal 300 amino acids of the pre-GP are identical to those of pre-sGP, but the C termini of each protein are unique. pre-GP is translocated to the endoplasmic reticulum (ER) by an N-terminal signal sequence, and anchored by an extremely short membrane-spanning sequence at the C-terminus. The protein is glycosylated in the ER and Golgi apparatus with both N-linked and O-linked glycans. Most of the O-linked glycans are located in a mucin-like region (rich in theronine, serine, and proline residues) located in the middle of the protein. pre-GP is then cleaved by a subtilisin/kexin-like convertase such as furin, leading to the formation of disulfide-bonded GP1,2. The smaller C-terminal cleavage fragment GP2 contains the transmembrane region that anchors GP to the membrane. Interestingly, proteolytic cleavage of pre-GP is not required for infectivity or for virulence, indicating that the uncleaved precursor can mediate receptor binding and fusion. The production of pre-sGP differentiates EBOV from Marburg viruses, which do not express this soluble protein. pre-sGP is also translocated into the ER, modified in the secretory (e.g., oligomerization, glycosylation) pathway, and cleaved by furin near the C-terminus to release a short peptide termed delta peptide. No biological properties have been attributed to delta peptide. Recent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis suggests that sGP forms a homodimer in a parallel orientation, held together by disulfide bonds between the most N-terminal and C-terminal cysteine residues. sGP circulates in the blood of acutely infected humans. Its exact function is still unknown; however, an interaction with the cellular and
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molecules are actually used in vivo or the degree of requirement for infection and disease. Folate receptor alpha was the first identified possible receptor but entry was also shown to occur in the absence of the molecule. C-type lectins such as DC-SIGN, DC-SIGNR, and hMGL were shown to be able to enhance binding but are not required for infection. Expression of members of the Tyro3 family converted the poorly susceptible Jurkat T cells into susceptible cells for particles pseudotyped with ZEBOV GP and enhanced ZEBOV infection. Receptor binding results in endocytosis into endosomes, possibly via clathrincoated pits and caveolae although some studies suggest that they may not be necessary. Acidification of the endosomes is necessary for fusion of the viral and endosomal membranes, which is mediated by a region in GP2. Proteolysis by cathepsins B and L in the acidic endosomes might be essential for infectivity. Transcription and genome replication seems to follow the general principles for Mononegavirales. There seems to
humoral host immune responses has been postulated. In contrast to GP which mediates endothelial cell (EC) activation and decreases EC barrier functions, sGP seems to have an anti-inflammatory role.
Viral Life Cycle The viral life cycle consists of several events: binding/ entry, uncoating, transcription, translation, genome replication, and packaging/budding (see Figure 2). EBOVs have selective tropism primarily for monocytes, macrophages, and dendritic cells (DCs), although other cell types such as fibroblasts, hepatocytes, and ECs can be infected. In contrast, lymphocytes generally do not support EBOV replication, which is believed to be due to the lack of expression of the viral receptor(s). Although several molecules have been identified as possible viral receptors in various in vitro systems, it is not clear if any of the identified
Attachment (folate receptor alpha; C-type lectins: DC-SIGN, DC-SIGNR, hGML; and Tyro3 family: Axl, Mer, DTK)
Endocytosis (caveolae and clathrin, GP cleavage by cathepsins B and L)
Uncoating
(−) ER
Transcription ------
polyA
------
Replication
polyA ------ polyA
(GP/sGP through ER/Golgi, other viral proteins at cytosolic ribosomes)
Ribosomes
NP, VP35, VP40, VP30, VP24, L
Translation and processing Golgi
(−)
polyA
GP
Nucleus
------
(+)
Cytoplasm
Inclusion bodies
Assembly/budding Plasma membrane
(lipid rafts)
Figure 2 Ebola virus life cycle. The virus attaches to specific receptor(s) on the plasma membrane which leads to endocytosis via caveolae and clathrin-mediated pathways. The viral GP is cleaved in the endosome by cathepsins L and B. After uncoating, transcription and replication take place in the cytoplasm. All proteins except the GPs are translated on free ribosomes in the cytoplasm, while the glycoproteins are produced and modified in the ER and Golgi. Nucleocapsids formed in inclusion bodies interact with the matrix protein VP40 at the plasma membrane. Assembly and particle budding occurs at lipid rafts in the plasma membrane.
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be a gradual decrease in mRNA levels from 30 to 50 end of the genome. The encapsidated viral genome serves as the template for transcription, which in the case of EBOV requires the L proteins, VP35 and VP30. mRNA transcripts are monocistronic, capped, polyadenylated, and contain long noncoding regions at their 30 and/or 50 ends. Stem–loop structures at the 50 ends of mRNA transcripts may affect transcript stability, ribosome binding, and translation. The switch from transcription to replication seems to be triggered by an accumulation of viral proteins, especially NP, in the cytoplasm. Large amounts of NP are localized in inclusion bodies to which other viral proteins are recruited. These inclusion bodies may function as sites of RNP complex formation. Membrane/lipid rafts have been identified as platforms for the assembly of virions; GP trimers conveyed to the surface membrane have an affinity for these rafts that is associated with palmitoylation of the membrane-spanning anchor sequence. RNP complexes interact with VP40, which is deposited at the plasma membrane via the late retrograde endosomal pathway. Abolition of the late domains in VP40 blocks particle formation, but only partially affects viral replication, suggesting that other domains or proteins must be involved. VP24 is also associated with the plasma membrane and enhances particle formation, but a specific role in particle maturation has not been identified. GP trimers seem to interact with VP40 and/or VP24 to finalize the budding process.
Experimental Models Experimental hosts include monkeys (specifically rhesus and cynomolgus macaques), for which infection with ZEBOV is usually 100% lethal, guinea pigs (which show febrile responses 4–10 days after inoculation, but not uniform lethality), and newborn and immunocompromised mice when inoculated with wild-type virus. The resistance of the adult rodent models to EBOV infection has led to the production of guinea pig- and mouse-adapted ZEBOV strains (GPA-ZEBOV and MA-ZEBOV), produced through the serial passage of the virus through progressively older animals. These adapted strains are able to present uniform lethality in their respective hosts. MA-ZEBOV demonstrates reduced virulence in NHPs, whereas GPA-ZEBOV remains uniformly lethal in this model. Infected mice do not exhibit strong coagulation abnormalities (a hallmark of EBOV infection in humans and NHPs) and also slightly differ in other clinical symptoms. Mice, however, represent a good screening model for studies of antiviral and host immune responses. Infected guinea pigs do present coagulation defects that more closely resemble a human or an NHP infection. For growth in cell culture, primary monkey kidney cells and monkey kidney cell lines (e.g., Vero) are often used, but EBOV can replicate in many mammalian cell types including human ECs and monocytes/macrophages.
Clinical Features Host Range and Experimental Models Natural Hosts and Geographic Distribution EBOVs typically infect humans and nonhuman primates (NHPs); ZEBOV and SEBOV appear to be the main causes of lethal infections in humans, and thus are of primary public health concern. EBOVs appear to be indigenous to the tropical rain forest regions of central Africa (with the exception of REBOV), as indicated by the geographic locations of known outbreaks and seroepidemiological studies. The discovery of REBOV in the Philippines suggests the presence of a filovirus in Asia. Many species have been discussed as possible natural hosts; however, no nonhuman vertebrate hosts or arthropod vectors have yet been definitely identified. Epidemiological data have suggested monkeys as a potential reservoir of filoviruses; however, the high pathogenicity of EBOV for NHPs does not generally support such a concept. Similarities in biological properties to other viral hemorrhagic fever agents, such as ‘Old World’ arenaviruses, favor a chronic infection of an animal that regulates survival of the viruses in nature. More recently, viral RNA and virus-specific antibodies could be detected in African fruit bat species, suggesting that bats might be a reservoir for EBOV.
Nonspecific, flu-like symptoms such as fever, chills, and malaise appear abruptly in infected individuals after an incubation period that ranges from 2 to 21 days, but on average lasts 4–10 days. Subsequently, multisystemic symptoms such as prostration, anorexia, vomiting, chest pain, and shortness of breath develop. Macropapular rash associated with varying degrees of erythema may also occur and is a valuable differential diagnostic feature. At the peak of the disease, vascular dysfunction signs appear ranging from petechiae, echymoses, and uncontrolled bleeding at venipuncture sites, to mucosal bleeding and diffuse coagulopathy. Massive blood loss is atypical, although it may happen in the gastrointestinal tract, and is not sufficient to lead to death. Fatal cases develop shock, multiorgan failure, and coma with death occurring between days 6 and 16. Survivors can have multiple sequelae such as hepatitis, myelitis, ocular disease, myalgia, asthenia, and psychosis. The mortality and severity of symptoms are viral species dependent, with ZEBOV causing 60–90% and SEBOV 50–60% lethality. Viremia in fatal cases can reach peak levels of 109 genomes ml1, while survivors have peak levels of about 107 genomes ml1. Viral antigen can be found systemically, although it is most abundant in the spleen and liver.
Ebolavirus
Diagnosis of EHF is based on the detection of virusspecific antibodies, virus particles, or particle components. The procedures are the same as for Marburg hemorrhagic fevers.
Pathogenicity Most of the information available regarding pathogenicity is derived from ZEBOV infections in humans and animal models. Death seems to be the result of systemic shock due to vascular dysfunction, which is caused by a complex interaction of the immune system with vascular physiology. Three major processes – increase in vascular permeability, disseminated intravascular coagulation, and impaired protective immunological responses – are the main events that lead to shock and death. The first two processes are the product of a series of events starting with infection of primary target cells (monocytes/macrophages (M ) and
Virus replication and activation (tissue factor, pro-inflammatory cytokines)
63
DCs) and later leading to activation and decrease of barrier function of ECs, while the third is still being actively investigated (see Figure 3). Infected M are strongly activated, secreting proinflammatory molecules such as interleukin 6(IL-6), tumor necrosis factor alpha (TNF)-a, IL-1b, nitric oxide (NO), and spread the infection systemically. In contrast, infection of DCs results in impaired activation, with no upregulation of co-stimulatory molecules such as major histocompatibility complexes (MHCs) I and II, CD80, CD86, and CD40. Early interaction with primary target cells is independent of virus replication and current models suggest that GP in the repetitive context of a particle is required for activation, possibly by binding and cross-linking cellular receptors. Activation of ECs by mediators such TNF-a and NO is believed to be the main cause for the decrease in EC barrier function. TNF-a, NO, and pro-inflammatory cytokines increase vascular permeability and EC surface adhesion molecule expression, which are
Virus replication and impaired function
Apoptosis of T and NK cells (no virus replication)
T cell Monocyte/ macrophage
DC Immune evasion and systemic replication − type I IFN antagonism
d
an
d
se
bl ee
ea cr
di
In
ng
Shock
ag
Monocyte/ macrophage
Co
lity
bi
ea
ul op
at
rm
hy
pe
ECs
ECs EC activation (by cytokines, viral particles, coagulopathy)
EC and macrophage activation (tissue factor, fibrin formation, disseminated intravascular coagulopathy)
Figure 3 Ebola virus pathogenesis. Shock, the final event in severe/lethal cases, is caused by three processes, which influence each other: systemic viral replication and immune evasion, increase in vascular permeability, and coagulopathy. Infection of primary target cells such as monocytes/macrophages and DCs results in systemic spread of the virus and differential activation. Monocytes/macrophages are activated to produce pro-inflammatory cytokines and tissue factor (TF), while DC activation is impaired, leading to poor protective immune responses. Type I interferon responses are also inhibited by virus-encoded inhibitors (VP35 and VP24). Despite no infection of T- and natural killer (NK) cells, there is extensive apoptosis in those cell types. ECs are activated by pro-inflammatory cytokines and virus, which leads to increased permeability. TF expression induces coagulopathy, which is also able to increase inflammation.
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necessary in extravasation of immune cells in inflamed tissue. Although these pro-inflammatory cytokines are an integral part of a normal, localized immune response by attracting and activating immune cells to the site of infection, in the context of a systemic and extensive response, they have a negative effect by inducing shock. ECs also serve as target cells and infection results in activation as indicated by upregulation of adhesion molecule expression followed by cytolysis. However, in vivo EC destruction can only be observed at the end stage of the disease. Infection of M also results in the expression of tissue factor (TF), which can impair the anticoagulant– protein-C pathway by downmodulating thrombomodulin. TF is believed to be an important protein in the development of disseminated intravascular coagulopathy (DIC), as inhibition of TF delays death in NHPs and even protects 33% of the infected animals. TNF-a can also induce TF expression in ECs, which would further amplify coagulopathy and DIC. Coagulopathy is known to enhance inflammation, and this could lead to a vicious cycle where inflammation enhances coagulopathy, which in turn would amplify inflammation. Rapid systemic viral replication is an integral part of the pathogenesis, as infection and even binding of particles results in not only M activation but also DC impairment. ZEBOV infection in humans and NHPs results in rapid massive viremia and high titers in various organs, especially spleen and liver. This correlates with an impaired innate and adaptive immune response. However, high viremia and lethality are only seen in guinea pigs and mice after host adaption or infection of various immune-deficient mice, suggesting that evasion of immune responses plays a pivotal role in pathogenesis. Mice lacking the IFN-a/b receptor or STAT1, the main signaling molecule in response to type I IFN, are extremely susceptible to ZEBOV. Treatment of NHPs with IFN-a did not protect the animals but delayed death, suggesting that type I IFN may be an important innate molecule in delaying viral replication. Interestingly, MA-ZEBOV is less sensitive to IFN-a treatment in murine macrophages compared to wild-type ZEBOV, and analysis of genomic mutations in GA- and MA-ZEBOV indicated that mutations in NP and VP24 are sufficient for a lethal phenotype. VP24 inhibits responses to interferon in vitro, but it is not clear yet if the mutations in the adapted strains are required for type I IFN evasion or other functions. ZEBOV is also able to inhibit IFN gene induction through VP35, and deletion of the region in VP35 involved in IFN antagonism results in a highly attenuated virus. Furthermore, comparative in vitro gene microarray analysis demonstrated a correlation between cytotoxicity and IFN antagonism, which was strongest with ZEBOV and weakest with REBOV. Adaptive immune responses are also impaired during infection. As mentioned above, upregulation of co-stimulatory molecules is inhibited in DCs, which reduces activation of T cells. There is also a dramatic drop in number of T and
natural killer (NK) cells despite the fact that lymphocytes do not support ZEBOV replication. It is believed that lymphopenia is caused by ‘bystander’ apoptosis, although the mechanism is not well understood. Activation of T cells could also be inhibited by treatment in vitro with a 17amino-acid peptide present in GP2, which has homology to a known immunosuppressive peptide in the retroviral Gag protein; yet there is no evidence that the complete ZEBOV GP has the same capabilities.
Treatment The current treatment of EHF is strictly supportive, involving fluid and electrolyte replenishment and pain reduction. Due to the remote location of the outbreaks and limited resources available in the affected regions, treatment options have not been tested in patients. Several experimental treatment strategies have been successful in the rodent models, but failed in the NHP model, which is considered the most accurate in modeling human disease. Therapeutic antibodies are still considered a shortterm solution despite varying success in animal models and humans. Convalescent serum was used in a limited number of patients during the Kikwit 1995 ZEBOV outbreak but the success is a matter of dispute. Passive immunization with hyperimmune horse serum resulted in protection of hamadryl baboons, whereas it only delayed death in cynomolgus macaques. Monoclonal antibody treatment is successful in rodent models but has failed in preliminary NHP studies. Currently the most feasible and promising approach relates to interference with coagulation using the recombinant nematode anticoagulant protein c2 (rNAPc2). Administration of the drug, which is already in clincial trials for other applications, as late as 24 h post infection resulted in 33% survival in the rhesus macaque model. Even more potent seems to be post-exposure treatment with a recombinant vesicular stomatitis virus (VSV) expressing the ZEBOV GP, which resulted in 50% protection when given 30 min post infection, but the mechanism of post-exposure protection is not yet understood. It is expected that approval of this attenuated replicationcomponent vector will be difficult. The recent advances in the understanding of EBOV pathogenesis and replication will open new avenues for intervention therapy. Novel antiviral strategies such as viral gene silencing through specific siRNA, cathepsin inhibition, and functional domain interference with small peptides showed promise in tissue culture and partially also in rodent models. Strategies targeting host responses are important alternative options and include anticytokine therapy and modulation of coagulation pathways. It should be noted that more classical approaches such as ribavirin treatment, which has been successfully used to treat other VHFs, are not indicated for EHF.
Echoviruses
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Vaccines
Acknowledgments
There are no approved vaccines against EBOV. Similar to therapeutic approaches, many initial vaccination strategies were successful in rodents but failed in NHPs. Nevertheless, there are several promising experimental strategies. Nonreplicating adenoviruses expressing the ZEBOV GP and NP were able to induce sterile immunity in cynomolgus macaques within 28 days or more post vaccination, either alone in a single shot approach or in combination with DNA priming and adenovirus boost. Although DNA priming is not necessary for protection of NHPs, it may be required to overcome the problem of preexisting immunity to human adenoviruses. DNA vaccination alone against EBOV is already in Phase I clinical trial, where it was shown to be safe and effective in inducing humoral and cellular immune responses. Recombinant VSV expressing ZEBOV GP also induced sterile immunity when given 28 days prior to challenge of cynomolgus macaques, but was also able to protect 50% of rhesus macaques when administered 30 min post challenge. These results, together with the fact that prior immunity against the vector is extremely low, and the only target of neutralizing antibodies, VSV G protein, is removed from the vaccine vector, indicate that this platform may be successful in humans if licensing for the replication-competent vector can be achieved. Human parainfluenza virus-based vectors and VLPs are successful in protecting rodents, and preliminary results suggest that they even show efficacy in NHPs. Safety testing of vaccine vectors and the establishment of immune correlates is a priority for all these and future vaccine platforms. Note : Investigation of an ongoing hemorrhagic fever outbreak in southwestern Uganda revealed what appears to be an additional distinct species of Ebola virus associated with this outbreak. Preliminary genome sequence analysis suggests the most closely related Ebola virus species would be the Coˆte d’Ivoire ebolavirus. Initial outbreak investigations suggest that infection with this newly discovered virus is associated with a lower case fatality than infections with Zaire ebolavirus (60–90%) or Sudan ebolavirus (50–60%) (T. G. Ksiazek, Centers for Disease Control and Prevention, Atlanta, GA, United States, personal communication).
The Public Health Agency of Canada (PHAC), Canadian Institutes of Health Research (CIHR), and CBRNE (Chemical, Biological, Radiological & Nuclear) Research and Technology Initiative (CRTI), Canada, supported work on filoviruses at the National Microbiology Laboratory of the Public Health Agency of Canada. See also: Marburg Virus.
Further Reading Feldmann H, Geisbert TW, Jahrling PB, et al. (2005) Filoviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, and Ball LA (eds.) Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, pp. 645–653. San Diego, CA: Elsevier Academic Press. Feldmann H, Jones SM, Klenk HD, and Schnittler HJ (2003) Ebola virus: From discovery to vaccine. Nature Reviews Immunology 3: 677–685. Feldmann H, Jones SM, Schnittler HJ, and Geisbert TW (2005) Therapy and prophylaxis of Ebola virus infections. Current Opinion in Investigational Drugs 6: 823–830. Geisbert TW and Hensley LE (2004) Ebola virus: New insights into disease aetiopathology and possible therapeutic interventions. Expert Reviews in Molecular Medicine 6: 1–24. Geisbert TW and Jahrling PB (2004) Exotic emerging viral diseases: Progress and challenges. Nature Medicine 10(supplement 12): S110–S121. Hensley LE, Jones SM, Feldmann H, Jahrling PB, and Geisbert TW (2005) Ebola and Marburg viruses: Pathogenesis and development of countermeasures. Current Molecular Medicine 5: 761–772. Hirsch M (ed.) (1999) Journal of Infectious Diseases 179(supplementum 1): S1–S288. Jasenosky LD and Kawaoka Y (2004) Filovirus budding. Virus Research 106: 181–188. Paragas J and Geibert TW (2006) Development of treatment strategies to combat Ebola and Marburg viruses. Expert Reviews of Anti-Infective Therapy 4: 67–76. Pattyn SR (ed.) (1978) Ebola Virus Hemorrhagic Fever. Amsterdam: Elsevier/North-Holland Biomedical Press. Reed DS and Mohamadzadeh M (2007) Status and challenges of filovirus vaccines. Vaccine 25: 1923–1934. Sanchez A, Geisbert TW, and Feldmann H (2007) Marburg and Ebola viruses. In: Knipe DM, Howley PM Griffin DE, et al. (eds.) Fields Virology, 5th edn., pp. 1409–1448. Philadelphia: Kluwer/Lippincott Williams and Wilkins. Walsh PD, Abernethy KA, Bermejo M, et al. (2003) Catastrophic ape decline in western equatorial Africa. Nature 422: 611–614. Zaki SR and Goldsmith CS (1999) Pathologic features of filovirus infections in humans. Current Topics in Microbiology and Immunology 235: 97–116.
Echoviruses T Hyypia¨, University of Turku, Turku, Finland ã 2008 Elsevier Ltd. All rights reserved.
History and Classification Echoviruses belong to the species Human enterovirus B (HEV-B), in the genus Enterovirus of the family
Picornaviridae. Original classification of picornaviruses was based mainly on physicochemical properties and pathogenesis in experimental animals. At the beginning of the twentieth century, poliomyelitis was transmitted to
History Ebola virus (EBOV) first emerged as the causative agent of two major outbreaks of viral hemorrhagic fever (VHF) occurring almost simultaneously along the Ebola River in Democratic Republic of Congo (DRC, formerly Zaire) and Sudan in 1976 (see Table 1). Over 500 cases were reported, with case fatality rates (CFRs) of 88% and 53%, respectively. It was later recognized that these two outbreaks were caused by two distinct species of EBOV (Zaire ebolavirus and Sudan ebolavirus). In 1989, a novel virus, Reston ebolavirus (REBOV), was isolated from naturally infected cynomolgus macaques (Macaca fascicularis) imported from the Philippines into the United States. All shipments except one were traced to a single supplier in the Philippines; however, the actual origin of the virus and mode of contamination for the facility have never been ascertained. While pathogenic for naturally and experimentally infected monkeys, limited data indicate that REBOV may not be pathogenic for humans as animal caretakers were infected without producing clinical symptoms. In 1994, the first case of Ebola hemorrhagic fever (EHF) occurred in western Africa in the Tai Forest Reserve in Coˆte d’Ivoire (Ivory Coast). An ecologist was infected by performing a necropsy on a dead chimpanzee whose troop had lost several members to infection with Coˆte d’Ivoire ebolavirus (CIEBOV). A single seroconversion was later documented, suggesting another nonfatal human case in nearby Liberia. Zaire ebolavirus (ZEBOV) reemerged in Kikwit, DRC, in 1995, causing a large EHF outbreak with 81% CFR. Sudan ebolavirus (SEBOV) reemerged in 2000–01 in the Gulu District in northern Uganda. There were over 425 cases (53% CFR), making it the largest EHF epidemic documented so far. Starting in 1994, an endemic focus of ZEBOV activity became obvious in the northern boarder region of Gabon and the Republic of Congo (RC) with multiple small EHF outbreaks over the past decade. Most infections there have been associated with the hunting and handling of animal
carcasses (mainly great apes). In addition, ZEBOV has virtually decimated the chimpanzee and gorilla populations in those areas. At least three laboratory exposures to EBOV have occurred; one in Russia (2004) was fatal.
Taxonomy and Classification Filoviruses are classified in the order Mononegavirales, a large group of enveloped viruses containing nonsegmented, negative-sense (NNS) RNA genomes. The family Filoviridae is separated into two distinct genera, Marburgvirus and Ebolavirus. The genus Ebolavirus is subdivided into four species – Zaire ebolavirus, Sudan ebolavirus, Coˆte d’Ivoire ebolavirus, and Reston ebolavirus. Filoviruses are classified as maximum containment (biosafety level 4 (BSL-4)) agents as well as category A pathogens based on their generally high mortality rate, person-to-person transmission, potential aerosol infectivity, and absence of vaccines and chemotherapy.
Biological and Physical Properties of Virion EBOV particles are pleomorphic, appearing as U-shaped, 6-shaped, circular forms, or as long filamentous, sometimes branched forms varying greatly in length (up to 14 000 nm), but have a uniform diameter of 80 nm (see Figures 1(a) and 1(b)). EBOV virions purified by ratezonal gradient centrifugation are bacilliform in outline and show an average length associated with peak infectivity of 970–1200 nm. Except for the differences in length, EBOVs seem to be very similar in morphology. Virions contain a helical ribonucleoprotein complex RNP or nucleocapsid roughly 50 nm in diameter bearing crossstriations with a periodicity of approximately 5 nm, and a dark, central axial space 20 nm in diameter running
57
58
Ebolavirus
Table 1
Ebola hemorrhagic fever (EHF) outbreaks from 1976 to 2005
Ebola species
Year
Outbreak location (country)
Place of origin
Human cases (% case fatality rate)
Zaire ebolavirus
1976 1977 1994 1995 1996 1996
Yambuku (DRC) Tandala (DRC) Ogooue-Invindo province (Gabon) Kikwit (DRC) Mayibout (Gabon) Booue (Gabon); Johannesburg (South Africa) Ogooue-Invindo province (Gabon); Cuvette region (RC) Cuvette region (RC); Ogooue-Invindo province (Gabon) Mboma and Mbandza (RC) Etoumbi and Mbomo in Cuvette region (RC) Kampungu (DRC) Nzara, Maridi, Tembura, Juba (Sudan) Nzara, Yambio (Sudan) Gulu District in Mbarrara, Masindi (Uganda) Yambio Country (Sudan) Tai Forest (Ivory Coast) Liberia (Liberia) Reston, Virginia (also Pennsylvania and Texas) (USA) Siena (Italy) Alice, Texas (USA)
DRC DRC Gabon DRC Gabon Gabon
318 (88) 1 (100) 51 (60) 315 (79) 37 (57) 61 (74)
Gabon?a
124 (79)
RC?a
143 (90)
RC RC
35 (83) 12 (75)
DRC Sudan Sudan Uganda
Ongoing 284 (53) 34 (65) 425 (53)
Sudan Ivory Coast Liberia?a Philippinesb
17 (41) 1 (0) 1 (0) 4 (0)c
Philippines Philippines
0d 0d
2001–02 2002–03 2003 2005
Sudan ebolavirus
Coˆte d’Ivoire ebolavirus Reston ebolavirus
2007 1976 1979 2000–01 2004 1994 1995 1989 1992 1996
a
Place of origin unconfirmed. Reston virus has only been traced to a single monkey-breeding facility in the city of Calamba, the Philippines, which was depopulated in 1996 and is no longer in operation. c Mortality in monkeys was estimated at 82%. d Only monkeys were infected in these outbreaks; no reported human cases. DRC, Democratic Republic of the Congo; RC, Republic of the Congo. b
the length of the particle. The RNP complex is composed of the genomic RNA and the RNA-dependent RNA polymerase (L), nucleoprotein (NP), and virion proteins 35 and 30 (VP35 and VP30). A lipoprotein unit-membrane envelope derived from the host cell plasma membrane surrounds it. Spikes approximately 7–10 nm in length, spaced apart at 10 nm intervals, are visible on the virion surface and are formed by the viral glycoprotein (GP). Virus particles have a molecular weight of approximately 3–6 108 Da and a density in potassium tartrate of 1.14 g cm–3. Virus infectivity is quite stable at room temperature. Inactivation can be performed by ultraviolet (UV) light and g-irradiation, 1% formalin, bpropiolactone, and brief exposure to phenolic disinfectants and lipid solvents, like deoxycholate and ether.
Properties of Genome The EBOV genome consists of a molecule of linear, nonsegmented, negative-stranded RNA which is noninfectious,
not polyadenylated, and complementary to viral-specific messenger RNA. The genome amounts to c. 1.1% of the total virion. EBOV genomes are 19 kbp in length and fairly rich in adenosine and uridine residues. Genomes show a linear gene arrangement in the order 30 leader–NP–VP35–VP40–GP–VP30–VP24–L–50 trailer (see Figure 1(b)). All genes are flanked at their 30 and 50 ends by highly conserved transcriptional start (30 -CUnCnUnUAAUU-50 ) and termination signal sequences (30 UAAUUCUUUUU-5), respectively, all of which contain the pentamer 30 -UAAUU-50 . Most genes are separated by intergenic sequences variable in length and nucleotide composition. A feature of all EBOV genomes is the fact that some intergenic regions overlap by the conserved pentamer (UAAUU) sequence. ZEBOV and SEBOV show three such overlaps within the intergenic sequences of VP35/VP40, GP/VP30, and VP24/L, whereas REBOV shows only two between VP35/VP40 and VP24/L. Extragenic leader and trailer sequences are present at the 30 and 50 genome ends. These sequences are complementary at their very extremities, showing the potential to form
Ebolavirus
59
(a) Spikes Nucleocapsid 80 nm
Envelope
NP
VP35
VP40
GP
Matrix
L
VP30 VP24
5⬘
3⬘ IR Leader
Overlap
Edit site
Overlap
Gene noncoding regions Extragenic and intergenic region (IR) sequences
Overlap
Trailer
Nucleoproteins Polymerase complex Membrane associated Nonstructural (sGP)
(b) Figure 1 Particle morphology. (a) Transmission electron microscopy of Ebola virus particle. Both graphs show images of Vero E6 cells infected with Zaire ebolavirus. (b) Ebola virus particle structure and genome organization. The upper part provides a scheme of the virus particle separated into four components. The inner core consists of the nucleocapsid which is a structure formed by the single-stranded, negative-sense RNA genome associated with the two nucleoproteins (NP, VP30) and the polymerase complex (L and VP35). The matrix is built by the viral matrix protein VP40 and a minor component VP24. The envelope is derived from the infected cell during assembly/budding. The spikes consist of a homotrimer of the glycoprotein (GP).
stem–loop structures. Phylogenetic analyses on the basis of the GP gene of the different Ebola species show a 37–41% difference in their amino acid and nucleotide sequences. Analysis within one species shows remarkable genetic stability between strains (the variation in nucleotide sequences has been shown to be <7% and even <2% among distinct ZEBOV strains), unexpected for an RNA virus, but highly indicative that these viruses have reached a high degree of fitness to fill their respective niches.
Properties of Viral Proteins Virions contain seven structural proteins with presumed identical functions for the different viruses (see Table 2). The electrophoretic mobility patterns of these proteins are characteristic for each species.
RNP Complex and Matrix Proteins Four proteins are associated with the viral RNP complex: NP, L, VP30, and VP35 (see Figure 1(b)). These proteins are involved in the transcription and replication of the genome. NP and VP30 represent the major and minor nucleoproteins, respectively. They interact strongly with the genomic RNA molecule, and are both phosphoproteins. VP30 is also a zinc-binding protein that behaves as a transcriptional activator. The L and VP35 proteins form the polymerase complex. The L protein, like other L proteins of NNS RNA viruses, represents the RNA-dependent RNA polymerase. Motifs linked to RNA (template) binding, phosphodiester bonding (catalytic site), and ribonucleotide triphosphate binding have all been described. VP35 appears to behave in a mode similar to that of the phosphoproteins found in other NNS RNA viruses, acting as a cofactor that affects the mode of RNA synthesis (transcription vs
60
Ebolavirus
Table 2
Ebola virus proteins: functions and localization
Gene ordera
Ebola virus proteins
Protein function (localization)
1 2
Nucleoprotein (NP) Virion protein 35 (VP35)
3 4 (primary) 4 (secondary) 5
Virion protein 40 (VP40) Secreted glycoprotein (sGP) (nonstructural) Glycoprotein (GP) Virion protein 30 (VP30)
Major nucleoprotein, RNA genome encapsidation (component of RNP complex) Polymerase complex cofactor, type I interferon antagonist (component of RNP complex) Matrix protein, virion assembly, and budding (membrane associated) May have immunomodulatory role (secreted)
6
Virion protein 24 (VP24)
7
Polymerase (L)
a
Receptor binding and membrane fusion (membrane associated) Minor nucleoprotein, RNA binding, transcriptional activator (component of RNP complex) Minor matrix protein, virion assembly, type I interferon antagonist (membrane associated) RNA-dependent RNA polymerase, enzymatic portion of polymerase complex (component of RNP complex)
Gene order refers to 30 –50 gene arrangement as shown in Figure 1(b).
replication). VP35 is also known to be a type I interferon (IFN) antagonist; it blocks IFN-a/b expression by inhibiting IFN regulatory factor 3 (IRF-3). VP40 functions as the viral matrix protein and represents the most abundant protein in the virion. It plays a number of roles in viral infection related to assembly and budding of the viral particles. The production of VP40 by itself is sufficient to initiate the budding process for the production of viruslike particles (VLPs); however, the production of these particles is greatly enhanced by the addition of GP and NP. Recent studies have shown that EBOV hijacks the cellular protein machinery in order to mediate assembly and budding from the cellular membranes. These functions have been associated with overlapping late domain sequences (PTAP and PPEY) found in VP40. Late domains interact with cellular proteins such as Tsg101 and Nedd4 and affect a late step in the budding process. VP24 is thought to be a secondary (minor) matrix protein that, unlike VP40, is only incorporated into virions in small amounts. It has an affinity for the plasma membrane and perinuclear region of infected cells. The precise role of VP24 in replication is unclear; however, VP24 is a known type I IFN antagonist. Unlike VP35, VP24 blocks the translocation of phosphorylated STAT1 into the nucleus, subverting the antiviral response. More recently, VP24 has been associated with adaptation in rodent hosts. Glycoproteins The structural GP is a type I transmembrane protein inserted into the membrane as a trimer (see Figure 1(b)). It functions in viral entry, influences pathogenesis, and acts as the major viral antigen. The GP gene shows two open reading frames (ORFs) encoding for the precursors of GP (pre-GP) and a nonstructural secreted glycoprotein (pre-sGP), which is the primary product of this gene. Translation of pre-GP can only be achieved through
mRNA editing, where one adenosine residue is added at a seven-uridine-stretch template sequence, resulting in a frameshift of the primary ORF. GP is cytotoxic when expressed at higher levels, leading to the hypothesis that mRNA editing may be evolutionarily related to the control of overexpression of this protein. The N-terminal 300 amino acids of the pre-GP are identical to those of pre-sGP, but the C termini of each protein are unique. pre-GP is translocated to the endoplasmic reticulum (ER) by an N-terminal signal sequence, and anchored by an extremely short membrane-spanning sequence at the C-terminus. The protein is glycosylated in the ER and Golgi apparatus with both N-linked and O-linked glycans. Most of the O-linked glycans are located in a mucin-like region (rich in theronine, serine, and proline residues) located in the middle of the protein. pre-GP is then cleaved by a subtilisin/kexin-like convertase such as furin, leading to the formation of disulfide-bonded GP1,2. The smaller C-terminal cleavage fragment GP2 contains the transmembrane region that anchors GP to the membrane. Interestingly, proteolytic cleavage of pre-GP is not required for infectivity or for virulence, indicating that the uncleaved precursor can mediate receptor binding and fusion. The production of pre-sGP differentiates EBOV from Marburg viruses, which do not express this soluble protein. pre-sGP is also translocated into the ER, modified in the secretory (e.g., oligomerization, glycosylation) pathway, and cleaved by furin near the C-terminus to release a short peptide termed delta peptide. No biological properties have been attributed to delta peptide. Recent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis suggests that sGP forms a homodimer in a parallel orientation, held together by disulfide bonds between the most N-terminal and C-terminal cysteine residues. sGP circulates in the blood of acutely infected humans. Its exact function is still unknown; however, an interaction with the cellular and
Ebolavirus
61
molecules are actually used in vivo or the degree of requirement for infection and disease. Folate receptor alpha was the first identified possible receptor but entry was also shown to occur in the absence of the molecule. C-type lectins such as DC-SIGN, DC-SIGNR, and hMGL were shown to be able to enhance binding but are not required for infection. Expression of members of the Tyro3 family converted the poorly susceptible Jurkat T cells into susceptible cells for particles pseudotyped with ZEBOV GP and enhanced ZEBOV infection. Receptor binding results in endocytosis into endosomes, possibly via clathrincoated pits and caveolae although some studies suggest that they may not be necessary. Acidification of the endosomes is necessary for fusion of the viral and endosomal membranes, which is mediated by a region in GP2. Proteolysis by cathepsins B and L in the acidic endosomes might be essential for infectivity. Transcription and genome replication seems to follow the general principles for Mononegavirales. There seems to
humoral host immune responses has been postulated. In contrast to GP which mediates endothelial cell (EC) activation and decreases EC barrier functions, sGP seems to have an anti-inflammatory role.
Viral Life Cycle The viral life cycle consists of several events: binding/ entry, uncoating, transcription, translation, genome replication, and packaging/budding (see Figure 2). EBOVs have selective tropism primarily for monocytes, macrophages, and dendritic cells (DCs), although other cell types such as fibroblasts, hepatocytes, and ECs can be infected. In contrast, lymphocytes generally do not support EBOV replication, which is believed to be due to the lack of expression of the viral receptor(s). Although several molecules have been identified as possible viral receptors in various in vitro systems, it is not clear if any of the identified
Attachment (folate receptor alpha; C-type lectins: DC-SIGN, DC-SIGNR, hGML; and Tyro3 family: Axl, Mer, DTK)
Endocytosis (caveolae and clathrin, GP cleavage by cathepsins B and L)
Uncoating
(−) ER
Transcription ------
polyA
------
Replication
polyA ------ polyA
(GP/sGP through ER/Golgi, other viral proteins at cytosolic ribosomes)
Ribosomes
NP, VP35, VP40, VP30, VP24, L
Translation and processing Golgi
(−)
polyA
GP
Nucleus
------
(+)
Cytoplasm
Inclusion bodies
Assembly/budding Plasma membrane
(lipid rafts)
Figure 2 Ebola virus life cycle. The virus attaches to specific receptor(s) on the plasma membrane which leads to endocytosis via caveolae and clathrin-mediated pathways. The viral GP is cleaved in the endosome by cathepsins L and B. After uncoating, transcription and replication take place in the cytoplasm. All proteins except the GPs are translated on free ribosomes in the cytoplasm, while the glycoproteins are produced and modified in the ER and Golgi. Nucleocapsids formed in inclusion bodies interact with the matrix protein VP40 at the plasma membrane. Assembly and particle budding occurs at lipid rafts in the plasma membrane.
62
Ebolavirus
be a gradual decrease in mRNA levels from 30 to 50 end of the genome. The encapsidated viral genome serves as the template for transcription, which in the case of EBOV requires the L proteins, VP35 and VP30. mRNA transcripts are monocistronic, capped, polyadenylated, and contain long noncoding regions at their 30 and/or 50 ends. Stem–loop structures at the 50 ends of mRNA transcripts may affect transcript stability, ribosome binding, and translation. The switch from transcription to replication seems to be triggered by an accumulation of viral proteins, especially NP, in the cytoplasm. Large amounts of NP are localized in inclusion bodies to which other viral proteins are recruited. These inclusion bodies may function as sites of RNP complex formation. Membrane/lipid rafts have been identified as platforms for the assembly of virions; GP trimers conveyed to the surface membrane have an affinity for these rafts that is associated with palmitoylation of the membrane-spanning anchor sequence. RNP complexes interact with VP40, which is deposited at the plasma membrane via the late retrograde endosomal pathway. Abolition of the late domains in VP40 blocks particle formation, but only partially affects viral replication, suggesting that other domains or proteins must be involved. VP24 is also associated with the plasma membrane and enhances particle formation, but a specific role in particle maturation has not been identified. GP trimers seem to interact with VP40 and/or VP24 to finalize the budding process.
Experimental Models Experimental hosts include monkeys (specifically rhesus and cynomolgus macaques), for which infection with ZEBOV is usually 100% lethal, guinea pigs (which show febrile responses 4–10 days after inoculation, but not uniform lethality), and newborn and immunocompromised mice when inoculated with wild-type virus. The resistance of the adult rodent models to EBOV infection has led to the production of guinea pig- and mouse-adapted ZEBOV strains (GPA-ZEBOV and MA-ZEBOV), produced through the serial passage of the virus through progressively older animals. These adapted strains are able to present uniform lethality in their respective hosts. MA-ZEBOV demonstrates reduced virulence in NHPs, whereas GPA-ZEBOV remains uniformly lethal in this model. Infected mice do not exhibit strong coagulation abnormalities (a hallmark of EBOV infection in humans and NHPs) and also slightly differ in other clinical symptoms. Mice, however, represent a good screening model for studies of antiviral and host immune responses. Infected guinea pigs do present coagulation defects that more closely resemble a human or an NHP infection. For growth in cell culture, primary monkey kidney cells and monkey kidney cell lines (e.g., Vero) are often used, but EBOV can replicate in many mammalian cell types including human ECs and monocytes/macrophages.
Clinical Features Host Range and Experimental Models Natural Hosts and Geographic Distribution EBOVs typically infect humans and nonhuman primates (NHPs); ZEBOV and SEBOV appear to be the main causes of lethal infections in humans, and thus are of primary public health concern. EBOVs appear to be indigenous to the tropical rain forest regions of central Africa (with the exception of REBOV), as indicated by the geographic locations of known outbreaks and seroepidemiological studies. The discovery of REBOV in the Philippines suggests the presence of a filovirus in Asia. Many species have been discussed as possible natural hosts; however, no nonhuman vertebrate hosts or arthropod vectors have yet been definitely identified. Epidemiological data have suggested monkeys as a potential reservoir of filoviruses; however, the high pathogenicity of EBOV for NHPs does not generally support such a concept. Similarities in biological properties to other viral hemorrhagic fever agents, such as ‘Old World’ arenaviruses, favor a chronic infection of an animal that regulates survival of the viruses in nature. More recently, viral RNA and virus-specific antibodies could be detected in African fruit bat species, suggesting that bats might be a reservoir for EBOV.
Nonspecific, flu-like symptoms such as fever, chills, and malaise appear abruptly in infected individuals after an incubation period that ranges from 2 to 21 days, but on average lasts 4–10 days. Subsequently, multisystemic symptoms such as prostration, anorexia, vomiting, chest pain, and shortness of breath develop. Macropapular rash associated with varying degrees of erythema may also occur and is a valuable differential diagnostic feature. At the peak of the disease, vascular dysfunction signs appear ranging from petechiae, echymoses, and uncontrolled bleeding at venipuncture sites, to mucosal bleeding and diffuse coagulopathy. Massive blood loss is atypical, although it may happen in the gastrointestinal tract, and is not sufficient to lead to death. Fatal cases develop shock, multiorgan failure, and coma with death occurring between days 6 and 16. Survivors can have multiple sequelae such as hepatitis, myelitis, ocular disease, myalgia, asthenia, and psychosis. The mortality and severity of symptoms are viral species dependent, with ZEBOV causing 60–90% and SEBOV 50–60% lethality. Viremia in fatal cases can reach peak levels of 109 genomes ml1, while survivors have peak levels of about 107 genomes ml1. Viral antigen can be found systemically, although it is most abundant in the spleen and liver.
Ebolavirus
Diagnosis of EHF is based on the detection of virusspecific antibodies, virus particles, or particle components. The procedures are the same as for Marburg hemorrhagic fevers.
Pathogenicity Most of the information available regarding pathogenicity is derived from ZEBOV infections in humans and animal models. Death seems to be the result of systemic shock due to vascular dysfunction, which is caused by a complex interaction of the immune system with vascular physiology. Three major processes – increase in vascular permeability, disseminated intravascular coagulation, and impaired protective immunological responses – are the main events that lead to shock and death. The first two processes are the product of a series of events starting with infection of primary target cells (monocytes/macrophages (M ) and
Virus replication and activation (tissue factor, pro-inflammatory cytokines)
63
DCs) and later leading to activation and decrease of barrier function of ECs, while the third is still being actively investigated (see Figure 3). Infected M are strongly activated, secreting proinflammatory molecules such as interleukin 6(IL-6), tumor necrosis factor alpha (TNF)-a, IL-1b, nitric oxide (NO), and spread the infection systemically. In contrast, infection of DCs results in impaired activation, with no upregulation of co-stimulatory molecules such as major histocompatibility complexes (MHCs) I and II, CD80, CD86, and CD40. Early interaction with primary target cells is independent of virus replication and current models suggest that GP in the repetitive context of a particle is required for activation, possibly by binding and cross-linking cellular receptors. Activation of ECs by mediators such TNF-a and NO is believed to be the main cause for the decrease in EC barrier function. TNF-a, NO, and pro-inflammatory cytokines increase vascular permeability and EC surface adhesion molecule expression, which are
Virus replication and impaired function
Apoptosis of T and NK cells (no virus replication)
T cell Monocyte/ macrophage
DC Immune evasion and systemic replication − type I IFN antagonism
d
an
d
se
bl ee
ea cr
di
In
ng
Shock
ag
Monocyte/ macrophage
Co
lity
bi
ea
ul op
at
rm
hy
pe
ECs
ECs EC activation (by cytokines, viral particles, coagulopathy)
EC and macrophage activation (tissue factor, fibrin formation, disseminated intravascular coagulopathy)
Figure 3 Ebola virus pathogenesis. Shock, the final event in severe/lethal cases, is caused by three processes, which influence each other: systemic viral replication and immune evasion, increase in vascular permeability, and coagulopathy. Infection of primary target cells such as monocytes/macrophages and DCs results in systemic spread of the virus and differential activation. Monocytes/macrophages are activated to produce pro-inflammatory cytokines and tissue factor (TF), while DC activation is impaired, leading to poor protective immune responses. Type I interferon responses are also inhibited by virus-encoded inhibitors (VP35 and VP24). Despite no infection of T- and natural killer (NK) cells, there is extensive apoptosis in those cell types. ECs are activated by pro-inflammatory cytokines and virus, which leads to increased permeability. TF expression induces coagulopathy, which is also able to increase inflammation.
64
Ebolavirus
necessary in extravasation of immune cells in inflamed tissue. Although these pro-inflammatory cytokines are an integral part of a normal, localized immune response by attracting and activating immune cells to the site of infection, in the context of a systemic and extensive response, they have a negative effect by inducing shock. ECs also serve as target cells and infection results in activation as indicated by upregulation of adhesion molecule expression followed by cytolysis. However, in vivo EC destruction can only be observed at the end stage of the disease. Infection of M also results in the expression of tissue factor (TF), which can impair the anticoagulant– protein-C pathway by downmodulating thrombomodulin. TF is believed to be an important protein in the development of disseminated intravascular coagulopathy (DIC), as inhibition of TF delays death in NHPs and even protects 33% of the infected animals. TNF-a can also induce TF expression in ECs, which would further amplify coagulopathy and DIC. Coagulopathy is known to enhance inflammation, and this could lead to a vicious cycle where inflammation enhances coagulopathy, which in turn would amplify inflammation. Rapid systemic viral replication is an integral part of the pathogenesis, as infection and even binding of particles results in not only M activation but also DC impairment. ZEBOV infection in humans and NHPs results in rapid massive viremia and high titers in various organs, especially spleen and liver. This correlates with an impaired innate and adaptive immune response. However, high viremia and lethality are only seen in guinea pigs and mice after host adaption or infection of various immune-deficient mice, suggesting that evasion of immune responses plays a pivotal role in pathogenesis. Mice lacking the IFN-a/b receptor or STAT1, the main signaling molecule in response to type I IFN, are extremely susceptible to ZEBOV. Treatment of NHPs with IFN-a did not protect the animals but delayed death, suggesting that type I IFN may be an important innate molecule in delaying viral replication. Interestingly, MA-ZEBOV is less sensitive to IFN-a treatment in murine macrophages compared to wild-type ZEBOV, and analysis of genomic mutations in GA- and MA-ZEBOV indicated that mutations in NP and VP24 are sufficient for a lethal phenotype. VP24 inhibits responses to interferon in vitro, but it is not clear yet if the mutations in the adapted strains are required for type I IFN evasion or other functions. ZEBOV is also able to inhibit IFN gene induction through VP35, and deletion of the region in VP35 involved in IFN antagonism results in a highly attenuated virus. Furthermore, comparative in vitro gene microarray analysis demonstrated a correlation between cytotoxicity and IFN antagonism, which was strongest with ZEBOV and weakest with REBOV. Adaptive immune responses are also impaired during infection. As mentioned above, upregulation of co-stimulatory molecules is inhibited in DCs, which reduces activation of T cells. There is also a dramatic drop in number of T and
natural killer (NK) cells despite the fact that lymphocytes do not support ZEBOV replication. It is believed that lymphopenia is caused by ‘bystander’ apoptosis, although the mechanism is not well understood. Activation of T cells could also be inhibited by treatment in vitro with a 17amino-acid peptide present in GP2, which has homology to a known immunosuppressive peptide in the retroviral Gag protein; yet there is no evidence that the complete ZEBOV GP has the same capabilities.
Treatment The current treatment of EHF is strictly supportive, involving fluid and electrolyte replenishment and pain reduction. Due to the remote location of the outbreaks and limited resources available in the affected regions, treatment options have not been tested in patients. Several experimental treatment strategies have been successful in the rodent models, but failed in the NHP model, which is considered the most accurate in modeling human disease. Therapeutic antibodies are still considered a shortterm solution despite varying success in animal models and humans. Convalescent serum was used in a limited number of patients during the Kikwit 1995 ZEBOV outbreak but the success is a matter of dispute. Passive immunization with hyperimmune horse serum resulted in protection of hamadryl baboons, whereas it only delayed death in cynomolgus macaques. Monoclonal antibody treatment is successful in rodent models but has failed in preliminary NHP studies. Currently the most feasible and promising approach relates to interference with coagulation using the recombinant nematode anticoagulant protein c2 (rNAPc2). Administration of the drug, which is already in clincial trials for other applications, as late as 24 h post infection resulted in 33% survival in the rhesus macaque model. Even more potent seems to be post-exposure treatment with a recombinant vesicular stomatitis virus (VSV) expressing the ZEBOV GP, which resulted in 50% protection when given 30 min post infection, but the mechanism of post-exposure protection is not yet understood. It is expected that approval of this attenuated replicationcomponent vector will be difficult. The recent advances in the understanding of EBOV pathogenesis and replication will open new avenues for intervention therapy. Novel antiviral strategies such as viral gene silencing through specific siRNA, cathepsin inhibition, and functional domain interference with small peptides showed promise in tissue culture and partially also in rodent models. Strategies targeting host responses are important alternative options and include anticytokine therapy and modulation of coagulation pathways. It should be noted that more classical approaches such as ribavirin treatment, which has been successfully used to treat other VHFs, are not indicated for EHF.
Echoviruses
65
Vaccines
Acknowledgments
There are no approved vaccines against EBOV. Similar to therapeutic approaches, many initial vaccination strategies were successful in rodents but failed in NHPs. Nevertheless, there are several promising experimental strategies. Nonreplicating adenoviruses expressing the ZEBOV GP and NP were able to induce sterile immunity in cynomolgus macaques within 28 days or more post vaccination, either alone in a single shot approach or in combination with DNA priming and adenovirus boost. Although DNA priming is not necessary for protection of NHPs, it may be required to overcome the problem of preexisting immunity to human adenoviruses. DNA vaccination alone against EBOV is already in Phase I clinical trial, where it was shown to be safe and effective in inducing humoral and cellular immune responses. Recombinant VSV expressing ZEBOV GP also induced sterile immunity when given 28 days prior to challenge of cynomolgus macaques, but was also able to protect 50% of rhesus macaques when administered 30 min post challenge. These results, together with the fact that prior immunity against the vector is extremely low, and the only target of neutralizing antibodies, VSV G protein, is removed from the vaccine vector, indicate that this platform may be successful in humans if licensing for the replication-competent vector can be achieved. Human parainfluenza virus-based vectors and VLPs are successful in protecting rodents, and preliminary results suggest that they even show efficacy in NHPs. Safety testing of vaccine vectors and the establishment of immune correlates is a priority for all these and future vaccine platforms. Note : Investigation of an ongoing hemorrhagic fever outbreak in southwestern Uganda revealed what appears to be an additional distinct species of Ebola virus associated with this outbreak. Preliminary genome sequence analysis suggests the most closely related Ebola virus species would be the Coˆte d’Ivoire ebolavirus. Initial outbreak investigations suggest that infection with this newly discovered virus is associated with a lower case fatality than infections with Zaire ebolavirus (60–90%) or Sudan ebolavirus (50–60%) (T. G. Ksiazek, Centers for Disease Control and Prevention, Atlanta, GA, United States, personal communication).
The Public Health Agency of Canada (PHAC), Canadian Institutes of Health Research (CIHR), and CBRNE (Chemical, Biological, Radiological & Nuclear) Research and Technology Initiative (CRTI), Canada, supported work on filoviruses at the National Microbiology Laboratory of the Public Health Agency of Canada. See also: Marburg Virus.
Further Reading Feldmann H, Geisbert TW, Jahrling PB, et al. (2005) Filoviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, and Ball LA (eds.) Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, pp. 645–653. San Diego, CA: Elsevier Academic Press. Feldmann H, Jones SM, Klenk HD, and Schnittler HJ (2003) Ebola virus: From discovery to vaccine. Nature Reviews Immunology 3: 677–685. Feldmann H, Jones SM, Schnittler HJ, and Geisbert TW (2005) Therapy and prophylaxis of Ebola virus infections. Current Opinion in Investigational Drugs 6: 823–830. Geisbert TW and Hensley LE (2004) Ebola virus: New insights into disease aetiopathology and possible therapeutic interventions. Expert Reviews in Molecular Medicine 6: 1–24. Geisbert TW and Jahrling PB (2004) Exotic emerging viral diseases: Progress and challenges. Nature Medicine 10(supplement 12): S110–S121. Hensley LE, Jones SM, Feldmann H, Jahrling PB, and Geisbert TW (2005) Ebola and Marburg viruses: Pathogenesis and development of countermeasures. Current Molecular Medicine 5: 761–772. Hirsch M (ed.) (1999) Journal of Infectious Diseases 179(supplementum 1): S1–S288. Jasenosky LD and Kawaoka Y (2004) Filovirus budding. Virus Research 106: 181–188. Paragas J and Geibert TW (2006) Development of treatment strategies to combat Ebola and Marburg viruses. Expert Reviews of Anti-Infective Therapy 4: 67–76. Pattyn SR (ed.) (1978) Ebola Virus Hemorrhagic Fever. Amsterdam: Elsevier/North-Holland Biomedical Press. Reed DS and Mohamadzadeh M (2007) Status and challenges of filovirus vaccines. Vaccine 25: 1923–1934. Sanchez A, Geisbert TW, and Feldmann H (2007) Marburg and Ebola viruses. In: Knipe DM, Howley PM Griffin DE, et al. (eds.) Fields Virology, 5th edn., pp. 1409–1448. Philadelphia: Kluwer/Lippincott Williams and Wilkins. Walsh PD, Abernethy KA, Bermejo M, et al. (2003) Catastrophic ape decline in western equatorial Africa. Nature 422: 611–614. Zaki SR and Goldsmith CS (1999) Pathologic features of filovirus infections in humans. Current Topics in Microbiology and Immunology 235: 97–116.
Echoviruses T Hyypia¨, University of Turku, Turku, Finland ã 2008 Elsevier Ltd. All rights reserved.
History and Classification Echoviruses belong to the species Human enterovirus B (HEV-B), in the genus Enterovirus of the family
Picornaviridae. Original classification of picornaviruses was based mainly on physicochemical properties and pathogenesis in experimental animals. At the beginning of the twentieth century, poliomyelitis was transmitted to