Ebola virus: new insights into disease aetiopathology … · Ebola virus: new insights into disease...

of 25/25
Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press http://www.expertreviews.org/ Ebola virus: new insights into disease aetiopathology and possible therapeutic interventions 1 expert reviews in molecular medicine Ebola virus: new insights into disease aetiopathology and possible therapeutic interventions Thomas W. Geisbert and Lisa E. Hensley Thomas W. Geisbert (corresponding author) Chief, Department of Viral Pathology and Ultrastructure, Virology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702-5011, USA. Tel: +1 301 619 4803; Fax: +1 301 619 2290; E-mail: [email protected] Lisa E. Hensley Research Microbiologist, Virology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702-5011, USA. Tel: +1 301 619 4808; Fax: +1 301 619 2290; E-mail: [email protected] Institute URL: http://www.usamriid.army.mil Ebola virus (EBOV) gained public notoriety in the last decade largely as a consequence of the highly publicised isolation of a new EBOV species in a suburb of Washington, DC, in 1989, together with the dramatic clinical presentation of EBOV infection and high case-fatality rate in Africa (near 90% in some outbreaks), and the unusual and striking morphology of the virus. Furthermore, there are no vaccines or effective therapies currently available. Progress in understanding the origins of the pathophysiological changes that make EBOV infections of humans so devastating has been slow, primarily because these viruses require special containment for safe research. However, an increasing understanding of the mechanisms of EBOV pathogenesis, facilitated by the development of new tools to elucidate critical regulatory elements in the viral life cycle, is providing new targets that can be exploited for therapeutic interventions. Notably, identifying factors triggering the haemorrhagic complications that characterise EBOV infections led to the development of a strategy to modulate coagulopathy; this therapeutic modality successfully mitigated the effects of EBOV haemorrhagic fever in nonhuman primates. This review summarises our current understanding of EBOV pathogenesis and discusses various approaches to therapeutic intervention based on our current understanding of how EBOV produces a lethal infection.
  • date post

    19-Aug-2018
  • Category

    Documents

  • view

    216
  • download

    0

Embed Size (px)

Transcript of Ebola virus: new insights into disease aetiopathology … · Ebola virus: new insights into disease...

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    1

    expert reviewsin molecular medicine

    Ebola virus: new insights into disease

    aetiopathology and possible

    therapeutic interventions

    Thomas W. Geisbert and Lisa E. Hensley

    Thomas W. Geisbert (corresponding author)Chief, Department of Viral Pathology and Ultrastructure, Virology Division, United StatesArmy Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702-5011, USA.Tel: +1 301 619 4803; Fax: +1 301 619 2290; E-mail: [email protected]

    Lisa E. HensleyResearch Microbiologist, Virology Division, United States Army Medical Research Institute ofInfectious Diseases, Fort Detrick, MD 21702-5011, USA. Tel: +1 301 619 4808; Fax: +1 301 619 2290;E-mail: [email protected]

    Institute URL: http://www.usamriid.army.mil

    Ebola virus (EBOV) gained public notoriety in the last decade largely as aconsequence of the highly publicised isolation of a new EBOV species in asuburb of Washington, DC, in 1989, together with the dramatic clinicalpresentation of EBOV infection and high case-fatality rate in Africa (near 90%in some outbreaks), and the unusual and striking morphology of the virus.Furthermore, there are no vaccines or effective therapies currently available.Progress in understanding the origins of the pathophysiological changes thatmake EBOV infections of humans so devastating has been slow, primarilybecause these viruses require special containment for safe research. However,an increasing understanding of the mechanisms of EBOV pathogenesis,facilitated by the development of new tools to elucidate critical regulatoryelements in the viral life cycle, is providing new targets that can be exploitedfor therapeutic interventions. Notably, identifying factors triggering thehaemorrhagic complications that characterise EBOV infections led to thedevelopment of a strategy to modulate coagulopathy; this therapeutic modalitysuccessfully mitigated the effects of EBOV haemorrhagic fever in nonhumanprimates. This review summarises our current understanding of EBOVpathogenesis and discusses various approaches to therapeutic interventionbased on our current understanding of how EBOV produces a lethal infection.

  • Report Documentation Page Form ApprovedOMB No. 0704-0188Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering andmaintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information,including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, ArlingtonVA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if itdoes not display a currently valid OMB control number.

    1. REPORT DATE 21 SEP 2004

    2. REPORT TYPE N/A

    3. DATES COVERED -

    4. TITLE AND SUBTITLE Ebola virus: new insights into disease aetiopathology and possibletherapeutic interventions, Expert Reviews of Molecular Medicine 6:1 - 24

    5a. CONTRACT NUMBER

    5b. GRANT NUMBER

    5c. PROGRAM ELEMENT NUMBER

    6. AUTHOR(S) Geisbert, TW Hensley, LE

    5d. PROJECT NUMBER

    5e. TASK NUMBER

    5f. WORK UNIT NUMBER

    7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) United States Army Medical Research Institute of Infectious Diseases,Fort Detrick, MD

    8. PERFORMING ORGANIZATIONREPORT NUMBER RPP-04-322

    9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITORS ACRONYM(S)

    11. SPONSOR/MONITORS REPORT NUMBER(S)

    12. DISTRIBUTION/AVAILABILITY STATEMENT Approved for public release, distribution unlimited

    13. SUPPLEMENTARY NOTES The original document contains color images.

    14. ABSTRACT Ebola virus (EBOV) gained public notoriety in the last decade largely as a consequence of the highlypublicised isolation of a new EBOV species in a suburb of Washington, DC, in 1989, together with thedramatic clinical presentation of EBOV infection and high case-fatality rate in Africa (near 90% in someoutbreaks), and the unusual and striking morphology of the virus. Furthermore, there are no vaccines oreffective therapies currently available. Progress in understanding the origins of the pathophysiologicalchanges that make EBOV infections of humans so devastating has been slow, primarily because theseviruses require special containment for safe research. However, an increasing understanding of themechanisms of EBOV pathogenesis, facilitated by the development of new tools to elucidate criticalregulatory elements in the viral life cycle, is providing new targets that can be exploited for therapeuticinterventions. Notably, identifying factors triggering the haemorrhagic complications that characteriseEBOV infections led to the development of a strategy to modulate coagulopathy; this therapeutic modalitysuccessfully mitigated the effects of EBOV haemorrhagic fever in nonhuman primates. This reviewsummarises our current understanding of EBOV pathogenesis and discusses various approaches totherapeutic intervention based on our current understanding of how EBOV produces a lethal infection.

    15. SUBJECT TERMS Filovirus, Ebola Reston, Zaire, pathogenesis, review, therapy

    16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT

    SAR

    18. NUMBEROF PAGES

    24

    19a. NAME OFRESPONSIBLE PERSON

    a. REPORT unclassified

    b. ABSTRACT unclassified

    c. THIS PAGE unclassified

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    2

    expert reviewsin molecular medicine

    Ebola virus (EBOV) infections are usually themost severe of those caused by the viruses oflethal haemorrhagic disease in humans. Clinicalsymptoms appear suddenly after an incubationperiod of 2 to 21 days (Ref. 1). Common presentingcomplaints include high fever, chills, malaise andmyalgia (Refs 2, 3, 4, 5, 6, 7). As the diseaseprogresses, there is evidence of multisystemicinvolvement, and manifestations includeprostration, anorexia, vomiting, nausea,abdominal pain, diarrhoea, shortness of breath,sore throat, oedema, confusion and coma (Refs 2,3, 4, 5, 6, 7). Abnormalities in blood coagulationand fibrinolysis are manifested as petechiae,ecchymoses, mucosal haemorrhages, anduncontrolled bleeding at venipuncture sites(Refs 2, 3, 4, 5, 6, 7); however, massive loss of bloodis atypical and, when present, is largely restrictedto the gastrointestinal tract. In fact, even in thesecases, blood volume loss is insufficient to accountfor death. The presence of a maculopapular rashis a prominent feature (Refs 2, 3, 4, 5, 6, 7), but isnot pathognomonic for EBOV haemorrhagic fever(HF). Fulminant EBOV infection typically evolvesto shock, convulsions, and, in most cases, diffusecoagulopathy (Refs 2, 3, 4, 5, 6, 7). Death usuallyoccurs 69 days after the onset of clinicalsymptoms. It should be noted that evidence ofasymptomatic EBOV infection was documentedin a small group of individuals during arecent outbreak (Ref. 8), but the clinical andepidemiological relevance of this observation isuncertain.

    EpidemiologyEBOV was first recognised during near-simultaneous explosive outbreaks in 1976 insmall communities in the former Zaire [nowthe Democratic Republic of the Congo (DRC)](Ref. 6) and Sudan (Ref. 5). There was significantsecondary transmission through reuse ofunsterilised needles and syringes, and nosocomialcontacts. These independent outbreaks involvedserologically distinct viral species: Zaire EBOV(ZEBOV) and Sudan EBOV (SEBOV). The ZEBOVoutbreak involved 318 cases and 280 deaths (88%mortality), while the SEBOV outbreak involved284 cases and 151 deaths (53% mortality). Since1976, EBOV has appeared sporadically in Africa,causing several small to mid-size outbreaksbetween 1976 and 1979. In 1995, there was a largeepidemic of ZEBOV HF involving 315 cases, withan 81% case fatality rate, in Kikwit, a community

    in the former Zaire (Ref. 1). Meanwhile, between1994 and 1996, there were smaller outbreakscaused by ZEBOV in Gabon (Ref. 9). Morerecently, Uganda, Gabon and the DRC sufferedlarge epidemics of viral HF attributed to EBOV.The most recent outbreak in the DRC alsoinvolved a catastrophic decline in populations ofgreat apes, which are thought to have a role intransmission to humans (Ref. 10).

    In 1989, a third species of EBOV, RestonEBOV (REBOV), appeared in Reston, VA, USA,in association with an outbreak of viral HFamong cynomolgus monkeys (Macaca fascicularis)imported to the USA from the Philippine Islands(Ref. 11). Hundreds of monkeys were infected(with high mortality) in this episode, but nohuman cases occurred (although four animalcaretakers seroconverted without overt disease).Epizootics in cynomolgus monkeys recurred atother facilities in the USA and Europe through1992, and again in 1996. A fourth species of EBOV,Ivory Coast EBOV (ICEBOV), was identified inCte dIvoire in 1994; this species was associatedwith chimpanzees and only one (nonfatal) humaninfection was identified (Ref. 12).

    Very little is known about the natural historyof EBOV. Implication of animal reservoirs andarthropod vectors has been aggressively soughtwithout success. (See Ref. 13 for a more detaileddiscussion of the epidemiology of EBOV.)

    TaxonomyEBOV belongs to the family Filoviridae, whichcomprises filamentous, enveloped, nonsegmented,negative-sense RNA viruses (reviewed in Ref. 13;see also http://www.ncbi.nlm.nih.gov/ICTV).The family Filoviridae is divided into two genera:Marburgvirus and Ebolavirus. The Marburgvirusgenus contains a single species: Lake Victoriamarburg virus (LVMARV). The Ebolavirus genusconsists of the four species of EBOV discussedabove: ZEBOV, SEBOV, REBOV and ICEBOV.

    EBOV: structure and protein functionsEBOV particles contain an ~19 kb, singlenegative-stranded, linear RNA genome that isnoninfectious. The genome encodes sevenstructural proteins, with a gene order of: 3' leader,nucleoprotein (NP), virion protein (VP) 35 (VP35),VP40, glycoprotein (GP), VP30, VP24, polymeraseL protein, and 5' trailer (Ref. 14). Four of theseproteins NP, VP30, VP35 and L associate withthe genomic RNA in a ribonucleoprotein complex,

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    3

    expert reviewsin molecular medicine

    while the three remaining proteins (GP, VP24and VP40) are associated with the membrane. GPis the surface glycoprotein that forms the spikeson the virion and is the effector for receptorbinding and membrane fusion (Refs 15, 16). GP issynthesised as a precursor molecule, GP0; this ispostranslationally cleaved by furin or a furin-likeendoprotease into two subunits GP1 and GP2 which are linked by disulphide bonds to forma heterodimer (Refs 17, 18). Homotrimers ofGP1GP2 form the virion spikes. The primarygene product of the EBOV GP gene is not theGP, but rather a smaller nonstructural secretedGP (sGP), which is efficiently released frominfected cells (Refs 19, 20). The function of sGPhas not been fully delineated, but it appearsthat expression of sGP might protect againstcytotoxicity (Ref. 21). VP40 functions as a matrixprotein and is responsible for the formation ofthe filamentous particles (Ref. 22). VP24 is a minorviral protein whose functions remain unknown,but recent data indicate that VP24 possessesstructural features consistent with a functionas a viral matrix protein and suggest that VP24might have a role in viral assembly and budding(Refs 23, 24, 25).

    EBOV receptors and entry mechanismThe viral entry process consists of three sequentialstages: attachment, co-receptor binding andfusion. Viral fusion can occur at the host cellplasma membrane or viruses can exploit thehost cells endocytic machinery to access thecytoplasm, depending on the characteristics of theviral fusion protein. The entry of filoviruses intohost cells has not been fully described, but is notthought to occur by direct fusion with the plasmamembrane (Ref. 26). The endocytic routes bywhich extracellular ligands, including viruses, areinternalised into the cell include clathrin-coatedvesicles, caveolae, macropinosomes, and otherpathways that presently are poorly characterised.One study suggested that filoviruses use caveolaeto gain entry into host cells (Ref. 27); however,subsequent work by others showed that caveolaeare not required for EBOV entry (Ref. 28). It isknown that EBOV entry is mediated by thebinding of transmembrane viral GP to cell-surfacereceptors (Ref. 15). Currently, four different typesof cell-surface receptors have been proposed toplay a role in EBOV entry: the 1 integrin receptors(Ref. 29); the folate receptor (Ref. 30); thedendritic-cell-specific intercellular adhesion

    molecule (ICAM)-3-grabbing nonintegrin(DC-SIGN) and DC-SIGN-related (DC-SIGNR)factors (Refs 31, 32, 33); and a human macrophagegalactose- and N-acetylgalactosamine-specificC-type lectin (hMGL) (Ref. 34). Although folatereceptor was originally thought to play animportant role in EBOV entry (Ref. 30), recentstudies show that this receptor is not required forEBOV infection (Refs 28, 35), questioning its roleas a key factor in EBOV entry. As EBOV has abroad cell tropism, infecting a wide range of celltypes (Refs 36, 37, 38, 39, 40), it is highly likelythat EBOV exploits several molecules for entryinto host cells. In support of this view, Takada andcolleagues recently proposed that EBOV uses avariety of different C-type lectins for efficiententry into host cells (Ref. 34).

    EBOV replication and transcriptionEBOV replication and transcription is describedonly briefly here; for a more detailed account, seerelevant book chapters on this subject (Refs 13, 41).Replication and transcription of EBOV occursexclusively in the host cell cytoplasm. Because thenegative-strand EBOV RNA genomes cannot beemployed as templates by the DNA-dependentRNA polymerases of the host cell, EBOV encodesits own RNA-dependent RNA polymerase, theL protein. The L protein is thought to control allcatalytic functions that are required for replicationand transcription. Fusion of the viral membranewith the host cell membrane releases the EBOVribonucleoprotein complexes (the viral RNA andthe proteins NP, VP35, VP30 and L) into thecytoplasm. The RNA genome is then transcribedinto mRNAs to generate the EBOV proteins, whilereplication leads to synthesis of a replicativeintermediate. This full-length antigenomic RNA,which is a copy of the negative-strand RNAgenome, then functions as a template for EBOVRNA genome synthesis. Lastly, the nascentribonucleoprotein complexes are assembled withEBOV structural proteins at the plasma membraneor intracellular membranes.

    Animal models of EBOV HFAnimal models have been invaluable for studyingthe pathogenesis of numerous infectious diseasesas well as for testing the efficacy of experimentalprophylactic and therapeutic vaccine and/or drugregimens. The development of animal modelsfor EBOV HF has been particularly challengingbecause the pathophysiology of human EBOV HF

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    4

    expert reviewsin molecular medicine

    has not been clearly defined. This shortcoming isprimarily due to the limited number of casesbeing managed in a medical setting equipped forboth safe and exhaustive clinical laboratoryevaluations.

    Mice, guinea pigs, and several nonhumanprimate species have been employed to modelEBOV HF (reviewed in Refs 42, 43). In addition tothe encumbrances caused by a paucity of humandata, comparison of the disease pathogenesisamong these groups is difficult because ofdifferences in age, route of infection, doseadministered, and the nature of the challengevirus itself. As an example, the challenge doseappears to have a profound effect on the courseof disease in nonhuman primates. Cynomolgusmacaques exposed by intramuscular injectionwith a low challenge dose of ZEBOV [10 plaque-forming units (pfu)] succumbed to infection812 days after challenge (mean = 9.8 days),whereas cynomolgus monkeys exposed byintramuscular injection to a high dose (1000 pfu)of the exact same ZEBOV isolate died 58 daysafter challenge (mean = 6.3 days) (Refs 44, 45). Inhuman cases, route of infection ostensibly affectsthe disease course and the outcome. The meanincubation period for cases of ZEBOV known tobe due to injection was 6.3 days, versus 9.5 daysfor contact exposures (Ref. 46). Moreover, thecase-fatality rate in this 1976 ZEBOV outbreakwas 100% (85 of 85) in cases associated withinjection compared with ~80% (119 of 149) in casesof known contact exposure (Ref. 46). Generalfeatures of disease pathogenesis associated withZEBOV infection in mice, guinea pigs, andnonhuman primates are compared with humanZEBOV infection in Table 1.

    RodentsGuinea pigs and mice have been the primaryrodent models employed to study EBOV HF(Refs 47, 48, 49, 50). Serial adaptation is requiredto produce lethal disease in rodents becauseisolates obtained from fatal primate infectionsrarely produce severe illness in rodents on initialexposure. Although rodents have unquestionablyserved as important models of EBOV infection,we recently showed that rodent models of EBOVHF are not ideal for studying human EBOV HF(Ref. 44); also, others have suggested thatguinea pigs are inadequate for evaluating thepathogenesis of human EBOV HF (Ref. 47). Morespecifically, mice do not exhibit the coagulation

    abnormalities that characterise primate EBOVinfections (Refs 44, 51). The development ofcoagulopathy in EBOV-infected guinea pigs isuncertain, with findings varying among studies(Refs 44, 48, 50, 51). Also, bystander lymphocyteapoptosis, which is associated with human andnonhuman primate EBOV infections (Ref. 52),has yet to be reported in EBOV-infected mice orguinea pigs (Refs 49, 50, 53), although a systematicinvestigation is needed to determine the incidenceof lymphocyte apoptosis in the rodent models.Although the value of rodents for evaluatingthe efficacy of immune- and/or coagulation-modulating therapies is of course in doubt, theseobservations do not imply that rodent models ofEBOV infection have no utility whatsoever.Rodents can serve an important role for evaluatingthe antiviral efficacy of candidate therapies againstEBOV infection, and genetically engineered miceclearly have utility for evaluating specific hostpathogen interactions.

    Nonhuman primatesNot unexpectedly, clinical disease and relatedpathology in nonhuman primates infected withEBOV appear to closely resemble featuresdescribed for human EBOV HF. Several primatespecies have been employed to model ZEBOV HF,including African green monkeys (Chlorocebusaethiops) (Refs 39, 54, 55, 56, 57), cynomolgusmacaques (Refs 44, 45, 55, 58, 59, 60, 61, 62, 63),rhesus macaques (Macaca mulatta) (Refs 38, 44, 54,64, 65), and hamadryad baboons (Papio hamadryas)(Refs 56, 57, 66, 67, 68). Similar pathological featuresof ZEBOV infection have been documentedamong these species; however, a few pathologicalfeatures differ. Most notably, African greenmonkeys do not present with the macular rashthat is characteristic of disease in macaques andbaboons (Refs 38, 54, 55, 61, 65, 66); importantly,this rash also appears to be a prominent featureof human disease (Refs 2, 5, 6). Lymphoiddepletion and impairment of the microcirculationas evidenced by formation of fibrin thrombi invisceral organs is seen to various degrees. Species-specific differences in the appearance ofcoagulopathies have been reported: fibrindeposition is seen in vessels of African greenmonkeys whereas haemorrhages are seen inbaboons (Ref. 56). Species-specific differences incoagulopathy among cynomolgus and rhesusmacaques experimentally infected with ZEBOVhave not been observed (Ref. 63).

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    5

    expert reviewsin molecular medicine

    Table 1. Comparison of disease pathogenesis of Ebola virus infection inanimal models and humansa

    Element / Feature Mouse Guinea pig Macaque Human

    Time to deathb 57 days 812 days 510 days Up to 30 days

    Fever No Yes Yes Yes

    Anorexia Yes Yes Yes Yes

    Peak viraemia 7.9 log10 pfu/ml 5.2 log10 pfu/ml 6.9 log10 pfu/ml 6.5 log10 pfu/ml

    Thrombocytopaenia Yes Yes Yes Yes

    Bleedingc None Rare Occasional Occasional

    Macular rash No No Yes Yes

    DIC (biochemical None Equivocal Yes Yesevidence)

    DIC (fibrin deposits) None Few Abundant NE

    Lymphopaenia Equivocal Yes Yes Yes

    Lymphocyte apoptosis NE NE Yes Yes

    Permissive host cells Monocytes, Monocytes, Monocytes, Monocytes,macrophages, macrophages, macrophages, macrophages,dendritic cells, dendritic cells, dendritic cells, dendritic cells,fibroblasts, fibroblasts, fibroblasts, fibroblasts,hepatocytes, hepatocytes, hepatocytes, hepatocytes,adrenal cortical adrenal cortical adrenal cortical endothelial cells,cells, endothelial cells, endothelial cells, endothelial epithelial cellscells, epithelial cells, epithelial cells, epithelialcells cells cells

    Cytokines/chemokines MCP-1, TNF- NE IFN-, IL-6, IFN-, IL-2,(increased circulating IL-18, MIP-1, IL-6, IL-10,levels) MIP-1, MCP-1, TNF-

    TNF-

    a The table lists symptoms and characteristics of disease pathogenesis observed during ZEBOV infection ofmice, strain 13 guinea pigs, nonhuman primates of the genus Macaca, and humans (summary of data fromRefs 1, 2, 3, 6, 7, 8, 13, 38, 40, 42, 43, 44, 45, 46, 49, 50, 51, 52, 53, 55, 58, 59, 60, 61, 62, 63, 64, 65, 76,77, 87, 96, 159).b For animal models of ZEBOV HF, the time to death after experimental infection varies among studies andlargely depends on the dose of ZEBOV employed. For human cases, information regarding time to death isdifficult to assess because dose, route of exposure, and time of exposure are often unknown. In caseswhere route and time of exposure were documented, disease course appears to be more rapid in casesassociated with injection than in cases of known contact exposure.c Bleeding manifestations in nonhuman primates and humans are infrequent and include bleeding of thegums, haematemesis, bleeding from the rectum and/or bloody stools, haematuria, epistaxis, and bleeding atpuncture sites.Abbreviations: DIC, disseminated intravascular coagulation; IFN, interferon; IL, interleukin; MCP, monocytechemoattractant protein; MIP, macrophage inflammatory protein; NE, not systematically evaluated; TNF,tumour necrosis factor.

    Few studies have evaluated the pathogenesisof SEBOV in nonhuman primates (Refs 55, 69, 70).

    The disease course in rhesus and cynomolgusmacaques appears slower (by several days) than

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    6

    expert reviewsin molecular medicine

    that seen in ZEBOV infections (Refs 55, 69), andrates of survival appear consistent with humandisease. SEBOV infection was not lethal in asmall cohort of African green monkeys, nor wasREBOV (Ref. 55). Similar to SEBOV, and unlikeZEBOV, the disease course in REBOV-infectedcynomolgus monkeys is protracted (Ref. 71).Bystander lymphocyte apoptosis is proposed tobe the mechanism of lymphoid depletion innonhuman primates experimentally infected withREBOV as well as ZEBOV (Ref. 52). Little isknown about the pathogenesis of ICEBOV innonhuman primates, apart from its high lethalityin chimpanzees (Pan troglodytes verus) (Ref. 72).

    Coagulopathy is a prominent feature ofEBOV infection of nonhuman primates. Somereviewers have argued that fibrin deposition isnot ubiquitous in EBOV-infected primates, citingoriginal studies (e.g. Refs 56, 57) reporting thatboth viral strain and nonhuman species can affectthe prominence of fibrin deposits. However, theappearance of fibrin deposits is only one ofseveral indicators of a dysregulated coagulationresponse. Other indicators of coagulopathyinclude consumption of clotting factors, increasesin clotting times, increases in levels of fibrindegradation products, and thrombocytopaenia.A more extensive review of previous ZEBOVstudies in nonhuman primates reveals evidenceof coagulopathy in nearly every case, althoughthose correlates can vary with species. Forexample, for ZEBOV-infected baboons, dramaticchanges were noted in blood-clotting parameters,including marked increases in fibrin degradationproducts, but fibrin deposits were not a prominentfeature (Ref. 57). This finding conclusively showsthat elevated levels of fibrin were being formedat some point during the course of infection.

    Pathogenesis of EBOV HF: the hostresponse to infection

    Understanding the kinetics of hostpathogenrelationships, and identifying critical pathogeneticprocesses, are important for the rationaldevelopment of therapeutic interventions. Amodel representing our current understanding ofEBOV pathogenesis in primates is shown inFigure 1. EBOV infection of humans andnonhuman primates is characterised by markedlymphopaenia and severe degeneration oflymphoid tissues (Refs 13, 61), and defects in thecoagulation system. With a view to understandingthese events, this section reviews the interactions

    between EBOV and the cells of lymphoid tissues,such as monocytes/macrophages, lymphocytesand dendritic cells, as well as cells that areimportant in maintaining the coagulation system,such as endothelial cells.

    EBOV and monocytes/macrophagesThe predilection of EBOV for monocytes/macrophages and their importance in EBOVpathogenesis are well documented (Refs 37, 40,61, 73, 74). Recently, during a temporal study innonhuman primates, monocytes/macrophageswere shown to be among the cells initially infected(Ref. 61). As monocytes/macrophages are usuallythe cells that elicit the response cascade in theacute phase of inflammation (Ref. 75), their earlyinfection represents an effective strategy forevading the host defence system as well asfacilitating dissemination of the virus. EBOVinfection of mononuclear phagocytes triggers acascade of events involving cytokines/chemokinesand oxygen free radicals (Refs 74, 76); it is thoughtthat the consequence of these events, rather thandirect viral infection, causes much of the observedpathology (Refs 73, 74, 76). Recent studies showedthat EBOV infections might be further exacerbatedby upregulation of anti-apoptotic genes neuronal apoptosis inhibitory protein (NAIP) andcellular inhibitor of apoptosis protein 2 (cIAP2) in ZEBOV-infected monocytes/macrophages(Ref. 61). This suggests that EBOV has evolvedan additional mechanism to resist host defensesby inducing these protective transcripts in cellsthat it infects.

    EBOV and lymphocytesDespite the significant lymphocyte destructionassociated with EBOV infections, lymphocytesdo not support production of progeny virus(Refs 37, 52). Extensive lymphocyte apoptosisappears to be critical to the pathogenesis ofEBOV in humans and nonhuman primates(Refs 52, 77). Recently, we showed that apoptosisof bystander lymphocytes, previously describedin end-stage tissues, occurred early in thedisease course in intravascular and extravascularlocations (Ref. 61). Use of fluorescent caspaseinhibitors to detect apoptosis indicated inductionof apoptosis primarily among the CD8+ and CD16+

    subsets of circulating lymphocytes (Refs 61, 78).The mechanism(s) underlying lymphocyteapoptosis has been unclear but is likely to beinduced through multiple pathways. Analysis of

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    7

    expert reviewsin molecular medicine

    Figure 1. Paradigm showing key events in Ebola virus pathogenesis in primates. Ebola virus (EBOV)infection of monocytes/macrophages and dendritic cells appears central to much of the observed pathology.EBOV infection induces monocytes/macrophages to release a multitude of soluble factors including tissuefactor and cytokines/chemokines, triggering a host of downsteam effects. Tissue factor leads to activation ofthe coagulation cascade, fibrin formation and development of disseminated intravascular coagulation. Therelease of cytokines/chemokines indirectly contributes to apoptosis of bystander lymphocytes by upregulatinga variety of genes with pro-apoptotic functions. In addition, EBOV-induced dysfunction of dendritic cells likelyimpairs costimulatory signals important for rescuing activated T cells. The end result of these events is loss ofhomeostasis and dysregulation of the host immune response.

    peripheral blood mononuclear cell (PBMC)gene expression in EBOV-infected cynomolgusmonkeys showed temporal increases in tumournecrosis factor (TNF)-related apoptosis-inducingligand (TRAIL) and Fas transcripts, revealing

    possible pro-apoptotic mechanisms (Refs 61, 76).In addition, bystander lymphocyte apoptosismight be associated with dysfunction of dendriticcells induced by EBOV infection (discussedbelow).

    Paradigm showing key events in Ebola virus pathogenesis in primatesExpert Reviews in Molecular Medicine 2004 Published by Cambridge University Press

    Tissue factor

    Coagulationcascade

    Disseminatedintravascular coagulation

    CytokinesChemokines

    Dysregulatedcostimulation

    Monocyte/macrophage

    Dendritic cell

    Other host cellEbola virus

    Activation ofdeath receptors

    Apoptosis ofbystander lymphocytes

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    8

    expert reviewsin molecular medicine

    EBOV and dendritic cellsIn a recent temporal study in nonhuman primates,dendritic cells in lymphoid tissues wereidentified as early and sustained targets ofZEBOV, indicating their important role in theimmunosuppression characteristic of EBOVinfections (Ref. 61). This finding is of particularimportance as others have shown that ZEBOVinfects human monocyte-derived dendritic cellsand impairs their function (Ref. 79). Specifically,monocyte-derived dendritic cells exposed toZEBOV failed to secrete pro-inflammatorycytokines, did not upregulate costimulatorymolecules including B7-1 and B7-2, andstimulated T cells poorly. Apoptosis of bystanderlymphocytes during EBOV infections might resultfrom the lack of costimulatory signals or from theengagement of death receptors or ligands such asFas or TRAIL. As an example, dendritic cellsprevent Fas-mediated T-cell apoptosis throughcostimulatory rescue signals (Ref. 80). Therefore,it is possible that EBOV-induced dysfunction ofdendritic cells impairs costimulatory signalsimportant for both rescue of activated T cellsand/or for the proper development of T-cellresponses. In addition, the rapid induction ofTRAIL, and possibly Fas, in ZEBOV-infectedmacrophages and dendritic cells (Ref. 76) suggeststhat these might be key factors in the observedbystander apoptosis of lymphocytes in EBOV-infected nonhuman primates.

    EBOV and endothelial cellsRecent studies suggested that the EBOV GP is themain determinant of vascular cell injury andconsequently that direct EBOV-replication-induced structural damage of endothelial cellstriggers the haemorrhagic diathesis (Refs 81, 82).Dysregulation of endothelial cell functions cancause a wide range of vascular effects that lead tochanges in vascular permeability or haemorrhage(Refs 83, 84). Vascular damage can be induced byimmunological mechanisms and/or by directinfection of the vascular tissue. Several microbialdiseases are characterised by severe vascularlesions attributed to direct microbial-replication-induced damage to endothelial cells. For example,intracellular replication of Rickettsia rickettsii, theaetiological agent of Rocky Mountain spottedfever, directly induces lethal injury to hostendothelial cells, causing pathophysiologicalchanges including thrombosis, haemorrhage andvasculitis (Ref. 85). Another example is Nipah

    virus infection, where a systemic vasculitis withextensive thrombosis was attributed to infection,damage and necrosis of endothelial cells (Ref. 86).The aetiology of the haemorrhagic diatheses infatal cases caused by the filoviruses MARV andEBOV was searched for in tissues from initialoutbreaks in 1967 and 1976, respectively, but novascular lesions were identified (Ref. 87).Nonetheless, there has been much speculation thatEBOV-replication-induced structural damage ofendothelial cells triggers the haemorrhagicdiathesis.

    In a recent study, and consistent with theoriginal histology observations of Murphy in fatalhuman cases (Ref. 87), it was demonstrated thatZEBOV infection of endothelial cells does notextensively disrupt the architecture of the vascularendothelium in ZEBOV-infected cynomolgusmonkeys (Ref. 62). Although ZEBOV replicatedin endothelial cells of these animals, endothelialcell infection was only seen focally at late stagesof disease, after the onset of the haemorrhagicabnormalities that characterise EBOV HF(Refs 61, 63). Although ultrastructural evidenceof endothelial cell activation and disruption wasobserved, it is likely that the vasoactive effects onendothelial cells are mediated indirectly becausethese changes were not associated with thepresence of intracytoplasmic EBOV antigens.Additionally, these data showed that whileZEBOV is capable of replicating in microvascularand macrovascular human endothelial cells invitro, replication does not directly inducecytopathology (Ref. 62).

    The subject of GP-mediated cytotoxicityand its relevance in pathogenesis is a highlycontroversial topic. Yang et al. reported that GPexpression caused cell death (Refs 81, 82), whereassubsequent work by other groups showed thatmost of the detached cells (>90%) were viable,suggesting that expression of GP interferes withcell attachment but does not trigger cell death(Refs 88, 89). Another group reported that thecytotoxic effect of GP is associated with its levelof expression (Ref. 21). In this study, using a novelreverse genetics system, Volchkov et al. generateda mutant virus in which the editing site of the GPgene was removed (Ref. 21). The mutant virus nolonger expressed sGP. Notably, the mutant wassignificantly more cytotoxic than wild-type virus,showing that cytotoxicity caused by GP isdownregulated by EBOV through transcriptionalRNA editing and expression of sGP. Thus, the

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    9

    expert reviewsin molecular medicine

    authors postulated that transcriptional editingof the GP gene might play an important role inEBOV pathogenesis by restricting cytotoxicityand thereby increasing viral loads andpromoting spread. The apparent protective effectof transcriptional editing and sGP is consistentwith results detailed above showing that EBOVreplication does not directly induce cytolysis inendothelial cells in vivo (Ref. 62).

    Effect of the pro-inflammatory responseCytokines and chemokines are soluble proteinsthat are generated and secreted in response to anassortment of attacks on a host organism,including microbial infection. Cytokines andchemokines function in a pleiotropic manner,acting on many different types of cells tomodulate the hosts immune response. Whilecytokines and chemokines typically apply theirantimicrobial actions locally, for example in areasof infection, cytokines and chemokines might alsoact systemically, and they commonly induce manyof the symptoms of infection (e.g. fever, myalgia).When present in high concentrations, cytokinesand chemokines can have toxic or even lethal effects.Indeed, studies of septic shock have associatedabnormal production of pro-inflammatorycytokines with disease severity and fatal outcome(Refs 90, 91, 92). For a detailed discussion of theclinical implications of dysegulated immuneresponses, see review articles on this subject(Refs 93, 94, 95).

    Primate EBOV infections are characterisedby a dysregulation of normal host immuneresponses. Increased levels of interleukin (IL)-10have been associated with fatality in previousZEBOV outbreaks (Refs 77, 96). One of thesestudies associated increased levels of interferon(IFN)- with fatalities (Ref. 96), whereas the otherstudy did not detect IFN-, IL-1, IL-8 or IL-12(Ref. 77). Moderate levels of IL-6 and TNF- wereassociated with fatal infection in the days beforedeath in the Gabon study (Ref. 77), and high levelsof TNF-, but not IL-6, were associated withfatality in the Kikwit investigation (Ref. 96). InZEBOV-infected nonhuman primates, increasedcirculating levels of IFN-, IL-6, monocytechemoattractant protein 1 (MCP-1), macrophageinflammatory protein (MIP)-1 and MIP-1 at theearly to mid-stages of disease, and IFN-, IL-18and TNF- at later stages of disease, wereobserved, whereas increased levels of IFN-, IL-4,IL-8, IL-10 or IL-12 were not detected (Ref. 61). In

    other studies, increased levels of IL-6, TNF-, IFN-, IL-2, IL-4, IL-8, IL-10, granulocytemonocytecolony-stimulating factor (GM-CSF), MCP-1, MIP-1 and RANTES (for regulated on activation,normal T cells expressed and secreted) weredetected in nonhuman primates naturally infectedwith REBOV, and the increase in circulatingcytokines was associated with an increase incirculating viral antigen (Ref. 97). However, thecontribution of a second virus, simian HF virus,demonstrated in nearly all REBOV outbreaks todate, remains unknown and potentiallyconfounds these results (Refs 11, 98).

    The ability of EBOV to modulate directly thehost pro-inflammatory response has yet to befully delineated. The EBOV protein VP35reportedly functions as a type I IFN (IFN-/)antagonist (Refs 99, 100, 101). Recent studies showthat VP35 prevents IFN regulatory factor (IRF-3)activation by inhibiting phosphorylation, and itis likely that VP35 prevents EBOV-inducedtranscription of IFN- (Ref. 100). Other studiessuggest that VP24 expression might also interferewith type I IFN signalling (Ref. 101). Althoughnot fully understood, the type I IFN responseappears to be a critical determinant of EBOVpathogenicity. Studies performed in mice showthat host resistance to ZEBOV infection ismediated by the type I IFN response (Ref. 102).Notably, mice lacking the IFN-/ receptor diedwhen challenged with a wild-type strain of ZEBOVthat is not lethal in immunocompetent mice.Furthermore, treatment of immunocompetentmice with anti-IFN-/ antibodies markedlyaccelerated the course of ZEBOV infection whenanimals were challenged with a lethal, adaptedstrain of ZEBOV.

    Dysregulation of the coagulation systemDisseminated intravascular coagulation (DIC)is a syndrome characterised by systemicintravascular activation of coagulation leading towidespread deposition of fibrin in the vasculature,which contributes to the development of multipleorgan failure (reviewed in Refs 103, 104). DIC isassociated with both bleeding and thromboticabnormalities, and, in fact, widespreadthrombosis and bleeding commonly occursimultaneously. Although DIC is often viewed asa prominent manifestation of EBOV infection inprimates, the presence of DIC in human filoviralinfections has been a controversial topic; culturalmores and logistical problems have hampered

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    10

    expert reviewsin molecular medicine

    systematic studies. Fibrin deposition has beendocumented at autopsy for MARV (Ref. 87);furthermore, clinical laboratory data suggest thatDIC is an important feature of human EBOV HF(Refs 2, 6). The coagulation picture is much clearerfor nonhuman primates. Numerous studies haveshown histological and biochemical evidence ofDIC syndrome during EBOV infection in a varietyof nonhuman primate species (Refs 37, 38, 39, 44,51, 55, 61, 63, 64, 65). The mechanism(s) fortriggering the coagulation abnormalities has notbeen fully delineated. New studies have begunto shed some light on the pathogenesis ofcoagulation system dysregulation and suggestthat development of coagulation abnormalitiesmight occur much earlier than previouslythought. Although it is likely that thecoagulopathy seen in EBOV HF is caused bymultiple factors, particularly during the laterstages of disease, recent data strongly implicatetissue factor (TF) expression/release from EBOV-infected monocytes/macrophages as key a factorthat induces the development of coagulationirregularities seen in EBOV infections (Ref. 63).

    Clinical implications/applicationsThere are no known effective treatments forhuman filoviral infections. At this time, treatingpatients infected with EBOV essentially consistsof intensive supportive care. Progress to developtherapies/treatments has been encumberedprimarily because these viruses require specialcontainment (Biosafety Level 4) for safe research,but also as a result of the sporadic and transitorynature of filoviral outbreaks and confinement toremote geographic locales. Nonetheless, therehave been a number of attempts in clinical settingsto improve the status of infected patients, andseveral strategies have been developed andtested in animal models (Table 2). The treatmentstrategies are best analysed when broken downinto the themes of direct antiviral approachesversus strategies to modulate the host immuneresponse.

    Antiviral approachesNeutralisation of virusFor EBOV, passive therapy was first used to treata laboratory worker who also received IFN(Ref. 105); he survived, but the role of immuneplasma in his recovery was not clear. SeveralRussian laboratory workers potentially exposedto EBOV were treated with anti-EBOV goat IgG

    and IFN (Ref. 68); the workers survived, but againthe role of the IgG in their recovery was uncertain.More recently, transfusion of convalescent-phasewhole blood to infected patients in the 1995 Kikwitoutbreak was anecdotally described as conferringincreased survival on treated patients (Ref. 106),but other explanations for survival of seven of theeight patients have been proposed (Refs 106, 107).Of particular concern is the observation that thepatient who died was treated 4 days after the onsetof symptoms, whereas the survivors were treated7 to 15 days after onset, suggesting that thesepatients might have been convalescing whentreated. Passive transfer of neutralising antibodiesprotected guinea pigs (Refs 58, 68) and hymadryadbaboons (Refs 66, 68, 108), and partially protectedmice (Ref. 58), from EBOV infection. In contrast,passive therapy of EBOV-infected cynomolgusmonkeys with a commercially available IgG fromhorses (hyperimmune to EBOV) delayed onset ofviraemia and clinical signs but was not protective,even though the equine product had a high logneutralisation index (Refs 58, 59). Successfultreatment of guinea pigs and hymadryad baboonscould relate to lower viraemias and infectiousviral burdens compared with those in monkeys.Some protective murine monoclonal antibodieswere effective therapeutically when administeredto mice after exposure to ZEBOV (Ref. 109). Also,recombinant monoclonal antibodies have beenproduced from convalescent human bone marrowand peripheral blood mononuclear cells (Ref. 110),and one of these recombinant human antibodiesprotected guinea pigs from lethal ZEBOVinfection (Ref. 111). However, recent testing ofthis antibody in monkeys showed that theadministration of the reagent delayed onset ofviraemia and clinical signs but did not protect theanimals from lethal ZEBOV disease (Ref. 112).

    Inhibition of membrane fusionEach stage of the viral entry process offers apotential target for interrupting viral replication.Inhibition of fusion as an antiviral modality hasrecently gained interest primarily based on the USFood and Drug Administrations approval of anovel fusion inhibitor (T-20, Enfuvirtide, Fuseon)for antiretroviral therapy in humans (reviewedin Ref. 113); Enfuvirtide is a peptide that mimicsa critical region within the viral envelopeglycoprotein gp41, thereby blocking gp41structural rearrangements at a transitionalprefusion conformation. The use of fusion

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    11

    expert reviewsin molecular medicine

    Table 2. Summary of treatment options for Ebola virus haemorrhagic fever

    Treatment option Feasibility and success

    Antiviral approachesAntibody therapy Passive immunisation using approaches including recombinant monoclonal

    antibodies and a polyclonal equine IgG consistently protected rodents from lethalZEBOV HF but failed to protect nonhuman primates. Technological advances toraise circulating antibody concentrations might improve success in primates.

    Fusion inhibitors Some inhibition in vitro but approach has not been tested in vivo.

    Transcription/replicationinhibitors

    Nucleoside analogues Ribavirin failed to protect guinea pigs and nonhuman primates from lethal ZEBOV HF.Antisense In vitro studies have been inconclusive. Advances in chemistry and formulation

    oligonucleotides of cell-penetrating peptides would enhance in vivo opportunities.Small interfering RNAs Not reported. Potential advantages over antisense oligonucleotides in efficiency

    and duration of effect. Specificity with minimal adverse effects is strength oftechnology; drawbacks include genetic variation in viruses and undevelopeddelivery systems. Not yet proven in vivo.

    Maturation inhibitorsFurin inhibitors In vitro data sparse but not encouraging. More studies needed to evaluate

    potential. Approval for HIV therapy in humans shows in vivo feasibility.Budding inhibitors In vitro data sparse but not encouraging. More studies needed.

    Modulation of the host immune responseInflammatory response modulators

    Type I IFNs Treatment encouraging in rodents, but failed to protect nonhuman primatesfrom lethal ZEBOV HF. Improvement of formulations and selective use ofIFN- subtypes with greater antiviral properties might have more utility.

    SAH inhibitors Adenosine analogues, but efficacy in mice linked to induction of type I IFNresponse. Failed to protect nonhuman primates from lethal ZEBOV HF.

    Anticytokine therapies Abnormal production of cytokines/chemokines is a notable feature of diseasein EBOV-infected primates. Anticytokine therapies used for several humandiseases and conditions present opportunities.

    Inhibitors of Problem will be specifically targeting approach to protect lymphocyteslymphocyte apoptosis without concurrently enhancing survival of EBOV-infected cells.

    Coagulation modulatorsTissue factor pathway Pilot study in small cohort of rhesus monkeys showed partial protection,inhibitors and delayed death in unprotected animals. Might have greatest utility in

    combinatorial approaches and/or in supportive care.Factor X inhibitors Blocking common pathway might be more beneficial than inhibiting a specific

    coagulation pathway. Success in treating deep-vein thrombosis with factor Xinhibitors in humans shows potential for mitigating effects of EBOV HF.

    Activated protein C Reduced levels of plasma protein C in ZEBOV-infected nonhuman primatesmirrors reduced levels associated with human sepsis. Success in treatinghuman sepsis with a recombinant product indicates possible utility for EBOV HF.

    Therapeutic vaccines Vesicular stomatitis virus-based ZEBOV vaccine showed promising resultswhen administered therapeutically to mice. Confirmatory studies in nonhumanprimates are critically needed to determine utility for future development.

    Combination therapiesDifficulty in protecting nonhuman primates using a variety of approachessuggests that improved survival might be realised by combining strategies thatsuppress viral replication with strategies that modulate inflammation andcoagulation, allowing the host time to mount an effective immune response.

    Abbreviations: EBOV, Ebola virus; IFN, interferon; HF, haemorrhagic fever; HIV, human immunodeficiencyvirus; ZEBOV, Zaire EBOV.

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    12

    expert reviewsin molecular medicine

    inhibitors against EBOV has not been thoroughlyexplored. Watanabe and colleagues recentlydemonstrated a model for EBOV GP2-mediatedmembrane fusion using pseudotyped viruses(Ref. 114). This model showed the importance ofa coiled-coil motif of GP2 in mediating membranefusion. Notably, an oligopeptide correspondingto the coiled-coil structure of GP2 competitivelyinhibited EBOV entry. However, others havenoted that the concentration required to attain50% inhibition (>1 mg/ml) would be difficult toachieve in sera of test animals (Ref. 115).

    Inhibition of transcription and replicationWith the exception of retroviruses, all RNA virusesincluding EBOV encode an RNA-dependentRNA polymerase to catalyse the synthesis ofnew genomes and mRNAs. Thus, these viralreplication enzymes (L polymerase for filoviruses)are primary targets for antiviral therapeutics. Thenucleoside analogue ribavirin is a broad-spectrumantiviral drug that can block the RNA polymeraseof many RNA viruses; however, ribavirin does noteffectively inhibit the replication of EBOV.Notably, ribavirin does not protect guinea pigsfrom lethal infection with EBOV nor did itprotect monkeys from lethal EBOV infection(Ref. 116). A similar compound, ribamidyl, wasunable to protect hamadryad baboons againstEBOV (Ref. 108). Other tactics to interfere withtranscription and replication include usingantisense oligonucleotides corresponding tosequences in the viral genomic RNA or mRNA.Preliminary studies employing this strategyto inhibit EBOV replication were mostlyunsuccessful (Ref. 115). However, recent studiesby Hartlieb et al. showed that the oligomerisationof EBOV VP30 could be dose-dependentlyinhibited by a synthetic peptide derived from thepresumed oligomerisation domain, suggestingthat VP30 oligomerisation might be a potentialtarget for antiviral drugs (Ref. 117).

    Interference with viral assembly,maturation, and buddingUnlike human immunodeficiency virus (HIV),where pharmacological blockade of a viralprotease was shown to have therapeutic efficacy,EBOV does not encode its own protease that couldserve as a target for an inhibitor. In fact, there is asingle proteolytic step described in EBOVreplication, involving the cleavage of GP0 intoGP1 and GP2 by a cellular furin-like protease

    (Refs 17, 18). In an important study, Neumann andcolleagues showed that a genetically engineeredZEBOV lacking the cleavage site was able toreplicate normally in vitro (Ref. 118), suggestingthat even if an inhibitor could block this step inmaturation, it would likely have a negligible effecton viral replication. Although replication of theZEBOV cleavage-site mutant was approximately1.0 log10 pfu/ml lower than wild-type ZEBOVin the same in vitro assay system (Ref. 118),ZEBOV infections in vivo are typically associatedwith very high viraemias, usually in excess of7.0 log10 pfu/ml (Ref. 61). Thus, this apparentmild attenuation of the ZEBOV cleavage-sitemutant would likely have little significance invivo. However, inhibition of cleavage mighthave other effects on pathogenesis that are notassociated with replication, and the potentialrole of furin inhibitors cannot be discounted(see Ref. 119 for a detailed review of furininhibitors). Specifically, Dolnik et al. recentlyshowed that TNF--converting enzyme (TACE),a member of the ADAM family of zinc-dependentmetalloproteinases, is involved in EBOV GPshedding, which might play an important rolein the pathogenesis of infection by efficientlyblocking the activity of virus-neutralisingantibodies (Ref. 120). Because furin is themajor proprotein convertase involved in thematuration/activation of TACE (Ref. 121),inhibition of furin could potentially interfere withTACE and thereby mitigate the purporteddeleterious consequences of GP shedding.

    Modulation of the host immune responseRegulation of the pro-inflammatoryresponseThe other primary theme for treatment of EBOVinfections has been modulation of the hostimmune response. This area has primarilyinvolved efforts to boost innate immunity.Treatment with exogenous type I IFNs has beenevaluated by several groups. A combination ofridostin (an IFN inducer) and reaferon (IFN-2a)prolonged the mean time to death of ZEBOV-infected guinea pigs (Ref. 122). A recombinantB/D form of human IFN- protected miceagainst a lethal challenge of ZEBOV, even whenadministered up to 2 days after infection (Ref. 115).

    S-adenosylhomocysteine hydrolase (SAH)inhibitors have a protective effect against ZEBOVinfection in mice (Refs 123, 124, 125). Theseadenosine analogues inhibit replication of

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    13

    expert reviewsin molecular medicine

    several DNA and RNA viruses, and theirantiviral efficacy has been ascribed to reducedmethylation of the 5' cap of viral mRNA by(guanine-7-)methyltransferase, which impedestranslation of viral transcripts. However, Braylinked the protective effect of these SAH inhibitorsin ZEBOV-infected mice to induction of a strongIFN- response (Ref. 102).

    As noted previously, strategies that haveshown efficacy in rodents are often ineffective innonhuman primates. Although SAH hydrolaseinhibitors can effectively protect mice from lethalZEBOV infection (Refs 123, 124, 125), studies havethus far been unable to demonstrate efficacy ofthese compounds in nonhuman primates (Ref. 126).Likewise, IFN- showed little beneficial effect incynomolgus monkeys treated with high doses ofrecombinant human IFN-2b immediately aftera high-dose challenge of ZEBOV (Ref. 59).

    Other approaches to modulation of the hostimmune response include strategies to offset ormoderate the physiological effects of disease.Pretreating rhesus monkeys with antioxidants,such as vitamin E, failed to confer any beneficialor protective effect to ZEBOV-infected rhesusmonkeys (Ref. 64).

    Regulation of the coagulation systemTwo patients infected with MARV in 1975 weregiven vigorous supportive treatment andprophylactic heparin (Ref. 127). As both patientssurvived infection, heparin treatment wasattempted on a single patient during the originaloutbreak of ZEBOV in 1976 (Ref. 2). Unfortunately,there was no favourable outcome in this case.As noted above, ZEBOV infection inducesoverexpression of the procoagulant TF in primatemonocytes/macrophages (Ref. 63), suggestingthat TF-pathway inhibition might ameliorate theeffects of EBOV HF. Based on these data, it waspostulated that blocking factor VIIa (FVIIa)/TFmight be beneficial after EBOV infection (Ref. 65).In a preliminary study, nine ZEBOV-infectedmonkeys were treated with a protein thatprevents blood clotting recombinant nematodeanticoagulant protein c2 (rNAPc2) while threeZEBOV-infected monkeys were given a placebocontrol. Three of the nine treated animalssurvived, whereas all three animals that weregiven the placebo control died. Importantly, EBOVinfection is nearly 100% fatal in monkeys and killsup to 90% of humans infected with the virus, so a33% survival rate for one of the most virulent

    diseases known is significant and represents akey breakthrough. Other coagulation defectsassociated with EBOV infection of primatesinclude thrombocytopaenia (Refs 13, 61), althoughthe mechanism for this anomaly has not beendefined. Pretreating rhesus monkeys withthromboxane inhibitors or prostacyclin tocounteract platelet aggregation failed to conferany beneficial or protective effect to ZEBOV-infected rhesus monkeys (Ref. 64).

    Research in progress and outstandingresearch questions

    Antibody therapyPassive transfer of neutralising antibodiesprotects mice, guinea pigs and hymadryadbaboons from EBOV infection. However, passiveimmunotherapy in monkeys of the genus Macacahas been extremely disappointing. Recent dataindicate that high levels of shed GP are present inthe blood of EBOV-infected animals, suggestingthat circulating GP might play an importantrole in disease pathogenesis by blocking theactivity of EBOV-neutralising antibodies (Ref. 120).Although previous attempts were not completelysuccessful in macaques, several new approacheswarrant investigation before passive transfer ofantibodies is abandoned as a potential therapyfor EBOV HF. The availability of EBOV-immunemonkeys, and the development of improvedelectrophoresis-based separation technologies,should facilitate the preparation of high-qualityhyperimmune reagents. Passive therapy usinghomologous hyperimmune plasma shouldafford the opportunity to achieve much highercirculating antibody concentrations, which arelikely necessary to offset the extreme viraemiaobserved in the nonhuman primate models ofEBOV HF. Recently, there has been discussionabout the role of antibodies in enhancing EBOVinfection and potentially exacerbating disease(Refs 128, 129, 130). Although the significance ofimmunological enhancement has yet to bedocumented in vivo, any such demonstrationwould clearly require a re-evaluation ofimmunotherapeutic strategies.

    RNA interferenceSmall interfering RNAs (siRNAs) are powerful,sequence-specific reagents designed to suppressthe expression of genes in cultured mammaliancells through the process of RNA interference(RNAi) (Refs 131, 132). The small size of siRNAs,

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    14

    expert reviewsin molecular medicine

    compared with traditional antisense molecules,prevents activation of the mammalian dsRNA-inducible IFN system. These complexes recognisetheir single-stranded mRNA targets by matchingRNA sequences, and direct the cleavage anddestruction of any mRNAs that perfectly matchone of the duplex siRNA strands, therebypreventing mRNA translation. Intracellularsilencing complexes can be generated either byexogenously administered siRNA duplexes or bysiRNAs processed from precursor short-hairpin(sh) RNAs expressed in the nucleus by vector-based systems (Refs 133, 134, 135). Various RNAistrategies have been employed to test the efficaciesof siRNAs as potential therapeutic agents againstRNA viruses. Whereas most studies demonstratedinhibition of either viral entry or production invitro, recent studies documented that transgeneexpression can be suppressed in vivo. Specifically,the therapeutic potential of this technique wasdemonstrated by effectively targeting conservedregions of influenza virus genes in mice (Ref. 135).Although there have been no published studiesevaluating the efficacy of RNAi as a strategy forsuppressing EBOV replication either in vitro orin vivo, development and optimisation of thistechnology might lead to useful and effectivetreatments against EBOV HF. Indeed, the utilityof this approach as an effective strategy to combatEBOV HF will depend on overcoming a numberof obstacles, in particular the ability to developan efficient drug delivery system. Progress isbeing made in this area, as evidenced by the recentwork of Ge et al. illustrating the ability ofpolyethyleneimine (PEI), a cationic polymer, topromote the successful delivery of siRNA byintravenous administration in influenza-virus-infected mice (Ref. 135).

    Budding inhibitorsEBOV budding is mediated by two proline-richmotifs PPxY and PTAP within VP40 (Refs 136,137, 138, 139, 140). A full N-terminus of VP40 isrequired for budding to occur efficiently (Refs 137,139). Optimal budding is facilitated by interactionwith the cellular ubiquinating enzymes Nedd4and Tsg101 (Refs 138, 139, 140). Thus, drugs thatinterfere with these interactions are of obviousinterest. However, identifying compounds thathave therapeutic utility will be challenging,because it is likely that concentrations requiredto attain sufficient inhibition would be difficultto realise in sera of test animals. Furthermore, the

    utility of budding inhibitors as potential therapiesis called into question by recent work reportedby Ebihara and colleagues (Ref. 141). Specifically,rescue of ZEBOVs containing mutations in the latedomains of VP40 resulted in viral yields that wereonly 1.5 log10 pfu/ml less than wild-type ZEBOV;it is unlikely that such reduction would havemuch therapeutic benefit, considering theextremely high viral loads attained during in vivoZEBOV infections (Ref. 61).

    Immune modulatorsIFNsThe use of IFN- and inducers of type I IFNsas a therapy for treating EBOV infection hasbeen largely unsuccessful thus far. However, anincreased understanding of EBOV pathogenesisconcomitant with recent advances in theproduction of alternative IFN therapeuticpreparations might offer improved efficacy. Forexample, the EBOV VP35 protein reportedlyprevents EBOV-induced transcription of IFN-(Ref. 101). Moreover, induction of IFN- appearsto be a late event in disease pathogenesis ofZEBOV-infected nonhuman primates (Ref. 61).Therefore, therapeutic treatment of animals withrecombinant IFN- might successfully reduceEBOV viraemia and thus ameliorate the effects oflethal disease. Other potential therapies includethe use of universal type I IFNs and other IFNformulations that have increased bioavailability.

    Anticytokine interventionsThere have been relatively few attempts tomodulate the dysregulated cytokine/chemokineresponse that is a consistent feature among manyof the viral HFs. Treating MARV-infected guineapigs with desferal, an immunomodulator that isan IL-1 and TNF- antagonist, partially protecteda small cohort of animals (Ref. 142). In anotherstudy, treating MARV-infected guinea pigs withIL-1 receptor antagonist (IL-1RA) or anti-TNF-serum administered on days 4 to 7 after infectiondecreased the concentration of circulating TNF-and protected half of the animals from lethalinfection (Ref. 143). Anti-TNF therapy alsoreduced mortality in a lethal mouse model ofdengue virus infection (Ref. 144). Increasedplasma levels of TNF- have been shown inZEBOV-infected humans and nonhumanprimates (Refs 61, 77, 96). It would be interestingto determine whether anti-TNF therapy has anybeneficial effect in lethal animal models of EBOV

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    15

    expert reviewsin molecular medicine

    HF. Pro-inflammatory cytokines such as IL-6have been shown to effectively upregulate theprocoagulant TF expression on monocytes(Refs 93, 145). Elevations of plasma IL-6 have beenreported by day 4 postinfection in ZEBOV-infected monkeys (Ref. 61). Given the potentiallyimportant pathogenic role of IL-6 in EBOV HF,the in vivo neutralisation of IL-6 could havetherapeutic utility in ameliorating the lethal effectsof EBOV HF. Anti-IL-6 approaches can mitigatethe deleterious effects of endotoxin-induced sepsisin several animal models (Refs 146, 147, 148).Additional benefits might be realised by usingdrugs that target NF-B activity because many ofthese cytokines/chemokines can be upregulatedby NF-B.

    Inhibition of apoptosis of bystanderlymphocytesIt is likely that the marked apoptosis ofbystander lymphocytes contributes to theimmunosuppression that characterises EBOVinfection of primates. Therefore, successfulinhibition of bystander lymphocyte apoptosisoffers an attractive therapeutic target formitigating the lethal effects of EBOV HF. The exactmechanism(s) for triggering apoptosis pathwaysactivated during EBOV infection of primatesremains unknown. Although it will be importantto determine whether EBOV proteins exhibitpro-apoptotic activities, it is clear that increasedexpression of pro-apoptotic genes such as TRAILand Fas during EBOV infections are contributingfactors to the observed lymphocyte apoptosisand thus are obvious targets for interventionstrategies. Therapies directed to inhibit Fasand/or TRAIL function might have a beneficialeffect. These therapeutic interventions arecurrently being tested in other disease models.For example, treatment using a neutralising anti-TRAIL monoclonal antibody in HIV-infectedmice significantly reduced the development ofapoptotic CD4+ T cells (Ref. 149). Caspaseinhibitors have also shown some benefit inmurine models of sepsis (Ref. 150). However, thetargeted blockade of apoptosis to lymphocytes,and not to cell populations that harbour EBOV, isa formidable hurdle that must be overcome beforethis therapeutic strategy can be fully realised.

    Coagulation modulatorsDysregulation of the coagulation systemappears to be important to the development of

    haemorrhagic shock and death in fatal cases ofZEBOV. The first therapeutic treatment for EBOVHF was recently demonstrated in nonhumanprimates (Ref. 65). This treatment regimen wasbased on targeting the mechanism by which thecoagulation cascade was activated during EBOVHF. In particular, identifying the importance ofthe TF pathway provided a rational approach toidentify and utilise rNAPc2 in an attempt tomitigate the severe effects of EBOV infection. AsrNAPc2 primarily targets signalling throughthe extrinsic blood coagulation pathway,additional benefits might be realised by usinginhibitors of FX to target the intrinsic pathwayas well.

    In other recent studies, we showed rapid dropsin levels of plasma protein C in ZEBOV-infectednonhuman primates, with the initial decreaseobserved as early as the first day after ZEBOVchallenge (Ref. 63). This work suggests thatprotein C might be a critical component to theobserved coagulation dysfunction in ZEBOVHF. The protein C system is one of the mainanticoagulant mechanisms in blood (Ref. 151).Growing evidence also suggests that protein Chas direct anti-inflammatory properties andmodulating activity on cellular functions, likely byblocking NF-B nuclear translocation (Ref. 152).Reduced levels of protein C are found in themajority of patients with sepsis and are associatedwith an increased risk of death (Ref. 153). From atherapeutic perspective, treatment withrecombinant human activated protein Csignificantly reduced mortality in patients withsevere sepsis (Ref. 154). Clearly, identifyingprotein C abnormalities in primate models ofEBOV HF (which so closely parallel protein Canomalies in human coagulopathies) offers anideal target for chemotherapeutic interventions.

    Therapeutic vaccinesRecently, significant progress was made towardsthe development of an EBOV vaccine. Thus far,three EBOV vaccine platforms have been testedsuccessfully in nonhuman primates: (1) a primeboost strategy consisting of a DNA vaccineprime followed by an adenovirus (ADV) vaccineboost (Ref. 60); (2) an accelerated ADV vaccineconsisting of a single injection of ADV expressingEBOV proteins (Ref. 45); and (3), most recently, arecombinant vesicular stomatitis virus (rVSV)-based vaccine (Ref. 155). All of these candidatevaccines are based on ZEBOV and have shown

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    16

    expert reviewsin molecular medicine

    protective efficacy against homologous ZEBOVchallenge, but it is unknown whether the ZEBOVproteins incorporated into these vaccines will alsoconfer protection against other species of EBOV.Indeed, few studies have challenged animalsimmune to one species of EBOV with a differentspecies of EBOV. Small cohorts of nonhumanprimates immune to either REBOV or SEBOVwere only partially protected from lethal ZEBOVchallenge (Refs 55, 70), suggesting thatmultivalent vaccine approaches might benecessary.

    Postexposure vaccination has utility inpreventing or modifying disease for a number ofviral agents including rabies (reviewed in Ref. 156),hepatitis B (Ref. 157), and smallpox (Ref. 158).Recently, a ZEBOV vaccine based on rVSVprotected eight of ten mice when therapeuticallyadministered 30 min after receiving a lethal dose(>1000 LD50) of ZEBOV (Ref. 155). The mechanismof protection has not been determined. It is worthnoting the studies of Baize and colleagues on theimmune response in fatalities and survivorsduring two outbreaks of ZEBOV HF in Gabon(Ref. 77). Their results suggest that recoveryfrom EBOV HF is associated with early androbust humoral responses. Furthermore, cytotoxiccell activation was noted among PBMCs ofrecovering patients at the end of the disease. Thesame investigators subsequently reported thatinflammatory responses are critical determinantsin innate and adaptive immune responses(Ref. 159). They noted that the rapid release ofpro-inflammatory cytokines in asymptomaticZEBOV infections suggests that this responsemight be involved in controlling viral replicationand inducing specific immunity. If the outcomeof EBOV infection is mostly determined by hostresponses during the early stages of the incubationperiod, then interventions to augment thosedefences, such as therapeutic vaccines, might helpto prevent or ameliorate the disease.

    Concluding remarksThere is a critical need for the development ofeffective therapies to respond to outbreaks ofEBOV in Africa and to counter potential acts ofbioterrorism. In addition, a potential EBOVexposure involving a researcher at a US Armylaboratory (Ref. 160), and the recent death of aRussian scientist after an accidental exposure toEBOV (Ref. 161), emphasise the need for medicalcountermeasures for postexposure prophylaxis.

    Considering the aggressive nature of EBOVinfections, in particular the rapid andoverwhelming viral burdens, early diagnosis willplay a significant role in determining the successof any intervention strategy. Thus far, antiviralmodalities targeting EBOV replication havelargely been unsuccessful. Strategies showingsome promise in rodents have uniformly failedto protect nonhuman primates from lethal EBOVHF (Refs 58, 59, 111, 112, 116, 123, 124, 125, 126).Currently, among the most promising antiviralapproaches are siRNAs. Assessment of thisapproach in in vivo models hinges on the abilityto develop an efficient drug delivery system, andan intense effort is under way towards achievingthis goal.

    Results of in vivo studies performed to datesuggest that there might be no single treatmentto combat the lethal effects of EBOV HF. Successfultreatment might require combining severalbeneficial approaches, such as strategies targetingviral replication with modalities controlling themanifestations of disease. A beneficial effect usingan inhibitor, rNAPc2, of the TF pathway has beenobserved (Ref. 65). It will be important todetermine whether survival can be improved bycombining coagulation-modulating agents suchas rNAPc2 with agents that have antiviralproperties. Moreover, it will be important toevaluate whether recombinant activatedprotein C provides synergistic benefit withother coagulation-modulating agents, such asrNAPc2. Therapeutic vaccines might also haveutility in combination approaches. In somecases, therapeutic vaccines are administered inconjunction with other treatments includingimmune globulin to improve efficacy (reviewedin Ref. 156). It will be interesting to determinewhether the therapeutic efficacy of rNAPc2 canbe improved by employing this anticoagulantprotein in combination with therapeutic vaccines.Clearly, an increased understanding of themechanisms of EBOV pathogenesis will facilitateour ability to develop effective countermeasuresagainst this notoriously aggressive pathogen.

    Acknowledgements and fundingOpinions, interpretations, conclusions andrecommendations are those of the author and arenot necessarily endorsed by the US Army. Wethank the peer reviewers of this article for theircomments. The authors work on Ebola andMarburg viruses was supported by the Medical

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    17

    expert reviewsin molecular medicine

    Chemical/Biological Defense Research Program,US Army Medical Research and MaterialCommand (project numbers 02-4-4J-081, 02-4-4J-084, 03-4-7J-020, and 04-4-7J-012).

    References1 Khan, A.S. et al. (1999) The reemergence of Ebola

    hemorrhagic fever, Democratic Republic of theCongo, 1995. Commission de Lutte contre lesEpidemies a Kikwit. J Infect Dis 179 Suppl 1, S76-86, PubMed: 9988168

    2 Isaacson, M. et al. (1978) Clinical aspects of Ebolavirus disease at the Ngaliema hospital, Kinshasa,Zaire, 1976. In Ebola Virus Haemorrhagic Fever(Pattyn, S.R., ed.), pp. 15-20, Elsevier/North-Holland Biomedical Press, New York

    3 Piot, P. et al. (1978) Clinical aspects of Ebola virusinfection in Yambuku area, Zaire, 1976. In EbolaVirus Haemorrhagic Fever (Pattyn, S.R., ed.), pp.7-14, Elsevier/North-Holland Biomedical Press,New York

    4 Smith, D.H., Francis, F. and Simpson, D.I.H.(1978) African haemorrhagic fever in thesouthern Sudan, 1976: the clinical manifestations.In Ebola Virus Haemorrhagic Fever (Pattyn, S.R.,ed.), pp. 21-26, Elsevier/North-HollandBiomedical Press, New York

    5 (1978) Ebola haemorrhagic fever in Sudan, 1976.Report of a WHO/International Study Team.Bull World Health Organ 56, 247-270, PubMed:307455

    6 (1978) Ebola haemorrhagic fever in Zaire, 1976.Bull World Health Organ 56, 271-293, PubMed:307456

    7 Bwaka, M.A. et al. (1999) Ebola hemorrhagicfever in Kikwit, Democratic Republic of theCongo: clinical observations in 103 patients. JInfect Dis 179 Suppl 1, S1-7, PubMed: 9988155

    8 Leroy, E.M. et al. (2001) Early immune responsesaccompanying human asymptomatic Ebolainfections. Clin Exp Immunol 124, 453-460,PubMed: 11472407

    9 Georges-Courbot, M.C. et al. (1997) Isolation andphylogenetic characterization of Ebola virusescausing different outbreaks in Gabon. EmergInfect Dis 3, 59-62, PubMed: 9126445

    10 Walsh, P.D. et al. (2003) Catastrophic ape declinein western equatorial Africa. Nature 422, 611-614,PubMed: 12679788

    11 Jahrling, P.B. et al. (1990) Preliminary report:isolation of Ebola virus from monkeys importedto USA. Lancet 335, 502-505, PubMed: 1968529

    12 Le Guenno, B. et al. (1995) Isolation and partial

    characterisation of a new strain of Ebola virus.Lancet 345, 1271-1274, PubMed: 7746057

    13 Sanchez, A. et al. (2001) Marburg and Ebolaviruses. In Fields Virology (Knipe, D.M. andHowley, P.M., eds), pp. 1279-1304, LippincottWilliams & Wilkins, Philadelphia

    14 Sanchez, A. et al. (1993) Sequence analysis of theEbola virus genome: organization, geneticelements, and comparison with the genome ofMarburg virus. Virus Res 29, 215-240, PubMed:8237108

    15 Takada, A. et al. (1997) A system for functionalanalysis of Ebola virus glycoprotein. Proc NatlAcad Sci U S A 94, 14764-14769, PubMed: 9405687

    16 Ito, H. et al. (1999) Mutational analysis of theputative fusion domain of Ebola virusglycoprotein. J Virol 73, 8907-8912, PubMed:10482652

    17 Sanchez, A. et al. (1998) Biochemical analysis ofthe secreted and virion glycoproteins of Ebolavirus. J Virol 72, 6442-6447, PubMed: 9658086

    18 Volchkov, V.E. et al. (1998) Processing of theEbola virus glycoprotein by the proproteinconvertase furin. Proc Natl Acad Sci U S A 95,5762-5767, PubMed: 9576958

    19 Volchkov, V.E. et al. (1995) GP mRNA of Ebolavirus is edited by the Ebola virus polymeraseand by T7 and vaccinia virus polymerases.Virology 214, 421-430, PubMed: 8553543

    20 Sanchez, A. et al. (1996) The virion glycoproteinsof Ebola viruses are encoded in two readingframes and are expressed through transcriptionalediting. Proc Natl Acad Sci U S A 93, 3602-3607,PubMed: 8622982

    21 Volchkov, V.E. et al. (2001) Recovery of infectiousEbola virus from complementary DNA: RNAediting of the GP gene and viral cytotoxicity.Science 291, 1965-1969, PubMed: 11239157

    22 Noda, T. et al. (2002) Ebola virus VP40 drives theformation of virus-like filamentous particlesalong with GP. J Virol 76, 4855-4865, PubMed:11967302

    23 Huang, Y. et al. (2002) The assembly of Ebolavirus nucleocapsid requires virion-associatedproteins 35 and 24 and posttranslationalmodification of nucleoprotein. Mol Cell 10, 307-316, PubMed: 12191476

    24 Han, Z. et al. (2003) Biochemical and functionalcharacterization of the Ebola virus VP24 protein:implications for a role in virus assembly andbudding. J Virol 77, 1793-1800, PubMed:12525613

    25 Watanabe, S. et al. (2004) Production of novel

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    18

    expert reviewsin molecular medicine

    ebola virus-like particles from cDNAs: analternative to ebola virus generation by reversegenetics. J Virol 78, 999-1005, PubMed: 14694131

    26 Geisbert, T.W. and Jahrling, P.B. (1995)Differentiation of filoviruses by electronmicroscopy. Virus Res 39, 129-150, PubMed:8837880

    27 Empig, C.J. and Goldsmith, M.A. (2002)Association of the caveola vesicular system withcellular entry by filoviruses. J Virol 76, 5266-5270,PubMed: 11967340

    28 Simmons, G. et al. (2003) Folate receptor alphaand caveolae are not required for Ebola virusglycoprotein-mediated viral infection. J Virol 77,13433-13438, PubMed: 14645601

    29 Takada, A. et al. (2000) Downregulation of beta1integrins by Ebola virus glycoprotein:implication for virus entry. Virology 278, 20-26,PubMed: 11112476

    30 Chan, S.Y. et al. (2001) Folate receptor-alpha is acofactor for cellular entry by Marburg and Ebolaviruses. Cell 106, 117-126, PubMed: 11461707

    31 Alvarez, C.P. et al. (2002) C-type lectins DC-SIGNand L-SIGN mediate cellular entry by Ebolavirus in cis and in trans. J Virol 76, 6841-6844,PubMed: 12050398

    32 Lin, G. et al. (2003) Differential N-linkedglycosylation of human immunodeficiency virusand Ebola virus envelope glycoproteinsmodulates interactions with DC-SIGN and DC-SIGNR. J Virol 77, 1337-1346, PubMed: 12502850

    33 Simmons, G. et al. (2003) DC-SIGN and DC-SIGNR bind ebola glycoproteins and enhanceinfection of macrophages and endothelial cells.Virology 305, 115-123, PubMed: 12504546

    34 Takada, A. et al. (2004) Human macrophage C-type lectin specific for galactose and N-acetylgalactosamine promotes filovirus entry. JVirol 78, 2943-2947, PubMed: 14990712

    35 Sinn, P.L. et al. (2003) Lentivirus vectorspseudotyped with filoviral envelopeglycoproteins transduce airway epithelia fromthe apical surface independently of folatereceptor alpha. J Virol 77, 5902-5910, PubMed:12719583

    36 Baskerville, A. et al. (1985) Ultrastructuralpathology of experimental Ebola haemorrhagicfever virus infection. J Pathol 147, 199-209,PubMed: 4067737

    37 Geisbert, T.W. et al. (1992) Association of Ebola-related Reston virus particles and antigen withtissue lesions of monkeys imported to the UnitedStates. J Comp Pathol 106, 137-152, PubMed:

    159753138 Jaax, N.K. et al. (1996) Lethal experimental

    infection of rhesus monkeys with Ebola-Zaire(Mayinga) virus by the oral and conjunctivalroute of exposure. Arch Pathol Lab Med 120, 140-155, PubMed: 8712894

    39 Davis, K.J. et al. (1997) Pathology of experimentalEbola virus infection in African green monkeys.Involvement of fibroblastic reticular cells. ArchPathol Lab Med 121, 805-819, PubMed: 9278608

    40 Zaki, S.R. and Goldsmith, C.S. (1999) Pathologicfeatures of filovirus infections in humans. CurrTop Microbiol Immunol 235, 97-116, PubMed:9893381

    41 Muhlberger, E. (2004) Genome organization,replication, and transcription of filoviruses. InEbola and Marburg Viruses: Molecular andCellular Biology (Klenk, H.D. and Feldmann, H.,eds), pp. 1-26, Horizon Bioscience, Norfolk, UK

    42 Geisbert, T.W. et al. (2004) Filovirus pathogenesisin nonhuman primates. In Ebola and MarburgViruses: Molecular and Cellular Biology (Klenk,H.D. and Feldmann, H., eds), pp. 203-238,Horizon Bioscience, Norfolk, UK

    43 Bray, M. (2004) Pathogenesis of filovirus infectionin mice. In Ebola and Marburg Viruses:Molecular and Cellular Biology (Klenk, H.D. andFeldmann, H., eds), pp. 255-277, HorizonBioscience, Norfolk, UK

    44 Geisbert, T.W. et al. (2002) Evaluation innonhuman primates of vaccines against Ebolavirus. Emerg Infect Dis 8, 503-507, PubMed:11996686

    45 Sullivan, N.J. et al. (2003) Accelerated vaccinationfor Ebola virus haemorrhagic fever in non-human primates. Nature 424, 681-684, PubMed:12904795

    46 Breman, J.G. et al. (1978) The epidemiology ofEbola hemorrhagic fever in Zaire, 1976. In EbolaVirus Haemorrhagic Fever (Pattyn, S.R., ed.), pp.103-121, Elsevier/North-Holland BiomedicalPress, New York

    47 Ryabchikova, E. et al. (1996) Ebola virus infectionin guinea pigs: presumable role ofgranulomatous inflammation in pathogenesis.Arch Virol 141, 909-921, PubMed: 8678836

    48 Chepurnov, A.A. et al. (1997) [Change inbiochemical and hemostatic indicators in guineapigs upon administering Ebola viruspreparations]. Vopr Virusol 42, 171-175, PubMed:9304298

    49 Bray, M. et al. (1998) A mouse model forevaluation of prophylaxis and therapy of Ebola

  • Accession information: DOI: 10.1017/S1462399404008300; Vol. 6; Issue 20; 21 September 2004 Published by Cambridge University Press

    http://www.expertreviews.org/

    Eb

    ola

    vir

    us:

    new

    insi

    gh

    ts in

    to d

    isea

    se a

    etio

    pat

    ho

    log

    yan

    d p

    oss

    ible

    th

    erap

    euti

    c in

    terv

    enti

    on

    s

    19

    expert reviewsin molecular medicine

    hemorrhagic fever. J Infect Dis 178, 651-661,PubMed: 9728532

    50 Connolly, B.M. et al. (1999) Pathogenesis ofexperimental Ebola virus infection in guineapigs. J Infect Dis 179 Suppl 1, S203-217, PubMed:9988186

    51 Bray, M. et al. (2001) Haematological,biochemical and coagulation changes in mice,guinea-pigs and monkeys infected with a mouse-adapted variant of Ebola Zaire virus. J CompPathol 125, 243-253, PubMed: 11798241

    52 Geisbert, T.W. et al. (2000) Apoptosis induced invitro and in vivo during infection by Ebola andMarburg viruses. Lab Invest 80, 171-186,PubMed: 10701687

    53 Gibb, T.R. et al. (2001) Pathogenesis ofexperimental Ebola Zaire virus infection inBALB/c mice. J Comp Pathol 125, 233-242,PubMed: 11798240

    54 Bowen, E.T. et al. (1978) Ebola haemorrhagicfever: experimental infection of monkeys. TransR Soc Trop Med Hyg 72, 188-191, PubMed:418537

    55 Fisher-Hoch, S.P. et al. (1992) Pathogenicpotential of filoviruses: role of geographic originof primate host and virus strain. J Infect Dis 166,753-763, PubMed: 1527410

    56 Ryabchikova, E.I., Kolesnikova, L.V. and Luchko,S.V. (1999) An analysis of features ofpathogenesis in two animal models of Ebolavirus infection. J Infect Dis 179 Suppl 1, S199-202,PubMed: 9988185

    57 Ryabchikova, E.I., Kolesnikova, L.V. a