Ebola VirusesAnthony Sanchez, Centers for Disease Control and Prevention, Atlanta, Georgia, USA

A small group of exotic and mysterious viral agents that cause a severe haemorrhagic

disease in human and/or nonhuman primates.

Molecular Biology

Ebola viruses have evolved to occupy some unknownniche. The ability of these filoviruses to exist in the wild andcause disease in human and nonhuman primates derivesfrom the peculiarities and dynamics of the molecules thatare produced by these pathogens. Ebola viruses evolvedfrom a progenitor virus, which also gave rise to a multitudeof other human pathogens, such as Measles virus andRabies virus. The manner in which these viruses replicatetheir genomes and express proteins from their genes isgreatly influenced by the types of host cells they are capableof infecting. The characteristic effects of their molecularinteractions within the host dictates the type, severity andduration of infection. An important aspect in thepathogenesis of Ebola viruses is their ability to affect theimmune responses of human and/or nonhuman primatesand the wide range of tissues that support the growth ofthese viruses.

Genome organization

The single-stranded ribonucleic acid (RNA) genome of theZaire species of Ebola virus is nearly 19 000 bases in lengthand is very similar in organization to that of Marburg virus.The genome organization and gene structure is consistentwith those of paramyxoviruses and rhabdoviruses. Figure 1illustrates the genome with seven linearly arranged genes(Sanchez et al., 1993). At the extreme 3’- and 5’-ends areshort, extragenic regions (known as leader and trailersequences) that are important in the initiation andregulation of viral RNA synthesis. Genes are delineatedby conserved transcriptional signals for synthesis ofpositive-sense messenger RNA (mRNA) (a start site at

the 3’-end and a stop or polyadenylation site at the 5’-end),and adjacent genes are either separated by a shortintergenic region (also extragenic) or share short sequencesthat are limited to transcriptional signals (overlap).Intergenic regions andoverlaps alternate along the genomeof the Zaire species. The Reston species differs from Zairespecies in that the overlap between the glycoprotein andVP30 genes is absent and an intergenic region separatesthem. The sequence 3’-UUAAU is common to both startand stop signals and is positioned in the centre of overlaps.As seen in Figure 1, there is a large amount of noncodingsequence in the genome and its function in expression isunclear. Stem–loop structures are also predicted for thesequences at the beginnings of the genes and also theextreme 3’- and 5’-ends of the genome (also seen inMarburg virus). These structures are present in mRNA andmay improve their translation; comparable structures havenot been identified in viruses from other families in theorder Mononegavirales.

Expression of the glycoprotein gene andstructures of SGP and GP

As with many other viruses, products of the glycoproteingene of Ebola viruses play an important role in virus entryand pathogenesis. The organization of the glycoproteingenes of all Ebola viruses is unusual and differs fromsimilar genes of other viruses, including Marburg virus inthe same Filoviridae family. Figure 2 depicts the organiza-tion and expression strategy of anEbola virus glycoproteingene. This gene encodes two prominent proteins, a secretedglycoprotein (SGP) that is released from infected cells inlarge amounts as a homodimer, and a structural glycopro-tein (GP) that forms trimers of a membrane-anchoredheterodimer; GP trimers form the peplomers or ‘spikes’ onthe surface of virions. The nonstructural SGP is expressedin preference to the structural GP, with mRNA for GPproduced through a transcriptional editing mechanism(Sanchez et al., 1996). The editing event that leads to GPexpression is an insertion of a single extra adenosine at arun of seven adenosines in the middle of the coding region.This insertion connects the 0 and 2 1 frames that encodethe full-length GP and occurs in about 25% of thetranscripts. The coding frame shared by SGP and GPand the frame accessed by editing are seen in Figure 2. As a

Article Contents

Secondary article

. Molecular Biology

. Pathology and Pathogenesis

. Epidemiology

. Patient Management and Control

0 1921 3 4

Trailer

5 6 7 8 9 10 11 12 13 14 15 16 17 18

Leader IR

Overlap

IR Editsite

Overlap

IR

Overlap

NP

3’

VP35VP40 SGP/GPVP30 VP24 L

5’

Figure 1 Ebola virus (Zaire species) genomic RNA. Coding regions forseven linearly arranged genes are shown as hatched boxes. The scale at thebottom of the figure indicates units of sequence length numbered inkilobases. IR, intergenic region.

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consequence of this organization, SGP and GP share thesame N-terminal � 300 amino acids with unique C-termini, both in length and sequence.

Roles of SGP and GP molecules

The role of the SGP molecule is unclear, but it is knownthat SGP dimers are structurally very distinct frommultimers of GP (Sanchez et al., 1998). The SGP dimeralso has a different cell binding pattern, so it appears thattheir roles are separate (Yang et al., 1998). It is likely thatexpression of significant quantities of SGP is important inthe natural host and could function in establishing apersistent infection through interactions with certain whiteblood cells, or possibly binding soluble mediators pro-duced in response to infections. In acute human cases, SGPis circulating in the blood in fairly large amounts and maycontribute to the pathogenesis of Ebola virus infections ofhuman and nonhuman primates. Efforts are under way toexamine the interaction of SGP with cells and molecules inthe human body, and also to determine the three-dimensional structure of SGP to better understand theactions of this molecule.

The GP molecule has an important role in initiatinginfections, as the large peplomers covering the surface ofvirions function in the recognition of and attachment tospecific but currently unknown cell receptors (Wool-Lewisand Bates, 1998). The spike structures of Ebola virus havebeen shown to bind to endothelial cells and not tolymphocytes, which are not infected by Ebola virus. It isalso believed to cause fusion of virion and cell membranes,which results in the release of the nucleocapsid into thecytoplasm. Structural studies have shown that GP iscleaved at a single site in the N-terminal third of themolecule by furin, a cellular subtilisin/kexin-like conver-tase, into GP1 (N-terminal end) andGP2 that are joined by

a disulfide bond (Volchkov et al., 1998). GP1 is highlyglycosylated and contains most of the N- and O-linkedglycans present in the surface spike. GP1 projects awayfrom the surface of the virion and probably functions inreceptor binding. The glycosylation of GP1 affects thefolding of the molecule and may also be important inimmune evasion. Virions of Ebola viruses (and filovirusesin general) are resistant to neutralization by antibodyspecific for the surface glycoprotein, and this may be due tothe large amount of nonimmunogenic carbohydrate cover-ing GP1, accounting for 60% or more of the molecularweight (GP1=130kDa). GP2 contains an a-helical se-quence important in trimerization through the formationof coiled coils. The GP2 trimer forms a membrane-anchored, rod-like stalk that stabilizes the peplomer andcontains a sequence positioned near its N-terminus thathas been shown in vitro to promote fusion of membranes(fusion peptide) dependent on the presence of phosphati-dylinositol and calcium (Weissenhorn et al., 1998). Itwould thus appear that the role ofGP2 lies in the formationand stabilization of the virion spike and fusion of the virusand host cell membranes during infection. The structuralpicture that is being revealed for theEbola virusGP (aswellas that of Marburg virus) is very similar to the glycopro-teins of retroviruses and influenza viruses.

Pathology and Pathogenesis

Histology

In severe and fatal cases of Ebola virus infection, as withinfections with other haemorrhagic fever viruses, there isan extensive multiorgan involvement, with focal to wide-spread necrosis. There is no inflammation (i.e. infiltrationof neutrophils and other phagocytic cells) in tissuesinfected with Ebola virus, which has been suggested to bethe result of an immunosuppression induced by the virus.Immunohistochemical staining of the liver reveals massiveamounts of virus antigen in hepatocytes, Kupffer cells, andendothelial cells, and antigen is also deposited in theintercellular connective tissues; the reticuloendothelial cellsystem is heavily involved. The spleen is another targetorgan for the replication of Ebola virus, and in addition tolarge amounts of antigen, there is follicular necrosis and ageneral loss of structural organization. Examination oftissues from infectedhumans,monkeys andguineapigs hasshown that cells of the mononuclear phagocytic system(macrophages and monocytes) are infected by Ebola virus.It is possible that infection and replication in these celltypes early in the course of the disease may contribute tosome form of immunosuppression, especially if these andother cells are induced to secrete substances that disruptsignalling or are rendered ineffective in clearing the virusfrom the body.

0 frame–1 frame

mRNA synthesis

poly(A)

poly(A)

SGP and GP SGPGP

Unedited (75%)

Edited (25%)

SGP homodimer(secreted)

+A

Membrane-anchoredtrimer of GP heterodimers

(virion)

Translation andprocessing

Figure 2 Organization and expression of the glycoprotein genes of Ebolaviruses, showing the production of the secreted SGP homodimer as theprimary gene product from an unedited transcript, and the structural GPtrimer through a transcriptional editing event (single adenosine insertion).

Ebola Viruses

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Disruption of immune responses

It is known that an immunosuppression is established earlyin the course of infections of human and nonhumanprimates, and development of a strong cellular immuneresponse is critical to surviving Ebola virus infections.There is some indication that inactivated virions of Ebolavirus are capable of inhibiting the proliferation of Tlymphocytes, which would contribute to immunosuppres-sion. Humoral (antibody) responses do not appear to be asimportant to virus clearance, as virions of filoviruses aredifficult to neutralize (even when treated with high-titreanti-GP sera), and antibody levels are usually very low orundetectable at the critical time when cell-mediatedclearance of the virus either begins or fails to develop.

Based on results from histological and in vitro studies,Ebola viruses are capable of infecting endothelial cells.Infection of primary cell cultures of human umbilical veinendothelial cells with theZaire species of Ebola virus resultsin a disruption of the signalling processes in these cellswithout a shutdown of host cell protein synthesis. Thisdisruption suppresses the induction of immunomodula-tory genes (e.g., major histocompatibility complex (MHC)class I and class II, interleukin 6 (IL-6), intracellularadhesion molecule 1 (ICAM-1) etc.) that are important inhelping to clear the body of infections. The mechanism ofthis suppression has not been characterized, so disruptionof cellular functions by Ebola virus may occur at the cellmembrane, within the cytoplasm, at the nuclear mem-brane, or at any combination of these sites.

Epidemiology

Outbreaks of Ebola viruses that cause severe disease inhumans have originated in rural areas (tropical forests or

savannah) of what was formerly Zaire (now the Demo-cratic Republic of the Congo), Sudan and Cote d’Ivoire(Table 1). The original 1976 outbreaks in northern Zaireand southern Sudan involved twodifferent species of Ebolavirus that emerged simultaneously. The events or condi-tions that triggered these concurrent outbreaks areunexplained, and subsequent reemergences have failed toshed any light on the factors involved in precipitatingEbola outbreaks. The Reston species of Ebola virus, whichhas not been shown to be pathogenic for humans, has beenisolated from monkeys (Macaca fascicularis) native to thePhilippines. However, the Reston species has not beendetected in monkeys directly from the wild and has onlybeen associated with a single Philippine breeding/export-ing facility that has been the source of all outbreaks causedby the Reston species. The possibility that this virus wasintroduced into the facility through trafficking of nonhu-man primates (from Africa?) cannot be dismissed.

Because the natural reservoir of filoviruses remains amystery, one cannot easily predict the occurrence ofoutbreaks. Efforts to detect Ebola virus in animal speci-mens from the wild have not been successful. These effortshave been few and too limited to adequately address theproblem of identifying the natural host from animalsinhabiting the forests. Results of experimental infection ofbats indicate they can support the replication of Ebola virus(1995 Zaire isolate) and shed the virus without showingovert signs of disease. Chimpanzees in the wild are knownto be naturally infected with Ebola virus, and, fromepidemiological investigations in Cote d’Ivoire and Ga-bon, it would seem likely that other nonhuman primatesbecome infected as well. Ebola virus infections of thesenonhuman primates in Africa probably result from director indirect contact with the natural host, leading to

Table 1 Outbreaks of Ebola virus infections of humans and/or nonhuman primates

Ebola virus speciesa Year Locations of outbreaks Human cases (% mortality)

Zaire 1976 Yambuku, Zaire 318 (88)Sudan 1976 Nzara and Maridi, Sudan 284 (53)Zaire 1977 Tandala, Zaire 1 (100)Sudan 1979 Nzara and Yambio, Sudan 34 (65)Reston 1989 Reston, Virginia, USA 4 (0)

Calamba, PhilippinesReston 1992 Siena, Italy 0

Calamba, PhilippinesCote d’Ivoire 1994 Tai Forest, Cote d’Ivoire 1 (0)Zaire 1994 Minkouka, Gabon 49 (59)Zaire 1995 Kikwit, Zaire 315 (77)Reston 1996 Alice, Texas, USA 0

Calamba, PhilippinesZaire 1996 Makokou and Booue, Gabon 91 (73)

Johannesburg, South Africa

aOutbreak confirmed by virus isolation.

Ebola Viruses

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amplification of the virus and introductions into humanpopulations if these animals are encountered.

During the 1995 Kikwit outbreak, it was noted thatsecondary transmission occurred only through direct andclose contact with patients, and that aerosol transmissiondid not occur. The spread of disease among humans duringsome of the outbreaks in Africa, including the 1995 Kikwitepisode, was accelerated when cases were treated in localhospitals. The nosocomial spread of disease was attributedto the reuse of contaminated needles and the lack of properbarrier nursing procedures to protect patients and stafffrom contact with highly infectious body excretions andsecretions. These epidemics stopped or were slowed whenmedical care facilities ceased to function as sources ofinfectious virus, usually as a result of mortality among thestaff or when proper barrier nursing techniques anddecontamination practices were established. Becausespread of the virus requires contact with infected personsand/or body fluids, the risk of infection is easily decreasedby avoiding behaviours that subject persons to suchcontact.

Patient Management and Control

No specific treatment for filovirus haemorrhagic fever isavailable for use in the event of an outbreak. The antiviraldrug ribavirin, which is useful in treating other haemor-rhagic fever diseases such as Lassa fever and haemorrhagicfever with renal syndrome (HFRS) due to Hantaan virus,has no effect on filoviruses in vitro and has not beenconsidered for use in infected patients. Experimentaltreatment of infected monkeys with interferon failed toincrease survival of infection. Currently, only supportivecare is available for treating patients; the administration ofintravenous fluids and/or transfusions may be therapeutic.

The development of vaccines against Ebola virus hasbeen slow, and the use of killed virus preparations has notstimulated protective immune responses when evaluatedusing guinea-pig and monkey models. However, geneticimmunization, a technique that uses injections of plasmiddeoxyribonucleic acid (DNA) that programmes de novosynthesis of a foreign protein, has proven to be an effectivemethod of inducing protective immune responses inguinea-pigs (Xu et al., 1997). This technique mimics anactual infection by producing newly synthesized proteinwithin antigen-processing cells that stimulate the develop-ment of cytotoxic T cells capable of killing Ebola virus-

infected cells. Genetic immunization with DNA expressingthe GP, SGP or nucleoprotein of the Zaire species of Ebolavirus has proven effective in inducing immunity. It remainsto be seen if this method of vaccination, as well as otherrecombinant DNA-based strategies, can be successfullytransferred to monkeys and humans. If successful, thedevelopment of a safe and protective vaccinewould be veryimportant in safeguarding the lives of persons workingwith Ebola viruses in research facilities and persons at riskduring future outbreaks.

References

Sanchez A, Kiley MP, Holloway BP and Auperin DD (1993) Sequence

analysis of the Ebola virus genome: organization, genetic elements,

and comparison with the genomeofMarburg virus. Virus Research 29:

215–240.

Sanchez A, Trappier S, Mahy BWJ, Peters CJ and Nichol ST (1996) The

virion glycoproteins of Ebola viruses are encoded in two reading

frames and are expressed through transcriptional editing. Proceedings

of the National Academy of Sciences of the USA 93: 3602–3607.

Sanchez A, Yang Z-Y, Xu L et al. (1998) Biochemical analysis of the

secreted and virion glycoproteins of Ebola virus. Journal of Virology

72: 6442–6447.

Volchkov VE, Feldmann H, Volchkova VA, and Klenk H-D (1998)

Processing of the Ebola virus glycoprotein by the proprotein

convertase furin. Proceedings of the National Academy of Sciences of

the USA 95: 5762–5767.

WeissenhornW,CalderLJ,WhartonSA, Skehel JJ andWileyDC (1998)

The central structural feature of the membrane fusion protein subunit

from the Ebola virus glycoprotein is a long triple-stranded coiled coil.

Proceedings of the National Academy of Sciences of the USA 95: 6032–

6036.

Wool-Lewis RJ andBates P (1998)Characterization ofEbola virus entry

using pseudotyped viruses: identification of receptor-deficient cell

lines. Journal of Virology 72: 3155–3160.

Xu L, Sanchez A, Yang Z-Y, Nabel EG, Nichol ST and Nabel GJ (1997)

Genetic immunization for Ebola virus infection. Nature Medicine 4:

37–42.

Yang Z-Y, Delgado R, Xu L et al. (1998) Distinct cellular interactions of

secreted and transmembrane Ebola virus glycoproteins. Science 279:

1034–1037.

Further Reading

Feldmann H, SanchezA and Klenk H-D (1998) Filoviruses. In: Collier L

et al. (eds) Topley & Wilson’s Microbiology and Microbial Infections,

9th edn, vol. 1, pp. 651–664. London: Arnold.

Peters CJ, Sanchez A, Rollin PE, Ksiazek TG and Murphy FA (1996)

Filoviridae: Marburg and Ebola viruses. In: Fields BN, Knipe DM,

Howley PM et al. (eds) Fields Virology, 3rd edn, pp. 1161–1176.

Philadelphia: Lippincott-Raven.

Ebola Viruses

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