Ebola: Viral Hemorrhagic Fever
by Kim Farrell
First identified in 1976, Ebola hemorrhagic
fever is one of the two members of the family Filoviridae, the other being Marburg virus.There are four subtypes of the Ebola virus. Three subtypes,
Ebola-Zaire, Ebola-Sudan, and Ebola-Ivory Coast, cause disease in humans and
the fourth, Ebola-Reston, causes disease in non-human primates (see figure 1
for structure of Ebola virus) (1).
In Central Africa, Ebola virus
distribution appears to be contained within the geographic ecosystem associated
with the rain forest belt extending to forest-savanna ecotone. At this time,
there is no known reservoir for the virus. Outbreaks appear to be related to
humans entering the rain forest, which increases the risk of coming in contact
with a highly pathogenic strain. Ebola virus strains constantly circulate in
this eco-niche and populations from this area, mainly Pygmies and non-pygmies
in the vicinity, test positive for Ebola virus IgG, immunoglobulins. These
people are asymptomatic but have come in contact with a non-lethal strain of
the virus (2).
Ebola is spread through body fluid contact
and using contaminated needles. The symptoms of Ebola virus vary; within a few
days of infection, most patients will exhibit high fever, head and muscle
aches, stomach pain, fatigue and diarrhea. These are general flu-like symptoms
that can lead to miss diagnosis. Some patients will exhibit sore throat,
hiccups, rash, red and itchy eyes, vomiting blood and bloody diarrhea. Within
one week of infection, most patients will exhibit chest pain, shock and death.
Some patients will exhibit blindness and bleeding. Some people do recover even
with mortality rates as high as 90% for Ebola-Zaire and 50% for Ebola-Sudanese (1).
Diagnosis can be accomplished through a
variety of means. Early detection is done with enzyme-linked immunosorbent
assay (ELISA), IgG ELISA, polymerase chain reaction (PCR) and virus isolation.
For later detection, testing for IgM and IgG antibodies can be done. After a
patient dies, immunohistochemistry can be used for diagnosis (1).
There is no cure for the Ebola virus. Only
supportive therapy is available, which consists of balancing patient’s fluids
and electrolytes, maintaining their oxygen level and blood pressure, and
treating any other infection (1). Because there is no cure at this
time, many studies are in progress to find either a cure or a prophylactic
vaccination. Any research that is to be done must be done at a biosafety level
4 or done using pseudoviruses.
Fusion between the viral envelope and host
cellular membrane is essential for viral infection. The fusion domain of viral
proteins usually is made up of a run of hydrophobic amino acids (3).
The putative fusion domain of the Ebola virus was tested by substituting an
alanine, a non-polar, hydrophobic amino acid for a proline, an uncharged amino
acid, at position 533 or 537 (putative fusion domain-amino acids at 524 to
539,prolines are positioned at 533 and 537). This caused reduced infectivity.
While the substitution of an arginine, a positively charged, hydrophilic amino
acid, for a proline at the same positions almost completely stopped
infectivity. The substitution at 533 reduced the efficiency of glycoprotein
incorporation into the virus that was used for this study. Therefore, it was
concluded that the proline residue at position 537 in the Ebola glycoprotein
fusion peptide is important for fusion to the host cellular membrane (4).
Fusion requires Ca2+ to be present. The calcium ion does not aid in
depth of penetration or in the process of binding, but only the actual fusion
process. How Ca2+ does this is not known at this time. The host
cellular membrane must also contain phosphotidylinositol (PI) for maximum
fusion to cells (5).
The glycoprotein (GP) is a type 1
transmembrane protein and is on the surface of the infectious virion. It
functions in the attachment structure and entry of the virus into the host cell
(7). It is acted upon endoproteolytically to produce two sub-units
that have been designated GP1 and GP2. These sub-units
are joined together with a disulfide bond. This study suggests that in
wild-type Ebola a broad array of proteases will perform the cleaving (6).
The cleavage site that produces the two
sub-units is a furin site. “Furin is a widely expressed cellular protease that
cleaves proteins at a basic amino acid sites”(20). This site is
fairly conserved through out the Ebola viruses. The Reston species deviates
from this conservation in that it contains a lysine reside at the –4 position
when the others contain an arginine residue. Cleavage of the GP is not needed
for infectivity. But since the site is conserved in all the filoviruses,
cleavage must be needed at some point in the viral life cycle (6).
Fusion is purported to be a function of GP2,
which contains an internal fusion peptide flanked by cysteines (C10 and C55)
and are predicted to be joined by a disulfide bond. This is similar to the
oncovirus avian sarcoma/leukosis virus (ASLV) (8). There are more
convincing reasons for the use of ASLV as a prototype for the study of Ebola
fusion. There is the conservation of many putative functional domains such as a
central CX6CC motif, the potential coiled-coil, and the putative
fusion peptide. There is also a conserved stretch of basic residues in all
strains of Ebola virus that, in ASLV, constitute an endoproteolytic cleavage
site (21,22).
To create mature viral fusion proteins, the
ASLV envelope A must be processed proteolytically into surface (SU) and
transmembrane (TM) sub-units. The TM contains the fusion peptide. The SU and TM
are present in the env. A as trimers. As it matures, the fusion peptide is
positioned by the N terminus of the fusion-mediating sub-unit. In ASLV, the
fusion peptide is moved internally and exists as a looped structure. Recent
crystallographic data on the core of the virus GP2 (9,10)
shows that a disulfide bond joins C10 and C55 (8).
To test the importance of the cysteines,
they were mutated to serine residues. No combination of mutations prohibited
the proteolytic processing, trimer formation, receptor binding or virion
incorporation properties of env. A. Mutant combinations did cause a ~ 1,000
fold decrease in env. A infectivity as compared to the wild-type env. A. Also,
the ability to mediate fusion was significantly decreased (8).
This showed that C9 and C45 are required
for virus-
Figure 2. Active fusion conformation of
ASLV
The model for the active fusion
conformation of ASLV TM is predicted to be similar to that of the Ebola virus
GP2. Figure 2 shows the model for ASLV TM active fusion state. The A
picture shows the core monomer and picture B shows the trimer produced by TM.
These structures are based on crystal structures of the core fragment of Ebola
virus GP2 (9,10). The stars indicate the predicted
position of the proline within the fusion peptide. The barbell shapes indicated
disulfide-bonded cysteines. The C and N terminal a-helices are packed against each other
in the antiparallel position (8).
Entry of Ebola into cells is pH dependent;
weak bases inhibit entry. It has also been found the B and T lymphocytes are
unable to support Ebola virus entry. This is possibly due to the lack of a
functional viral receptor on their cells (11).
Infected cells secret a nonstructural
glycoprotein (SGP). SGP was once thought to be a decoy to draw the immune
response away form the infected cells by mimicking the structure of GP. This is
not the case, as suggested by research, due to the dimer structure of SGP (the
structure of glycoprotein, GP1 and GP2, is trimeric)(5).
SGP binds to neutrophils and not endothelial cells, just the opposite of the
cell-binding pattern found for GP. There are indications that SGP binding may
inhibit neutrophil activation, thus dampening inflammatory responses that are
essential for immune response. This is thought to contribute to the
pathogeneses of Ebola virus, especially considering the large amount of SGP in
the blood during acute infections and the lack of immune response (12).
Viral infections usually cause interferons
(INF), chemicals that are able to provide some protection against virus
invasion of the body or inhibit viral growth to be produced by infected cells
(13). The infected cells cannot be rescued but in this manner, they
protect nearby healthy cells. The interferon diffuses to nearby cells and
stimulates the synthesis of protein kinase R (PKR), which interferes with viral
replication in the healthy cells by inhibiting protein synthesis by the
ribosomes (13). INF also induces immune response genes such as those
coding for major histocompatibility complex class I (MHC I) and IFN regulatory
factor 1 (IRF-1)(14).
Ebola-Zaire blocks the signaling of IFN in
endothelial cells. Therefore, the virus does not start the process of the
production of MHC I, which interferes with cytotoxic T-cell production. PKR is
not produced as a result of the blockage (14).
To find if the antiviral pathway was
disrupted or if de novo protein synthesis was stopped, interleukin-1b (IL-1b) responsiveness was examined. IL-1b is a cytokine. Cytokines are chemical
mediators involved in cellular immunity (13). The IL-1b pathway is separate from that of IFN.
IL-6 is induced by IL-b but
not IFN-a or
IFN-g(23). IFN-a and IFN-g both share several signaling
molecules; i.e. Janus kinase-1 and signal transducers and activators of
transcription-1a (Jak/STAT). IL-1b pathway does not contain any Jak/STAT
signals. Thus, with the formation of IL-6, it was shown that the Jak/STAT
pathway is blocked or inhibited in some manner while the IL-1b pathway is not (14).
Mouse models are used to test possible
cures for Ebola infections. Unfortunately, mice are not generally susceptible
to Ebola and the virus must undergo eight-fold serial passage through mice to
produce a lethal virus (15). A mouse model can be produced but how
representative they are to human filovirus infection is not known.
Mouse models were employed to test
usefulness of a s-adenosyl-L-homocysteine
(SAH) hydrolase inhibitor as an antiviral therapy. Adenosine analogs possess
strong antiviral activity, including the ones tested, carbocyclic
3-deazaadenosine (C-c3Ado) and 3-deazaneplanocin A (c3-NpcA)(16).
Their antiviral capabilities are thought to occur through dampening of the
methylation of the 5’ cap of viral mRNA by inhibiting SAH hydrolase (17).
SAH is produced through cellular methylation reactions, which uses S-adenosylmethionine (SAM) as the methyl
group donor. Hydrolysis of SAH to adenosine and homocysteine keeps SAH at low
levels, which allows the methylation reaction to occur. As the SAH/SAM ratio
increases due to inhibition of both cellular and viral transmethylation
reactions. The dampened methylation of the 5’ cap of viral mRNA causes
inefficient translation of viral transcript (18).
This study found that a competent immune
system is required for C-c3Ado or c3-Npc to work. The
cure appears to have two separate events. First, antivirals keep the viral
replication below lethal threshold level in serum of approximately 106 pfu/ml.
Secondly; a protective immune response further suppresses viral reproduction
and eventually eliminates the virus. Improvement is seen in weight gain, which
coincides with dropping viral serum levels (16).
These antivirals worked best if the
mice were treated in 24-48 hours from infection. Antivirals administered 48
hours post infection showed 100% survival. These compounds appear to be most
effective after viral replication has begun, but before widespread circulation
of infection and extensive tissue damage (16).
Ebola is a negative-sense single stranded
RNA virus that appears to be native to rain forest and forest-savanna regions
of Africa. It is a viral hemorrhagic fever and has a very high mortality rate,
roughly 50%-90%. There is no known cure.
The immune response associated with Ebola
infection is little if any. The infection destroys the liver, spleen, lymph
nodes, and lungs and very high viremia is present (19).
Further study of the putative fusion
domain, which is similar to that of ASLV, will eventually lead to the
determination of the actual fusion domain. This will create the opportunity to
find a cure or find a prophylactic vaccine.
References
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Copyright © 2001 Kim Farrell and Koni Stone
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