Transcript Viral Hemorrhagic Fevers

Viral Hemorrhagic
Fevers
The Filoviruses
Steve
Vivian
Jenn
Live Footage
Ebola Virus
Outbreak
Viral Hemorrhagic Fevers
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Severe multi-system syndrome (multiple
organs affected).
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Vascular system is damaged and body loses the
ability to regulate itself.
Accompanied by hemorrhaging.
Many VHF viruses cause life threatening
diseases
Most have no established treatment or cure.
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VHF Caused by Viruses
of 4 Families
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Arenaviruses
Filoviruses (Ebola and Marburg)
 Work with filovirus species requires
Biosafety Level 4 (BSL-4) “space suit”
containment (HIV requires BSL-2
“space suit” containment).
Bunyaviruses
Flaviviruses
 Features
of these Viruses
RNA Viruses, covered in lipid
coating
Humans are not natural
reservoir, but can transmit virus
Viruses are restricted to areas of
their host species
Marburg
Caused by a zoonotic virus of the
filovirus family.
 Mortality Rate 23-25%
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First recognized in 1967 when
simultaneous outbreaks occurred in
Marburg and Frankfurt, Germany, and in
Belgrade, Yugoslavia.
 37 people became ill. The ill were
mostly laboratory workers and
medical personnel.
 Original people who became ill had
been exposed to the tissues of
African green monkeys, which were
imported from Uganda for research.
 Location:
 The
area to which the virus is native
is unknown, but it is believed to
include parts of Uganda, Western
Kenya, and Zimbabwe.
 Transmission
 The animal host to the Marburg
Virus is unknown, and so is the way
that the animal transmits the disease
to humans.
 People who have been exposed to
infected monkeys or their body fluids
have become infected in the past.
 Transmission
(continued)
 The disease is easily transmitted
between humans. Direct contact with
an infected person, or exposure to
their body fluids, are both ways by
which the disease is thought to be
transmitted.
Ebola
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Believed to be caused by a zoonotic
member of the filovirus family.
 Believed to be carried by an animal
host that is native to Africa.
Named after a river in the Democratic
Republic of Congo in Africa (formerly
Zaire) where first outbreak occurred in
1976.
First Ebola outbreak in Zaire (1976)- 318
Human cases, 88% of those people died
of the disease.
Great Plague of Athens
529 B.C.
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Thucydides wrote about a
disease with symptoms that
are very similar to Ebola,
including:
 High Fever, Vomiting,
Chest Pain, Diarrhea, fetid
breath and a bumpy red
rash.
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4 Types of Ebola Virus
 Ebola-Zaire
 Ebola-Sudan
 Ebola-Ivory Coast
 Ebola Reston
Electron Micrograph of Ebola Virus
All of these strains have been known to
cause disease in humans, except for
Ebola-Reston, which has only caused
disease in Non-Human Primates.
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Location:
 The Ebola Virus
has been
reported in the
Democratic
Republic of
Congo, Gabon,
Sudan, the Ivory
Coast, and
Uganda.
Map of Ebola Outbreaks in Africa

Ebola-Reston outbreak at primate
research facility in Virginia. Virus was
carried by monkeys that had been
imported from the Phillipines.
 4 humans were infected and developed
antibodies, but none of them became
sick
Map of Ebola Reston
Infected Monkeys in
Philippines
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Transmission
 It is believed that the first human
becomes infected with Ebola through
contact with an infected animal.
 An outbreak of Ebola-Zaire occurred
in Gabon in 1996 after people had
eaten an infected monkey.
 One Scientist who had conducted an
autopsy on a wild chimpanzee in the
Tai Forest in Ivory Coast was
diagnosed with Ebola-Ivory Coast in
1994.
 People
can become infected with Ebola
if they come into contact with the
blood or secretions of an infected
person.
 People can also become infected
through contact with objects, such as
needles, that are contaminated with
secretions or blood.
 In 1976, in England, a person
became infected with Ebola-Sudan
after being accidentally pricked with
a contaminated needle.
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Because the disease is easily transmitted,
Nurses must wear protective clothing
when interacting with a patient and must
dispose of all needles and other medical
supplies properly.
Nurses in
full
protective
gear in
Gabon, 2002
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Patients who have died of the disease
must be buried properly. Care must be
taken that there is no contact with the
deceased patient.
Burying Ebola Victim in Gabon, 2002
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The Ebola Virus takes a psychological toll
as well, causing devastated villagers to
search for scapegoats.
Posted, February 21, 2003 8:37 AM EST
Brazzaville, Congo- Congolese villagers have stoned and
beaten to death four teachers accused of casting an evil
spell to cause an outbreak of the deadly Ebola disease
that killed nearly 70 people, a local official said Friday.
(Reuters)
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Relief , joy and celebration after the
Gabonese Health Ministry declares that
the Ebola outbreak has ended. (May 6,
2002)
Pleomorphic
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Definition: having the ability to assume different
forms or shapes
Four main shapes: long filamentous, U shaped,
6 shaped, circular
Structure
• Filoviruses are non-segmented negative strand
viruses
• Composed of a helical ribonucleoprotein complex
(nucleocapsid)
• Covered with a host-cell derived membrane envelope
• Matrix between the envelope and nucleocapsid is
filled with a protein lattice
Viral Genome
Viral Genome
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Consists of seven genes
These genes encode seven proteins in the
Marburg virus and eight in the Ebola virus
Both viruses have a 3’ leader and 5’ trailer
sequence that is highly conserved and has a
high degree of complementarity
These extragenic seguences are important
in the initiation of transcription and
replication
Viral Proteins
* Ebola virus only
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NP: Nucleoprotein – the primary
structural protein associated with the
filovirus nucleocapsid
Viral Proteins
* Ebola virus only
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VP35: acts as a cofactor in transcription and
replication of the viral proteins
Viral Proteins
* Ebola virus only
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VP40: a matrix protein, also the most
abundant viral protein; may facilitate in the
budding process
Viral Proteins
* Ebola virus only
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GP: Glycoprotein – makes up the virion
spikes or peplomers and mediates entry into
host cells through receptor binding
Viral Proteins
* Ebola virus only
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sGP: secreted from the cell, present in large
amounts in the blood of Ebola victims; may
help to inhibit the immune response
Viral Proteins
* Ebola virus only
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VP30 – a minor nucleoprotein that may be
involved in securing the RNA to the C
terminus of NP
Viral Proteins
* Ebola virus only
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VP24: unknown function, possibly a matrix
protein
Viral Proteins
* Ebola virus only
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L: Polymerase L – acts as the polymerase
and is the largest and least abundant viral
protein
Interactions with Cells
Entrance into Cells
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Mediated by the Glycoprotein on the envelope surface
Structure of GP
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Precursor form GP0 is cleaved yeilding GP1 and
GP2, they are connected via one disulfide bond
Form a heterodimer
Peplomers are then formed by the association of
three of these heterodimers to form a homotrimer
GP2 is responsible for trimerization, fusion of the
envelope and cell, and has an immunosuppressive
motif
GP1 is the receptor binding domain
Receptor - Lipid Rafts
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Receptor, while for most cells unknown, appears to
be located in LIPID RAFTS : less fluid areas of
the membrane high in cholesterol and
glycosphingolipid; the cholesterol-binding caveolin
is also closely associated with the rafts
Endocytosis
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Caveolae are present in the plasma
membranes of most cell types and are
thought to form from lipid rafts
The major structural protein is caveolin, a
multipass protein
Caveolae invaginate and collect their cargo
proteins, but do not require the assembly of
a cytosolic protein coat
Endocytosis
Transcription
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Takes place in cytoplasm of cell
The seven genes are transcribed to produce
seven monocistronic polyadenylated
mRNA’s
Each gene has a conserved transcriptional
stop and start sequences
Transcripts are thought to have 5’ caps
Gene overlaps - function unknown
Unusual GP Transcription
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In the Ebola Virus the GP gene codes for GP and sGP
sGP is the primary gene product
Full length GP is expressed by transcriptional editing of a
single adenosine at a run of seven uridine residues in the
genomic RNA
Translation
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sGP and GP are
translated by
membrane bound
ribosomes and enter
the ER and follow the
exocytotic transport
route to the cell
surface
All other viral proteins
are translated by free
ribosomes in the
cytosol
Replication
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Build up of proteins
signals switch to
replication
Switch results in
synthesis of + sense
RNA as a template
Depletion of proteins
causes switch back to
transcription,
eventually equilibrium
Budding and Release
Invasion
Process
Monocytes
Immune System
Immune System Evasion
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Host dies with little evidence of an immune
response
Mitosis of lymphoid cells seen to decrease
2-3 days postinfection with eventual
apoptosis
Swelling of MPS and stromal cells and
eventual lysis
Dendritic (antigen-presenting) cells also
undergo apoptosis
Immune System Evasion
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sGP may bind to neutrophils and inhibit
their activation
sGP as a decoy? – absorb neutralizing
antibodies
VP35 may act as an IFN antagonist
GP2 may have an immunosuppressive role
Ebola shown to inhibit induction of
immunomodulatory and antiviral genes in
endothelial cells (MHC I, IFN, etc…)
Cytotoxicity, Hemorrhage,
and Shock
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GP expression causes downregulation of the
cell-surface expression of integrins – cell
rounding and detachment seen
Disruption of cell functions in general
Release of vasoactive agents (cytokines,
chemokines, histamines and peroxidases)
from infected monocytes and endothelial
cells could increase hemorrhaging and
induce shock
Risk Factors
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Travel to Asia or Africa
Handling of animal carcasses
Contact with sick animals or people
Arthropod bites within 21 days of onset of
symptoms
Transmission is highest during latter stages
of illness.
Reservoir Unknown
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Bats?
Birds?
Insects?
Plants?
Spread of Ebola
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Direct contact with the blood and/or
secretions of an infected person. (Family
setting)
Contact with objects, such as needles that
have been contaminated with infected
secretions. (Health care setting)
Ebola Invasion
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Effects innate immune system (non-specific
responses)
Enters through broken skin, mucous
membrane or exchange of bodily fluids
Can also enter through ingestion, inhalation,
injection of infectious material
Ebola Basics
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Incubation period of 2-21
days
Mortality rate of 50-90%
Some primary targets are
hepatocytes and
endothelial cells, which
produce the greatest
amounts of furin in the
human body
Stable at room temperature
but is destroyed at 60 0 C.
Also destroyed by gamma
and UV radiation, lipid
solvents, and bleach
Marburg Basics
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Incubation period of 2-14
days
Mortality rate of 23-25%
Stable at room
temperature but is
destroyed at 60 0 C.
Also destroyed by gamma
and UV radiation, lipid
solvents, and bleach
Unsuspecting Victim
Your eyes are red and itchy. You're feeling a
little stuffed up. Your body aches, and
you feel warm.
You think you have a case of the flu coming
on. Nothing a few good nights' rest
won't cure, you think. The next
morning, your head throbs. Your skin
aches. You feel feverish, and your chest
hurts. You can't get out of bed.
In the new few days, your fever skyrockets.
You are disoriented and delirious. You
throw up and notice the vomit is laced
with blood. Next, you feel a liquid
trickle out of your ears. With horror, you
realize that it's more blood. Soon blood
is seeping from your nose, gums, and
eyes. The pain is excruciating. Inside
your body, an army of microorganisms
eats away at your veins and arteries and
turns your organs into a sort of bloody
cottage cheese. In 36 hours, you're gone.
You die in a pool of blood that has
oozed out of every part of your body.
Early Symptoms
The first signs usually are:
 Prolonged fever that does not
respond to antimalarial
treatment or antibiotics
 Joint pains and generalized body
aches
 Headache
 Generalized weakness
 Nausea and vomiting
 Abdominal pain and diarrhea
 Fatigue
 Arthritis
 Sore throat
 Rash
 Hiccups
After 5-7 Days
The fever may get associated with
spontaneous bleeding e.g.:
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Bleeding from the gums, nose, eyes,
ears
Eyes may turn red (conjunctivitis)
Anal bleeding or bloody diarrhea,
gastrointestinal bleeding
Genital swelling (labia and scrotum)
Bleeding from the skin
Difficulty in swallowing and dryness
of the throat
Increased sense of pain in skin
Blood does not clot
Depression
Hypotensive shock
Liver damage
Treatment for Ebola and
Marburg
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Isolation and quarantine
Quick containment
No standard treatment
Supportive therapy, which includes balancing the
patient’s fluids and electrolytes, maintaining their
oxygen status and blood pressure, and treating
them for complicating infections.
Mechanical ventilation, renal dialysis, and
antiseizure therapy may be required.
What is the key to survival?
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Demise or improvement usually occurs
around day 7 to 11, when humoral antibody
response can be detected.
Known that patients who die usually have
NOT developed a significant immune
response to the virus at the time of death.
Convalescence and Recovery
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Slow – takes 5 weeks
or more
Can still harbor
infectious viral
particles up to 82
days after
presentation
Weight loss and
amnesia in early
stages of recovery
Survivors
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Psychosocial disturbances
Loss of memory
Central nervous system
disorders
Loss of hair, hair elasticity
and hair drying
Permanent hearing loss
Arthralgia
Uveitis
Orchitis
Death and Burial
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Villagers traditionally help
sick, transport and kiss the
dead
Wash and dress body for
the afterlife
Burial practices last days
Dead buried near home
Hiding of bodies
Clinical Diagnosis of
Ebola
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In early stages, it is difficult to diagnose
because early symptoms, such as red eyes
and a skin rash, are nonspecific and are
similar to other diseases that occur much
more frequently.
Use RT-PCR and ELISA to diagnose Ebola
and Marburg.
RT-PCR
Enzyme-linked immunosorbent
assays (ELISA)
Detects early immunoglobuling (Ig) M antibody responses to the VHFcausing agents
Challenges to Vaccine
Development
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In past, rare occurrences of Ebola and
remote locations of outbreak did not favor
vaccine development.
Also required industrial support, which was
not feasible since it was predicted there
would be no market for the vaccine.
However, this changed with the existing
threat of bioterrorism. Now Ebola is
categorized as a ‘List A agent’.
Ebola Protection Using
Immunotherapy
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Antibody titers against Ebola virus GPs are
detected in patients who recover. However,
serum from recovered patients did not
consistently protect against infection or
exhibit neutralization of virus replication in
cell culture.
Passive transfer of antibodies in animal
models only delays onset of symptoms and
does not alter overall survival.
Ebola Protection Using
Immunotherapy
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Monoclonal antibodies isolated from bone marrow
of recovered patients confer immune protection in
a murine model of Ebola virus infection and in
guinea pigs.
But it is relatively easy to protect against infection
in the mouse model and protection of guinea pigs
required a high dose of antibody administered
very close to the time of virus challenge.
Conclusion: antibodies alone do not provide
protective immunity in a natural context. May not
be applicable to humans.
Antiviral Therapy
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Use of S-adenosyl-L-homocysteine
hydrolase inhibitors.
The drugs are active in vitro and in mouse
and nonhuman primate models.
Drugs act through accumulation of Sadenosylhomocysteine and its selective
inhibition of virus methylation reactions.
Blood Transfusions
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In 1995 Zaire outbreak, 8 patients given
blood transfusions from individuals who
recovered from Ebola.
7 out of 8 patients survived.
However, study size was small and patients
were young so actual effectiveness remains
unknown.
Vaccine Development
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Vaccine made with heat inactivated virions but
level of protection was inconsistent. Guinea pigs
were partially protected in one study and 4 out of 5
baboons survived challenge.
However, inactivated virions did not induce
sufficient immunity in hamadryl baboons against a
lethal challenge.
May not be effective in humans.
Vaccine with eVLP
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Recently, vaccine developed that is based on
a single or combinations of virus-encoded
structural proteins to induce immunity.
After vaccination with eVLP (Ebola viruslike particles), mice developed high titers of
Ebola virus-specific antibodies, including
neutralizing antibodies. The mice were 100%
protected from Ebola virus.
Protective Materials
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Rubber boots which can be
disinfected
Washable trousers over the
boots
Washable jacket or blouses with
long sleeves which can hold at
the wrists
Rubber aprons which can be
disinfected or disposed
Two pairs of latex gloves for
certain manipulations or rubber
gloves (heavy duty gloves)
Masks
Protective eye wear
(glasses/goggles)
Headgear
BSL4 Safety Procedures
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BSL Clip
Weaponization

Characterized as “class A” weapons
because of their:
 High virulence
 Demonstrated aerosol infectivity in the
laboratory
 Ability to cause a state of intense fear
and anxiety

Most likely method of attack would be
aerosolization of virus into highly
infectious particles in the 1-5 micrometer
range. (particles remain suspended in air)
 Although protective measures are in
place to prevent contamination of
food and water supplies, it would be
impossible to ensure a constant
supply of purified air.
 The tremendous surface area of the
lungs make them highly susceptible
to microbial/viral infection.
A
large scale attack would seem
unlikely, as such an attack would
require correct conditions of wind,
temperature and humidity.
 Terrorists are much more likely to
carry out a smaller attack via a
vulnerable ventilation system.
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Difficulty in Detection
If terrorists were to carry out an
attack, it would be a week before
the first cases of illness appeared.
(not known if disease is
contagious in incubation phase)
 Even
after the illness appeared, it
would be very difficult to determine
that the illness was caused by a
filovirus because of:
 The similarity of the early
symptoms of Ebola and Marburg,
to those of other diseases
 The unlikelihood that a case of
these diseases would occur
outside of Central Africa.
 It
is during this period (the opening phase
of the outbreak) that the possibility of
transmission of the disease is greatest.
 Family members and health care
workers who are treating the diseased
person, but do not yet know the nature
of their illnesses have an extremely high
risk of contracting the virus.
 The Detection of a deliberately caused
outbreak will be based on the detection of
an usual number of severe illnesses within
a short period of time.
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Availability of filoviruses
 Samples of Marburg and Ebola were
distributed to many research laboratories
after their initial outbreaks in the 1960’s
and 1970’s. It is feared that samples may
have been stolen and distributed to
wealthy terrorist organizations.
 Aum Shrinkyo (Japanese cult responsible
for Sarin gas attack on Japanese subway)
has reportedly attempted to obtain the
Ebola virus by sending some of its
members down to Zaire in the early
1990’s.
 Independent
terrorist organizations
may have a sympathizer infiltrate a
BSL-4 lab with the intent of stealing
a filovirus sample or they may
attempt to purchase it from a
scientist working in the lab.
 Attempts are being made to
improve security at these labs, but
detecting the loss of the tiny
volume of seed material needed to
start a new culture is extremely
difficult.
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The Soviet Union reportedly took an
active interest in filoviruses and their
potential for use in bio-weapons.
 Soviet laboratories acquired a sample
of the Marburg Virus after the 1967
outbreak.
 Research reports published by
Russian investigators after the
collapse of the Soviet Union show a
strong interest in the stability of
freeze dried Marburg virus, and its
aerosol infectivity.
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Soviet Scientist Nikolai Ustinov
accidentally pricked himself in the finger
with a syringe that contained Marburg
Virus.
Ustinov began to vomit blood, pass
bloody black diarrhea, and sweat blood
from his pores.
Scientists kept this strain of Marburg,
named Strain U , after Ustinov and
processed it into inhalable dust.
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The strain was extremely potent.
 Upon testing, it was found that only
one to five particles of Variant U,
lodged into the lungs of a monkey, was
almost guaranteed to make the monkey
crash and die ( It takes 8000 spores of
Anthrax in the lungs to guarantee
death)
Conclusion
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In addition to the sickness and death that it
would cause, a filovirus attack would cause
tremendous disruption of social and
economic activity.
Ebola has the benefit (to terrorists) of
having a reputation for causing horrifying
illnesses.
Increased anxiety would occur if people
were to see the effects of the disease, as
well as the full protective gear that law
enforcements officials, doctors, and nurses
would be wearing.