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Hemorrhagic Fevers With a Concentration on Filoviruses Simon A Francis Susanna Epstein Medha Goyal History Hemorrhagic Fevers Definition: a severe multi-system syndrome . Vascular System Damaged Body regulation Impaired Accompanied by hemorrhage Four families of viruses

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hemorrhagic fevers

Hemorrhagic Fevers

With a Concentration on Filoviruses

Simon A Francis

Susanna Epstein

Medha Goyal

  • Hemorrhagic Fevers
    • Definition: a severe multi-system syndrome.
      • Vascular System Damaged
      • Body regulation Impaired
      • Accompanied by hemorrhage
    • Four families of viruses
      • Arenaviruses (Junin Virus)
      • Bunyaviruses (Nariovirus )
      • Flaviviruses (ie. Yellow Fever)
      • Filoviruses (Marburg & Ebola)
    • CDC Classification: BSL-4 Agent
  • Hemorrhagic Fevers (Continued)
    • RNA viruses covered in a lipid coating
    • Viruses are geographically restricted to areas where host species live
    • Humans are not natural reservoirs for these viruses
  • Filoviruses
    • Marburg Virus
      • A.K.A. (African Hemorrhagic Fever, Green Monkey Disease, Marburg Fever)
      • First outbreak (Marburg Germany, 1967)
      • laboratory workers infected by monkeys
      • Simultaneous outbreak in Hamburg, Germany and Belgrade, Yugoslavia (now Serbia)
      • 32 human cases
      • 31 primary one generation of secondary transmission
      • 23% of human mortality
      • Overall Mortality 23%-25%
  • Filoviruses (continued)
    • Ebola Virus
      • Named after a river in the Republic of Congo (Formerly Zaire)
      • First outbreak (Zaire 1967)
        • 318 human cases
        • 88% mortality
        • Disease spread by close personal contact in hospital setting (amplification)
      • Fatal in humans and non-human primates
      • Four subtypes (~80 nm in diameter)
        • Ebola-Zaire(990-1086 nm in length ), Ebola-Sudan (974-1063 nm in length), Ebola-Ivory Coast, Ebola-Reston (disease in non-human primates 1026-1083 nm in length )
  • Filoviruses (Continued)
    • Ebola (Continued)
      • Sudan international scientist arrived
        • To late to deal with virulent epidemics
          • Hospitals closed
          • Infected patients quarantined
        • Reconstructed data from survivors
  • Marburg
    • Europe Outbreaks
      • 1967
      • Hamburg and Marburg, Germany and Belgrade, Yugoslavia
    • Africa Outbreaks
      • 1975
        • Johannesburg, South Africa
        • 3 died
      • 1980
        • Western Kenya
        • 2 Died (Physician died in Nairobi)
      • 1987
        • Young man traveling extensively in Kenya
      • 1999-2000
        • Outbreak in Durba, Republic of Congo
        • Cases linked to workers in a gold mine
  • Ebola (types named after location)

This diagram was adapted from a WHO publication accompanied by the following note (edited): Phylogenic tree showing the evolutionary relationship of Ebola viruses (courtesy of A. Sanchez, Centers for Disease Control and Prevention [CDC]; derived from Georges-Courbet MC, Sanchez A, Lu CY, et al. Isolation and phylogenetic characterization of Ebola viruses causing different outbreaks in Gabon. Emerg Infect Dis 1997;3:59-62).

new york times
New York Times
  • February 15, 2003
  • World Briefing: Africa
  • CONGO REPUBLIC: EBOLA TOLL REACHES 51 The death toll from a suspected outbreak of the deadly Ebola virus in Congo Republic has crept up to 51, and people have begun fleeing into dense forest to escape what some believe to be an evil spell. The authoritieshave tried to impose tight restrictions on movement in the hope of preventing the spread of the outbreak, the second reported in little over a year in the country's remote northwest. It is thought to have been caused by the consumption of infected monkey meat.    (Reuters)
  • Ebola Virus
    • No carrier state (reservoir Unknown)
    • Researchers Hypothesize that it is Zoonotic
    • Unpopular theory that plant may be the reservoir of the virus
    • Human to Human Transmission through contact of contaminated secretions.
  • Ebola-Reston
    • Occurred in the U.S (Reston, VA)
    • Occurred in African Green Monkeys
    • Why only to non-human primates?
      • Four scientist found to have antibody for the disease
    • Circumstantial Evidence of airborne transmission
      • Spread within and between rooms (national center for Infectious Diseases)
  • Marburg Virus
    • Transmission from animal host unknown
    • Human to Human (Close Contact and change of fluids highly suspect source of transmission)
  • Not known
  • May be
    • A rare species
    • One that usually does not contact clinical host
    • If contact is made the virus may not be easily transmitted
  • Hypothesize
    • Bats
    • Plants
filoviruses general facts
Filoviruses General Facts
  • Replication:
    • Not fully understood. Created by budding of the surface of their host cells (Susanna)
  • Structure:
    • Pleomorphic: Long sometimes branched filament shaped like a “6”, “U” or a circle.
    • Each Virion contains one molecule of single stranded, negative sense RNA (Susanna)
  • Symptoms of Ebola and Marburg (Medha)
incubation death periods
  • Ebola Incubation at 2 – 21 Days
  • Marburg Incubation: : 3-9 days
  • VHF (in general) - Death Between 7 – 16 Days
  • Ebola Death Ensues as Early as 2 Days after expression of symptoms
morphology and structure
Morphology and Structure
  • Filamentous or Bacillus form
  • Vary greatly in length (up to 14000 nm)

Mean Unit Length

Marburg- 860 nm

Ebola- 1200 nm

  • Uniform diameter of 80 nm

Composed of:

  • Ribonucleoprotein complex (nucleocapsid)
  • Matrix
  • Envelope studded with peplomers (10 nm long)
filovirus genome
Filovirus Genome
  • Nonsegmented, negative-strand RNA
  • Filovirus Genome – 19 kb
  • 7 Genes
  • Nucleotide and amino acid differences b/w

Marburg and Ebola – 55%

Ebola – 37 to 41%

  • Overlaps

1 in Marburg genome –

VP30 to VP24

2 in Ebola genome-

VP35 to VP40 & GP to VP30

Glycoprotein Gene

Marburg- GP gene encodes 1 product

1 open reading frame (0)

Ebola- GP gene encodes 2 products

2 open reading frames (0 & -1)

connected by the insertion of 1 additional A

at a series of 7 U’s on the genomic RNA

The 7 sequentially arranged genes are transcribed into

Ebola - 8 major mRNAs

(7 structural proteins and 1 nonstructural protein)

Marburg – 7 major mRNAs

(7 structural proteins)

filovirus proteins
Filovirus Proteins
  • Ebola and Marburg

encode 7 structural proteins

  • Ebola

encodes 1 nonstructural protein

  • Two Main Categories

Associated with the nucleocapsid

transcription and replication of viral genome

Associated with the envelope

assembly of virus

receptor binding and virus entry

Ribonucleocapsid Proteins

1. Nucleoprotein (NP) Gene 1

2. Viral Protein 35 (VP35) Gene 2

3. Viral Protein 30 (VP30) Gene 5

4. Polymerase L Gene 7

  • Matrix Proteins

5. Viral Protein 40 (VP40) Gene 3

6. Viral Protein 24 (VP24) Gene 6

  • Membrane Protein

7. Glycoproteins (GP) Gene 4

  • Secreted Protein

8. Secretory Glycoprotein (sGP) Gene 4

  • Primary structural protein associated with the nucleocapsid
  • Hydrophobic N-terminal half

Binds genomic RNA

  • Hydrophilic C-terminal half

(Variable b/w Marburg and Ebola)

Interacts with matrix proteins

VP30- Minor structural protein associated with the nucleocapsid
  • Polymerase L- Transcription and Replication

(largest and least abundant protein)

  • VP35- Cofactor in transcription and Replication

(Cofactor in polymerase complex)

  • VP40- Matrix protein

Virus assembly and budding

Forms hexamers when it contacts the plasma membrane which confers added stability during assemebly.

(most abundant protein)

  • VP24- Minor Matrix Protein

Possibly uncoats virus during infection

expression of glycoprotein
Expression of Glycoprotein
  • Transcriptional RNA Editing (occurs only in Ebola)

At a series of 7 U’s on the genomic RNA template

insert a non-template-coded adenosine

20% of GP mRNA is edited

GP with 680 amino acids

80% of GP mRNA is not edited

sGP with 370 amino acids

gp structural
GP (Structural)
  • Formation of GP1-GP2 Heterodimer

In the trans-Golgi, the precursor molecule (GP0) is post-translationally cleaved by furin at yielding a heterodimer, (GP1-GP2)

Furin cleavage site (Arg-Arg-X-Arg/LYS-Arg)

(Marburg, cleavage site is more toward N-terminus)

Heterodimer is linked together by one disulfide bond, a cysteine bridge

GP1 Molecule

C-terminus: hydrophilic, highly glycosylated

Sequences for receptor recognition and binding

N-terminus: hydrophobic

Connects GP1 to GP2 by a disulfide bond

  • GP2 Molecule

Fusion peptide near near its N-terminus

Capable of inserting itself in plasma membranes

Believed to mediate the fusion of the host and virus membranes

  • Functions of GP

Forms the Virion Peplomers (Surface Spikes)

Trimers of the GP1-GP2 Heterodimer,

probably assembled in the ER

Mediates viral entry

by receptor binding and membrane fusion

sgp non structural
sGP (non-structural)
  • Formation and Structure


Synthesized from GP mRNA using the conventional ORF

Produced from a precursor molecule cleaved by furin near the C-terminus, Precursor molecule  SGP and Delta Peptide

Homodimer is linked in anti-parallel orientation by 2 disulfide bonds between the 1st and 6th cysteines on separate molecules

virus entry and replication in host cells
Virus Entry and Replicationin Host Cells
  • Viral surface spikes recognize and bindsurface receptors of host
  • Virus enters cell via endocytosis
  • Release of nucleocapsid into cytoplasm
  • Transcription

viral RNA  polyadenylated, monocistronic mRNA

  • Translation and buildup of viral proteins, primarily NP
  • Budding and release of viruses
  • Host Cell – dies

intracytoplasmic vesiculation, mitochondrial swelling,

organelle breakdown

molecules mediating filovirus entry
Molecules mediating filovirus entry


  • Asialoglycoprotein Receptor

(Found exclusively in hepatocytes)

Recognizes glycoproteins displaying N-linked sugar chains with terminal galactose residues


  • Integrins

N-glycosylated transmembrane cell surface receptors

Ebola and Marburg

  • Human folate receptor-

Co-factor expressed on cell-surface

reverse transcription system
Reverse TranscriptionSystem

Volchkov et al. contructed 2 recombinant EBOV clones

1. pFL-EBOVe+

antigenomic cDNA clone with authentic editing site

2. pFL-EBOVe-

antigenomic cDNA clone with mutated editing site

Eliminated editing site using site-directed mutagenesis

Editing site in middle of GP gene AAAAAAA

EBOV polyadenylation signal ATTAAGAAAAAA



1. Both pFL-EBOVe+and pFL-EBOVe-

Showed typical filovirus structure

Possessed similar infectivity and virus production

2. Visible Cytopathic Effects

pFL-EBOVe+: 4-6 days after infection

But there was still an intact monolayer at day 8

pFL-EBOVe- : 3-4 days after infection

5-6 days after infection, cell rounding was complete

3. Differences in expression of the GP gene

Wild Type – 1/5 GP, 4/5 sGP

pFL-EBOVe+- 1/5 GP, 4/5 sGP

pFL-EBOVe- - no sGP expression,

increase in GP expression

The increase in GP expression by pFL-EBOVe-

no simultaneous increase in virus release

most of GP synthesized were immature precursors

-with sugar side chains high in mannose

-sensitive to treatment with endoglycosidase H

-GP transport was arrested in ER or early Golgi

  • No Transcriptional RNA Editing

Over-expression of GP

Exhausts Cell Host Machinery

Eventual Cell Death

  • So GP expression and cytotoxicity can be down-regulated by virus through transcriptional RNA editing and sGP expression
pathogenesis of ebov infection
Pathogenesis of EBOV Infection
  • sGP

1. Inhibits early activation of neutrophils

-Binds to neutrophils via CD16b cell surface receptor

-CD16b activates neutrophils via lateral membrane interaction with CR3

2. Adsorbs neutralizing antibodies

  • GP

1. Specific region of GP induces cytotoxic effects in endothelial cells

-Rapid release of vasoactive agents from infected cells

-Induces cell rounding and detachment from extracellular matices

-Increases cell membrane premeability

2. Proteolytic activation of GP0 precursor via cleavage

-EBO-Z GP cleaved by furin

-Prerequisite for fusion between viral envelope and host cell membrane

-Enables virus to replicate in host systematic infection

3. Two sequences contribute to evasion of host immunity

-Possible immunosuppressive sequence in GP2 molecule

-Amino acid sequence at amino terminus suppresses lymphocyte mitogen-stimulated proliferation in vitro

Destruction of the Immune System

1. Infects mononuclear phagocytes and fibroblastic reticular system (associated with lymph nodes)

- Failure of early T-cell activation

-Disrupts antigen trafficking and cytokine production

-Extensive apoptosis of blood leukocytes

-Lymphopenia (reduction in lymphocyte #) and severe damage to lymphoid tissue

2. Macrophages and circulating monocytes help transmit virus to other tissues

3. VP35 protein – Type 1 IFN Antagonist

-Combats the host interferon response

possibly enhancing the replicative ability of the virus

key to surviving ebola
Key to Surviving Ebola

French National Institute- Early Immune Response

1996 – Two large outbreaks in Gabon

Compared immune responses

  • Survived

-IgG response against virus’s protein coat early on

-Cleared circulating antigen

-Activated cytotoxic T-cells

  • Died

-No IgG response

-Barely detectable levels of IgM

pathology of marburg and ebola
Pathology of Marburg and Ebola


  • Striking lesions usually in liver, spleen and kidney
  • Necrosis prominent in liver, lymph tissue, and spleen
  • little inflammatory response
  • Viral particles invade phagocytic cells
pathology of marburg and ebola53
Pathology of Marburg and Ebola


  • Liver and spleen become enlarged with excess blood
  • Hemorrhage in the gastrointestinal tract, pleural, pericardial and peritoneal spaces and into the renal tubules with deposition of fibrin
  • Abnormalities in coagulation parameters suggest that disseminated intravascular coagulation is a terminal event.
  • There is usually also profound leukopenia in association with bacteremia.

Example of cervical tissues with

severely enlarged and hemorrhagic lymph nodes from a laboratory Rhesus monkey.

differential diagnosis
High Fever

Severe prostration

Diffuse maculopapular rash





Loss of apetite

Differential Diagnosis


  • Coagulation Studies
  • Serologic Study
  • Leukopenia (Low White Blood Cell Count)
  • Thrombocytopenia (Low Platelet Count)
  • Low Electrolytes
  • lymphopenia followed by neutrophilia


  • ELISA (Enzyme-Linked Immunosorbent Assay)
  • Can’t differentiate Ebola strains with electron microscope
  • Indirect fluorescent antibody test (IFAT)
  • Western blot analysis
  • Radioimmunoprecipitation assay
  • skin biopsies


  • Purified, inactivated filovirus antigens pre-coated onto an ELISA plate
  • If the patient has filovirus, serum contains antibodies to the filovirus antigens, and those antibodies will bind to the antigens on the plate. Test can be specific to IgM or IgG.
  • Anti-human Ig coupled to Chromogen enzyme binds IgM or IgG.
  • Color change indicates positive test
clinical course
Clinical Course
  • 2-21 Day incubation time
  • 50-90% Mortality
  • Variability of clinical presentations complicate early detection and management
  • Non-specific prodrome typically lasts < 1 week
  • 10 to 12 days after the onset of disease, the sustained fever may break, with improvement and eventual recovery of the patient.
  • 1-2 weeks after onset of symptoms: Death often preceded by hemorrhagic diathesis, shock, multi-organ system failure
  • Direct contact with the blood, secretions, organs or semen of infected persons:
    • study of risk factors among family members in Kikwit. 27 surviving members were interviewed. Determined that those who had direct contact or who were exposed during late hospital phase were at very high risk. Those who had no physical contact with infected members remained uninfected.
    • Transmission through semen occurs up to 80 days after clinical recovery.
  • Handling ill or dead infected people or chimpanzees
  • Health care workers have frequently been infected while attending patients- Kikwit outbreak.
  • Neonatal
  • Droplets or small filovirus particle aerosols confirmed in alveoli of monkeys during Reston outbreak.
current therapy
Current Therapy
  • Essentially supportive and directed toward ensuring adequate tissue delivery of oxygen, nutritional support, and hydration.
  • Hemorrhage is managed by replacement of blood, platelets, and clotting factors
  • Passive transfer of human antibodies has remained inconclusive in its benefit to Marburg and Ebola.
  • Ribavirin is a synthetic nucleoside analog that is of use in treating Lassa fever and Arenavirus, but there is unfortunately no benefit in Ebola virus infection.
  • Study shows:
    • hyperimmune globulin effective in protecting experimentally infected baboons if administered at the same time as virus, the treatment was ineffective if delayed for 3 days. Hyperimmune globulin should, however, be considered for prophylactic use in laboratory or nosocomial accidents although humans may require antibodies with higher specificity.
  • Inconclusive evidence- whether whole blood transfusion from convalescent patients are effective.
    • Kikwit epidemic- 8 convalescent patients were given blood transfusions. 7/8 survived. Administered late, patients had better prognosis for survival, lack of controls.
comorbidity pregnancy
Comorbidity: Pregnancy
  • Illness from Ebola is generally more severe in pregnant women, with more serious hemorrhagic and neurologic complications
  • Case fatality rate of pregnant women was 95.5% versus 77% in nonpregnant infected persons
  • Spontaneous abortion is frequent, with fetal losses reported to be as high as 23%–66%
  • All infants born to mothers with EHF ultimately died within 19 days of delivery
potential vaccines
Potential Vaccines
  • Dutch biotechnology company, Crucell, is to collaborate with the US National Institute of Allergy and Infectious Diseases (NIAID) to develop the first vaccine against the Ebola virus.
    • The new vaccine uses DNA encoding three Ebola glycoproteins and one nucleoprotein, followed by a boost with a replication-defective adenovirus expressing Ebola antigens (Nature408, 605; 2000). It is the first vaccine to generate protective immunity against Ebola in non-human primates.
  • Scientists at the US Army Medical Research Institute of Infectious Diseases report a simple method for generating Ebola virus–like particles. (JAMA March 2002)
  • A researcher has discovered a link between HIV and Ebola virus: Both viruses use the same method to spread through the human body. Aids Alert - 01-Feb-2002
vaccine contd
Vaccine Contd.
  • Reverse genetics vs Forward genetics
    • genetics that is concerned with genetic material whose nucleotide sequence is known -analyzes its contribution to the phenotype of the organism by varying the nucleotide sequence

-observe the results of such variation in the living organism, in living cells, or in vitro on macromolecules

ebola from scratch
Ebola from scratch
  • In January 2002 scientists reported making Ebola using "reverse genetics".
    • They took the virus's genome plus pieces of DNA coding for the key viral proteins and added them to cells. These proteins then kick start the replication process.
  • Good News: DNA is more stable and easily manipulated. Method provides in-depth understanding of viral mechanism of action that may lead to a vaccine/cure.
  • Bad News: People acquire ability to synthesize Ebola
  • History of Ebola Weaponization
    • Soko Asafra head of Aum Shinrikyo cult 1992
    • Russians


  • Ustinov conducted basic research on the Marbur Virus studying its potential as a weapon
  • Long term goal: To see if Marburg Virus could be loaded into special biological warheads on the Soviets MIRV missiles.
  • MIRV has multiple warheads which are directed at different targets
  • They at the time where designed to be loaded with strategic/operational smallpox virus, black death, and anthrax.
  • Variant U was born


  • Fall 1991 Marburg Variant U becomes a strategic operational Bioweapon
  • Russians loaded virus into 10 separate cone-shaped warheads on the MIRV.
    • Cooling system inside each warhead keeps the virus alive during heat of atmosphere.
    • Biowarheads parachuted over the target city
      • Certain altitude they break apart
      • Each warhead burst a spray of more than a hundred oval bomblets each the size of a small cantaloupe
      • Cantaloupes fly and then split again in an overlapping manner releasing a haze of bio-particles that quickly become invisible.
  • Finding the Reservoir
  • Need to know more about Transmission
    • From animal to man
    • Ways of putting it into food
  • Aerosolization
    • Possible
    • Stable Base needed (difficult)
why use filoviruses as bioweapon
Why Use Filoviruses as Bioweapon
  • Filoviruses (Ebola in particular)
    • Rates of fatality
    • Deaths
      • Terror of dying
      • Ignorance of the general public
        • Think that they can die by being in same room with person
      • Much not known by general physicians and scientist
genetic engineering
Genetic Engineering
  • At Marburg and Ebola conference talk about creation of genetically engineered Ebola virus
    • Volchkov of Claude Bernard University in France and Institute for Virology in Marburg
    • Powerful Molecular tool to analyze how these viruses cause disease.
      • Constructed DNA Molecule (Complimentary Nucleotide sequence to Ebola)
      • Introduced complementary sequence in cultured cell lines with genes coding for 4 key Ebola proteins
      • Cells proceeded to make new Ebola RNA
      • Resulting in fully infectious when transferred to new cell lines
  • Military Definition: The creation of genetically altered viruses and/or bacteria to enhance their power as weapons
  • Reasons for use in Filoviruses
    • To make the virus stronger
    • Mixtures
      • Ebolapox
        • Smallpox is an extremely lethal virus highly contagious in the air.


  • Produces a form of smallpox called blackpox
  • Blackpox is the most severe type of Smallpox.
  • In BLACKPOX infection the skin does not develop blisters
  • Instead the skin becomes dark all over
  • Blood vessels leak, resulting in severe internal hemorrhaging
  • Therefore, EBOLAPOX would not only provide the Terrorist with the hemorrhages and high mortality rate of the Ebola virus, but in addition as an added bonus would incorporate into its operational effectiveness the incredibly high contagiousness of Smallpox
ways to protect against ebola
Ways to protect against Ebola
  • Government
    • Biosafety Department
    • CDC & other agencies
    • Debriefing of experts
    • Public health education
  • Weaponeers
  • Public policy and preventative measures (Medha)


  • Hospitalization and Isolation of Patients
  • Quarantine Areas if Necessary
  • Protective Measures (Gloves, Gowns, Face Shields, Masks, Eye Protection)
  • Disinfect Bedding, Utensils, Excreta (Heat or Chemicals)
  • Burn Used Articles
isolation criteria
Isolation Criteria

Decision-making for isolation is based upon 4 main factors:

1. Potential harm to life

2. Potential harm to critical systems

3. Potential harm to property

4.Topography & meteorological considerations

isolation precautions
Isolation Precautions
  • Direct Contact with infected blood, body fluids to be avoided.
  • Airborne transmission rare, but cannot be conclusively excluded- negative pressure room and HEPA respirator.
  • Laboratory Precautions- BSL 4
  • Personal Protective Equipment
  • Post-Mortem Practices (Kikwit

And Uganda)

prevention and control
Prevention and Control
  • Reservoir remains unknown
  • Clarify roles of GP, sGP, and

other viral proteins.

  • Multivalent vaccine needed

(4 strains of Ebola)

  • Health infrastructures strengthened

especially in Africa

  • Surveillance improved to prevent potential spread of an epidemic.