Ebola Virus Pathogenesis:

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Ebola Virus Pathogenesis Lessons learned from
my Post-doc experience at CDC
Amy L. Hartman, Ph.D.
IDM 2004 Viral Pathogenesis November 20, 2008
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RNA Virus Families
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Filoviruses Ebola and Marburg
From Frederick Murphy, CDC
From Fields Virology 5th Ed. Ch. 40
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Genomic organization of Filoviridae
VP30
VP24
L
VP40
GP
NP
VP35
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3
19 Kb
Membrane-associated proteins
RNP-complex-associated proteins
Intergenic region
Leader/trailer sequences
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From Fields Virology 5th ed.
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Filovirus Genome replication
Replication (viral RdRp)
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Experimental models

• Unique properties of (-)ssRNA viruses makes
  genetic manipulation difficult
• Need for RdRp in the virion for rep/transc.
• How can Ebola genome be manipulated?

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Reverse genetics systems for (-)ssRNA viruses

• Goal to make mutant viruses
• This method will
• make a desired mutation in a viral gene
• introduce it into the viral genome
• generate recombinant virus with the desired
  mutation
• look at the resulting phenotype

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Forward Genetics
Observable phenotype
sequencing
genotype
Reverse Genetics
Genotype (mutation)
Introduce into sequence
phenotype?
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Reverse genetics system for generation of
recombinant Ebola virus
Full-length genomic cDNA plasmid
x
Ebola
trailer
T7
Rib
leader
Ebo cRNA
NP, L, VP35, VP30 proteins
Ebola proteins
Ebo mRNAs
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Take home message

• Reverse genetics is the only way to generate
  mutations in the genome of (-)ssRNA viruses
• Can be applied to segmented (-)ssRNA viruses

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Geographic distribution of Filovirus outbreaks
Ebola Zaire Ebola Sudan and Marburg
hemorrhagic fever outbreaks
Kenya 80, 87
DRC 98
Uganda 67
Angola 05
Zimbabwe 75
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(No Transcript)
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From Fields Virology 5th ed.
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Clinical features of Ebola and Marburg
Hemorrhagic Fever
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From Fields Virology 5th ed.
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Characteristics of Ebola virus pathogenesis

• Subversion of innate and adaptive immune
  responses
• Induction of harmful inflammatory responses
• Direct tissue necrosis
• Death
• Multi-organ failure
• Severe septic shock-like syndrome

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Infection and spread of Ebola Virus
Zampieri et al. Nat. Immunol. 8(11)1159
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Filovirus specificity

• Pantropic
• Most cells can be infected
• Exception lymphocytes

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Receptor for Ebola virus entry?

• C-type lectins
• ICAM3
• DC-SIGN
• L-SIGN
• TREM
• Heparin-sulphage proteoglycan (HSPG)
• Folate receptor-alpha
• Others?

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Mohamadzadeh et al. Nat. Rev. Immunol. 7556
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Important cytokines secreted by macrophages TNF
IL-6
Fig. 2.39 6th ed.
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TNF-? release friend or foe?
Fig. 2.45 6th ed.
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Tissue Factor (TF) the key to coagulation
abnormalities in Ebola infections?

• TF over-expression in Ebola infection
• TF pro-coagulation pathway
• Decreased Protein C expression in Ebola infection
• Protein C anti-coagulation pathway

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DIC (disseminated intravascular coagulation)

• Small blood clots form in vessels (TF)
• consume all the available coagulation proteins
  and platelets
• normal coagulation is disrupted
• abnormal bleeding
• disrupt normal blood flow to organs
• Multi-organ failure

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Hallmark of fatal cases immunosupression
Zampieri et al. Nat. Immunol. 8(11)1159
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Immunomodulatory properties of Ebola Virus

• VP35
• Inhibits activation (phosphorylation) of the
  transcription factor Interferon Regulatory Factor
  (IRF)-3
• Inhibits activation of protein kinase R (PKR)
• RNA-silencing suppressor (RSS) to inhibit RNAi
• VP24
• Inhibits nuclear translocation of phospho-STAT1
• Immunosuppressive domain (ISD) within GP
• Decreased activation of CD4/CD8 T cells and Th1
  cytokine secretion
• Increased apoptosis

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Animal models of Filovirus infections

• Mouse
• Adaptation required
• ip infection necessary
• no hemmorrhage
• Guinea pig
• Adaptation required
• Non-human primate
• Very sensitive

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Filoviral vaccine candidates
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Opportunities at CDC

• ASM/CCID Post-doctoral fellowship
  (research-based)
• http//www.asm.org/Education/index.asp?bid15497
• Emerging Infectious Disease fellowship
  (research-based)
• http//www.aphl.org/profdev/fellowships/Pages/defa
  ult.aspx
• Epidemic Intelligence Service (epi-based)
• http//www.cdc.gov/eis/index.htm
• Other CDC fellowships
• http//www.cdc.gov/employment/pubhealth.htm