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Transcript of Molecular Mechanisms Underlying Marburg and Ebola … · Molecular Mechanisms Underlying Marburg...
Molecular Mechanisms UnderlyingMarburg and Ebola VirusPathogenicity
Hans-Dieter KlenkInstitut für VirologiePhilipps Universität Marburg
Brussels, 21. 6. 2004
Viral Hemorrhagic Fever (VHF)
Definition
• unprecisely defined clinical syndrome • high fever, capillary leakage, bleeding tendency, shock • frequent liver involvement, sometimes jaundice • DD: severe malaria, typhoid fever, shigellosis ("diarrhée rouge"),
leptospirosis, rickettsial infections, viral hepatitis, meningococcal infections,gram-negative sepsis
Suspected VHF
• visit to endemic area during incubation period • unexplained high fever (>38,5°C), edema, hemorrhages, jaundice, CNS
symptomes • contact with other VHF patients, infectious material
Public health impact
• high case fatality rates (<90%) • imported infections • bioterroristic potential
Hämorrhagische Fieber-Viren - IFamilie Flaviviridae
Flavivirus Gelbfieber Gelbfieber
Dengue Typen 1, 2, 3, 4 Dengue, DHF
Omsk HF-Virus Omsk HF
Kyasanur Forest-Virus Kyasanur Forest-Krht.
Familie BunyaviridaePhlebovirus Rift Valley Fever Virus Rifttal-Fieber
Nairovirus Krim-Kongo-Virus Krim-Kongo HF (CCHF)
Hantavirus Hantaan HF mit renalem
Seoul Syndrom (HFRS)
Puumala
Dobrava / Belgrad
Sin Nombre u.a. neu- Hantavirus - Lungen-
weltliche Hantaviren syndrom (HPS)
Nephropathia epidem.
Hämorrhagische Fieber-Viren - IIFamilie Arenaviridae
Arenavirus Lassa Lassa-Fieber
Junin Argentinisches HF
Machupo Bolivianisches HF
Guanarito Venezuelanisches HF
Sabia Brasilianisches HF
Familie FiloviridaeFilovirus Marburg Marburg-Krankheit
Ebola-Zaïre Ebola-Krankheit
Ebola-Sudan Ebola-Krankheit
Ebola-Reston (nicht humanpathogen)
Ebola-Côte d’Ivoire Ebola-Krankheit
Ausbrüche
Ebola 1976/77/79
Marburg1980/87
Lake Victoria
Ebola 1976
Ebola 1995
DR Congo
Sudan
Kenya
Uganda
Kikwit
Mt. Elgon
Yambuku
Entebbe
WatsaNzara Juba
Marburg 1999
Gulu
Ebola 2000/01
Marburg 1975
Ebola 1994
GabonRep.Congo
Mekambo
Ebola 2001/02/03
Ebola 1995/96
Ebola 2004
MaridiYambio
Filovirus outbreaksVirus Subtype Year Location human cases (deaths)
Marburg
1967 1975 1980 1987 since 1998
Germany/ Yugoslavia Zimbabwe Kenia Kenia Democratic Republic Congo
32321
>90
(7)(1)(1) (1)
(>50)
Ebola Sudan Zaire Zaire Sudan Reston Reston Reston Zaire Ivory Coast Zaire Zaire Zaire Reston Sudan
1976 1976 1977 1979 1989 1992 1992 1994 1994 1995 1996 1996 1996 2000 2002 2003 2003 2004
Sudan Zaire Zaire Sudan USA Philippines Italy Gabon Ivory Coast Kikwit (Zaire) Gabon Gabon USA Uganda Gabon / Congo-Brazaville Congo-Brazaville Congo-Brazaville Sudan
284318
134
400
unknown1
31550 54
0425 33
1431130
(141)(280)
(1)(22)(0)
(0)(243)
(25)(41)
(224)(24)
(128)(11)(7)
Hospital Gulu
Ebola Outbreak, Uganda, October – December 2000Victims among the medical staff, Lacor Hospital Gulu
Dr. Matthew Lukwiya, 4 nurses, 4 assistant nurses, 3 student nurses
Filovirus disease
Pathogenetic mechanisms
- Severe hemorrhagic manifestation- Marked, hepatic involvement- Disseminated intravascular coagulopathy- Shock syndrome
- Vascular leakage- Dysregulation of cytokine release- Immune suppression
80nm
L
VP30
GPVP24
VP35VP40
RNA-GenomNP
sGP
5’Marburg 3’ 35 40 3024 LNP GP
GP40 30 24Ebola 3’ 5’NP 35 L
Filoviruses
MononegaviralesRhabdoviridaeParamyxoviridaeBornaviridaeFiloviridae
Marburgvirus (MBGV)Ebolavirus (EBOV)
Zaire EBOVSudan EBOVIvory Coast EBOVReston EBOV
Transcription
Replication
Translation
Assembly
Formation of nucleocapsids
β1 Integrin receptorDC-SIGNC-type lectinsFolate receptor ?
ASGP-R
MBGV
EBOV
Replication cycle of filoviruses
L
VP30
GPVP24
VP35VP40
RNA-GenomNP
sGP
5’Marburg 3’ 35 40 3024 LNP GP
GP40 30 24Ebola 3’ 5’NP 35 L
VP35
IFN-βIFN-α/β
IFNAR
IFN-γ
IFNGRVirus Jak1 tyk2 Jak1 Jak2
Antiviral Gene Produkts
STAT1:STAT1
EXTRACELLULAR
CYTOPLASM
NUCLEUS
STAT1:STAT2:IRF-9(ISGF-3)
GAS promoterISRE promoterIFN-β promoter
ATF-2/c-JunIRF-3NF- κB
Interferon Antiviral Pathways
VP35
VP 35 inhibits activation of interferon regulatory factor 3Basler et al., J.Virol 77, 7945 (2003)
L
VP30
GPVP24
VP35VP40
RNA-GenomNP
sGP
5’Marburg 3’ 35 40 3024 LNP GP
GP40 30 24Ebola 3’ 5’NP 35 L
GP
RRKR
GP1 GP2
TDSP
CC C C*C*CC
FD
CT× ×
C CC CCC
× ×× ×× × × ×
MBGV
EBOV RTRR
GP1 GP2
TDSP
CC CCC
FDCT× × ××× × ××
C CC CC
××× ×× ××
37
53
512 557
602609610
MD× ×××××××××
MD
AH AH× ×
601608609511 556
501
435
××AH AH
C*C*
MBGV
RRKR435
TACE
Furin
C
C C
C
602
C
C
512
557
609
37
GP1
610
EBOV
RRKR501
TACE
Furin
C
C C
C
601
C
C
511
556
608 609
53
GP1
Functional domains of GP
GP has an internal fusion peptide
Virus entryReceptor bindingMembrane fusion
Cytotoxicity (endothelia)Yang et al., Nature Med. 6, 886-889 (2000)
Biological functions of GP
Furin
TACE (ADAM17)
S Pro Cat Mid Cys TC
S Pro Cat Dis Cys T CEGF
Proteases involved in GP processing
Subtilisin-like eukaryotic serine protease (pro- proteinconvertase)TGN localizationCleavage site: R-X-K/R-R, R-X-X-R, R-X-X-X-X-RSubstrates: peptide hormones, receptors, adhesion molecules,
neurotransmittersmany viral envelope proteins
Zink dependent metalloproteaseCell surface localizationCleavage site: close to membrane (sheddase), no specific motifSubstrates: release of TNFα, IL-6 receptor, TNF receptors,
l-selectin, IL-1 receptor, ectodomain (sAPPα) of amyloidal precursor protein (APP) (α-secretase)
Mono/Mac
Endothelial cell
The Role of TACE in Pathogenesis
Infection
Immuno-suppression
Antibody decoy(Dolnik et al., 2004)
GP sheddingInduction(Relman et al.,unpublished)
TACETNF α shedding
RBCEndothelial leakage
Coagulation disorders(Feldmann et al., 1996) Shock
Filovirus Vaccines
Virus Strategy Model Response Reference
EBOV DNA (NP, sGP, GP)
guinea pig humoral, T-cell
Xu et al., Nat. Med. 4, 37-49, 1998
EBOV DNA (NP, GP) mouse T-cell Vanderzanden et al., Virology 246, 134-144, 1998
MBGV/EBOV VEE replicon (GP, NP)
guinea pig mouse monkey
T-cell Hevey et al., Virology 251, 28-37, 1998
MBGV Baculovirus (GP) guinea pig Hevey et al., Virology 239, 206-216, 1997
EBOV DNA (GP, NP)-Vector (GP, NP)
monkey humoral, T-cell
Sullivan et al., Nature 408, 605-609, 2000
EBOV rec.VSV mouse n.d. Garbutt et al., unpublished
Research Aims
B. Mechanisms of Pathogenesis
1. Functional significance of GP cleavage by furin
2. Role of VP40 late domains in virus maturation
3. Function of sGP
4. Role of apoptosis of bystander lymphocytes
5. Dysregulation of cytokine response
6. Role of the innate immune response in pathogenesis
and host tropism
7. Dysregulation of coagulation pathway
Research AimsC. Therapeutic and Prophylactic Interventions
1. Antiviral approaches- Inhibition of membrane fusion- Inhibition of transcription and replication (siRNA, anti-sence
oligunucleotides- Inhibition of host proteases (furin, TACE)
2. Modulation of host response mechanisms Regulation of proinflammatory response (IFN, IFN inducers, S-adenosylhomocysteine hydrolase. TACE inhibition)
- Regulation of coagulation cascade (inhibition of factor VIIa / tissue factor by rec. nematode anticoagulant protein c2)
3. Antibody therapy
4. Vaccination
Infrastructure
Emergency network• Clinical units• BSL4 laboratories
BSL4 laboratories• Standardize safety regulations, GLP• Lab must be manageable (avoid safety overkill, restrain bureaucracy)
• Combine diagnostic surveillance and research• Link to academic environment• Provide animal experimentation• Facilitate exchange of scientific material