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

Fledermäuse

Filovirus infections are zoonosesThe filovirus reservoir is not known

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

A. Natural Reservoirs of Marburg and Ebola Viruses

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