filoviridae 2008 pdf[1]

Family Filoviridae

How the story startedAugust 1967

•Marburg, Frankfurt, Belgrade.•Primary cases: Lab workerswith contact to tissue of imported monkeys (Uganda).•Secondary cases: relatives, health care workers.•32 cases, CFR: 25%.

Werner Slenczka Rudolf Siegert 100 nm

Vervet or Green Monkeys( Cercopithecus aethiops )

polio vaccineproduction

Siegert R, Shu HL, Slenczka W. 1968. Demonstration of the "Marburg virus" in the patient. Dtsch Med Wochenschr. Mar 26;93(12):616-9.

Siegert R, Shu HL, Slenczka W, Peters D, Muller G. 1967. On the etiology of an unknown human infection originating from monkeys. Dtsch MedWochenschr. 1967 Dec 22;92(51):2341-3.

Marburg Virus

1967 Uganda / Germany-Yugoslavia1975 Zimbabwe/South Africa1980,1987,1992 Kenya1999-2000 D.R.Kongo2004-2005 Angola / over 200 dead, children before 5.

Emerging/Re-emerging virus

~5-7 years intervals

Ebola VirusThe Ebola virus was first identified in Sudan and in a nearby region of Zaire (now Democratic Republic of the Congo) in 1976.

Sudan - 284 infected people with 117 deathsZaire - 318 cases and 280 deathsZaire 1977Sudan 1979

A large epidemic occurred in Kikwit, Zaire in 1995 with 315 cases, 244 fatal outcomes.

Single human case of Ebola haemorrhagic fever and several cases in chimpanzees in Côte d'Ivoire in 1994-95.

(Subtype Ivory Coast)

Gabon has been affected by three epidemics between1994 and 1997: the area is difficult to access, withpopulations in small settlements located several miles apart from each other.

Ebola virus infections were not reported again until the autumn of 2000 when an outbreak occurred in northern Uganda.

2002 : Gabon and DR Congo 2003-2004 : DR Congo.

2004: Sudan

Ebola Virus

Excluding the most recent outbreak, approximately 1,600 cases with over 1,000 deaths have been documented since the virus was discovered.

In 1989, 1990 and 1996, Ebola-Reston, was isolated in monkeys being held in quarantine in Reston (Virginia), Pennsylvania, and Texas USA. In 1992 in Sienna Italy.

Ebola-related filoviruses were isolated from cynomolgus monkeys (Macacca fascicularis) imported into the United States of America from the Philippines in 1989.

In the Philippines, Ebola-Reston infections occurred in the quarantine area for monkeys intended for exportation, near Manila.

A number of the monkeys died and at least four persons were infected, although none of them suffered clinical illness (USA).

Ebola-Reston

Filoviridae

Marburg and Ebola virus

African origin

Filoviridae

Marburg and Ebola virus

filovirus belt

Angola

Sudan

KeniaGabon DR.Kongo

(Zaire)l.Victoria

Zimbabwe

UgandaIvoryCoast

Filovirus EpisodesFilovirus Episodes ((AfricaAfrica))

Filovirus EpisodesFilovirus Episodes Rainforests of the worldRainforests of the world

Filoviridae

Marburg and Ebola virus

African origin

Hemorrhagic Fever, Lethality up to 90%

Therapy: No

Vaccine: No

Human and nonhuman victimsNatural reservoir unknown

Democratic Republic of Congo

Durba, DRC

by Pierre Rollin, CDC Atlanta

Goroumbwa gold mine near Durba

by Pierre Rollin, CDC Atlanta

Official entrance of the mine

by Pierre Rollin, CDC Atlanta

Inofficial entrances

by Pierre Rollin, CDC Atlanta

Isolation ward, Durba 1999

Colebunders et al., 2004

Isolation ward, Durba 1999

Sequence analyses of different MARV isolates (CDC, Atlanta; NICD,

Johannesburg):

• Up to 20% nucleotide difference between theisolates.

• 9 different introductions of Marburg virus duringthe Durba outbreak

- direct contact with the blood - secretions - semen of infected persons. Transmission through semen may occur up to seven weeks after clinical recovery.

- transmission through handling ill or dead infected chimpanzees.

- health care workers have frequently been infected while attending patients (nosocomial infection).

In the 1976 epidemic in Zaire, every Ebola case caused by contaminated syringes and needles died.

Transmission

Epidemiological studies: infection is not transmitted by the aerosol route

Experimental infection of non-human primates: aerosol transmission can cause infection

Transmission

Has not yet been identified. - rodents were suspected. - plant virus may have caused the infection of vertebrates.- laboratory observation has shown that bats experimentally infected with Ebola do not die and this has raised speculation that these mammals may play a role in maintaining the virus in the tropical forest.- great apes have been the source of infection for humans. They, like humans, are infected directly from the natural reservoir or through a chain of transmission from the natural reservoir.

Extensive ecological studies are currently under way to identify the reservoir of Ebola and Marburg viruses.

Natural Reservoir

• Bats?

Natural hosts of filoviruses

by Pierre Rollin, CDC Atlanta

Marburg virus in species collected in Durba

Phylogenetic Analysis of MARV basedon nested PCR results from bat samples

Natural Reservoir

No virus isolatedSome are IgM positiveSome others are PCR positive.

Fruit bats.Leroy et al., Science 2006

E P I D E M I C A L E R T A N D R E S P O N S E

Marburgvirus outbreak, Angola 2005

“Terrified people had attacked aid workers”

“Number of health workers had fled out of fear of catching the disease”

“No signs of abating”“We clearly don't know the dimensions of the outbreak”

“Hiding sick relatives out of fear that they will be infected if taken to the hospitals, thereby increasing the chance the disease will spread”

Two cases have occurred in Luanda, which has an international airport, raising the specter that the disease could spread.

Isolation ward

E P I D E M I C A L E R T A N D R E S P O N S E

Convalescences ward

E P I D E M I C A L E R T A N D R E S P O N S E

Convalescent, 5 years, Pedreira02.05.05: positive

07.05.05: negative

08.05.05: negative

E P I D E M I C A L E R T A N D R E S P O N S E

Health education

Pathogenesis of filovirus infections

day 1infection

day 3-5liver, spleen

day 5-7pantropicinfection

Marburg virus hemorrhagic fever

ConjunctivitisEnanthema

ThrombocytopeniaDiarrhea

ExanthemaHepatitis

Hemorrhages

Symptoms:

Incubation 2 to 21 daysSudden onset of the following symptoms: - fever - weakness - muscle pain - headache - sore throat

Late symptoms:- vomiting - diarrhea - limited kidney and liver functions - internal and external bleeding / hemorrhage (70% cases)

Terminal symptoms:- shock / severe blood loss- anuria- tachypnea

Death 6-9 days

Early symptoms / similar to those of many other viral diseases

Fields Virology

Hemorrhagic Signs

- puncture site bleedings- bloody stool - hematuria- gum bleedings- hemoptysis- hematemesis- petechia- epistaxis- hematoma

Geisbert et al., 2004

Hemorrhagic Signs

Vaccination against Filoviruses

Starting Point (≈1988):Monkeys, immunized with inactivated Marburg or Ebola virus.Challenge infection with Marburg virus oder Ebola virus.

⇒50% of the animals survive the challenge Infection.

Even surviving animals did not develop a robust immunityagainst subsequent infections.

Animal models for filovirus disease

• Nonhuman primates (cynomolgous/ maccaques)

• Guinea pigs• Mice

only after adaptation of virus

• Vaccination of guinea pigs with recombinant surfaceprotein of Ebola virus.

Animals developed antibodies against the surfaceprotein.

• Challenge with infectious Ebola virus.• 50 - 90% of guinea pigs survived.⇒ Antibodies against the surface protein are able to

mediate protection against Ebola virus infection.

Which viral proteins mediate protectionagainst filoviral disease?

• Vaccination of guinea pigs with recombinant nucleoprotein.Animals developed antibodies agianst nucleoprotein.

• Challenge with infectious Ebola virus.• All animals died.⇒ Antibodies against the nucleoprotein do not play a

role in protection against Ebola virus infection.

Which viral proteins mediate protectionagainst filoviral disease?

• Vaccination of mice with DNA vaccines expressing thenucleoprotein of Ebola virus.

Animals develop antibodies and cytotoxic T cells directedagainst the Nucleoprotein

• Challenge with Ebola virus.• Survival rate: 80%.⇒ Cellular immune response is involved in defense

against filoviral infection.

Induction of cellular immunity

Combination of DNA vaccination and useof recombinant viruses

• 1. Immunisation of nonhuman primates with GP- and NP-expressing DNA-Vaccine

• 2. Boostering with GP-expressing recombinant Adenoviruses

• Challenge Infection with Ebola-Virus

Survival rate: 100%

Sullivan et al., Nature, 2000

Copyright ©2003 by the National Academy of SciencesWarfield, Kelly L. et al. (2003) Proc. Natl. Acad. Sci. USA 100, 15889-15894

Ebola virus-like particles as vaccinecandidates

Warfield, Kelly L. et al. (2003) Proc. Natl. Acad. Sci. USA 100, 15889-15894

Ebola virus-like particles as vaccine cadidates

• Immunisation of mice with VLPs.

• 2x booster immunisations.

• Challenge with Ebola virus

Warfield, et al. (2003) Proc. Natl. Acad. Sci. USA 100, 15889-15894

Ebola virus-like particles as vaccinecandidates

Recombinant vesicular stomatitis virusas vaccine against filoviruses

N P M G L

N P M GP L

replace gene encoding „G“ by Ebolavirus GP

VSVgenome

VSVgenome

VSV G

virion

EBOV GP

virion

Recombinant VSV expressing filoviralsurface proteins as vaccine candidates

ZEBOV

SEBOV

MARV

MARV Popp

MARV ZEBOV

Treatment of Marburg virushaemorrhagic fever using recombinant

VSV

Daddario et al., Lancet, 2006

Immunize monkeys 20 min after infection withMarburg virus

Currently no vaccines for human use or treatments are available

Severe cases require intensive supportive care, as patients are frequently dehydrated and in need of intravenous fluids.

Experimental studies involving use of hyper-immune sera on animals demonstrated no protection against the disease after interruption of therapy.

In the past : “Inactivated” vaccines, Rec. Vaccinia virus Sindbis virus and baculoviruses expressing GP did not protect experimental animals form Ebola challenge.

Recent developments: Adenovirus expressing GP and NP, VSV virus expressing GP, and DNA vaccine based on GP can protect monkeys form Ebola and Marburg diseases.

Therapy & Vaccine

“The authorities of the Congo had confirmed that gorillas were infected with Ebola virus. The illness already killed 80% of gorillas of the region.”

BSL P4 Laboratory in Lyon

Ebola and Marburg virusesVirion structure

Genomic RNA

membrane

Surface glycoprotein: GP

Proteins associated withviral RNA:

. Nucleoprotein(NP)

. VP30

. VP35

. Polymerase(L)

Matrix proteinsVP40 et VP24

Virion proteins

VP40VP35VP30

NP

VP24GP2

GP1

Filovirus Proteins

48,33636,527,631,83738,7EBOV/Zaire – MBGV%

49,134,535,725,528,937,438,8EBOV/Reston – MBGV%

74,881,468,149,373,167,668,8EBOV/Reston – EBOV/ZAIRE%

2332254278682304330696MBGV (aa)

2213252289676327341740EBOV-Zaire (aa)

2214252289677332330740EBOV-Reston (aa)

LVP24VP30GPVP40VP35NPHomology %

Genes

Virion Structure

Enveloped, NNS RNA viruses, linear gene orderLength 790-970 nm, Diameter 80nm7 structural proteins (1 nonstructural EBOV)Single surface glycoprotein GP, forms spikes

cross section

Fields Virology

Taxonomy of Filoviruses

Genome organization of filoviruses

~19 kbnegative orientation7 genesgene overlapscis-active elements in leader and trailer

GP

NP 35 40 GP 30 24 L

N P M L

F HN L

L

G L

G L

F SH HN LN P M

N P M

N P M

N P M

N P M

G LN P M

F H

NS1 NS2 SH G F 22K

Conservedvariable

Conserved

filovirus

rubulavirus

Family Genera Genome

Filoviridae

Paramyxoviridae

Rhabdoviridae

Bornaviridae

paramyxovirus

pneumovirus

morbillivirus

lyssavirus

vesiculovirus

bornavirus

Infection

Ebola hemorrhagic fever: Clinical aspects and histopathology

Dendritic cellsMonocytes/Macrophages

FibroblastsEndothelial cellsEpithelial cells

Hepatocytes

Primary symptoms→ Syndrome pseudo-grippal→Abdominal pain, headache, diarhea, vommiting,

3 to 21 Days

Hemorrhage

Dysfunction of organs

Death

10 days

Infection

Ebola hemorrhagic fever: Clinical aspects

Primary symptoms→ Syndrome pseudo-grippal

Abdominal pain, headache, diarhea, vommiting

3 to 21 days

Hemorrhage

Death

10 days

Dysfunction of organs

Asymptomatic infection

Leroy et al., Lancet, 2000.Leroy et al., Clin. Exp. Immunol, 2001.

Asymptomatics

Survie

Convalescence

SurvivalsClinical symptoms

7-10 days

, NK cells

Fas/Fas-L cascadeCD4+, CD8+, CD16+

Fibrin deposition

Monocytes and lymphocytes depletion in bone marrow,

Abnormalities in vascular permeability /

fluid distribution problems Geisbert et al., 2004

Comparison of disease pathogenesis of Ebola virus infection in animal models and humans

IFN-a, IL-2, IL-6, IL-10, TNF-a

Guinea pig Macaque HumanElement / Feature

Time to death 8–12 days 5–10 days Up to 30 daysPeak viraemia 5.2 log10 pfu/ml 6.9 log10 pfu/ml 6.5 log10 pfu/ml

DIC (fibrin deposits) Few Abundant NELymphopaenia Yes Yes Yes

Lymphocyte apoptosis NE Yes YesPermissive host cells Monocytes,

macrophages, dendriticcells, fibroblasts,

hepatocytes, adrenal cortical cells, endothelial

cells, epithelial

Monocytes, macrophages, dendritic cells,

fibroblasts, hepatocytes, adrenal

cortical cells, endothelial cells,

epithelial

Monocytes, macrophages, dendritic cells,

fibroblasts, hepatocytes,

endothelial cells, epithelial cells

Cytokines/chemokines(increased circulating levels)

NE IFN-a, IL-6, IL-18, MIP-1a, MIP-1b, MCP-1, TNF-a

Geisbert and Hensley 2004

Viral Structure

From latin filum

Pleomorfic

800-1080nm X 80nm

Non-segmented

(–) strand RNA

19 Kb

NucleoproteinL (pol)

VP30

VP35

VP24

VP40Glycoprotein

Ebola and Marburg viruses

VP40VP35VP30

NP

VP24GP2

GP1

Virion structure

Genomic RNA

membrane

Surface glycoprotein: GP

Proteins associated withviral RNA:

. Nucleoprotein(NP)

. VP30

. VP35

. Polymerase(L)

Matrix proteinsVP40 et VP24

NP 35 40 GP 30 24 L

N P M L

F HN L

L

G L

G L

F SH HN LN P M

N P M

N P M

N P M

N P M

G LN P M

F H

NS1 NS2 SH G F 22K

ConservedvariableConserved

Filoviridae filovirusParamyxoviridae pneumovirus

rubulavirusparamyxovirus

morbillivirus

Rhabdoviridae lyssavirus

vesiculovirus

Bornaviridae bornavirus

Family Genera Genome

MONONEGAVIRALES

BORNAVIRIDAE PARAMYXOVIRIDAE FILOVIRIDAE RHABDOVIRIDAE

Marburg virus Ebola virus

Zaire (ZEBOV)

Sudan (SEBOV)

Ivory Coast (IVEBOV)

Reston (REBOV)

Filovirus Proteins

48,33636,527,631,83738,7EBOV/Zaire – MBGV%

49,134,535,725,528,937,438,8EBOV/Reston – MBGV%

74,881,468,149,373,167,668,8EBOV/Reston – EBOV/ZAIRE%

2332254278682304330696MBGV (aa)

2213252289676327341740EBOV-Zaire (aa)

2214252289677332330740EBOV-Reston (aa)

LVP24VP30GPVP40VP35NPHomology %

Genes

Functions of viral proteinsFunctions of viral proteins

NP

L

24

30

GP

40

35

5’NP L2430GP40353’

sGP

Major nucleocapsid protein, RNA protection, part of polymerase complex

Second nucleocapsid protein, part of polymerase complex, INF-antagonist

Major matrix protein,trigger and facilitate budding, RNP traffic ?

Single surface glycoprotein, attachment and fusion.......

Non structural, secreted, ………?

Nucleocapsid protein, transcriptional factor

Viral polymerase

Membrane associated, RNP assembly, host adaptation factor ..

Genome Organization /Genes ExpressionGenome Organization /Genes Expression

Gene

ORF (complementary sense)3’ 5’

ORF5’ 3’

polyACap

Transcription

mRNA

5’NP L2430GP40353’(-)Genome

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

IR 5

VP35NPVP40

5’3’

GP VP24VP30 L

LeaderOverlap

OverlapIR 5 IR 143Overlap Trailer

VP35NP VP405’

IR 9

GPVP24

VP30L

OverlapIR 4 IR 5 Trailer

3’

Leader IR 107

Ebola

Marburg

IR 5

Replication signalsReplication signals

5’NP L2430GP40353’

Genomic RNA:

Leader Trailer

Replication Signals….?

GCCUGUGUG

UUUUU

CUU

U

C

UU

CU UAAAA

AUCCU

AGAAAACACAC

GCUUAU

3’

vRNA (-) strand

5’

ACC

UGUGUG

UUUUU

C UC

U

U

UU

ACUUAAAU

A

AA

AAAAACACACGC

GUU 3’

GU

AAUUU

..

vRNA (+) strand

.. 5’

G C C U G U G U G U U U U U C UU U C U U C U U A A A A A UCC

U AGA A A A C A C A C G C U

U G G A C A C A C A A A A A A AG A G A AA

A U U U U U AA A

U U U UU

G U G U G C G AUAUU

UA

3’

5’

......

......

vRNA (-) strand

NPGP

VP40

VP24 VP30

VP35

L

Leader

Trailer

3’ 5’(-)

(+) 5’ 3’

Genome

Anti-genome

Leader Trailer

NP gene L gene

Volchkov et al., 2000 J.Gen Virol.

3’ 5’

1 55 81 128 469

NPDispensable3-8x(UN5)

Weik et al., 2005 J.Virol.

Spacer

PE1 PE2

UN5 – hexamers, N is any nucleotide

TSS

Modified Rule of six

Proposed structure of the EBOV replication promoter

Replication / Transcriptional signalsReplication / Transcriptional signals

5’NP L2430GP40353’

Virus specific mRNAs:NP

L2430

GP40

35

Genomic RNA:Start signals:

Stop signals: UAAUUUAAUA

CUUUUUUUUUUU L gene

CUCCUUCUAAUU

AU

UAAUUConsensus

Leader Trailer

NP/VP35: UAAUUCUUUUUU GAUUA CUACUUCUAAUU

VP35/VP40: CUACUUC UAAUU CUUUUU

VP40/GP: UAAUUCUUUUUU AGCCG CUACUUCUAAUU

VP30/VP24: UAAUUCUUUUU 144 CUACUUCUAAUU

GP/VP30: CUACUUC UAAUU CUUUUU

VP24/L: CUACUUC UAAUU CUUUUUUAAUUCUUUUUUCGGA

Start VP24IRStop VP30

Stop NP Start VP35IR

Start VP40 Stop VP35

Stop VP40 Start GPIR

Start VP30 Stop GP

Stop VP24(1) Start L Stop VP24(2)IR

GP VP30 VP40 VP35 VP24 L NP

NP CUCCUUCUAAUU

VP35 CUACUUCUAAUU

VP40 CUACUUCUAAUU

GP CUACUUCUAAUU

VP30 CUUCUUCUAAUU

VP24 CUACUUCUAAUU

L CUCCUUCUAAUU

Transcriptional start signal

Virus specific mRNAs

NP

L24

30GP

4035

5’NP L2430GP40353’

Genomic RNA

3’NP L2430GP40355’

Anti-genomic RNA

Replication

Transcription (viral NP,VP35,VP30 and L)

(+)

(+)

(-)

Transcription

Translation

Replication

ReplicationN

N

Replication/Transcriptionof Mononegavirales (Example VSV)

Nucleus

Endosome

Replication cycle of Marburg virus

Nucleus

Nucleocapsid

Transcription/Translation

Mühlberger et al., 1998

Replication cycle of Marburg virus

LVP30

NP

VP35

VP24

VP40

Translation

AAAAAn

AAAAAn

GP

AAAAAn

AAAAAn

AAAAAn

AAAAAn

AAAAAn

AAAAAn

AAAAAn

TranscriptionNP, VP35, L

Nucleus

Nucleocapsid

30

L35

NP

NP

Replication/Assembly

Mühlberger et al., 1998

Replication and assembly of nucleocapsids

Formation of inclusions

Kolesnikova et al., 2000

NP

MBGV

30L35

NP

NPNP

Filoviral genome and minigenome

VP35 VP30NP Lleader trailer

CAT minigenome481 nts 730 nts

VP24VP40 GP

Filoviral artificial replication and transcription system

Filoviral artificial replication and transcription system

NP

35

L

30

MG

NP

35

L

30MG

T7 transcriptionmRNA

transcriptionreplication

minigenome

MARVNC genes

Transfection

minigenomeCAT

translation

30L35

NP

Protein requirements for transcription and replication

Marburg virus:

Ebola virus:replication

transcription

NP L35

30+

NP L35

NP L35

replication + transcription

Rescue of infectious filoviruses fromcDNA

NP

35

L

30

NP

35

L

30FL

T7 transcriptionmRNA

encapsidationtranscriptionreplication

MARVNC genes

Transfection

translation

30L35

NP

genome

FL Plasmid encoding theentire genome of filoviruses+

Release of infectious viruses

Protein requirements for transcription and replication

Marburg virus:

Ebola virus:replication

transcription

NP L35

30+

NP L35

NP L35

replication + transcription

Functions of viral proteinsFunctions of viral proteins

NP

L

24

30

GP

40

35

5’NP L2430GP40353’

sGP

Major nucleocapsid protein, RNA protection, part of polymerase complex

Second nucleocapsid protein, part of polymerase complex, INF-antagonist

Major matrix protein,trigger and facilitate budding, RNP traffic ?

Single surface glycoprotein, attachment and fusion.......

Non structural, secreted, ………?

Nucleocapsid protein, transcriptional factor

Viral polymerase

Membrane associated, RNP assembly, host adaptation factor ..

RNPInclusion bodies

ER

Filovirus replication cycle

Golgi

. GP

ReplicationTranscription

Proteinsynthesis

Virions

Budding

GP

VP40, VP24

NP, VP35,

VP30, L

Release into cytoplasmEndosomes/

membrane fusionacid pH

GP

Cells of mononuclear phagocytic system :Monocytes, Macrophages, and Dendritic cells; Hepatocytes;Fibroblasts;Endothelial cells.

Virus targets:

Comparison of disease pathogenesis of Ebola virus infection in animal models and humans

IFN-a, IL-2, IL-6, IL-10, TNF-a

Guinea pig Macaque HumanElement / Feature

Time to death 8–12 days 5–10 days Up to 30 daysPeak viraemia 5.2 log10 pfu/ml 6.9 log10 pfu/ml 6.5 log10 pfu/ml

DIC (fibrin deposits) Few Abundant NELymphopaenia Yes Yes Yes

Lymphocyte apoptosis NE Yes YesPermissive host cells Monocytes,

macrophages, dendriticcells, fibroblasts,

hepatocytes, adrenal cortical cells, endothelial

cells, epithelial

Monocytes, macrophages, dendritic cells,

fibroblasts, hepatocytes, adrenal

cortical cells, endothelial cells,

epithelial

Monocytes, macrophages, dendritic cells,

fibroblasts, hepatocytes,

endothelial cells, epithelial cells

Cytokines/chemokines(increased circulating levels)

NE IFN-a, IL-6, IL-18, MIP-1a, MIP-1b, MCP-1, TNF-a

Geisbert and Hensley 2004

Fields Virology

Genome Organization /Genes ExpressionGenome Organization /Genes Expression

Translation

LVP30

NP

VP35

NP,VP35, LVP30

VP24

VP40

AAAAAn

AAAAAn

AAAAAn

AAAAAn

AAAAAn

AAAAAn

AAAAAn

Transcription AAAAAn

AAAAAn

GPTranscription and TranslationER

Replication

NP,VP35, LVP30

(-) strand (+) strand

NP,VP35, LVP30

(-) strand

TranscriptionReplication

L

VP30NP

VP35

NP / NP

NP / VP35

NP / VP30

VP35 / L

NP / VP35 / L

Interaction

Interaction of Nucleocapsid proteins

Gene

ORF (complementary sense)

TStartS TStopS

3’ 5’

ORF5’ 3’

polyACap

Transcription

mRNA

5’NP L2430GP40353’(-)Genome

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

IR 5

VP35NPVP40

5’3’

GP VP24VP30 L

LeaderOverlap

OverlapIR 5Stop

IR 143Overlap Trailer

VP35NP VP405’

IR 9

GPVP24

VP30L

OverlapIR 4 IR 5 Trailer

3’

Leader IR 107

Ebola

Marburg

IR 5

Virus specific mRNAs

NP

L24

30GP

4035

5’NP L2430GP40353’

Genomic RNA

3’NP L2430GP40355’

Anti-genomic RNA

Replication

Transcription (viral NP,VP35,VP30 and L)

(+)

(+)

(-)

Replication / Transcriptional signalsReplication / Transcriptional signals

5’NP L2430GP40353’

Virus specific mRNAs:NP

L2430

GP40

35

Genomic RNA:Start signals:

Stop signals: UAAUUUAAUA

CUUUUUUUUUUU L gene

CUCCUUCUAAUU

AU

UAAUUConsensus

Leader Trailer

NP/VP35: UAAUUCUUUUUU GAUUA CUACUUCUAAUU

VP35/VP40: CUACUUC UAAUU CUUUUU

VP40/GP: UAAUUCUUUUUU AGCCG CUACUUCUAAUU

VP30/VP24: UAAUUCUUUUU 144 CUACUUCUAAUU

GP/VP30: CUUCUUC UAAUU CUUUUU

VP24/L: CUCCUUC UAAUU CUUUUUUAAUUCUUUUUUCGGA

Start VP24IRStop VP30

Stop NP Start VP35IR

Start VP40 Stop VP35

Stop VP40 Start GPIR

Start VP30 Stop GP

Stop VP24(1) Start L Stop VP24(2)IR

GP VP30 VP40 VP35 VP24 L NP

NP CUCCUUCUAAUU

VP35 CUACUUCUAAUU

VP40 CUACUUCUAAUU

GP CUACUUCUAAUU

VP30 CUUCUUCUAAUU

VP24 CUACUUCUAAUU

L CUCCUUCUAAUU

Transcriptional start signal

Replication Signals….?

GCCUGUGUG

UUUUU

CUU

U

C

UU

CU UAAAA

AUCCU

AGAAAACACAC

GCUUAU

3’

vRNA (-) strand

5’

ACC

UGUGUG

UUUUU

C UC

U

U

UU

ACUUAAAU

A

AA

AAAAACACACGC

GUU 3’

GU

AAUUU

..

vRNA (+) strand

.. 5’

G C C U G U G U G U U U U U C UU U C U U C U U A A A A A UCC

U AGA A A A C A C A C G C U

U G G A C A C A C A A A A A A AG A G A AA

A U U U U U AA A

U U U UU

G U G U G C G AUAUU

UA

3’

5’

......

......

vRNA (-) strand

NPGP

VP40

VP24 VP30

VP35

L

Leader

Trailer

3’ 5’(-)

(+) 5’ 3’

Genome

Anti-genome

Leader

Trailer

NP gene L gene

Volchkov et al., 2000 J.Gen Virol.

3’ 5’

1 55 81 128 469

NPDispensable3-8x(UN5)

Weik et al., 2005 J.Virol.

Spacer

PE1 PE2

UN5 – hexamers, N is any nucleotide

TSS

Modified Rule of six

Proposed structure of the EBOV replication promoter

Polymorphism of the GP Gene

ORF I*TGGGAAACTAAAAAAAAACCTCACTAGAAAAATTC... (6A or 9A, ssGP)

E T K K P H Stop

. .

W

ssGP

ORF I* ssGP

K

N L T R K IE T K K

Expression strategy of the EBOV glycoproteins

C

WORF I-II

GG AA T T C

Transcription + RNA editingGP mRNA’s:

ORF I ORF I-II

sGPGP

GP gene: ORF I

ORF II

TGGGAAACTAAAAAAA-CCTCACTAGAAAAATTC... (7A, sGP)

TGGGAAACTAAAAAAAACCTCACTAGAAAAATTC... (8A, GP)

Editing site

W E T K K T S L E K F . .ORF I

Transmembrane region

Signal peptide

GP

sGP Cytoplasmictail

~80%~20%

7A 8A

+A

Transcription of Ebola virus GP gene

- cysteine residue

- potential N-linked glycans

S-S - disulfide linkage

GP1,2 trimersform spikes

Glycoproteins of Ebola virus

Δ-peptide, highly O-glycosylated

GP1

Cleavage site RTRR501

sGP

S-S

Cleavage site RVRR324

GP2

Δ

N

N

C

CSS

SSΔ

Δ

PM

PM

53 306

306 53

sGP dimer,

Cleavage of EBOV GP is inhibited by RVKR-cmk

040604h 4h 4h

8% gelInhibitor (µM)80 25 0 0 0 0

Chase (min/h)

15% gel

GP1

preGP

preGPer

Mock

0 20 40 9060 2h 4h 4h

Cells Medium

220

97,466

46

30GP2

0

EBOV glycoprotein GP is proteolytically processed in two subunits

SP TDCT

0 100 200 300 400 500 600 700a.a

RTRR501

FD

C CCCC C C CCC CC

S - S

GP1 GP2GP1,2

PathogenicityHuman

++++++

+ (++)-

+++

Monkey+++++

+ (++)+ / +++++

EBOV-Zaire -R-T-R-R-EBOV-Sudan -R-S-R-R-EBOV-Ivory Coast-R-K-R-R-EBOV-Reston -K-Q-K-R-MBGV -R-R-K-R-

Virus species -1-4 -2-3

*

COOH

NH2

S S

GP1

GP2

Volchkov et al., 1998

Eukaryotic subtilisin-like endoprotease Furin

• expressed in most mammalian cells• type I transmembrane protein• accumulates in the trans Golgi network• cleaves and activates precursor proteins:

cellular proteinsviral glycoproteinsbacterial toxinsat the C-terminus of

-R-X-K/R-R--1-2-3-4

Cleavage by Furin

Virus Glycoprotein Cleavage site

Orthomyxoviridae

Retrovirus

Paramyxoviridae

TogaviridaeBornaviridae

Filoviridae

A/FPV/Rostock/34 (H7) HA KKRKKR GL

Measles virus F SRRHKR FA

Ebola virus GP GRRTRR EA Marburg virus GP YERRKR SIEbola (Reston) GP TRKQKR FA

Sindbis virus p62 SGRSKR SV

Flaviviridae Denge 2 virus prM HRREKR SV

HIV-I env(gp160) VQREKR AV

(R x K/R R )

BDV GP84 LKRRRR DT

Family-1-2-3-4

MoMuLv TM HIV GP41Influenza HA2EBOV GP2 HTLV1 GP21SV5 F1

Viral fusion proteins: structural similarityViral fusion proteins: structural similarity

NHNH22 COOHCOOHFusion

Domain (FD)

Cleavage

Amphipathichelices

RTRR501

Transmembrane Domain (TD)

EBOV

GP2

GP1

Cleavage

Transmembrane subunit GP2 of Ebola virus

EBOV GP2 Rous Sarcoma TMGP1

Glycosyl

Glycosyl

Disulfide Loop

AmphipathicHelix

Furincleavage site

ChargedHelix

FusionPeptide

MBGV I . . H . . . . . . T . . . . . . K V . . . . . . .

EBOV L N R K A I D F L L Q R W G G T C H I L G P D C C I

RSV Q . . A . . . . . . L A H . H G . E D V A G M . . F

ASV Q . . A . . . . . . L A H . H G . E D I A G M . . F

M-MULV Q . . R G L . L . F L K E . . L . A A . K E E . . F

FeLV Q . . R G L . I . F L Q E . . L . A A . K E E . . F

HTLV-I Q . . R G L . L . F W E Q . . L . K A . Q E Q . . F

ARV Q . . R G L . L . T A E Q . . I . L A . Q E K . . F

BAEV Q . . R G L . L . T A E Q . . I . L A . Q E K . . F

“Immunosuppressive-like motif “

Mechanism of membrane fusion

Shedding of Soluble Glycoprotein GP of Ebola Virus

GP2

GP1

cells

TM

TACE

GP1,2Δ trimer, recognized by monoclonal virus neutralizing antibodies

KTLPD QGD

KTLPD VGD

KTLPE QGD

KTLPL QGD

KTLPV QGD

KTLAD QGD

KTVPD QGD

KILPD QGD

VTLPD QGD

WT

0 100 200 300 400 500

TACE

% GP release

TACE: TNF-α converting enzyme

GP1

GP2Δ

supernatant

soluble GP1,2Δ-complexes

TM

C M P S

GP2

ΔTmGP2C - cellsM - mediumP – pellet S - supernatant

Dolnik et al EMBO J 2004

Surface GP of EBOV shed from virus-infected cells following cleavage by the cellular metalloprotease TACE.

TACE

COOH

NH2S S

GP1

GP2

Ectodomain shedding is a mechanism to escape control by hosts immune system. Shed GP present in the blood of infected animals and blocks virus neutralizing activity of antibodies / Decoy function

NITDKIDQIIHDFVDKTLPD QGDNDNWWTGWRQ

RTRRAsn563 Asn618

Tm. regionFusionpeptide

GP2

Cyt. tail

Cleavage site

Ver

o

9d 5d 293T

GP2

GP2∆

Serum

9d

TNF-α-CONVERTING ENZYME TACE (ADAM-17)

ADAM (A Disintegrin And Metalloprotease)

Zinc-dependant metalloprotease

Type-I membrane protein

Plasma membrane, intensive recycling

Substrates: TNF-α precursor, TNF-receptors, TGF- α, L-selektin,

APP, IL-6 receptor, Notch 1 receptor and others

Pro

Metalloproteinase

DisInt

Cys-rich

EGF

TM

Cytosolic

Reverse Genetics of Filoviruses

Virus WT

Virus MutantSequencing

Virus WT

Search for mutated genes

Mutagenesis Virus replication, pathogenicity …

Virus Rescue(recombinant virus)

Traditional Genetics:

Reverse genetics:

Reverse Genetics

Virus mutants

Reverse Genetics

3’ 5’NP L2430GP4035

Ebola virus: 18,959 n

EBOV mini genome

T7 promoter Ribozyme

CATCATNP 35 30 L

J Virol 73, 1999

BSR T7/5

T7 RNA pol.J Virol 73, 1999

Vector 2,0

T7 promoter Ribozyme

LL2424GPGP 303040403535NPNP

EBOV full-length antigenome (pFL-EBOV)

Construction of the EBOV full-length antigenome

Sal I (GTSal I (GTAAGAGATT))C CC C

Sal I (GTSal I (GTCCGAGACC))

40403535NPNP LL2424GPGP 3030 LLGPGP

Sma I Cla I Cla I Sac II Sac II Sac I

+ +

NP

35

L

30

Transfection

NP35

L

30

30

NC genes(T7 promoter)

Recombinant Ebola virus(cross section)

pFL-EBOV

T7 promoter Ribozyme

LL2424GPGP 303040403535NPNP

pFL-EBOV

L

35NP

BSR T7/5cells

(+) vRNA

3 4

1

5

(-) vR

NA

6

(+) vRNA

mRNA`s

1

2

T7

T7

T7

T7

Reverse Genetics System for Ebola virus allows genetic manipulation of infectious virus by introducing single or multiple mutations in particular regions of viral genome.

Volchkov et al Science 2001

GP 2

preGP

Anti-GP2

WTGG NMLRWTRS RK

VP40

Anti-VP40

Multiple-step replication cycles in Vero cells

1

2

3

4

5

6

7

1 2 3 4 5 6 7

Days

Viru

s tit

ers

log 1

0(p

fu/m

l)

8

1

2

3

4

5

6

7

1 2 3 4 5 6 7 8

1

2

3

4

5

6

7

1 2 3 4 5 6 7 8

RRTRR RRTGGRRTLR

DaysDays

RKTRR

100%9%7%20%15%12%

100%

GG R T G GNM R T N MLR R T L RRS R T R SRK K T R R

-4 -1-2-3

TGGGAAACTAAAAAAA CCTCACT

TGGGAAACTAAGAAGAACCTCACT

recEBOVe+ -

recEBOVe- -K K N L TW E T

-. . .

. . .. . .

. . .

. . . . . .

LL2424GPGP 303040403535NPNP

recEBOVe+55’’ 33’’

sGPsGP

LL2424GPGP 303040403535NPNP

recEBOVe-

55’’ 33’’

«No editing » – « No sGP » mutant

GP ORF -

Recombinant Ebola virus variant (No editing)

GPGP

GPGP sGPsGP

+A(Transcriptional RNA editing)

GP1

sGP

recE

BO

Ve+

recE

BO

Ve-

Moc

k

2 31

4days 7days6days 8days

Mock

recEBOVe-

recEBOVe+

3days

Increase in cell rounding, detachment and death

recEBOVe+

recEBOVe-

Cytotoxicity of recEBOVs:Individual plaques

5 days p.i.

Expression of GP is down-regulated through the mechanism of the transcriptional RNA editing and expression of non-structural sGP

Editing of the GP gene of EBOV is an important pathogenicity factor.Reducing expression of GP, it is likely to enhance virus loads and promote spread of infection in the organism

sGP is not essential for replication of Ebola virus in cell culture, however this does not exclude sGP playing role in infection in humans or in the yet unknown natural host