Family Filoviridae

How the story started

August 1967
•Marburg, Frankfurt, Belgrade.
•Primary cases: Lab workers
with 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 polio vaccine
( Cercopithecus aethiops ) production
Siegert R, Shu HL, Slenczka W, Peters D, Muller G. 1967. On the etiology
of an unknown human infection originating from monkeys. Dtsch Med
Wochenschr. 1967 Dec 22;92(51):2341-3.

Siegert R, Shu HL, Slenczka W. 1968. Demonstration of the "Marburg

virus" in the patient. Dtsch Med Wochenschr. Mar 26;93(12):616-9.
 Marburg Virus

Emerging/Re-emerging virus

~5-7 years intervals

1967 Uganda / Germany-Yugoslavia

1975 Zimbabwe/South Africa
1980,1987,1992 Kenya
1999-2000 D.R.Kongo
2004-2005 Angola / over 200 dead, children
before 5.
Ebola Virus
The 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 deaths

Zaire - 318 cases and 280 deaths
Zaire 1977
Sudan 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)
Ebola Virus Gabon has been affected by three epidemics between
1994 and 1997: the area is difficult to access, with
populations 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

Excluding the most recent outbreak, approximately 1,600 cases with over
1,000 deaths have been documented since the virus was discovered.
Ebola-Reston
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).
 Filoviridae
Marburg and Ebola virus

African origin
 Filoviridae
Marburg and Ebola virus

filovirus belt
 Filovirus Episodes (Africa)

Sudan

Ivory
Coast
n da
g a Kenia
Gabon DR.Kongo U
(Zaire)
l.Victoria

Angola

Zimbabwe
Filovirus Episodes Rainforests of the world
 Filoviridae
Marburg and Ebola virus

African origin

Hemorrhagic Fever, Lethality up to 90%

Human and nonhuman victims
Natural reservoir unknown
Therapy: No
Vaccine: No
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 the

isolates.
• 9 different introductions of Marburg virus during
the Durba outbreak
Transmission
- 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

Experimental infection of non-human primates: aerosol

transmission can cause infection

Epidemiological studies: infection is not transmitted by the

aerosol route
Natural Reservoir 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 hosts of filoviruses

• Bats?
by Pierre Rollin, CDC Atlanta
Marburg virus in species collected in
Durba
Phylogenetic Analysis of MARV based
on nested PCR results from bat samples
Natural Reservoir Fruit bats.
Leroy et al., Science 2006

No virus isolated
Some are IgM positive
Some others are PCR
positive.
Marburgvirus outbreak, Angola 2005

E P I D E M I C A L E R T A N D R E S P O N S E
 “Terrified people had attacked
aid workers”

“Number of health workers

had fled out of fear of catching
the disease”
“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.
“We clearly don't know the dimensions of the outbreak”
“No signs of abating”
 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, Pedreira
02.05.05: 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 1 day 3-5 day 5-7

infection liver, spleen pantropic
infection
Marburg virus hemorrhagic fever

Conjunctivitis
Enanthema
Thrombocytopenia
Diarrhea
Exanthema
Hepatitis

Hemorrhages
Symptoms:

Incubation 2 to 21 days
Sudden onset of the following symptoms:
- fever
- weakness Early symptoms / similar
- muscle pain to those of many other
- headache viral diseases
- sore throat
Terminal symptoms:
Late symptoms: - shock / severe blood loss
- vomiting - anuria
- diarrhea - tachypnea
- limited kidney and liver functions
- internal and external bleeding Death 6-9 days
/ hemorrhage (70% cases)
Fields Virology
Hemorrhagic Signs
- puncture site bleedings
- bloody stool
- hematuria
- gum bleedings
- hemoptysis
- hematemesis
- petechia
- epistaxis
- hematoma
Hemorrhagic Signs

Geisbert et al., 2004

 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 immunity

against subsequent infections.
 Animal models for filovirus disease

• Nonhuman primates (cynomolgous/

maccaques)
• Guinea pigs only after adaptation of virus
• Mice
 Which viral proteins mediate protection
against filoviral disease?

• Vaccination of guinea pigs with recombinant surface

protein of Ebola virus.
Animals developed antibodies against the surface
protein.
• 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 protection
against 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.
 Induction of cellular immunity

• Vaccination of mice with DNA vaccines expressing the

nucleoprotein of Ebola virus.
Animals develop antibodies and cytotoxic T cells directed
against the Nucleoprotein
• Challenge with Ebola virus.
• Survival rate: 80%.
⇒ Cellular immune response is involved in defense
against filoviral infection.
 Combination of DNA vaccination and use
of 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

 Ebola virus-like particles as vaccine
candidates

Warfield, Kelly L. et al. (2003) Proc. Natl. Acad. Sci. USA 100, 15889-15894
Copyright ©2003 by the National Academy of Sciences
Ebola virus-like particles as vaccine cadidates

• Immunisation of mice with VLPs.

• 2x booster immunisations.

• Challenge with Ebola virus

Warfield, Kelly L. et al. (2003) Proc. Natl. Acad. Sci. USA 100, 15889-15894
 Ebola virus-like particles as vaccine
candidates

Warfield, et al. (2003) Proc. Natl. Acad. Sci. USA 100, 15889-15894
 Recombinant vesicular stomatitis virus
as vaccine against filoviruses
VSV
genome N P M G L

replace gene encoding „G“ by Ebolavirus GP

VSV
N P M GP L
genome

VSV G EBOV GP

virion virion
Recombinant VSV expressing filoviral
surface proteins as vaccine candidates

SEBOV MARV Popp

ZEBOV MARV

MARV ZEBOV
 Treatment of Marburg virus
haemorrhagic fever using recombinant
VSV

Immunize monkeys 20
min after infection with
Marburg virus

Daddario et al., Lancet, 2006

 Therapy & Vaccine
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.
“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 viruses
Virion structure
Surface glycoprotein: GP

membrane
Matrix proteins
VP40 et VP24

Genomic RNA

Proteins associated with

viral RNA:

. Nucleoprotein(NP)
. VP35
. VP30

. Polymerase(L)
Virion proteins

GP1
NP

VP40
VP35
VP30

VP24
GP2
 Filovirus Proteins
Genes
NP VP35 VP40 GP VP30 VP24 L
Homology %

EBOV-Reston (aa) 740 330 332 677 289 252 2214

EBOV-Zaire (aa) 740 341 327 676 289 252 2213

MBGV (aa) 696 330 304 682 278 254 2332

EBOV/Reston – EBOV/ZAIRE
68,8 67,6 73,1 49,3 68,1 81,4 74,8
%

EBOV/Reston – MBGV
38,8 37,4 28,9 25,5 35,7 34,5 49,1
%

EBOV/Zaire – MBGV
38,7 37 31,8 27,6 36,5 36 48,3
%
 Virion Structure

cross
section

Enveloped, NNS RNA viruses, linear gene

order
Length 790-970 nm, Diameter 80nm
7 structural proteins (1 nonstructural
EBOV)
Single surface glycoprotein GP, forms
spikes
Fields Virology
Taxonomy of Filoviruses
Genome organization of filoviruses

GP

~19 kb
negative orientation
7 genes
gene overlaps
cis-active elements in leader and trailer
 Family Genera Genome

Filoviridae filovirus NP 35 40 GP 30 24 L

Paramyxoviridae pneumovirus NS1 NS2 N P M SH G F 22K L

rubulavirus N P M F SH HN L

paramyxovirus N P M F HN L

morbillivirus N P M F H L

Rhabdoviridae lyssavirus N P M G L

vesiculovirus N P M G L

Bornaviridae bornavirus N P M G L

variable
Conserved Conserved
 Ebola hemorrhagic fever: Clinical aspects and histopathology

Infection

3 to 21 Days

Primary symptoms Dendritic cells

→ Syndrome pseudo-grippal Monocytes/Macrophages
→Abdominal pain, headache, diarhea, vommiting,

Hepatocytes

Fibroblasts
Endothelial cells
Hemorrhage Epithelial cells

Dysfunction of organs

10 days

Death
 Ebola hemorrhagic fever: Clinical aspects

Infection

3 to 21 days

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

Leroy et al., Lancet, 2000.

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

7-10 days Asymptomatics

Hemorrhage Clinical symptoms

Survivals

Dysfunction of organs
Convalescence

10 days

Death Survie
Monocytes and lymphocytes
depletion in bone marrow,

Fibrin deposition

, NK cells
CD4+, CD8+, CD16+
Fas/Fas-L cascade

Abnormalities in vascular
permeability /
fluid distribution problems Geisbert et al., 2004
Comparison of disease pathogenesis of Ebola virus infection in animal models and humans

Element / Feature Guinea pig Macaque Human

Time to death 8–12 days 5–10 days Up to 30 days
Peak viraemia 5.2 log10 pfu/ml 6.9 log10 pfu/ml 6.5 log10 pfu/ml
DIC (fibrin deposits) Few Abundant NE
Lymphopaenia Yes Yes Yes
Lymphocyte apoptosis NE Yes Yes
Permissive host cells Monocytes, Monocytes, Monocytes,
macrophages, dendritic macrophages, macrophages,
cells, fibroblasts, dendritic cells, dendritic cells,
hepatocytes, adrenal fibroblasts, fibroblasts,
cortical cells, endothelial hepatocytes, adrenal hepatocytes,
cells, epithelial cortical cells, endothelial cells,
endothelial cells, epithelial cells
epithelial
Cytokines/chemokines NE IFN-a, IL-6, IL-18, IFN-a, IL-2, IL-6,
(increased circulating MIP-1a, MIP-1b, IL-10, TNF-a
levels) MCP-1, TNF-a

Geisbert and Hensley 2004

Viral Structure
VP24
From latin filum
Non-segmented
L (pol)
Glycoprotein
Nucleoprotein
(–)Pleomorfic
VP40RNA
strand
VP30

800-1080nm
19 Kb X 80nm
VP35
 Ebola and Marburg viruses
Virion structure
Surface glycoprotein: GP

membrane
Matrix proteins
VP40 et VP24
GP1
NP
Genomic RNA

VP40
VP35 Proteins associated with
VP30 viral RNA:

VP24
. Nucleoprotein(NP)
GP2
. VP35
. VP30

. Polymerase(L)
 MONONEGAVIRALES

BORNAVIRIDAE PARAMYXOVIRIDAE FILOVIRIDAE RHABDOVIRIDAE

Marburg virus Ebola virus

Family Genera Genome Zaire (ZEBOV)

Filoviridae filovirus NP 35 40 GP 30 24 L
Sudan (SEBOV)
Paramyxoviridae pneumovirus NS1 NS2 N P M SH G F 22K L

rubulavirus N P M F SH HN L
Ivory Coast (IVEBOV)
paramyxovirus N P M F HN L

morbillivirus N P M F H L
Reston (REBOV)
Rhabdoviridae lyssavirus N P M G L

vesiculovirus N P M G L

Bornaviridae bornavirus N P M G L

Conserved
variable Conserved
 Filovirus Proteins
Genes
NP VP35 VP40 GP VP30 VP24 L
Homology %

EBOV-Reston (aa) 740 330 332 677 289 252 2214

EBOV-Zaire (aa) 740 341 327 676 289 252 2213

MBGV (aa) 696 330 304 682 278 254 2332

EBOV/Reston – EBOV/ZAIRE
68,8 67,6 73,1 49,3 68,1 81,4 74,8
%

EBOV/Reston – MBGV
38,8 37,4 28,9 25,5 35,7 34,5 49,1
%

EBOV/Zaire – MBGV
38,7 37 31,8 27,6 36,5 36 48,3
%
 Functions of viral proteins
3’ NP 35 40 GP 30 24 L 5’

NP
Major nucleocapsid protein, RNA protection, part of polymerase complex
35
Second nucleocapsid protein, part of polymerase complex, INF-antagonist

40
Major matrix protein,trigger and facilitate budding, RNP traffic ?

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

sGP
Non structural, secreted, ………?
30
Nucleocapsid protein, transcriptional factor

24
Membrane associated, RNP assembly,
host adaptation factor ..
L
Viral polymerase
Genome Organization /Genes Expression
Genome (-) 3’ NP 35 40 GP 30 24 L 5’

Gene
3’ 5’
ORF (complementary sense)

Transcription

5’ 3’
Cap ORF polyA
mRNA
Ebola VP30 VP24
L
VP40 GP
NP VP35 5’
3’
Overlap IR 143Overlap
Trailer
IR 5
Leader IR 5 Overlap

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

Marburg
VP24 L
NP VP35 VP40 GP VP30
5’
3’
IR 5
Leader IR 9 IR 4 IR 5 IR 107 Overlap Trailer
 Replication signals

Genomic RNA:

3’ NP 35 40 GP 30 24 L 5’

Leader Trailer
 Replication Signals….?
NP gene L gene
Genome
(-) 3’ 5’
Leader Trailer

(+) 5’ 3’
Anti-genome

vRNA (-) strand

Leader
C UA NP VP35 VP
3’
G CC UG UGUGU UUUUC UUUCUU C U
UA A AA AU
C G
A A A AC AC ACGCU ...... 40
GP
5’
UGG AC ACACA AAAAA GAAGA A AU U U U UA U U U UG UG UGCG A...... 3 0
A
UA UU
A A A U L VP24 VP
Trailer U
5’. 5’
. .
U .
A U
U U A 3’
U G C
C G 3’ U
G C
G C C
C G U
U C G
C G
A U A U
C G vRNA (-) strand C G vRNA (+) strand
A U A U
C G C G
A U A U
A U A U
A U A U
A U A U
A G U C U U A UC U
U U U U
C U A C
C U A U
U C A U
A U A U
A U
AA U C A
U A A U CU
A Volchkov et al., 2000 J.Gen Virol.
A U A
 Proposed structure of the EBOV replication promoter

PE1 PE2
1 55 81 128 469
TSS
3’ 3-8x(UN5) Dispensable NP 5’

Spacer
Modified Rule of six

UN5 – hexamers, N is any nucleotide

Weik et al., 2005 J.Virol.

 Replication / Transcriptional signals
Genomic RNA: A
U
Start signals: CUCCUUCUAAUU
Consensus
UAAUU

3’ NP 35 40 GP 30 24 L 5’

Leader Trailer

Stop signals: UAAUUCUUUUU

Virus specific mRNAs: UAAUAUUUUUU L gene
NP
35
40
GP
30
24
L
 Stop NP IR Start VP35

NP/VP35: UAAUUCUUUUUU GAUUA CUACUUCUAAUU

Start VP40 Stop VP35

VP35/VP40: CUACUUC UAAUU CUUUUU

Stop VP40 IR Start GP
VP40/GP: UAAUUCUUUUUU AGCCG CUACUUCUAAUU
Start VP30 Stop GP

GP/VP30: CUACUUC UAAUU CUUUUU

Stop VP30 IR Start VP24
VP30/VP24: UAAUUCUUUUU 144 CUACUUCUAAUU

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

VP24/L: UAAUUCUUUUUUCGGA CUACUUC UAAUU CUUUUU

NP CUCCUUCUAAUU
VP35 CUACUUCUAAUU
VP40 CUACUUCUAAUU
GP CUACUUCUAAUU
VP30 CUUCUUCUAAUU
VP24 CUACUUCUAAUU
L CUCCUUCUAAUU

Transcriptional start signal

GP VP30 VP40 VP35 VP24 L NP

Anti-genomic RNA

(+) 5’ NP 35 40 GP 30 24 L 3’

Replication
Genomic RNA

(-) 3’ NP 35 40 GP 30 24 L 5’

Transcription
Virus specific mRNAs (viral NP,VP35,VP30 and L)
(+)
NP
35
40
GP
30
24
L
 Replication/Transcription
of Mononegavirales (Example VSV)

Transcription N

Translation

Replication

N
Replication
 Replication cycle of
Marburg virus

Nucleus

Endosome
 Replication cycle of
Marburg virus
GP

AAAAAn AA
AA
AAAAAn A n

Nucleus AAAAAn
AAAAAn Translation
NP, VP35, L
AAAAAn
Transcription AAAAAn
AAAAAn
Transcription/Translation
AAAAAn

NP VP24
L
VP30
Nucleocapsid VP35 VP40
Mühlberger et al., 1998
 Replication and assembly of
nucleocapsids

Replication/Assembly
NP 30

NP
Nucleus L
35

Nucleocapsid

Mühlberger et al., 1998

 Formation of inclusions

NP 30
NP L
NP 35

MBGV

NP

Kolesnikova et al., 2000

 Filoviral artificial replication and
transcription system

Filoviral genome and minigenome

leader trailer
NP VP35 VP40 GP VP30 VP24 L

481 nts 730 nts

CAT minigenome
 Filoviral artificial replication and
transcription system
NP MG minigenome

35 Tr
a ns
L fec
tio
n
30
MARV
NC genes 30
35 MG
NP
L

T7 transcription CAT

mRNA minigenome
translation
transcription
30
NP
replication
L
35
Protein requirements for transcription
and replication

Marburg virus:
replication + NP 35 L
transcription

Ebola virus:
replication NP 35 L

transcription NP 35 L + 30
 Rescue of infectious filoviruses from
cDNA
NP
Plasmid encoding the
+ FL
35 Tr
a
entire genome of filoviruses
ns
L fec
tio
n
30
MARV
NC genes 30
35 FL
NP
L

T7 transcription
mRNA genome Release of
translation infectious viruses
encapsidation
30
NP
transcription
L replication
35
Protein requirements for transcription
and replication

Marburg virus:
replication + NP 35 L
transcription

Ebola virus:
replication NP 35 L

transcription NP 35 L + 30
 Functions of viral proteins
3’ NP 35 40 GP 30 24 L 5’

NP
Major nucleocapsid protein, RNA protection, part of polymerase complex
35
Second nucleocapsid protein, part of polymerase complex, INF-antagonist

40
Major matrix protein,trigger and facilitate budding, RNP traffic ?

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

sGP
Non structural, secreted, ………?
30
Nucleocapsid protein, transcriptional factor

24
Membrane associated, RNP assembly,
host adaptation factor ..
L
Viral polymerase
 Filovirus replication cycle

sm

N VP3
l a

P, 0
Protein y top

V ,L
to c Endosomes/

P3
synthesis se i n

5,
elea membrane fusion
R
acid pH
Replication
Transcription
Budding
ER VP4
0, V

Inclusion bodies
Golgi P24

. RNP GP
GP

Virions
GP
Virus targets: Cells of mononuclear phagocytic system :
Monocytes, Macrophages, and Dendritic cells;
Hepatocytes;
Fibroblasts;
Endothelial cells.
Comparison of disease pathogenesis of Ebola virus infection in animal models and humans

Element / Feature Guinea pig Macaque Human

Time to death 8–12 days 5–10 days Up to 30 days
Peak viraemia 5.2 log10 pfu/ml 6.9 log10 pfu/ml 6.5 log10 pfu/ml
DIC (fibrin deposits) Few Abundant NE
Lymphopaenia Yes Yes Yes
Lymphocyte apoptosis NE Yes Yes
Permissive host cells Monocytes, Monocytes, Monocytes,
macrophages, dendritic macrophages, macrophages,
cells, fibroblasts, dendritic cells, dendritic cells,
hepatocytes, adrenal fibroblasts, fibroblasts,
cortical cells, endothelial hepatocytes, adrenal hepatocytes,
cells, epithelial cortical cells, endothelial cells,
endothelial cells, epithelial cells
epithelial
Cytokines/chemokines NE IFN-a, IL-6, IL-18, IFN-a, IL-2, IL-6,
(increased circulating MIP-1a, MIP-1b, IL-10, TNF-a
levels) MCP-1, TNF-a

Geisbert and Hensley 2004

Fields Virology
Genome Organization /Genes Expression
 ER

Transcription and Translation GP

AAAAAn AA
AA
AAAAAn An
AAAAAn
AAAAAn Translation
NP,VP35, L
AAAAAn
VP30
Transcription AAAAAn
AAAAAn
AAAAAn

NP VP24
L
VP30
VP40
VP35
 Replication

NP,VP35, L NP,VP35, L
VP30 VP30

(-) strand (+) strand

(-) strand
Interaction of Nucleocapsid proteins
NP L
VP30
VP35

Interaction

Transcription NP / NP

Replication
NP / VP35

NP / VP30

VP35 / L

NP / VP35 / L
Genome (-) 3’ NP 35 40 GP 30 24 L 5’

Gene
3’ 5’
ORF (complementary sense)

Transcription
TStartS TStopS

5’ 3’
Cap ORF polyA
mRNA
Ebola VP30 VP24
L
VP40 GP
NP VP35 5’
3’
Overlap IR 143Overlap
Trailer
IR 5
Leader IR 5 Overlap Stop

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

Marburg
VP24 L
NP VP35 VP40 GP VP30
5’
3’
IR 5
Leader IR 9 IR 4 IR 5 IR 107 Overlap Trailer
Anti-genomic RNA

(+) 5’ NP 35 40 GP 30 24 L 3’

Replication
Genomic RNA

(-) 3’ NP 35 40 GP 30 24 L 5’

Transcription
Virus specific mRNAs (viral NP,VP35,VP30 and L)
(+)
NP
35
40
GP
30
24
L
 Replication / Transcriptional signals
Genomic RNA: A
U
Start signals: CUCCUUCUAAUU
Consensus
UAAUU

3’ NP 35 40 GP 30 24 L 5’

Leader Trailer

Stop signals: UAAUUCUUUUU

Virus specific mRNAs: UAAUAUUUUUU L gene
NP
35
40
GP
30
24
L
 Stop NP IR Start VP35

NP/VP35: UAAUUCUUUUUU GAUUA CUACUUCUAAUU

Start VP40 Stop VP35

VP35/VP40: CUACUUC UAAUU CUUUUU

Stop VP40 IR Start GP
VP40/GP: UAAUUCUUUUUU AGCCG CUACUUCUAAUU
Start VP30 Stop GP

GP/VP30: CUUCUUC UAAUU CUUUUU

Stop VP30 IR Start VP24
VP30/VP24: UAAUUCUUUUU 144 CUACUUCUAAUU

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

VP24/L: UAAUUCUUUUUUCGGA CUCCUUC UAAUU CUUUUU

NP CUCCUUCUAAUU
VP35 CUACUUCUAAUU
VP40 CUACUUCUAAUU
GP CUACUUCUAAUU
VP30 CUUCUUCUAAUU
VP24 CUACUUCUAAUU
L CUCCUUCUAAUU

Transcriptional start signal

GP VP30 VP40 VP35 VP24 L NP

 Replication Signals….?
NP gene L gene
Genome
(-) 3’ 5’
Leader

Trailer
(+) 5’ 3’
Anti-genome

vRNA (-) strand

Leader
C UA NP VP35 VP
3’
G CC UG UGUGU UUUUC UUUCUU C U
UA A AA AU
C G
A A A AC AC ACGCU ...... 40
GP
5’
UGG AC ACACA AAAAA GAAGA A AU U U U UA U U U UG UG UGCG A...... 3 0
A
UA UU
A A A U L VP24 VP
Trailer U
5’. 5’
. .
U .
A U
U U A 3’
U G C
C G 3’ U
G C
G C C
C G U
U C G
C G
A U A U
C G vRNA (-) strand C G vRNA (+) strand
A U A U
C G C G
A U A U
A U A U
A U A U
A U A U
A G U C U U A UC U
U U U U
C U A C
C U A U
U C A U
A U A U
A U
AA U C A
U A A U CU
A Volchkov et al., 2000 J.Gen Virol.
A U A
 Proposed structure of the EBOV replication promoter

PE1 PE2
1 55 81 128 469
TSS
3’ 3-8x(UN5) Dispensable NP 5’

Spacer
Modified Rule of six

UN5 – hexamers, N is any nucleotide

Weik et al., 2005 J.Virol.

Polymorphism of the GP Gene
 Expression strategy of the EBOV glycoproteins
GP gene:
ORF I
ORF II

Transcription + RNA editing

GP mRNA’s:

sGP ORF I ~80%

GP ORF I-II ~20%
ssGP ORF I*
GAT C GA T C
Editing site +A
TGGGAAACTAAAAAAA-CCTCACTAGAAAAATTC... (7A, sGP)
ORF I W E T K K T S L E K F . . 7A 8A
TGGGAAACTAAAAAAAACCTCACTAGAAAAATTC... (8A, GP)
ORF I-II W E T K K N L T R K I . .
TGGGAAACTAAAAAAAAACCTCACTAGAAAAATTC... (6A or 9A, ssGP)
ORF I* W E T K K K P H Stop

ssGP
sGP Cytoplasmic
tail
GP
Signal Transmembrane
peptide region
Transcription of Ebola virus GP gene
 Glycoproteins of Ebola virus

Cleavage site RTRR501

PM

GP1 GP2
S-S
GP1,2 trimers
form spikes

sGP Δ
53 306 Δ
N S S
C
Cleavage site Δ S S
C N
306 53
RVRR324

- cysteine residue
- potential N-linked glycans
S-S - disulfide linkage
Δ-peptide, highly O-glycosylated sGP dimer,
PM
 EBOV glycoprotein GP is proteolytically processed in two subunits

Mock
0 0 20 40 60 90 2h 4h 4h
4h 4h 4h 60 40 0 Chase (min/h)
220
preGP 97,4
GP1 66

preGPer 46

30
GP2
80 25 0 0 0 0 Cells Medium
Inhibitor (µM) 15% gel
8% gel

Cleavage of EBOV GP is inhibited by RVKR-cmk

 RTRR501

C CCC C C C C
CC CC

CT
SP FD TD

0 100 200 300 400 500 600 700a.a

GP1 GP2
GP1,2
S-S

GP1 Pathogenicity
Virus species Human Monkey
-4 -3 -2 -1
EBOV-Zaire -R-T-R-R- +++ +++
EBOV-Sudan -R-S-R-R- +++ ++
SS EBOV-Ivory Coast-R-K-R-R- + (++) + (++)
NH2 GP2 * EBOV-Reston -K-Q-K-R- - + / ++
MBGV -R-R-K-R- +++ +++

COOH

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 proteins
viral glycoproteins
bacterial toxins
at the C-terminus of
-4 -3 -2 -1
-R-X-K/R-R-
 Cleavage by Furin

Family Virus Glycoprotein Cleavage site

-4 -3 -2 -1
(R x K/R R )
Orthomyxoviridae A/FPV/Rostock/34 (H7) HA KKRKKR GL
Paramyxoviridae Measles virus F SRRHKR FA
Filoviridae Ebola virus GP GRRTRR EA
Marburg virus GP YERRKR SI
Ebola (Reston) GP TRKQKR FA
Bornaviridae BDV GP84 LKRRRR DT
Togaviridae Sindbis virus p62 SGRSKR SV
Flaviviridae Denge 2 virus prM HRREKR SV
Retrovirus HIV-I env(gp160) VQREKR AV
 Viral fusion proteins: structural similarity
EBOV Cleavage

GP1 RTRR Fusion

501
Domain (FD)
NH2 COOH
GP2
Amphipathic
helices Transmembrane
Cleavage Domain (TD)

EBOV GP2 SV5 F1 MoMuLv TM Influenza HA2 HTLV1 GP21 HIV GP41
 Transmembrane subunit GP2 of Ebola virus

GP1 EBOV GP2 Rous Sarcoma TM

Disulfide Loop

Glycosyl

Amphipathic
Helix

Charged Furin
Helix cleavage site

Glycosyl
Fusion
Peptide
 “Immunosuppressive-like motif “

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
MBGV I . . H . . . . . . T . . . . . . K V . . . . . . .
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
Mechanism of membrane fusion
 Shedding of Soluble Glycoprotein GP of Ebola Virus
supernatant
cells
GP1
GP1 GP1,2Δ trimer, recognized by
monoclonal virus neutralizing
soluble GP1,2Δ- antibodies
complexes C M P S
GP2Δ
GP2 GP2
TACE
ΔTmGP2
TM TM
TACE C - cells
M - medium
P – pellet
KTLPD QGD WT
TACE: TNF-α converting enzyme KTLPD VGD
S - supernatant

KTLPE QGD
KTLPL QGD
KTLPV QGD
KTLAD QGD
KTVPD QGD
KILPD QGD
VTLPD QGD
0 100 200 300 400 500
% GP release
Dolnik et al EMBO J 2004
 Surface GP of EBOV shed from virus-infected cells following cleavage
by the cellular metalloprotease TACE.
Vero
GP2

293T
RTRR Fusion
9d 9d 5d Tm. region
peptide Asn
563 Asn618
GP2 Cyt. tail
GP2∆

NITDKIDQIIHDFVDKTLPD QGDNDNWWTGWRQ
Serum
Cleavage site
GP1

Ectodomain shedding is a mechanism to escape

NH2 SS
GP
2 TACE control by hosts immune system.
Shed GP present in the blood of infected animals
COOH
and blocks virus neutralizing activity of
antibodies / Decoy function
TNF-α-CONVERTING ENZYME TACE (ADAM-17)

Pro DisInt EGF Cytosolic

Metalloproteinase Cys-rich TM

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
Reverse Genetics of Filoviruses
 Reverse Genetics
Traditional Genetics:

Virus WT
Sequencing
Virus Mutant
Search for mutated genes

Reverse genetics:

Virus WT
Virus Rescue
(recombinant virus)
Virus mutants

Mutagenesis Virus replication,

pathogenicity …
 Reverse Genetics
Ebola virus: 18,959 n
3’ NP 35 40 GP 30 24 L 5’

T7 promoter Ribozyme

CAT
NP 35 30 L

EBOV mini genome J Virol 73, 1999

BSR T7/5

T7 RNA pol.
J Virol 73, 1999
 Construction of the EBOV full-length antigenome

Sal I (GTCGAC)
Sma I Cla I Cla I Sac II Sac II Sac I

NP 35 40 GP GP 30 24 L L
+ +

C C
Sal I (GTAGAT)
T7 promoter Ribozyme

NP 35 40 GP 30 24 L

Vector 2,0
EBOV full-length antigenome (pFL-EBOV)
Reverse Genetics System for Ebola virus allows genetic manipulation of
infectious virus by introducing single or multiple mutations in particular
regions of viral genome.
T7 promoter Ribozyme
NP 35 40 GP 30 24 L

n pFL-EBOV
it o
c
sfe
NP a n pFL-EBOV
Tr
T7 1
35 NP BSR T7/5 T7
35
cells A
30 30 N
T7 A ) vR
L 1 T7 N (+
L
) vR 5
NC genes mRNA`s (+ 6
(T7 promoter) 2 L
30 4 A
35 3 N Recombinant
NP vR
(-) Ebola virus
(cross section)
Volchkov et al Science 2001
 Multiple-step replication cycles in Vero cells
RRTRR RRTLR RRTGG RKTRR
7 7 7
Virus titers log10 (pfu/ml)

6 6 6

5 5 5

4 4 4

3 3 3

2 2 2

1 1 1

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
Days Days Days

-4 -3 -2 -1 RS WT LR RK GG WT NM
GG R T G G preGP

NM R T N M Anti-GP2
100% 100%
LR R T L R 15% 20% 7% 9%
12%
RS R T R S
GP 2
RK K T R R
VP40

Anti-VP40
 Recombinant Ebola virus variant (No editing)
recEBOVe+
5’ 3’
NP 35 40 GP 30 24 L

recEBOVe+
recEBOVe-
Mock
sGP GP
(Transcriptional RNA editing) +A
1 2 3
recEBOVe+ - . . . TGGGAAACTAAAAAAA - CCTCACT . . . GP1

GP ORF - . . . W E T K K N L T ...
recEBOVe- - . . . TGGGAAACTAAGAAGAACCTCACT . . . sGP

recEBOVe- «No editing » – « No sGP » mutant

5’ 3’
NP 35 40 GP 30 24 L

GP sGP
 Increase in cell rounding, detachment and death

3days 4days 6days 7days 8days

Mock

recEBOVe+

recEBOVe-
 Cytotoxicity of recEBOVs:
Individual plaques

recEBOVe+

recEBOVe-

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