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Family Filoviridae
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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
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Vervet or Green Monkeys( Cercopithecus aethiops )
polio vaccineproduction
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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.
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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
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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)
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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.
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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
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Filoviridae
Marburg and Ebola virus
African origin
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Filoviridae
Marburg and Ebola virus
filovirus belt
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Angola
Sudan
KeniaGabon DR.Kongo
(Zaire)l.Victoria
Zimbabwe
UgandaIvoryCoast
Filovirus EpisodesFilovirus Episodes ((AfricaAfrica))
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Filovirus EpisodesFilovirus Episodes Rainforests of the worldRainforests of the world
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Filoviridae
Marburg and Ebola virus
African origin
Hemorrhagic Fever, Lethality up to 90%
Therapy: No
Vaccine: No
Human and nonhuman victimsNatural reservoir unknown
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Democratic Republic of Congo
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Durba, DRC
by Pierre Rollin, CDC Atlanta
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Goroumbwa gold mine near Durba
by Pierre Rollin, CDC Atlanta
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Official entrance of the mine
by Pierre Rollin, CDC Atlanta
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Inofficial entrances
by Pierre Rollin, CDC Atlanta
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Isolation ward, Durba 1999
Colebunders et al., 2004
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Isolation ward, Durba 1999
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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
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- 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
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Epidemiological studies: infection is not transmitted by the aerosol route
Experimental infection of non-human primates: aerosol transmission can cause infection
Transmission
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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
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• Bats?
Natural hosts of filoviruses
by Pierre Rollin, CDC Atlanta
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Marburg virus in species collected in Durba
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Phylogenetic Analysis of MARV basedon nested PCR results from bat samples
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Natural Reservoir
No virus isolatedSome are IgM positiveSome others are PCR positive.
Fruit bats.Leroy et al., Science 2006
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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
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“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.
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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
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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
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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
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Health education
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Pathogenesis of filovirus infections
day 1infection
day 3-5liver, spleen
day 5-7pantropicinfection
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Marburg virus hemorrhagic fever
ConjunctivitisEnanthema
ThrombocytopeniaDiarrhea
ExanthemaHepatitis
Hemorrhages
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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
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Fields Virology
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Hemorrhagic Signs
- puncture site bleedings- bloody stool - hematuria- gum bleedings- hemoptysis- hematemesis- petechia- epistaxis- hematoma
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Geisbert et al., 2004
Hemorrhagic Signs
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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.
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Animal models for filovirus disease
• Nonhuman primates (cynomolgous/ maccaques)
• Guinea pigs• Mice
only after adaptation of virus
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• 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?
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• 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?
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• 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
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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
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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
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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
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Warfield, et al. (2003) Proc. Natl. Acad. Sci. USA 100, 15889-15894
Ebola virus-like particles as vaccinecandidates
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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
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Recombinant VSV expressing filoviralsurface proteins as vaccine candidates
ZEBOV
SEBOV
MARV
MARV Popp
MARV ZEBOV
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Treatment of Marburg virushaemorrhagic fever using recombinant
VSV
Daddario et al., Lancet, 2006
Immunize monkeys 20 min after infection withMarburg virus
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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
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“The authorities of the Congo had confirmed that gorillas were infected with Ebola virus. The illness already killed 80% of gorillas of the region.”
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BSL P4 Laboratory in Lyon
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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
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Virion proteins
VP40VP35VP30
NP
VP24GP2
GP1
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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
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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
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Fields Virology
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Taxonomy of Filoviruses
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Genome organization of filoviruses
~19 kbnegative orientation7 genesgene overlapscis-active elements in leader and trailer
GP
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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
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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
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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
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, 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
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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
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Viral Structure
From latin filum
Pleomorfic
800-1080nm X 80nm
Non-segmented
(–) strand RNA
19 Kb
NucleoproteinL (pol)
VP30
VP35
VP24
VP40Glycoprotein
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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
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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)
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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
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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 ..
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Genome Organization /Genes ExpressionGenome Organization /Genes Expression
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Gene
ORF (complementary sense)3’ 5’
ORF5’ 3’
polyACap
Transcription
mRNA
5’NP L2430GP40353’(-)Genome
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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
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Replication signalsReplication signals
5’NP L2430GP40353’
Genomic RNA:
Leader Trailer
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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.
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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
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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
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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
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GP VP30 VP40 VP35 VP24 L NP
NP CUCCUUCUAAUU
VP35 CUACUUCUAAUU
VP40 CUACUUCUAAUU
GP CUACUUCUAAUU
VP30 CUUCUUCUAAUU
VP24 CUACUUCUAAUU
L CUCCUUCUAAUU
Transcriptional start signal
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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)
(+)
(+)
(-)
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Transcription
Translation
Replication
ReplicationN
N
Replication/Transcriptionof Mononegavirales (Example VSV)
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Nucleus
Endosome
Replication cycle of Marburg virus
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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
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Nucleus
Nucleocapsid
30
L35
NP
NP
Replication/Assembly
Mühlberger et al., 1998
Replication and assembly of nucleocapsids
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Formation of inclusions
Kolesnikova et al., 2000
NP
MBGV
30L35
NP
NPNP
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Filoviral genome and minigenome
VP35 VP30NP Lleader trailer
CAT minigenome481 nts 730 nts
VP24VP40 GP
Filoviral artificial replication and transcription system
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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
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Protein requirements for transcription and replication
Marburg virus:
Ebola virus:replication
transcription
NP L35
30+
NP L35
NP L35
replication + transcription
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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
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Protein requirements for transcription and replication
Marburg virus:
Ebola virus:replication
transcription
NP L35
30+
NP L35
NP L35
replication + transcription
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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 ..
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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
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Cells of mononuclear phagocytic system :Monocytes, Macrophages, and Dendritic cells; Hepatocytes;Fibroblasts;Endothelial cells.
Virus targets:
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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
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Fields Virology
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Genome Organization /Genes ExpressionGenome Organization /Genes Expression
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Translation
LVP30
NP
VP35
NP,VP35, LVP30
VP24
VP40
AAAAAn
AAAAAn
AAAAAn
AAAAAn
AAAAAn
AAAAAn
AAAAAn
Transcription AAAAAn
AAAAAn
GPTranscription and TranslationER
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Replication
NP,VP35, LVP30
(-) strand (+) strand
NP,VP35, LVP30
(-) strand
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TranscriptionReplication
L
VP30NP
VP35
NP / NP
NP / VP35
NP / VP30
VP35 / L
NP / VP35 / L
Interaction
Interaction of Nucleocapsid proteins
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Gene
ORF (complementary sense)
TStartS TStopS
3’ 5’
ORF5’ 3’
polyACap
Transcription
mRNA
5’NP L2430GP40353’(-)Genome
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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
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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)
(+)
(+)
(-)
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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
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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
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GP VP30 VP40 VP35 VP24 L NP
NP CUCCUUCUAAUU
VP35 CUACUUCUAAUU
VP40 CUACUUCUAAUU
GP CUACUUCUAAUU
VP30 CUUCUUCUAAUU
VP24 CUACUUCUAAUU
L CUCCUUCUAAUU
Transcriptional start signal
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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.
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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
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Polymorphism of the GP Gene
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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
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Transcription of Ebola virus GP gene
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- 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,
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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
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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
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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
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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
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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
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Transmembrane subunit GP2 of Ebola virus
EBOV GP2 Rous Sarcoma TMGP1
Glycosyl
Glycosyl
Disulfide Loop
AmphipathicHelix
Furincleavage site
ChargedHelix
FusionPeptide
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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 “
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Mechanism of membrane fusion
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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
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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
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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
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Reverse Genetics of Filoviruses
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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
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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
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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
+ +
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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
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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
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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
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4days 7days6days 8days
Mock
recEBOVe-
recEBOVe+
3days
Increase in cell rounding, detachment and death
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recEBOVe+
recEBOVe-
Cytotoxicity of recEBOVs:Individual plaques
5 days p.i.
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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