1

EBOLA AND MARBURG VIRUSES

HUMAN ANIMALHUMAN-ANIMAL INTERFACES

Douanier Rousseau

C.Goldsmith/S.Zaki

Second FAO-OIE-WHO consultation: Influenza

and other Emerging Zoonotic Diseases

Verona April 2010

Mononegavirales, Filoviridae, Filoviruses : 5 distinct Ebola strains : Sudan, Zaire, Reston, Côte

Ebola and Marburg Haemorrhagic Fevers

d'Ivoire, Bundibugyo2 lineages for Marburg : POP, RAVN

Reservoir: bats are strongly suspectedBSL 4 Incubation 2-21 daysCase Fatality Ratio 24-89%Case Fatality Ratio 24 89%Treatment : no specific treatment available,

careful rehydration, supportive, intensive care,

Vaccines in development

2

09 DRC 1999

07 DRC 1999

05 DRC 1999

Ravn Kenya 1987

1 1

1

1

Complete genome analysis:Filoviruses

Marburg 21% nt

Popp Uganda 1967

Zaire 1976

Ozolins Zimbabwe 1975

Musoka Kenya 1980

Zaire 1995

1379c Angola 2005

1

1

1

1

800 yrs

Reston 1989

Cote d’Ivoire 1994

Bundibugyo 2007

Zaire 1976

Sudan 2000

1

1

1

1

Ebola

32% nt

39% nt

Towner et al., PLoS Pathogens, 21 Nov 2008

3

Yambuku‘76

Nzara‘76

Zaire’79 80

Zaire‘95

I. Coast’96 97

CentralAfrican Republic

‘99 T t l

Summary of tropical African animals tested for evidence of Ebola Zaire, 1976-1999

Mammals Chiroptera 7 652Mega 4 41 125 19 189Micro 174 422 414 4 1014

Rodents 131 309 661 1759 283 163 3306Insectivores 7 53 114 398 56 628Carnivores 27 28 55Primates 267 12 27 312

Birds 67 184 533Reptiles 5 33 127 282 165

‘76 ‘76 ’79-80 ‘95 ’96-97 ‘99 Total

Pourrut et al., 2005 Microbes and Infection 7:1005-14

Reptiles 5 33 127 282 165Total vertebrates 157 499 1664 2814 1642 242 7018Arthropods 2318 27,843 30,161Test:

Virus isolation 0/2475 0/499 0/1664 0/30,657 0/1642 0/242 0/37,179

*Colobus monkey

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Bats Bats –– the likely reservoir for the the likely reservoir for the Marburg and Ebola virusesMarburg and Ebola viruses

Epidemiologic dataEpidemiologic dataSudan ebolavirus

1976 cases; 1st 6 cases were cotton factory employees who worked in a room where bats roosted

Cote d’Ivoire ebolavirus1994; chimpanzees which developed EHF had been feeding in a fig tree together with fruit bats for two weeks before developing the disease

Reston ebolavirus1989-96; all outbreaks in primate facilities link back to a single primate export facility in the Philippines which was a former fruit orchard and animals potentially exposed to fruit bats

Marburg virus1967 imported monkeys from Ugandan locations where fruit bats prevalent1975 tourists slept in rooms where insectivorous bats were present Zimbabwe1980 and 1987; two patients in Kenya both visited a cave inhabited by batsshortly before becoming ill1998-2000; Durba DRC outbreak linked to gold mine with infected bats2007; Kitaka Uganda, two cases link to mine with infected fruit bats2008; Python cave Uganda, two cases linked to cave with infected fruit bats

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Bats Bats –– the likely reservoir for the the likely reservoir for the Marburg and Ebola virusesMarburg and Ebola viruses

Direct lab dataDirect lab dataZaire ebolavirus

Detection of virus-specific antibody and genome RNA in 3 speciesDetection of virus specific antibody and genome RNA in 3 species of bats in Gabon

1996; Experimentally infected fruit bats shown to replicate ebolavirus without developing overt disease

Marburg virusDetection of virus-specific antibody, and genome RNA predominantly in Egyptian fruit bats (Rousettus aegyptiacus) inpredominantly in Egyptian fruit bats (Rousettus aegyptiacus) in Gabon, Durba DRC, Kitaka Uganda and Python Cave, Uganda

Multiple virus isolates from Kitaka and Python caves

Multiple diverse virus genetic lineages co-existing in R. aegyptiacus colonies; no correlation between genetic and geographic distances – likely reflecting mobility and large meta-population of host

Filovirus infection of batsFilovirus infection of bats

Marburg virus in R. aegyptiacus• virus infection (PCR) 1.6 – 5.1 %• past infection (IgG) 2.3 – 20.5 %

Zaire ebolavirus in 3 species of bats• virus infection (PCR) 2.8 – 19%• past infection (IgG) 6.8 – 23%

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Proposed mechanisms of Ebola virustransmission to wildlife, domestic animals

(or humans) from bats

•Competition for fruit between chiropterans and non-human primates leads to spatiotemporal clustering of frugivorousprimates leads to spatiotemporal clustering of frugivorous animals leading to an increased likelihood of spillover

•Infectious virus in:• saliva

-Nipah virus (Chua et al., 2002; Reynes et al., 2005)-Hendra virus (in horse saliva-Williamson et al., 1998)

• feces (guano) or urine(g )-Nipah virus (Chua et al., 2002)-Hendra virus (Williamson et al., 1998)-Ebola virus (experimentally infected bats Swanepoel et al., 1996)-Menangle virus (Hooper et al., 2000)

• birthing fluids (blood, placental tissues etc)-Hendra virus (Young et al., 1997; Halpin et al., 2000)

Suspected modes of transmission to humans

• Contact with reservoir (bats): hunters, miners, ecologists, tourists

• Contact with secondary hosts (pigs, primates): hunters, farmers slaughter house workersfarmers, slaughter house workers

• Contact with human (patients): family, nosocomial infections, burial practices

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02Uganda 2007sep09DRC 1999may

Rav Kenya1987aug04DRC 1999may03DRC 1999apr32DRC 2000aug19DRC 2000feb34DRC 2000aug07DRC 1999may06DRC 1999may14DRC 2000jan30DRC 2000aug01DRC 1999apr24DRC 2000apr23DRC 2000mar29DRC 2000jul27DRC 2000jul

Diversity Marburg complete genome

Uganda ‘07 humanSequences

(21% diverse)

Ravn lineage

27DRC 2000jul26DRC 2000may17DRC 2000feb16DRC 2000feb12DRC 2000jan20DRC 2000feb21DRC 2000feb22DRC 2000may18DRC 2000feb13DRC 2000jan28DRC 2000jul05DRC 1999may15DRC 2000feb

33DRC 2000aug25DRC 2000apr01Uganda 2007jul

Ozo Zimbabwe 1975feb

Durba DRC1998-2000

21.5%

09Angola 2005apr08Angola 2005apr04Angola 2005mar07Angola 2005apr05Angola 2005mar02Angola 2005mar01Angola 2005feb06Angola 2005apr03Angola 2005mar11Angola 2005may10Angola 2005apr

Pop Germany1967aug02DRC 1999apr

Mus Kenya 1980jan

50 changes

Main lineage

Angola 20050.07%

Bats- Aug ‘07

02U 2007.7

09d_1999.401k_1987.6Bat 44*

Bat 188*Bat 276Bat 288Bat 328

Bat 1013Bat 782Bat 982*

01U 2007.6Bat 772

Bat 29107d_1999.406d_1999.3

14d_2000.130d_2000.601d_1999.3

Bat 331*

02U 2007.7

09d_1999.401k_1987.6Bat 44*

Bat 188*Bat 276Bat 288Bat 328

Bat 1013Bat 782Bat 982*

01U 2007.6Bat 772

Bat 29107d_1999.406d_1999.3

14d_2000.130d_2000.601d_1999.3

Bat 331*

Uganda Bat Sequencesconcatenated

NP and VP35 frags (~700bp) Bats- May ‘08

Miners- Aug ’07Sept ‘07

Ravn lineage

Bat 331*Bat 371*Bat 427Bat 88304d_1999.303d_1999.332d_2000.619d_2000.134d_2000.6

24d_2000.323d_2000.2

29d_2000.527d_2000.5

17d_2000.116d_2000.112d_2000.020d_2000.118d_2000.121d_2000.226d_2000.405d_1999.413d_2000.028d 2000.5

Bat 331*Bat 371*Bat 427Bat 88304d_1999.303d_1999.332d_2000.619d_2000.134d_2000.6

24d_2000.323d_2000.2

29d_2000.527d_2000.5

17d_2000.116d_2000.112d_2000.020d_2000.118d_2000.121d_2000.226d_2000.405d_1999.413d_2000.028d 2000.5

•Bats sequencesidentical or near identicalto those foundin infectedminers

_22d_2000.2

15d_2000.125d_2000.3

33d_2000.702z_1975.1

96g2006.231g2005.1

48g2005.301k_1980.1

01g_1967.602d_1999.3

09a_2005.304a_2005.206a_2005.303a_2005.207a_2005.305a_2005.202a_2005.201a_2005.210a_2005.308a_2005.311a_2005.4

1 change

Maximum Parsimony

_22d_2000.2

15d_2000.125d_2000.3

33d_2000.702z_1975.1

96g2006.231g2005.1

48g2005.301k_1980.1

01g_1967.602d_1999.3

09a_2005.304a_2005.206a_2005.303a_2005.207a_2005.305a_2005.202a_2005.201a_2005.210a_2005.308a_2005.311a_2005.4

1 change1 change

Maximum Parsimony

Main lineage

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Kitaka mine--population 112,000

5000 actively infectedbats at any one time

(~5% actively infected)

Geographic distribution of Rousettus aegyptiacus in Africa

•Geographic distribution encompassesthe location of all known Marburgthe location of all known Marburgoutbreaks

•Reproduces twice a year-(~75-80% of adult females pregnant)

•Reproductive capacity combined withthe large colony sizes (>100,000) predicts large meta-populations

•Can have long life span up toCan have long life span, up to25 years in captivity

•Up to nine subspecies withinAfrica and Asia

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Pathogenesis in different species and ability to transmit diseases

Bats: unknownNon human primates:

laboratory: death in 5 to 7 days, very few survivorswild: death, some survivors (Gabon 2004 13% chimps IgG)

Excretion…

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Animal model of disease and vaccine model prediction

Rapid systemic spread with high viremiaInfection and necrosis of macrophages and dendritic

cellsRelease of proinflammatory mediators, leading to

increased vascular permeability and shockMassive lymphocyte apoptosis.

These changes are seen in non-human primates, guinea pigs and mice… BUT

12 No. of deathsOther source

Suspected Ebola hemorrhagic fever by source of infection,

Zaire, 1995

0

2

4

6

8

10Ot e sou ceMW 29yo MNA 45yo F

0

9-Apr

13-Apr

17-Apr

21-Apr

25-Apr

29-Apr

3-May7-M

ay

11-M

ay

15-M

ay

19-M

ay

23-M

ay

27-M

ay

31-M

ay4-J

un8-J

un12

-Jun

16-Ju

n

20-Ju

n

24-Ju

n

Date of death (1995)

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2008 Disease activity in pigs in 2008 Disease activity in pigs in PhilippinesPhilippines

• Porcine Respiratory and Reproductive Syndrome (PRRS) virus has caused respiratory failure in neonates or abortions in sows during sporadic PRRS outbreaks worldwide (also known as Blue Ear pig disease)

•• PRRSV is RNA virus, contains a ss positive-sense RNA genome (15kb); genus

arterivirus (same genus as SHFV !)

• Since 2006 large outbreaks of more virulent atypical PRRS swept through large areas of China and Vietnam – major economic impact

• Disease outbreaks in pigs in Bulacan, Philippines May 2007 through mid-2008. Increase in morbidity/mortality consistent with atypical PRRS, similar to Vietnam and China

• PRRS or swine influenza suspected

• Samples from 4 farms sent to USDA Plum Island facility Oct 2008 for testing• PRRS identified, but also Circo 2 virus. PRRSV does not grow on Vero cells

• One pig tissue yields a virus isolate growing on Vero cells.

• Panviral microarray developed in-house at Plum Island provides evidence of Reston ebolavirus

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Pig disease symptomsPig disease symptoms• High fever 41C• Labored breathing• Labored breathing• thumping• Coughing, nasal discharge• Loss of appetite, Diarrhea• Skin hemorrhage/reddish discoloration

S f d i b t iti• Some found in recumbent position

• High nursery house and growing house mortalities• Sows previously affected by high fever and

abortions

Ebola Reston virus: Hypothesis of transmission

Ebola Reston virus: Hypothesis of transmission

Fruit bats Secondary

Transmission

Human

Food Chain

Pigs: Amplification

Healthcare workers

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QuestionsQuestionsSource of virus infecting the pigs ?How common and widespread is the problem ?

Does Reston ebolavirus cause disease in pigs(alone or in combination with PRRSV) ?

Is there pig to pig transmission ? – Geelong

To what extent are humans getting infected down the Farm to Table chain ?to Table chain ?

Any evidence of human disease associated with Reston ebolavirus infection?

One World, One Health 

•CDC – SPB, IDPB

•Uganda Virus Research Institute (UVRI)

Serena Reeder

Brian Amman

Jon Towner

•NICD – South Africa

•WHOAlan Kemp

Stuart Nichol

Tom KsiazekPierre Rollin

Bob Swanepoel

Eileen Farnon Andy Comer Pierre Formenty Tara Sealy

Cesar Albarino

John KayiwaStephen Balinandi

Chris Paddock