00 grace workshop cover.ppt [kompatibilitätsmodus]

Educational WorkshopEW07: Viruses and the respiratory tract – current and future issues

arranged with the European Respiratory Society (ERS) for the EU-funded Network of Excellence Genomics to Combat Resistance against Antibiotics in Community-acquired LRTI in Europe (GRACE)

Convenors: Roger Finch (Nottingham, UK)g ( g , )Lia van der Hoek (Amsterdam, NL)

Faculty: Alberto Papi (Ferrara IT)Faculty: Alberto Papi (Ferrara, IT)Marco Contoli (Ferrara, IT)Alison Bermingham (London, UK)Lia van der Hoek (Amsterdam, NL)Albert Osterhaus (Rotterdam, NL)

Papi - Viral interactions with respiratory tract in health and disease

Viral interactions with respiratory tract in health and Viral interactions with respiratory tract in health and diseasedisease

Marco Contoli, MD, PhDMarco Contoli, MD, PhDProf. Alberto Papi, MDProf. Alberto Papi, MD

Research Centre on Asthma and COPDResearch Centre on Asthma and COPDSection of Respiratory MedicineSection of Respiratory Medicine

Department of Clinical and Experimental MedicineDepartment of Clinical and Experimental MedicineUniversity of Ferrara University of Ferrara -- ItalyItaly

Respiratory viruses with impact on the lung

•• rhinovirusesrhinoviruses•• enterovirusesenteroviruses

•• influenza viruses A & Binfluenza viruses A & B

Respiratory virusesRespiratory viruses

•• coronavirusescoronaviruses

•• parainfluenza virusesparainfluenza viruses•• respiratory syncytial virusrespiratory syncytial virus

•• adenovirusesadenoviruses

3


•• Virus culturesVirus cultures•• SerologySerology•• Antigen detection (immunofluorescence, Antigen detection (immunofluorescence,

Detection methodsDetection methods

hemagglutination…)hemagglutination…)

•• PCR and related methodologiesPCR and related methodologies

Do Do viralviral infectionsinfections in in earlyearly life life protecprotecagainstagainst asthmaasthma??againstagainst asthmaasthma??

•• ≥≥2 episodes of “common cold” 2 episodes of “common cold” beforebeforeage 1 yr decrease risk of age 1 yr decrease risk of

th b 7th b 7

Respiratory Infections in Respiratory Infections in Infancy May Protect Against Development of AsthmaInfancy May Protect Against Development of Asthma

asthma by age 7asthma by age 7by ~50%by ~50%

•• Other viral infections Other viral infections -- eg, eg, herpes, varicella, measles herpes, varicella, measles --also protectivealso protective

(Illi S et al. BMJ. 2001)(Illi S et al. BMJ. 2001)

4


Do viral infections in early life induce Do viral infections in early life induce asthma?asthma?

Children who had Children who had ≥≥2 older siblings or attended day care during first 6 2 older siblings or attended day care during first 6 mo of life had increased risk of wheeze early in life but decreased risk mo of life had increased risk of wheeze early in life but decreased risk later.later.

P=0.01P=0.012.72.7

e ris

ke

risk

zing

zing

Effect of Day Care in Infancy and Number of Older Effect of Day Care in Infancy and Number of Older Siblings on Asthma RiskSiblings on Asthma Risk

(Ball TM et al. N (Ball TM et al. N EnglEngl J Med. 2000)J Med. 2000)

P=0.03P=0.03P=0.001P=0.001 P<0.001P<0.001

P<0.001P<0.0011.01.0

0.40.4

11 22 33 44 55 66 77 88 99 1010 1111 1212 1313 1414

Adj

uste

d re

lativ

eA

djus

ted

rela

tive

of fr

eque

nt w

hee

of fr

eque

nt w

hee

Age (yr)Age (yr)

0.10.1

Resolving the paradoxResolving the paradox…..Resolving the paradox...……..Resolving the paradox...…

5


100000<

100

1000

10000

in G

ene

Cop

ies

Deficient type 1 immune response in RSV bronchiolitisDeficient type 1 immune response in RSV bronchiolitisDeficient type 1 immune response in RSV bronchiolitisDeficient type 1 immune response in RSV bronchiolitis

0.03

0.04

0.05

0.06

0.07

0.08

ILIL--4

/IFN

4/IF

N--gg

0

1

10

Day 1-2 Day 5-7

F pr

ote

Day 1-2 Day 5-7

BronchiolitisURTI

Legg et al, AJRCCM 2003Legg et al, AJRCCM 2003

0

0.01

0.02

0.03

URTI Bronchiolitis

Infections and asthma:Infections and asthma:the apparent paradoxthe apparent paradox

Th1Th1Th1Th2

CompetentCompetent immune system immune system developsdevelops appropriate appropriate responsesresponses

toto infectionsinfections in in earlyearly lifelife

CompetentCompetent immune system immune system developsdevelops appropriate appropriate responsesresponses

toto infectionsinfections in in earlyearly lifelife

RV RV BronchiolitisBronchiolitisRV RV BronchiolitisBronchiolitis

Th2Th2

ViralViral infectionsinfectionsand and

asthmaasthma exacerbationsexacerbations

6


Virus associated asthma exacerbationsVirus associated asthma exacerbations

ChildrenChildren AdultsAdults

Positive virus detectionPositive virus detection

No virus detectedNo virus detected

(Johnston et al. BMJ 1995)(Johnston et al. BMJ 1995) (Corne et al. Lancet 2002)(Corne et al. Lancet 2002)

Two thirds of viruses detected are rhinovirusesTwo thirds of viruses detected are rhinoviruses

(Reviewed in Contoli, Johnston,(Reviewed in Contoli, Johnston, PapiPapi et al et al ClinClin Exp Allergy 2005 )Exp Allergy 2005 )

MechanismsMechanisms ofof virus virus inducedinduced asthmaasthma exacerbationexacerbation: : In vivo In vivo humanhuman modelsmodels

est s

core

88

1212

1616

orni

ng P

EF

0

5

10

Tota

l che

Normals Asthmatics

--44

00

44

% c

hang

e in

mo

-20

-15

-10

-5

-4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Day post experimental RV16 infection

(Message SD, Mallia P, Contoli M, Papi A, Johnston SL et al. PNAS 2008)

20

40

60Asthmatic patientsNormal subjects

otal

che

stpt

om s

core

MechanismsMechanisms ofof virus virus inducedinduced asthmaasthma exacerbationexacerbation: : In vivo In vivo humanhuman modelsmodels

4 6 8 10

0

Nasal lavage virus load(Log10 copies/mL)

Tosy

mp

(Message SD, Mallia P, Contoli M, Papi A, Johnston SL et al. PNAS 2008)

7


Role of innate immune rersponse in virus induced asthma exacerbations

RespiratoryRespiratory virus virus inductioninduction ofof alphaalpha--, beta, beta-- and and lambdalambda--interferonsinterferonsin in bronchialbronchial epithelialepithelial cellscells

Khaitov, Laza-Stanca, Edwards, Walton, Rohde, Contoli, Papi, Stanciu, Kotenko,S. L. Johnston Allergy 2009

Role of deficient type III interferonRole of deficient type III interferon--λλ production in production in asthma exacerbationsasthma exacerbations

**2,500

2,000*

#

IFNIFN λλ1 in Bronchial Epithelial Cells1 in Bronchial Epithelial Cells

5

6 ***

xpre

ssio

n/µ

g R

NA)

RV16 infection in Bronchial RV16 infection in Bronchial Epithelial CellsEpithelial Cells

(Contoli, Message, Papi, Johnston et al. Nat Med 2006)

M RV16

Normal

1,500

1,000

500

0

(pg/

ml)

M RV16

Asthmatic

IFN

-λ

Normal Asthmatic3

4

RV1

6 vR

NA

ex(lo

g 10

copi

es/

8


10

-5

0

5

in F

EV1

se fr

om b

asel

ine

valu

e)

20

30

40

cold

sco

re

Role of deficient type III interferonRole of deficient type III interferon--λλ production in production in asthma exacerbationsasthma exacerbations

0 25 50 75 100 125 150 175 200-20

-15

-10

IFN-λ (pg/ml)

Fall

(per

cent

dec

reas v

0 50 100 150 2000

10

IFN-λ (pg/mL)

Tota

l c


Role of deficient type III interferonRole of deficient type III interferon--λλ production in production in asthma exacerbations: asthma exacerbations: ex vivo resultsex vivo results

200

300

-8 le

vel i

n B

AL

pg/m

l)

3

4

5

in s

putu

mn-

epith

elia

l cel

ls)

0 25 50 75 100 125 150 175 2000

100

IFNIFN--λλ(pg/ml)(pg/ml)

Inte

rleuk

in-

(p

0 50 100 150 2000

1

2

IFNIFN--λλ (pg/ml)(pg/ml)

Eosi

noph

ils

(per

cent

of n

o n


Antiviral Immune Response in AsthmaAntiviral Immune Response in Asthma

*

3

4

5

on fr

om b

asel

ine

RN

A ex

pres

sion

*

1.5

2.0

2.5

3.0

on o

f apo

ptos

isge

from

bas

elin

e)

((WarkWark, , JohnstonJohnston etet al. J al. J ExpExp Med 2005)Med 2005)

0

1

2

N NA A

Medium RV16

Fold

indu

ctio

in IF

N- β

mR

0

0.5

1.0

N NA A

Medium RV16

Indu

ctio

(fold

cha

ng

9


Viral Viral ReplicationReplication

ProPro--inflammatoy cytokinesinflammatoy cytokines(RANTES, IP(RANTES, IP--10, IL10, IL--6, IL6, IL--8)8)

IFNIFN--ββIFNIFN--λλ

IFNsIFNs

ISGF3ISGF3

Th1/Th2Th1/Th2

Conclusions: Conclusions: state of the artstate of the art

LPSLPS

vRNAvRNACytoplasm

Cytoplasm

NucleusNucleus

IRFIRF--3/73/7

NFNF--kBkB

ApoptosisApoptosisp53p53

PKRPKR

PKRPKRMxMx

OASOASADARADAR

Inhibition ofInhibition ofViral Viral

ReplicationReplicationEpi

thel

ial c

ells

Epi

thel

ial c

ells

Mac

roph

ages

Mac

roph

ages

SymtomsSymtoms Partecipants with Partecipants with asthma (n=76)asthma (n=76)

Healthy partecipants Healthy partecipants (n=76)(n=76)

URTURTSeverity (score)Severity (score)Duration (days)Duration (days)

2 (02 (0--12)12)3 (03 (0--11)11)

2 (02 (0--7)7)3 (03 (0--17)17)

LRTLRT

Frequency, severity and duration of rhinovirus infections in asthmatic Frequency, severity and duration of rhinovirus infections in asthmatic and nonand non--asthmatic individuals: a longitudinal cohort studyasthmatic individuals: a longitudinal cohort study

•• Respiratory infection in asthamatic patients are more severe. Respiratory infection in asthamatic patients are more severe. •• They are more prone to clinically severe infections They are more prone to clinically severe infections

Severity (score)Severity (score)Duration (days)Duration (days)

1 (01 (0--17)17)2.5 (02.5 (0--35)35)

0 (00 (0--7)*7)*0 (00 (0--22)†22)†

Data are median (range). *p=0.001; †p=0.005(Corne et al. Lancet 2002)(Corne et al. Lancet 2002)

Epidemiological relationship between the common cold and Epidemiological relationship between the common cold and exacerbation frequency in COPDexacerbation frequency in COPD

1

2

d fre

quen

cy/y

rd

frequ

ency

/yr

**

20304050607080

ciat

ed w

ith e

xac/

ciat

ed w

ith e

xac/

olds

ratio

%ol

ds ra

tio %

0

Tota

l col

Tota

l col

InfreqInfreq FreqFreq

010

Col

ds a

ssoc

Col

ds a

ssoc

Tota

l co

Tota

l co

InfreqInfreq FreqFreq

Exacerbation frequency in chronic obstructive pulmonary disease is associated Exacerbation frequency in chronic obstructive pulmonary disease is associated with an increased frequency of acquiring the common cold, with an increased frequency of acquiring the common cold, rather than an increased propensity to exacerbation once a cold has been acquired.rather than an increased propensity to exacerbation once a cold has been acquired.

(adapted from Hurst et al. ERJ 2005)(adapted from Hurst et al. ERJ 2005)

10


Detection Detection ofof rhinovirusrhinovirus in in inducedinduced sputumsputum at at exacerbationexacerbation ofof COPDCOPD

10

15

20

25

%

3

4

5

6

ptom

sco

re

((SeemungalSeemungal etet al. ERJ 2000)al. ERJ 2000)

0

5

10

1

2

3Sy

mp

RespiratoryRespiratory virusesviruses, , symptomsymptom and and inflammatoryinflammatory markers in markers in acute acute exacerbationexacerbation and and stablestable COPDCOPD

3

4

mpt

om c

ount

10

15

om c

ount

reco

very

tim

e

((SeemungalSeemungal etet al. AJRCCM 2001)al. AJRCCM 2001)

1

2

Tatl

daily

sym

5

Med

ian

daily

tota

l sym

pto

VirusesVirusesNo pathogenNo pathogen

24%24%21%21%

Viruses and bacteria in COPD exacerbationsViruses and bacteria in COPD exacerbations

Viruses &Viruses &BacteriaBacteria

BacteriaBacteria25%25%30%30%

Papi, Fabbri & Johnston et al. AJRCCM 2006

11

Papi - Viral interactions with respiratory tract in health and diseaseop

hils

ophi

lsugug 6

8

10 **** ******

****

**

Eosinophils increased only in virus related AEEosinophils increased only in virus related AE

Virus Virus & Bacteria No pathogenBacteria

Sput

um E

osin

oSp

utum

Eos

ino

101066/m

g pl

u/m

g pl

u

00

2

4

6

EE EE EE EESS SS SS SS

Papi, Fabbri & Johnston et al. AJRCCM 2006

ConclusionsConclusions

•• Viral infections are the most frequent Viral infections are the most frequent cause of asthma and COPD exacerbationscause of asthma and COPD exacerbations

•• Impaired antiviral response in asthmaImpaired antiviral response in asthmaImpaired antiviral response in asthmaImpaired antiviral response in asthma

•• The role of viral infections is The role of viral infections is understimated in COPDunderstimated in COPD

12

Contoli - Upper respiratory tract viral infections with impact on the lung

Upper Respiratory tract viral infections with impact on Upper Respiratory tract viral infections with impact on the lungthe lung

Marco Contoli, MD, PhDMarco Contoli, MD, PhDProf. Alberto Papi, MDProf. Alberto Papi, MD



•• rhinovirusesrhinoviruses•• enterovirusesenteroviruses

•• influenza viruses A & Binfluenza viruses A & B

Respiratory virusesRespiratory viruses

•• coronavirusescoronaviruses

•• parainfluenza virusesparainfluenza viruses•• respiratory syncytial virusrespiratory syncytial virus

•• adenovirusesadenoviruses

•• Virus culturesVirus cultures•• SerologySerology•• Antigen detection (immunofluorescence, Antigen detection (immunofluorescence,

Detection methodsDetection methods

hemagglutination…)hemagglutination…)

•• PCR and related methodologiesPCR and related methodologies

13


Virus associated asthma exacerbationsVirus associated asthma exacerbations

ChildrenChildren AdultsAdults

Positive virus detectionPositive virus detection

No virus detectedNo virus detected

(Johnston et al. BMJ 1995)(Johnston et al. BMJ 1995) (Corne et al. Lancet 2002)(Corne et al. Lancet 2002)

Two thirds of viruses detected are rhinovirusesTwo thirds of viruses detected are rhinoviruses

(Reviewed in Contoli, Johnston,(Reviewed in Contoli, Johnston, PapiPapi et al et al ClinClin Exp Allergy 2005 )Exp Allergy 2005 )

Experimental InfectionExperimental InfectionLocalization of RVs in the bronchiLocalization of RVs in the bronchi

•• Rhinovirus infects lower Rhinovirus infects lower airwaysairways

(Papadopoulos, Papi, Johnston J Infect Dis 2000)(Papadopoulos, Papi, Johnston J Infect Dis 2000)

Experimental infection: Experimental infection: B B -- ImmunologyImmunology

MacrophagesTh1 cellsTh1 cells

Dendriticcells

T cellsEosinophilsBasophils

Th2 cellsTh2 cells

•• In vitroIn vitro:– A defective type 1 response to rhinovirus in

atopic asthma. (Papadopoulos, Papi, Johnston et al. (Papadopoulos, Papi, Johnston et al. Thorax 2002)Thorax 2002)

•• In vivo:In vivo:– RV infection in atopic subjects is associated to

low Th1/Th2 antiviral responses (Gern 2000 (Gern 2000 AJRCCM)AJRCCM)

Neutrophils

14


Neutrophils activation chemotaxis

Eosinophils survival, chemotaxis

Macrophage

IL-8, Groα

IL-1β, MIP-1α, MCP-1, TNFα

GM-CSF, Eotaxin, RANTES, MIP-1α

Experimental infection: Experimental infection: C C -- InflammationInflammation

T lymphocytes activation, chemotaxis

virusMacrophages

NK cells activation

,

RANTES, IL-6

IFNα / β, MIP-1α

MHC I, ICAM-1,

VCAM-1 IFNα / β

•• In asthmatic subjectsIn asthmatic subjectsrhinovirus infection induces rhinovirus infection induces prolonged prolonged eosinophil eosinophil infiltration of the bronchial infiltration of the bronchial mucosa mucosa (Fraenkel et al. AJRCCM 1995)

Experimental infection: Experimental infection: C C -- InflammationInflammation

• Increased levels of eosinophil cationic protein were found in the sputum sputum of RV- infected subjects(Grunberg et al. AJRCCM 1997)

Inflammatory common pathway : Inflammatory common pathway : Adhesion moleculesAdhesion molecules

•• Epithelial ICAMEpithelial ICAM--1 1 expression is increased expression is increased in asthmain asthma

Surface epithelial Surface epithelial ICAMICAM--1 increases 1 increases afterafter allergenallergen challengechallenge

(Vignola, Am Rev Respir Dis 1993)(Vignola, Am Rev Respir Dis 1993)

(Bentley et al. JACI 1993)(Bentley et al. JACI 1993)

15


Common Common pathwaypathway: : AdhesionAdhesion moleculesmolecules•• ICAMICAM--1 is the cellular receptor for 90% RV1 is the cellular receptor for 90% RV•• Rhinovirus increases epithelial ICAMRhinovirus increases epithelial ICAM--11

(Papi, Johnston J Biol Chem 1999)(Papi, Johnston J Biol Chem 1999) (Grunberg, et al Clin Exp Allergy (Grunberg, et al Clin Exp Allergy 2000)2000)

RhinovirusRhinovirus

ICAMICAM--11

II--kB degradationkB degradation

InflammatoryInflammatoryResponseResponse

ICAMICAM--11

Rhinovirus infection of the airway epithelial cellsRhinovirus infection of the airway epithelial cellsIn vitro modelsIn vitro models

XX

Oxidant formationOxidant formation

II kB degradationkB degradation

NF-kB

NF-kB

(Papi A, Johnstion SL. FASEB J 2002)

XX

XX Reducing agentsReducing agents

Papi A, Contoli M, Pinamonti S et al; J Biol Chem 2008

16


Mucus Mucus hypersecretionhypersecretion InflammatoryInflammatory

cell recruitmentcell recruitmentand activationand activation

PlasmaPlasmaleakageleakage

Causes of virusCauses of virus--induced airway obstructioninduced airway obstruction

AirwayAirwayHyperresponsivenessHyperresponsiveness

and activationand activation

Neural activationNeural activation

VirusVirus--infected infected epitheliumepithelium

(Contoli (Contoli etet al. al. ClinClin ExpExp AllergyAllergy 2005)2005)

Effect of viral infection on Glucocorticoid ResponsivenessEffect of viral infection on Glucocorticoid Responsiveness

GCS

GR

mRNA

Cell membrane

hsp90

mRNA

+GRE - GRE

nucleus

Steroid responsivetarget genes

RhinovirusRhinovirus and and childhoodchildhood asthmaasthmaChildhoodChildhood OriginOrigin ofof AsThmaAsThma (COAST)(COAST)

50

60

70OR=10OR=10

OR=8OR=8

First 3 First 3 yearsyears ofof lifelife

year

sye

ars

(%)

(%)

0

10

20

30

40

Neither RSV only RV only RV & RSV

OR=2.6OR=2.6

OR=1OR=1

WheezingWheezing illnessesillnesses

Asth

ma

Ast

hma

at 6

at

6 yy

(Jackson (Jackson etet al. AJRCCM 2008)al. AJRCCM 2008)

17


Relative Relative contributioncontribution ofof rhinovirusrhinovirus wheezingwheezing illnessesillnesses and and aeroallergenaeroallergen sensitizationsensitization toto riskrisk ofof asthmaasthma at 6 at 6 yearsyears

OR (95% CI)

RV wheezing Aeroallergen

(Jackson (Jackson etet al. AJRCCM 2008)al. AJRCCM 2008)

RV wheezing Aeroallergen sensitization

First year of life 2.8 (1.4 – 5.6) 3.6 (1.7 – 7.7)

Third year of life 25.6 (8.2 –79.6)

3.4 (1.7 – 6.9)

MechanismsMechanisms byby whichwhich viralviral infectioninfection can can leadlead toto ananincreasedincreased Th2 Th2 responseresponse (mouse (mouse modelmodel))

VirusVirusSendaiSendai

TypeType I interferon I interferon receptorreceptor--dependentdependent expressionexpression ofof high high affinityaffinity IgEIgE receptotreceptot

CrossCross--linking Fclinking FcεεRI RI

((GraysonGrayson etet al. J al. J ExpExp MedMed 2007)2007)

DendriticDendritic cellscells

CCL28CCL28

IL-13 producing CD4+ T-cell

100

150

200

250

500

1000

1500

2000

pg/m

l pg/ml

ModulatoryModulatory effecteffect ofof viralviral infectioninfection on on allergenallergen inducedinduced Th2 Th2 inflammationinflammation, ,

followingfollowing in vitro in vitro stimulationstimulation withwith japanesejapanese cedarcedar pollenpollen

0

50

0

500

ILIL--44 ILIL--55

((LiuLiu etet al. J al. J MedMed VirolVirol 2007)2007)

18



ViralViral infectionsinfections impact on the impact on the lunglung::•• InflammatoryInflammatory effectseffects•• SteroidSteroid sensitivitysensitivity•• ImmunologicalImmunological consequencesconsequences

19

Contoli - Viral pneumonia – primary and co-pathogenic infections

Viral pneumonia – primary and co-pathogenic infectionsViral pneumonia – primary and co-pathogenic infections

Marco Contoli, MD, PhDMarco Contoli, MD, PhD



Pneumonia: Pneumonia: definitiondefinitionPneumonia: Pneumonia: definitiondefinition

Acute infectious disease

Documentation of abnormalities at chest radiografy

Frequent association with ifi i t t

Acute infectious disease

Documentation of abnormalities at chest radiografy

Frequent association with ifi i t taspecific respiratory symptoms

Possibly associated with systemic inflammatory symtoms

No correlations with aetiology, radiology and clinical severity

aspecific respiratory symptoms

Possibly associated with systemic inflammatory symtoms

No correlations with aetiology, radiology and clinical severity

2008

20


Epidemiological classification of pneumoniaEpidemiological classification of pneumonia

Community-acquired

pneumonia (CAP)

Hospital-acquired, and ventilator-

associatedpneumonia( ) p

Correlations with pathogensCorrelations with pathogens

Therapeutical implicationsTherapeutical implications

(ATS statement 2005 and 2001)(ATS statement 2005 and 2001)

EpidemiologyEpidemiology

Coxiella burnetii (2%)Chlamydia pneumoniae (12%)

Mycoplasma pneumoniae (8%)Haemophilus.influenzae (4%)

Legionella spp (5%)Gram- ,enterobatteri (3%)

Chlamydia psittaci(2%)

Staphylococcus

Virus (8%)

CommunityCommunity--acquiredacquired pneumonia: pneumonia: aetiologyaetiologyCommunityCommunity--acquiredacquired pneumonia: pneumonia: aetiologyaetiology

aureus (2%)

Streptococcus pneumoniae (28%)

Unknown (24.5%)

21


ImprovedImproved DiagnosisDiagnosis ofof the the EtiologyEtiology ofof CommunityCommunity--AcquiredAcquiredPneumonia Pneumonia withwith RealReal--Time Time PolymerasePolymerase ChainChain ReactionReaction

8101214161820

Conv

PCR

No.

Of p

atie

nts

0246

(Templeton et al. Clin Inf Dis 2005)

IncidenceIncidence and and characteristicscharacteristics ofof viralviral communitycommunity--acquiredacquiredpneumonia in pneumonia in adultsadults


Of 304 patients with CAP, a viral diagnosis was made in 88 (29%)

10

12

14

0

2

4

6

8

10

%

(Jennings et al. Thorax 2008)

ViralViral InfectionInfection in in AdultsAdults HospitalizedHospitalized WithWith CommunityCommunity--AcquiredAcquiredPneumonia : Pneumonia : PrevalencePrevalence, , PathogensPathogens, and , and PresentationPresentation

Of 193 patients with CAP, a viral diagnosis was made in 29 (15%)

6

7

8

case

s

0

1

2

3

4

5

(Johnstone et al. Chest 2008)

Num

ber o

f c

22


SARS: SARS: Severe Acute Respiratory SyndromeSevere Acute Respiratory Syndrome

(Li et al Nature 2003) (Li et al Nature 2003) (Holmes (Holmes etet al. NEJM 2003)al. NEJM 2003)

SARS CoV

Avian Influenza A (H5N1) Virus Infection in Humans

Influenza A H5N1

(NEJM 2008)

2009 H1N1 Influenza2009 H1N1 Influenza

Febrile respiratory illness in children from Febrile respiratory illness in children from southern California caused by infection southern California caused by infection with a novel influenza A (H1N1) viruswith a novel influenza A (H1N1) virus

H1N1 was isolated in 47 cases of rapidly H1N1 was isolated in 47 cases of rapidly progressive severe pneumonia that progressive severe pneumonia that

Influenza A H1N1

p g pp g presulted in 12 known deathsresulted in 12 known deaths

By June 11, 2009, nearly 30,000 cases of By June 11, 2009, nearly 30,000 cases of 2009 H1N1 virus had been confirmed 2009 H1N1 virus had been confirmed across 74 countries = across 74 countries = pandemic pandemic influenzainfluenza

Sullivan S J et al. Mayo Clin Proc. 2010;85:64-76

23


CDC CDC EstimatesEstimates ofof 2009 H1N1 Cases and 2009 H1N1 Cases and RelatedRelated HospitalizationsHospitalizations and and DeathsDeaths fromfrom AprilApril 2009 2009 -- JanuaryJanuary 16, 2010, 16, 2010, ByBy AgeAge GroupGroup

ClinicalClinical manifestationsmanifestations: : ifiifi ff ti lti l ??specificspecific forfor aetiologyaetiology??

ManagmentManagment ofof communitycommunity--acquiredacquired pneumoniapneumonia

SignsSigns and and symptomssymptoms cannotcannot predictpredict the the atiologicatiologic agentagentatiologicatiologic agentagent

(Halm et al. NEJM 2003)

24


ViralViral InfectionInfection in in AdultsAdults HospitalizedHospitalized WithWith CommunityCommunity--AcquiredAcquiredPneumonia : Pneumonia : PrevalencePrevalence, , PathogensPathogens, and , and PresentationPresentation

60

80

100

Viral

Bacterial

%


0

20

40

Cough Sputum Dyspnea

Bacterial

Mixed

Unknown



(Jennings et al. Thorax 2008)

4

5

TNFa IL6 PCT CRP

ug/ml Fatal Outcome

Cytokines & Hormokines upon InfectionCytokines & Hormokines upon Infection

adapted from Meisner M, J Lab Med 1999Dandona P, et al. J Clin Endocrinol Metab 1994

Harbarth S, AJRCCM 2001Becker KL, J Clin Endocrinol Metab 2004

0

1

2

3

0 12 24 36 48 60 72

Endotoxin ivEndotoxin iv

h

25


„Healthy“ Calcitonin

Control Sepsis

Spleen

LungLiverKidneyAdrenal

White Blood CellsPerit. Macrophage

Thyroid

Calcitonin in healthy people

Small IntestineColon

Heart

BrainSpine

MuscleSkinVisceral Fat

Adrenal

PancreasStomach

Testes

Müller B, et al. JCEM 2001

InfectionCTpr

incl PCT

Control Sepsis

Spleen

LungLiverKidneyAdrenal

White Blood CellsPerit. Macrophage

Thyroid

Calcitonin precursors (CTpr) in infected patients

Infection incl. PCT

Small IntestineColon

Heart

BrainSpine

MuscleSkinVisceral Fat

Adrenal

PancreasStomach

Testes

Müller B, et al. JCEM 2001

(Christ-Crain, Muller et al. Lancet 2004)

26


ComorbiditiesComorbidities and CAP and CAP etiologyetiology

30

40

50

60

70

Viral

Bacterial

%


0

10

20

30

CD RD IFS

Bacterial

Mixed

Unknown

ComorbiditiesComorbidities and CAP severityand CAP severity

Vedi anche jama 2009

CAP severity and CAP severity and etiologyetiology

(Templeton et al. Clin Inf Dis 2005)(Jennings et al. Thorax 2008)

27


CAP severity and CAP severity and etiologyetiology

Postmortem lung specimens of 77 fatal H1N1 cases documented Postmortem lung specimens of 77 fatal H1N1 cases documented coinfection in 22 cases (29%)coinfection in 22 cases (29%)

Streptococcus pneumoniae predominatedStreptococcus pneumoniae predominated(10 cases) (10 cases)

These findings highlight the importance of:These findings highlight the importance of:•• early detection and treatment of bacterial pneumonia in early detection and treatment of bacterial pneumonia in patients with 2009 H1N1 influenzapatients with 2009 H1N1 influenza•• pneumococcal vaccine for those in whom it is indicated.pneumococcal vaccine for those in whom it is indicated.

CDC CDC MMWR MMWR MorbMorb MortalMortal WklyWkly Rep.Rep.2009;58(38):10712009;58(38):1071--1074.1074.

Interaction between bacteriaInteraction between bacteria and virusand virusi l i t t t i f tii l i t t t i f tiin lower respiratory tract infectionsin lower respiratory tract infections

Criterion for exacerbation: increase over baseline in LRT symptom score of >2 for 2 days

Rhinovirus infections in COPD

Upper & lower respiratory tract scores

(Mallia, Johnston et al. Respir Res 2006)(Mallia, Johnston et al. Respir Res 2006)

28


A human experimental model of rhinovirus inducedexacerbations of Chronic Obstructive Pulmonary Disease

A human experimental model of rhinovirus inducedexacerbations of Chronic Obstructive Pulmonary Disease

Preliminary data indicates that in vivo experimental RV infection in COPD patients leads to increased bacterial load in

h i

(Mallia P, Message S, Gielen V, Contoli M, Papi A, Johnston SL, et al. Submitted)(Mallia P, Message S, Gielen V, Contoli M, Papi A, Johnston SL, et al. Submitted)

the airways


Viral infection is an understimated cause of Viral infection is an understimated cause of pneumoniapneumonia

Lack of specific pharmacological treatmentLack of specific pharmacological treatment

Role of prevention: vaccinationRole of prevention: vaccination

Interaction between virus and bacteria deserves Interaction between virus and bacteria deserves further investigationsfurther investigations

29

The best option for virus discovery, VIDISCA combined with high

throughput sequencing

Michel de Vries1, Marta Canuti1, Nuno R. Faria1, Maarten F. Jebbink1, Angela C.M. Luyf 2,

Barbera DC van Schaik2, Marja Jakobs3, Richard Molenkamp4, Martin Deijs1, Frank Baas3,

Lia van der Hoek1

1 Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity

Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam. 2 Department of Clinical

Epidemiology, Biostatistics and Bioinformatics, Bioinformatics Laboratory, Academic Medical Center of the

University of Amsterdam. 3 Department of Neurogenetics, Academic Medical Center of the University of

Amsterdam, 4 Laboratory of Clinical Virology, Department of Medical Microbiology, Center for Infection and

Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam.

In approximately 20% of the adults with respiratory tract illness no known pathogen can be detected. A

yet unknown virus could be the cause of the illness and a sequence independent amplifications

method such as virus discovery cDNA-AFLP (VIDSICA), can be used to identify the virus. The

VIDISCA method uses restriction enzyme recognition sites to digest all nucleic acids in a sample, a

ligation step to add adaptors, and a PCR with primers that anneal to the adaptors to amplify the input

viral DNA or viral RNA1. The method was successfully used to identify human coronavirus NL63

(HCoV-NL63 1) and picornaviruses 2. These discoveries were all from cultured virus isolates and not

from clinical samples, since the VIDISCA assay is not sensitive enough to detect viruses from

uncultured material. In theory the latest high throughput sequencing techniques may enhance

VIDISCA sensitivity and enable virus identification in cases where conventional VIDISCA may not.

In conventional VIDISCA a virus specific amplification product has to be visible and

recognizably different from the negative control PCR products. This means selection of virus specific

amplification by eye, whereas VIDISCA combined with high throughput sequencing does not need

recognizable visualization. With next generation sequencing large amounts of sequences are

generated, thus it allows everything in a sample to be sequenced, including background (mainly rRNA

amplification products) and potentially unknown viruses.

40

We evaluated this theoretical sensitivity improvement by sequencing VIDISCA amplified

products with the Roche 454-FLX system (VIDISCA-454, see figure). As input twelve randomly chosen

nasopharyngeal washings of patients all containing known respiratory viruses were used. The

outcome of the standard VIDISCA was compared to the results with VIDISCA-454. All samples were

analyzed double-blind to prevent biased analyzing of the sequences.

With Roche-454-FLX a total of 83135 sequences were generated of which 68 were viral

(HCoV-229E, HCoV-OC43, RSV, HRV, hMPV). Standard VIDISCA revealed the identity of only 1 virus

(HCoV-229E) in the 12 nasopharyngeal samples, whereas with the VIDISCA-454 the viruses in 6 of

the 12 samples could be correctly identified.

In conclusion, high throughput sequencing and VIDISCA increases the sensitivity of the

discovery method to a level that allows virus discovery directly from nasopharyngeal aspirates.

Reference List

1. van der Hoek L., Pyrc K, Jebbink MF et al. Identification of a new human coronavirus. Nat Med 2004;10(4):368-373.

2. de Vries M, Pyrc K, Berkhout R et al. Human parechovirus type 1, 3, 4, 5, and 6 detection in picornavirus cultures. J Clin Microbiol 2008;46(2):759-762.

41

Figure. Schematic overview of the steps in VIDISCA-454

42

Van der Hoek - Future diagnostic approaches to viral respiratory tract infections

Future diagnostic approaches to viral respiratory tract infections

Laboratory of Experimental Virology

Human bocavirus (HBoV)

Lia van der Hoek

We have so far…1. Influenzavirus A (H1, H3, H5, H7)2. Influenzavirus B3. Enterovirus4. Parechovirus5. Adenovirus6. RSV7. Rhinovirus type A/B8. PIV-19 PIV 2

Detection nowadays:

• (Real-time) RT-PCRs


9. PIV-210. PIV-311. PIV-412. HCoV-229E13. HCoV-OC4314. hMPV15. Bocavirus *16. HCoV-NL6317. HCoV-HKU1 *18. KIPyV *19. WUPyV *20. Rhinovirus type C *

• Luminex approach

Keep Ct into account with identification of the pathogen

* No culture system

What should we know

Koch’s postulates, causative role of disease:• The microorganism must be found in all organisms suffering from the disease, but not in

healthy organisms. • The microorganism must be isolated from a diseased organism and grown in pure culture. • The cultured microorganism should cause disease when introduced into a healthy

organism.


• The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

Problems- Only in organisms with the disease - Virus culture - Suitable animal model

43


Culture is needed in discovery

• Sequence independent amplifications• Identification• Genome characterisations• Worldwide spread• Association with disease


• Inhibition by antivirals ?• Replication kinetics ?• Pathogenicity ?• Koch’s postulates ?

Human bocavirus (HBoV-1)• Identified in 2005 within pools of human nasopharyngeal aspirates (Allander

et al PNAS 2005).

• The genomic DNA reference sequence is 5.299 nt in length.• Without flanking terminal hairpin structures.

• Infections have been found worldwide


• Respiratory samples• Serum, fecal and urine samples

• Frequently found in children under the age of 2 years• High frequency of co-infections with other respiratory viruses• Acute wheezing

• Despite the current knowledge, no in vitro or in vivo model has been established that supports replication.

Phylogeny of Parvovirinae


44


Human airway epithelial culture

• Tracheal or bronchial epithelial cells from donors

• Dedifferentiation

• Grow until confluence


• Grow until confluence

• Exposure to air on apical side

• Medium at basolateral side

• Differentiation (5 to 6 weeks)

Benefits of differentiated human airway epithelia (HAE)

– Morphologically and functionally resembles the human airways in vivo

– Susceptibility towards viruses coincides with


– Susceptibility towards viruses coincides with the degree of differentiation

Propagation of HBoV on HAE ?

HBoV1 replication on HAE: Apical release of the virus

d / m

l


Hours post-inoculation

Vira

l loa

d

Dijkman, Koekkoek, Molenkamp, Schildgen, van der Hoek J. Virology

45


Propagation? Yes

• Intracellular: spliced mRNA

• Release of virus particles only apical (so far…)


• Apical harvest can be passaged

• After first replication on HAE, replication in cell lines: ….. No (so far)

Transcript map of HBoV1


Dijkman, Koekkoek, Molenkamp, Schildgen, van der Hoek J. Virology 2009

And there is more to do…

• Type of cells infected• Receptor• Entry route• Release of the virus


• Antivirals• Find animal model

• Other unculterables: HCoV-HKU1, Rhinovirus type C, Polyomaviruses

46


With special thanks to:• AMC, Lab Experimental Virology Ronald Dijkman

University of Amsterdam Krzysztof PyrcAmsterdam, the Netherlands Maarten F. Jebbink

Martin DeijsMichel de Vries

• Municipal Health Service Amsterdam Wilma Vermeulen Oost


• Municipal Health Service Amsterdam, Wilma Vermeulen-OostThe Netherlands Ron Berkhout

• Bonn University, Germany Oliver Schildgen

47

Van der Hoek - Identifying novel respiratory viruses – the technology and examples from the GRACE approach


Identifying novel respiratory viruses

The technology and examples from the GRACE approachthe GRACE approach

Lia van der Hoek

Laboratory of Experimental Virology Dep. Medical Microbiology AMC

Amsterdam The Netherlands

Laboratory of experimental virology

Ronald DijkmanMichel de Vries

www.pathogendiscovery.com

Martin DeijsMaarten F. JebbinkKrzysztof PyrcMarta CanutiLia van der Hoek


In GRACE

Lower respiratory tract infections in adults

All known pathogens are tested

(bacterial, viral)

+/- 20% remains pathogen negative

Caused by an unknown virus?

48



Today

I: Introduction VIDISCA

II: Discovery of HCoV-NL63

III: VIDISCA-454 the GRACE approach


VIDISCA (Virus Discovery cDNA-AFLP)


VIDISCA: RNA and DNA viruses

Parvo B19 plasma(ss-DNA)

M - +

HIV-1 virus culture

(ss-RNA)M

M- + M - +

HBV plasma

(partially ds-DNA)

49



Part II

Virus discovery yexample: HCoV-NL63


7-month-old child with bronchiolitis, conjunctivitis and fever (Jan 2003)

Patient NL63

Routine diagnostics negative for all known respiratory viruses

RSV, Adenoviruses, Influenzavirus A and B, Parainfluenza virus types 1, 2 or 3, Rhinoviruses (PCR), Meta-pneumovirus (PCR), Enterovirus (PCR), Human coronaviruses HCoV-OC43 and HCoV-229E (PCR)

tMK cells: Cytopathic effect (CPE)


CPE on LLC-MK2 cells

control

NL63

50



VIDISCA on NL63

NL NLM P P 63 63 cC cC - - M

1 of the 16 selective PCRs:

In total 13 fragments with similarity to coronaviruses


Phylogenetic analysis

G1

HCoV-NL63

HCoV-229E

PEDV

FIPV

CCoV

PRCoV

G2

G3

TGEV

SARS-CoV

BCoV

HCoV-OC43

MHV

CoV-HKU1

TCoV

IBV

0.2


Not a recombinant

van der Hoek et al, Nature Medicine 2004

51



A previously unknown virus, what does it cause?

Further investigation of the virus

Treatment options


HCoV-NL63 worldwidePublished

The Netherlands (n=8) 1.4% van der Hoek et al 2004The Netherlands (n=5) 2.9% Fouchier et al 2004Australia (n=16) 2.1 % Arden et al 2005Japan (n=3) 2.5 % Ebihara et al 2005Canada (n=19) 3.6 % Bastien et al 2005USA (n=79) 8.8 % Esper et al 2005Belgium (n=7) 2.3 % Moes et al 2005France (n= 28) 9.3 % Vabret et al 2005Hong Kong (n= 15) 2.6 % Chiu et al 2005Canada (n=26) 2.1 % Bastien et al 2005Japan (n=5) 1 2 % Suzuki et al 2005Japan (n=5) 1.2 % Suzuki et al 2005Switzerland (n=6) 7.3% Kaiser et al 2005Germany (n=49) 5.2% van der Hoek et al 2005Canada (n=12) 3.0% Boivin et al 2005Korea (n=8) 1.6 % Choi et al 2006Italy (n=9) 1.1% Gerna et al 2006Hong Kong (n=53) 1.3% Lau et al 2006Sweden (n=12) 5.7% Koetz et al 2006Switzerland (n=6) 1.1% Garbino et al 2006Australia (n=6) 1.9 % Arden et al 2006Korea (n=14) 1.7% Han et al 2007USA (n=11) 1.0% Kuypers et al 2007Italy (n=1) 0.5% Pierangeli et al 2007Greece (n=2) 1.0 % Papa et al 2007Italy (n=13) 3.0 % Gerna et al 2007Korea (n=3) 1.3% Chung et al 2007USA (n=2) 0.7% Mahony et al 2007USA (n=4) 4.8% Kistler et al 2007Australia (n=8) 1.1% Lambert et al 2007


How common is 229E/NL63 infection?

-----: HCoV-229E antibodies___: HCoV-NL63 antibodies

Dijkman et al J. Clin Microb. 2008

52



Seroconversion 229E and NL63

HCoV-NL63HCoV-229E

Dijkman et al J. Clin Microb. 2008


So every child becomes infected, but in how many cases does it require

1. Hospitalization?p

2. Visit to the general practitioner/paediatrician?


The PRI.DE study (Parainfluenza- und Respiratory- Syncytial-Virus-

Infektionen in Deutschland)

Population based study of children under the age 3 years with LRTI

Lower Respiratory Tract Infections (LRTI):

Apnoea (under the age of 6 months)

Laryngotracheitis (croup)

Bronchitis

Bronchiolitis

Pneumonia

Visiting the hospital or peadiatrician*

Peadiatrician: out patient clinica. In Germany >95% of children have a peadiatrician

Multiple hospitals and practices in cities in the north (Hamburg), east (Dresden), south (Freiburg) and west (Bochum) of Germany

53



M&Ms

Samples: recruitment from November 1999 to October 2001. Total of 3654 respiratory samples

All tested for RSV, PIV-1, PIV-2, PIV-3, and influenza.

Randomized 1733 samples tested for HCoV-NL63 RNA by real-time RT-PCR

Laboratory of Experimental Virology Only high HCoV-NL63

Annual incidence of HCoV-NL63 in children (<3 yr)

7 per 1000 children (95% CI: 3 - 13 per 1000 children) visit the physician per year

Absolute number: 16.929 children per year in Germany

Hospitalization rate:

22 per 100.000 children (95% CI: 7 – 49 per 100.000 children) have to be hospitalized per year

Absolute number: 522 children per year in Germany

Rarely hospitalized, more often visit to the outpatient clinic

Van der Hoek et al J Clin Virol 2010


Croup associated with HCoV-NL63 and PIV

Confirmed by: Choi et al Clin. Inf. Dis. 2006 Han et al J. Clin Virol 2007Wu et al Eur. J. Pediatrics 2007

Van der Hoek et al PLoS Medicine 2005

54



Receptor for HCoV-NL63

Group 1 HCoV-229E CD13TGEV CD13FIPV CD13PrCoV CD13HCoV-NL63 ?

Group 2 MHV CAECAMSARS-CoV angiotensin converting enzyme 2 (ACE2)

HCoV-NL63HCoV-229E

PEDVFIPV

CCoVTGEVPRCV

SARS-CoVMHV

EqCoVHCoV-OC43BCoV

IBV

10099

100

78100

96

67100

10095

100

0.1

g g y ( )HCoV-OC43 Sialic acids

CD13CD13CD13CD13

ACE2CAECAMSialic acids


Overexpression of receptors

Hofmann et al PNAS 2005


Conclusions• The VIDISCA method is suitable for identification of RNA and DNA

viruses from Culture.

• HCoV-NL63 is a previously unknown human coronavirus and it has spread worldwide

• It circulates in the winter months

Nature Med 2004

Nature Med 2004Virology Journal 2004Emerg. Inf Dis 2005BioMed Central 2005

J.Mol Biol 2007

Nature Med 2004

• Every child becomes infected in youth

• Rarely requires hospital uptake

• Infection is associated with croup in children

• HCoV-NL63 is the only other coronavirus that uses the same receptor as SARS-CoV for entry

• HCoV-NL63 infections can be treated

PNAS 2005

PLoS Medicine2005

AAC 2006

J Clin Virol 2010

J. Clin Microbiology2008

55



BUT…..

The VIDISCA method is suitable for identification of RNA and DNA viruses from virus culture.

We want to detect viruses without the need to culture (direct from patient material)


Part III

VIDISCA-454The GRACE approach


VIDISCA454

56



Next Generation Sequencing

Clonal amplification by emulsion PCR: clonal on a bead

At the AMC: 454 sequencer of Roche.

Per run a maximum of 1.5 E6 beads can be used resulting in about 400.000 quality sequences.


monitor VIDISCA-454

Coxsackievirus B4 virus culture supernatant

12 Respiratory samples with known viruses


Virus culture VIDISCA-454

MID Nr. of sequences Viral sequences % of total

1 2805 2477 88.3

2 3890 3282 84.4

3 2369 2007 84.7

4 2782 2534 91.1

5 3056 2540 83.1

6 2271 2000 88.1

7 1429 1354 94.8

8 3830 2961 77.3

9 3018 2821 93.5

10 3040 2840 93.4

11 5216 4991 95.7

12 2891 2740 94.8

Total 36597 32547 88.9

57



Clinical sample VIDISCA-454

Twelve clinical samples containing known viruses

VIDISCA VIDISCA-454

Virus indentified 1 6

HCoV-229E

HCoV-229EHCoV-OC43

RSV (2X)Rhinovirus

hMPV


Conclusion

VIDISCA-454 in NPA is a very good virus discovery technique,

Ready to test GRACE samples for unknown viruses


Municipal Health Service Amsterdam, Wilma Vermeulen-OostThe Netherlands Ron Berkhout

Joke Spaargaren

University Hospital Freiburg, Germany Gabriela IhorstSt Joseph Hospital, Freiburg, Germany Johannes ForsterWyeth Pharma GmbH, Münster, Germany Gudula PetersonRuhr University Bochum, Germany Klaus Sure

Alexander StangKlaus Überla

University Hospital of Caen, France Astrid Vabret

AcknowledgmentsNL63discovery

NL63Croup+Burden of disease

University of Auckland, New Zealand Lea DengHoward A. Ross

University of California, San Diego, USA Chisato ShimizuSharon L. ReedJane C. Burns

Layola University, Maywoo, USA Susan C. BakerNorthwestern University, Chicago, USA Francesca Garcia

Anne H. RowleyStanford T. Shulman

Vanderbilt Univ. Med. Center, Nashville, USA Helen K.B.TalbotJohn V. Williams

Bonn University, Germany Marcel Müller

NL63Kawasakidisease

NL63evolution

NL63Serocon-version

58



AMC, Department of Neurogenetics Marja JakobsAMC, Amsterdam, The Netherlands Frank Baas

Department of Clinical Virology, AMC, Amsterdam, the Netherlands Richard Molenkamp

Clinical Epidemiology and biostatistics Barbara van Schaik

Acknowledgments II

Sequence facility

Clinicalsamples

IT-p gyAMC, Amsterdam, the Netherlands Angela Luijf

Laboratory of Experimental Virology Lia van der HoekAMC, Amsterdam, the Netherlands Michel de Vries

Krzysztof PyrcMaarten F. JebbinkRonald DijkmanMartin DeijsNuno M. FariaMarta CanutiLoes Jachimowski

VIDISCA

support

59

Osterhaus - Vaccine prevention of non-influenza viral respiratory disease:current and future

Vaccine prevention of non-influenza viral respiratory disease:

ESCMID 10-13 April 2010Vienna, Austria

viral respiratory disease:current and future.

Ab Osterhaus

Human respiratory tract disease- Associated pathogens -

540 respiratory GP patients , Netherlands, 1996-1997, culture and PCR (NIVEL)Bacteria, 16%

Mycoplasma pneumoniae, 1%Chlamydia pneumoniae, 1%

Parainfluenza virus, 1%,Adenovirus, 1%Coronavirus OC43, 2%Enterovirus, 4%

RSV, 5%

Influenza B virus, 9%

Influenza A (H3N2) virus, 14%Rhinovirus, 22%

Negative, 36%

Newly identified human respiratory viruses Newly identified human respiratory viruses in last 15 years alonein last 15 years alone

- AIV`s * influenza virus 1997…- Hendra-/NipahV paramyxovirus 2000…- hMPV * paramyxovirus 2001- SARS-CoV * coronavirus 2003- HCoV-NL63 * coronavirus 2004- HCoV-HKU1 coronavirus 2005- HBoV parvovirus 2005- KI/WU-PyV polyomavirus 2007- MelV (KamV) orthoreovirus 2007 (2009)- H1N1v influenza virus 2009- …

- animal origin * ErasmusMC involvement

71


NiVHeV

TuV CDV RPVMV

hPIV3bPIV3

SeVParamyxovirinae Respirovirus

MorbillivirusDNA Maximum likelihood, Polymerase ORF

PDV

Henipahvirus

Order Order MononegaviraleMononegavirales, family s, family ParamyxoviridaeParamyxoviridae

v.d.Hoogen et al., Nat.Med. 2001

0.1

hPIV1

NDV

MuVSV5hPIV2

hMPVhMPVAPV

hRSV

bRSV

SV41

LPMV

Pneumovirinae

y

RubulavirusMetapneumovirus

Pneumovirus Avulavirus

- (Young) children ~10 % of children with RTI

- Immunocompromised individuals (fatal cases!)

Newly discovered human paramyxovirushMPV

- Risk groups -

p ( )

- Elderly

- Normal individuals~5 % of RTI in community surveillance studies

Osterhaus and Fouchier, Lancet 2003v.d. Hoogen et al., JID 2003

v.d.Hoogen et al., Nat.Med. 2001

Human metapneumovirus- F protein -

-HMPV F protein is major determinant of protection

-Highly conserved2

3 4-Genetic lineages are highly

related antigenically

0.1

12

567

8

910

1112

13141516

A1

A2

B1

B2

F gene

72


Vaccine Animal model Authors

Soluble F protein Hamsters, cynomolgus macaques Herfst, 2007/2008

Soluble F protein and F DNA vaccine Cotton rats Cseke, 2007

FI HMPV Macaques cotton rats De Swart, 2007

Human metapneumovirus- Vaccine approaches -

FI-HMPV Macaques, cotton rats Yim, 2007

HI-HMPV Mice Hamelin, 2007

Cpts HMPV Hamsters, cynomolgus macaques Herfst, 2008

HMPV deletion mutants Hamsters, AGM Biacchesi, 2004/2005Buchholz, 2005

Chimeric HMPV / AMPV-C Hamsters, AGM Pham, 2005

B/HPIV3 expressing F Hamsters, AGM Tang, 2003/2005

Alphavirus replicon particlesexpressing F Mice, cotton rats Mok, 2008

T-lymphocyte vaccine Mice Herd, 2006

b/h PIV3

AvrII 104

N P/C M hPIV3 F hPIV3 HN L

AvrII 1774

b/hPIV3 expressing hMPV F gene

Tang R. S. et al., 2003, J Virol 77:10819-28

b/h hMPV F1

b/h hMPV F2N F P/C M hPIV3 F hPIV3 HN L

F N P/C M hPIV3 F hPIV3 HN L

MedImmune, Inc

b/hPIV3 expressing hMPV F gene

Virus a

Replication of bovine/human PIV3 expressing the hMPV Fprotein in position 1 or 2 of the PIV3 genome in hamsters.

Mean virus titer on day 4 post-infection (log10 TCID50/g tissue + S.E.) b, c

MedImmune, Inc

a Groups of six hamster were inoculated intranasally with 1 x 106 pfu ofindicated virus.

b Standard error.c TCID50 assays were read for CPE on Day 10 post-infection.

Virus a

b/h PIV3b/h hMPV F1b/h hMPV F2hMPV

4.8 + 0.2 5.6 + 0.65.3 + 0.5 5.7 + 0.45.7 + 0.5 4.6 + 0.35.3 + 0.1 3.6 + 0.3

Nasal turbinates Lungs

73

Osterhaus - Vaccine prevention of non-influenza viral respiratory disease:current and futureb/hPIV3 expressing hMPV F gene

Challenge virus:

hPIV3 hMPV

Mean virus titer on day 4 post-challengeMean virus titer on day 4 post-challenge

Hamsters immunized with bovine/human PIV3 expressing hMPV F protein are protected from challenge with hMPV or hPIV3.

MedImmune, Inc

Immunizing Virusa Nasal Turbinates Lungs Nasal Turbinates Lungs

b/h PIV3b/h hMPV F1b/h hMPV F2hMPVplacebo

a Virus used to immunize groups of six hamsters on Day 0.b On Day 28, the hamsters were challenged with 106 pfu of hPIV3 or hMPV/NL/1/00.ND = not determined.

Mean virus titer on day 4 post-challenge(log10 pfu/g tissue + S.E.)b

Mean virus titer on day 4 post-challenge(log10 TCID50/g tissue + S.E.)b

<1.3 + 0.2 <1.1 + 0.1 ND ND<1.3 + 0.1 <1.1 + 0.1 3.5 + 0.8 <0.5 + 0.2<1.2 + 0.1 <1.2 + 0.1 <0.9 + 0.4 <0.5 + 0.1

ND ND <0.8 + 0.3 <0.4 + 0.04.3 + 0.3 4.5 + 0.5 6.0 + 0.3 4.5 + 1.3

F subunit vaccine- Immunization / challenge -

-Vaccines:

* F1/00 (A1) Specol * F1/00 Iscom * F1/00 nonAdj

* F1/99 (B1) Specol * F1/99 Iscom * F1/99 nonAdj( ) p j

*Specol * Iscom * PBS

-Hamsters were immunized twice, 3 week interval (10ug F)

-Challenge with 106 TCID50 of recNL/1/00 (A1)

-Lungs and nasal turbinates were collected 4 dpi

F subunit vaccine- Immunization / challenge -

-- -- ----

---- -- --

--

9

0/g

NT 7

6

8

viral titersin NT

0

3

2

1

4

5

Log-

10 T

CID

50

74

Osterhaus - Vaccine prevention of non-influenza viral respiratory disease:current and futureF subunit vaccine- Immunization / challenge -

4

6

5

0/g

NT

virus titersin lungs

0

3

2

1Log-

10 T

CID

50

-- -- -- --

---- -- --

--

F1/99 S

F1/00 S

F1/99 IM

F1/00 IM

F1/99 A

F1/00 A

Subunit vaccines- Immunization / challenge – PRVN -

PRVN-assay:

Spec Spec IM IM nonA nonA

1/00 (A) 230 3563 163 2363 9 53

1/99 (B) 4546 1044 4816 963 9 11

Ratio A-B 3.4 2.5 4.8

Ratio B-A 20 30 1

Mean PRVN titers (8 animals / group), homologous titers are underlined

Cold-passaged viruses- cpNL/1/99 (B1) -

-NL/1/99 (B1) was passaged at gradually decreasing temperatures (25 ºC), pass 35 was sequenced

-Recombinant virus (HMPV11) was cloned and rescued

-Shut-off temperature ≥ 38 ºC in vitro

-Viruses attenuated in hamsters

-Four ts RSV mutations were found that can be introduced in recombinant HMPV

-Recombinant virus (NL/1/99 (B1) backbone) containing 3 mutations was cloned and rescued (HMPV/R3)

75

Osterhaus - Vaccine prevention of non-influenza viral respiratory disease:current and futureInfection of macaques- Waning immunity -

Van den Hoogen et al., J. Gen Virol, 2007

Immunization of macaques- F subunit / cptsHMPV11 -

-t=0 first imm, 10µg Fsol (Iscom) / 106 TCID50 HMPV11 / PBS

-t=28 second imm.

-t=84 heterologous challenge 106.5 TCID50

-1, 3, 5, 9 dpi collection of throat swabs / BAL samples

Immunization of macaques- PRVN -

1/99 (B1)Homol

PBS F/Iscom HMPV11

1/00 (A1)Heterol.

Homol.

76

Osterhaus - Vaccine prevention of non-influenza viral respiratory disease:current and futureImmunization of macaques- T-cell proliferation -

Cold-passaged viruses- Results challenge infection -

0/gr

am lu

ng 6

4

0/gr

am N

T 9

6

Imm: 106 TCID50

Chall: 107 TCID50

Vaccine

Log1

0 TC

ID50

2

0

Wt

PBS

HMPV

11HM

PV/R

30

Log1

0 TC

ID5

3

Vaccine Wt

PBS

HMPV

11HM

PV/R

3

Throatswabs

Immunization of macaques- Virus detection by Taqman -

BALsamples

77

Osterhaus - Vaccine prevention of non-influenza viral respiratory disease:current and futureHuman metapneumovirus- Conclusion / discussion -

-Cross-protection with adj. F subunit and cpts vaccines in hamsters-HMPV-specific responses were induced in macaques, but animals were not protected against infection-AGMs probably more suitable model, limited availability of reagents to study immunological responses-Ideal experimental animal model is not available, development of such a model may be needed

-Only transient protective immunity is induced after wt infection in macaques, so an effective vaccine should ideally be more immunogenic and protective than natural HMPV infection -Waning immunity: longer time frame between immunization and challenge infection should be considered for vaccination experiments

Paramyxovirus in SARS patientsParamyxovirus in SARS patients

Hong Kong researchers announce fingings of Paramyxoviruses in SARS patientsSimilar findings communicated from Canada through SARS etiology network

HCoV HKU- Phylogeny -

Rota et al., Science 2003v.d. Hoek et al., Nature Med., 2004Fouchier et al., PNAS 2004Woo et al., J.Virol., 2005

78


Cynomolgus macaques(Macaca fascicularis)

Simultaneous SCV-hMPVInfection-experiments:

•SCV•hMPV •SCV followed by hMPV

Fouchier et al.,Nature 2003Kuiken et al., Lancet 2004

2331 ∧ 10199 ∧ 9081 ∧ 10029 ∧

Gross pathology in aged macaques

3-5 yrsyoung

10 20

324 ∨ 334 ∨ 223 ∨ 507 ∨

de Lang et al., PLoS Path. 2007Smits et al., Plos Path. 2010

10-20 yrsaged

young-adult aged0

5

10

15

20

25

30

35

Gro

ss p

atho

logy

sco

re (%

)

4A low

4Dlow4D4A1A low

4B4C1A1B

Administration of pegylated IFN-alphain aged macaques

Haagmans et al., Nature Med. 2004Smits et al., Plos Path. 2010

4A low

4Dlow4D4A1A low

4B4C1A1B

79


Vaccine-induced enhancement of viral infections

Huisman et al., Vaccine 2009

Possible problems with “a candidate SARS vaccine”

• Lack of efficacy -- problems with animal coronavirus vaccines!

• Safety concerns• Safety concerns

-- antibody mediated enhancement (AME)example: FIP candidate vaccines!

-- predisposition for more serious diseaseexamples: i.a. RSV- and measles vaccines!

Antibody mediated enhancement in FIP

• Hallmarks:

- faster onset of diseasemore f lminant co rse of diseasemore fulminant course of disease

• Hypothesis:

- S-specific antibodies mediate infection of macrophages

80


Inactivated paramyxovirus vaccines: lessons for SARS?

1960’s: development of inactivated RSV and MV vaccines:formaldehyde-inactivated whole virus preparations (FI-RSV / FI-MV)precipitated with aluminium phosphate / - hydroxide

Vaccination induced short-lived antibody andinbalanced T cell responses

Vaccination predisposed infants for enhanced disease following subsequent natural infection with the respective viruses

→Immunopathology !!!!!

Adverse effects paramyxovirus vaccines

• Formalin-inactivated paramyxovirus vaccines adjuvanted with alum can predispose to hypersensitivity responses

• Similar pathology seen for FI-RSV, FI-MV, and FI-hMPV

• Differences:

• FI-RSV study: mild eosinophilic tracheobronchitis, but

two fatal cases (with low eosinophil counts!)

• FI-MV study: severe eosinophilic tracheobronchitis

• FI-hMPV study: eosinophilic alveolitis

Histopathology: infiltration of eosinophils

Eosinophilic tracheo-bronchitis

day 5

day 13

day 13

day 5

day 5

Blue: control animalsRed: FI-RSV primed animals

Eosinophilic bronchiolitis

Diffuse eosinophilic alveolitis

day 5

day 5

day 13

day 13

day 13

RL de Swart et al., J Virol 76: 11561 (2002)

81


Two FI-RSV animals died with hyperinflation

days 0-9 day 12

• Histopathology: no abnormalities• In vitro correlates: high IL-13 and IL-5 responders in the absence of

detectable IFN-γ responsesRL de Swart et al., J Virol 76: 11561 (2002)

Marshall and Enserink (Science 2004)

Ab

titer

400

600

800

1000

1200

1400

Human anti-SARS antibody ELISA titers in macaques 0 and 1 day after i.p. injection of different amounts of human SARS antiserum.

Antiviral effect of purified human SARS immunoglobulins in Macaques: protection and no indication for AME

Haagmans et al., in preparation

0

200

control 0.5 ml 5 ml 50 ml

SC

V T

CID

50/m

l

1e+2

1e+3

1e+4

1e+5

1e+6

1e+7

SARS CoV titres in the lungs of macaques treated 5 days earlier with different amounts of human SARS antiserum and infected 4 days earlier with SARS CoV

82


10e2

10e3

10e4

10e5

10e6

10e7

TCID

50/m

L

10e2

10e3

10e4

10e5

10e6

SAR

S-C

oV e

q / m

L

A B

Prophylactic treatment of ferrets with huMab C3104:SARS-CoV replication, shedding and pathology

LungPharynx

0

2

4

6

8

10

p=0.013

day 4

perc

enta

ge

10 p< 0.001

day 4 0 2 4

<10

0 2 4days .

C D

*

*

ter Meulen et al, Lancet 2004

Lung Pathology

A DNA vaccine induces protective immunity in miceYang et al., (Nature 2004)

Mice were immunised with plasmidsencoding different forms of the SARS CoV spike protein

NIAID-NIH study

SARS CoV spike protein expressed by attenuated Vaccinia virus protectively immunizes mice(Bisht et al., PNAS 2004)

Intranasal or intramuscularimmunisation of mice with MVA-Sat 0 and 4 weeks

Two days after intranasal challenge

NIAID-NIH study

y gSARS coV titers were determined in Nasal turbinates and in the lungs

83


Immunization of ferrets with modified vaccinia virus Ankara based recombinant vaccine against SARS CoV:challenge protection (Weingartl et al., J.V. 2004) Winnipeg study

Immunization of ferrets with modified vaccinia virus

Ankara based recombinant vaccine against SARS CoV:

enhanced inflammation in the liver (Weingartl et al., J.Virol. 2004)

MVA

rMVA-S

Naïve

PBS

Boosted VN response associated“periportal mononuclear panlobular hepatitis” NB: ADV?

Winnipeg study

Protective efficacy of BHPIV3 recombinants expressingSARS-CoV proteins in hamsters (Buchholz et al.,PNAS 2004)

NIAID-NIH study

84


Vaccination of ferrets with inactivated SARS-CoV

- BPL inactivated SARS CoV - in the presence or absence of aluminiumhydroxide (alum) or MF59 - SARS CoV challenge with 104 TCID50 SARS CoV

Haagmans et al

week 0 4

Necropsies were taken 5 days after challenge

immunization Challenge(alum group)

22

Challenge or(MF59 group)

7

2

3

4

5

6

b

P<0.05

AR

S C

oV E

q.og

10/g

tiss

ue)

200

300

400

500

a

VN

Ab

titer

Vaccination of ferrets withinactivated SARS-CoV in alum

Pharyngealswabs

VN-Abs


0

1

2

control 104 105 106

S (lo

1

2

3

4

5

6

7

c

control 104 105 106

SAR

S C

oV T

CID

50(lo

g 10/

g tis

sue)

P<0.05

01234567

d

control 104 105 106

SAR

S C

oV E

q.(lo

g 10/

g tis

sue)

P<0.05

0 5 10 15 20 250

100

weeks after vaccination

V

Lungs(titration)

Lungs(RT-PCR)

NB 106 ~ 1 μg

Vaccination of macaques with inactivated SARS CoV (w/wo alum)

- BPL inactivated SARS CoV - in the absence or presence of aluminium hydroxide - SARS CoV challenge with 107 TCID50 SARS CoV

week 0 4

Necropsies were carried out 5 days after challenge

immunization Challenge

19


85


250

500

750

1000

1250

a

VN

Ab

titer

500

1000

1500

b

IFN-γ IL-5

100

200

300

400

no adjuvant adjuvant

cytokine productio(pg/m

l)

Vaccination of macaques with inactivated SARS CoV (w/wo alum)

VN-Abs LST


0 5 10 15 200

250

weeks after vaccination

. . . . . .0 0

100

ctrl 106 107 ctrl 106 107

on

1

2

3

4

5

6

c

1

2

3

4

5

6


P<0.05 P<0.05

ctrl 106 107 ctrl 106 107

SAR

S C

oV T

CID

50 (l

og10

/g ti

ssue

)

0

1

2

3

4

5

6

d

0

1

2

3

4

5

6


ctrl 106 107 ctrl 106 107

SAR

S CoV Eq.

(log10 /g tissue)

P<0.05 P<0.05Lungs

(titration)Lungs

(RT-PCR)

Vaccination of macaques with inactivated SARS CoV:Virus neutralizing antibody titers after 2 vaccinations

64

128

256

VN ti

ter

Rotterdam studies

8

16

32

64

SAR

S C

oV V

D.L.

Vaccine - + ++ - + ++Adjuvant - - - + + +

Dose and adjuvant dependent VN antibody induction

Vaccination of macaques with inactivated SARS CoV:SARS CoV PCR titers in the lungs 5 days after challenge

1 0 4

1 0 5

1 0 6

50 /g

Rotterdam studies

1 0 0

1 0 1

1 0 2

1 0 3

1 0

SCV

TCID

5

D.L.

Vaccine - + ++ - + ++Adjuvant - - - + + +

**

Dose and adjuvant dependent protection!

86


Diffuse alveolar damage in virus controls

Peribronchiolar and perivascular lymphoid Peribronchiolar and perivascular lymphoid cuffing in macaques vaccinated with cuffing in macaques vaccinated with

inactivated SARSinactivated SARS--CoV with alumCoV with alum

30

35

40

45

50

ils p

er fi

eld

Eosinophil counts in the bronchus of Eosinophil counts in the bronchus of macaques vaccinated with inactivated macaques vaccinated with inactivated SARSSARS--CoV with alumCoV with alum

0

5

10

15

20

25

30

AG

865

AH

246

AH

444

AH

494

AH

547

AH

635

AH

639

AH

647

AG

958

AH

451

AH

531

AH

799

AF4

37

AH

081

AH

260

AH

638

AG

927

AG

940

AH

439

AH

514

AH

201

AH

622

AH

625

AI0

42

buffer adjuvant 10*5.9 virus 10*5.9 virus +adjuvant

10*6.9 virus 10*6,9 virus +adjuvant

Num

ber o

f eos

inop

h

87


IL-4

120

pt

IFN-Y400

t

IL-13

200

Presence of ILPresence of IL--13 mRNA in 13 mRNA in SARSSARS--CoV challenged vaccinated macaquesCoV challenged vaccinated macaques

PBS SARS CoV vaccine + SARS CoV0

40

80

rela

tive

tran

scri

pnu

mbe

r


100

200

300

rela

tive

tran

scri

pnu

mbe

r


50

100

150

rela

tive

tran

scri

ptnu

mbe

r

Since eradication smallpox:more animal poxvirus infections in humans?

Wolfs et al E I D 2002

cowpox

Pelkonen et al. E.I.D. 2003; 9:1458-1461

ProMED-mail 9 Jan 2003

Wolfs et al. E.I.D. 2002

monkeypox

cowpox

cowpox

7†

XX

C

Preventive smallpox vaccination with Elstree-RIVM against lethal MPXV infection

Plasma viral load (log10)

C: Control

0 5 10 15 20 25 30

3

4

5

6

XX

E

M/E

M/M

E*

Time after infection (days)

E: Elstree-RIVM

E*: Elstree-BN

M/E: MVA + Elstree-RIVM

M/M: MVA + MVA

Stittelaar et al 2005 JVI

88


Survival of monkeys subjected to different post-exposure treatments after lethal MPXV infection

75

100

III: Cidofovir [5 doses] (n=6)VI: HPMPO-DAPy [6 doses] (n=

IV: Cidofovir [6 doses] (n=6)

Time (days after exposure)

0 1 3 5 7 10 13 18 21 28

% S

urvi

val

0

25

50

II: Elstree (n=6)

V: HPMPO-DAPy [5 doses] (n=

I: Controls (n=17)*

V VVVVV VVVVV

V VVVV VV VVVV V

VI:V:IV:III:II:

Stittelaar et al., Nature, 2006

NiVNiVHeVHeV

TuV CDV RPVMV

hPIV3bPIV3

SeVParamyxovirinae Respirovirus

MorbillivirusDNA Maximum likelihood, Polymerase ORF

PDV

Henipahvirus

Order Order MononegaviraleMononegavirales, family s, family ParamyxoviridaeParamyxoviridae

Morbilliviruses: a continuing story!!!

Osterhaus et al., Nature 1988Jensen et al.,Science 2002

0.1

hPIV1

NDV

MuVSV5hPIV2

hMPVhMPVAPV

hRSV

bRSV

SV41

LPMV

Pneumovirinae

y

RubulavirusMetapneumovirus

Pneumovirus Avulavirus

Morbilliviruses crossing species barriers

Antarctic 1955: CDV in C b t l

CDV in Serengeti lionsVaccine 1994

Crabeater seals CDV in Baikal sealsNature 1988

CDV in Caspian sealsEID 2000

DMV in Med. monk sealsNature 1997

CDV in Jap. macaquesVet. Microbiol 1989

89


Morbilliviruses crossing species barriers

Antarctic 1955: CDV in C b t l

CDV in Serengeti lionsVaccine 1994

Crabeater seals CDV in Baikal sealsNature 1988

CDV in Caspian sealsEID 2000

DMV in Med. monk sealsNature 1997

CDV in Jap. MacaquesVet. Microbiol 1989

should we continue measles vaccination for ever?

Evaluation of new generation MV vaccines

Evaluation of alternative vaccination routes

90


Evaluation of alternative vaccination routes

CONCLUSIONS:CONCLUSIONS:

In the last decade alone a dozen new respiratory viruses have been identified in humansSeveral are the result of inter-species transmission The relative clinical impact of most is not yet knownThe relative clinical impact of most is not yet known Candidate vaccines are being developed with classical and novel technologies Novel administration routes (intranasal, dry powder) are exploredSafety and efficacy of most of candidate vaccines is not yet clear Rapid response is crucial to contain newly emerging viruses

Virology Bernadette van den HoogenMiranda de GraafTheo BestebroerMonique SpronkenLeo Sprong

Animal experimentsGeert van AmerongenRobert Dias-d’Ullois

PathologyThijs Kuiken

Roderick TangJeanne SchickliFiona FernandesLeenas Bicha

Acknowledgements: hMPV

MedImmune, Inc

p gChantal VerheyenOnno SchaapRob van LavierenEdwin FriesMartin SchuttenBert NiestersJan de JongEefje SchrauwenKarim HussainTheo HarmsenRik de SwartRon Fouchier

j

Electron MicroscopyBé Niemeyer

ClinicalGerard van DoornumJantijn FockensRonald de Groot

StatisticsWalter Beyer

Leenas BichaAurelia HallerRichard SpaeteNancy UlbrandtArnita BarnesKannaki SenthilJeanne SchickliRichard Spaete

James SimonRoel van EijkJeroen Maertzdorf

91

Osterhaus - Vaccine prevention of non-influenza viral respiratory disease:current and futureAcknowledgements: Rotterdam SARS studies

Dept. Virology,Erasmus MCTheo BestebroerBé NiemeyerGeorgina Aron

WHO & Members of the WHO SARS aetiology team

Centers for Disease Control & Prevention, Atlanta, USACentral Public Health Laboratory, London, UK

Public Health Laboratory Centre, Hongkong, SAR ChinaP i f W l H it l SAR ChiRobert Dias-d’Ullois

Gerard van DoornumMartin SchuttenBert NiestersGeert van AmerongenBart HaagmansByron MartinaThijs KuikenGuus RimmelzwaanRon FouchierAb Osterhaus

Prince of Wales Hospital, SAR ChinaQueen Mary Hospital, SAR China

Singapore General Hospital, SingaporeFederal Laboratories for Health Canada, Winnipeg, Canada

Health Canada, Ottawa, CanadaBernhard-Nocht Institute, Hamburg, Germany

Institut Pasteur, Paris, FranceNational Institute of Infectious Disease, Tokyo, Japan

Several industrial partnersJames Simon ViroNovative BV

Emerging

Department of Virology

Emergingvirus infections

Email: [email protected]

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