recent advances in the field of arbovirus diagnostics and ... viral diseases, may 23 rd /24 th, 2013...

Institut für neue und neuartige Tierseuchenerreger (INNT)Institute of Novel and Emerging Infectious Diseases

Martin H. Groschup

II cycle about zoonosis and emergent diseases vector-borne viral diseases, May 23rd/24th, 2013

Recent advances in the field of arbovirus diagnosticsand presentation of the current status of European research networks on emerging diseases


© ARGE FLI Riems

89 laboratories (each 60 m2)163 stables

BSL2, BSL3** BSL3enh. 54 (9x6) cattle + 72 (9x8) SR/P

BSL3ag 40 (8x5) cattle + 16 (2x8) SRP

BSL4


Inst. of Novel and Emer-ging Infectious Diseases

Head:

Prof. Dr. M.H. Groschup

NRL for ScrapieProf. Dr. M.H. Groschup

Dr. C. HoffmanDr. A. Buschmann

Transg. AnimalsSPF-Facility

Dr. M Keller.

NRLs for Japan Encephalitis /

West Nile Virus Dr. U. ZieglerDr. M. Keller


NRL for Equine Encephalomyelitis

Dr. M. Keller Prof. Dr. M.H. Groschup

NRL for Nipah / HendraDr. A. Buschmann


NRL for BSEProf. Dr. M.H. Groschup

Dr. C. HoffmanDr. A. Buschmann

Hepatitis E-VirusPD Dr. R. Ulrich

Dr. M. EidenProf.Dr.M.H.Groschup

Veterinary Branch of theNational Zoonoses Platform

Germany Prof. Dr. M H. Groschup

NRLs for Rift Valley Fever

Crimean Congo HFEbola

Prof. Dr. M.H. Groschup Dr. M. Eiden

NRL for Hantaviruses /

Network: Rodent associated pathogens

PD Dr. R. Ulrich

Deputy:Dr. A. Balkema-

Buschmann

Friedrich-Loeffler Institut, Isle of RiemsViruses and Prions

Institute of Molecular BiologyInstitute of InfectiologyInstitute of ImmunologyInstitute of Virus DiagnosticsInstitute of Novel and Emerging Infectious Diseases


Diagnosis of Arbovirus Infections

Virus isolation

Virus genome detection

Virus antigen detection

Antibody detecion (serology)



Virus isolation

Virus genome detectionPCR basedMicroarrayNGS




Lin Liu,* Yinhu Li, Siliang Li, Ni Hu, Yimin He, Ray Pong, Danni Lin, Lihua Lu, and Maggie Law. Comparison of Next‐Generation Sequencing Systems. J Biomed Biotechnol. 2012: 251364. doi: 10.1155/2012/251364 PMCID: PMC3398667

Beijing Genomics Institute



Virus isolation

Genome detectionPCR basedMicroarrayNGS



Flavivirus Usutu Virus


Emergence of Usutu virus in Germany

2010year USUV positive mosquito pools*

2010 1

2011 213

2012 18

*BNI, Schmidt-Chanasit

2011 2012


Mass mortality of wild birds in 2011Emergence of Usutu virus in southwest Germany


bird speciesnumber tested positive /

number of dead birds tested

Turdus merula (blackbird) 55 / 81 (67%)

Sturnus vulgaris (common starling) 1 / 7 (14%)

Serinus canaria domestica (canary) 2 / 2 (100%)

Strix nebulosa (great grey owl) 6 / 6 (100%)

Alcedo atthis (common kingfisher) 2 / 2 (100%)

Passer domesticus (house sparrow) 1 / 1 (100%)

Garrulus glandarius (Eurasian jay) 0 / 1

Carduelis chloris (greenfinch) 0 / 5

Turdus pilaris (fieldfare) 0 / 3

Perdix perdix (grey partridge) 0 / 2

TOTAL 67 / 110 (60%)

Carduelis chloris (greenfinch)

Sturnus vulgaris (common starling)

Becker et al., PLoS ONE, EID 2011Jöst et al., Am J Trop Med Hyg. 2011 BNI + KABS + FLI + NABU 2011

Mass mortality of wild birds in 2011Emergence of Usutu virus in southwest Germany


NGS sequencing platform (FLI: Martin Beer, Dirk Höper)Full-lenght sequencing (11 kbp)

more than 99% sequence homology with Austrian isolate (Vienna, 2001)

100% homology with a German mosquito isolate from year 2010



Virus isolation


Virus antigen detectionHAWBELISAIHC

Antibody detecion (serology)IFAELISAVNTHAI

Vertebrate and mosquito surveillance (FlaviV/AlphaV)



Competent vectors are present (e.g. Culex pipiens)Climate change

Wild birds (migratory and resident)

Horses

Mosquitoes

?

WNV surveillance in Germany2005-2009

Domestic fowlfree-ranged chickens, ducks, geese

http://www.google.ca/url?sa=i&rct=j&q=mosquito&source=images&cd=&docid=113q2Tg9RUZB_M&tbnid=WREpHoG8aE6Y_M:&ved=0CAUQjRw&url=http%3A%2F%2Fwww.beyondpesticides.org%2Fmosquito%2F&ei=Lo6FUfTaJoLXswb35oHAAg&psig=AFQjCNHr3dopD2C4qD1YNmod6xzBwWH-fA&ust=1367793549019907



Horses


Mosquitoes

WNV surveillance in Germany2005-2009

1.042 horses tested negative

962 poultry sera tested negative

not done

3.030 sera tested serologically, of which 74 were positive migratory birds

Seidowski et al. 2010, Vect. Borne Zoon. Dis, 2010 Oct;10(7):639-47

Ziegler et al. 2010, Arch. Virol. 2010 Jun;155(6):965-9




Horses


Mosquitoes

WNV surveillance in Germanysince 2010

546 sera were tested, of which 25 were positive migratory birds

Ziegler et al. 2012, Zoonoses and Public Health 59 Suppl 2:95-101

not done



Competitive WNV‐IgG‐ELISA

total reactive

Reactivityconfirmedby WNV‐IgM‐ELISA

WNVvaccinated

TBEV‐reactorsVNT

HE 84 4 ‐ 1 3

NI 221 2 ‐ 1 2

MV 565 3 0 ‐ ‐

SA 771 11 0 ‐ ‐

BW 3 0 ‐ ‐ ‐

BB 779 0 ‐ ‐ ‐

Total 2.423 20 0 2 5

Horse ‘sera‘ drawn from - sick animals in horse clinics - carcasses in rendering plant


Ziegler et al. 2013 submitted


Wild birds migratory and resident)

Horses


Mosquitoes


2.423 horses, of which 2.403 tested negative, 20 reactors were negative in IgM ELISA, VNT (2x vacc.

5x TBEV)Screening ongoing

546 sera tested serologically, of which 25 were positive migratory birds

not done




Virus isolation





Flavivirus Usutu virus

RVF virus


Countries with endemic disease and substantial outbreaks of RVF

Distribution and Transmission

http://www.cdc.gov/ncidod/dvrd/spb/mnpages/dispages/rvf/rvfmap.htm

Countries known to have some cases, periodicisolation of virus, or serologic evidence of RVF

www.mikroproduction.com

Potential vectors in Europe


• Family: Bunyaviridae; Genus: Phlebovirus

• (-)ss RNA; enveloped

• Zoonotic Arbo-Virus: transmission by mosquitoes(Culex, Aedes, Anopheles)

Habjan et al., 2009

Rift Valley fever virus (RVFV)

education.expasy.org/.../Bunyaviridae_virion.jpg


RVF serology ELISAs

• ID Screen Rift Valley Fever Competition multi-species (ID Vet)• ID Screen IgM Capture ELISA (ID Vet)

• BDSL Inhibition • BDSL Capture IgM

• OVI RVFV IgG• OVI RVFV IgM } Prototypes

education.expasy.org/.../Bunyaviridae_virion.jpg


Test sera

RVFV ΔGn

Protein G

Establishment of a novel Gn based ELISA


1952 samples tested in South Africa at the NICD (J. Paweska)

Sensitivity: 94,56%Specificity: 95,57%

Establishment of a novel Gn based ELISA



Virus isolation






RVF Virus, CCHFV


Enzootic transmission

horizontal transmission- between males and females

vertical transmission- transovarial- transstadial larvae

nymphs

adults

engorged females

eggs

Zooanthroponotictransmission

Human-to-humantransmission

Photos by Zati Vatansever, Kafkas University, Turkey

Crimean-Congo Hemorrhagic Fever Virus

http://www.hamburg-web.de/fotos/original/10111-kuh.jpg


dotted line: northern limit of distribution of H. marginatumblack dots: isolation of CCHFVwhite dots: detection of CCHFV-specific antibodies(Hubalek and Rudolf, 2012)

endemic areas


Year Albania Bulgaria Greece Kosovo Turkey1953-1974 1105/190 (17)1975-1996 279/32 (11)1985-1987 32/5 (17)

1995 46/7 (15)1996 9/5 (56)1997 20/4 (20) 0/0 (0)1998 15/3 (20) 1/0 (0)1999 5/2 (40) 3/2 (67)2000 10/1 (10) 1/0 (0)2001 7/0 (0) 18/5 (28) 31/7 (22)2002 56/12 (21) 14/3 (21) 17/0 (0)2003 14/2 (14) 6/3 (50) 133/6 (5)2004 18/6 (33) 16/2 (13) 249/13 (5)2005 14/2 (14) 11/5 (46) 266/13 (5)2006 7/2 (29) 4/2 (50) 438/27 (6)2007 2/0 (0) 713/33 (5)2008 6/1 (17) 1/1 (100) 5/1 (20) 1315/63 (5)2009 12/2 (17) 1300/62 (5)

EDENext - WP 2Emergence and spread of CCHFV

Human cases clinical cases / fatalities / rate


Company/Institute Type Species Principle D-SN, D-SP Price

Biological DiagnosticSupplies Limited(BDSL)

IgMELISA

humanmAk+Antigen+Serum

+Conjugatenoinformation

1000 Tests2750 Euro

VectorBest IgGELISA

humanAntigen+Serum

+Conjugateno information

96 Well179 USD

Gentaur IgG, IgMELISA

humanAntigen+Serum

+Conjugateno information

2x96 Well1597 Euro

Euroimmun IgM, IgGIFA

humantransfected Cells (Gc, N)

+Serum+Conjugate

IgM97%, 98%IgG90%, 100%

20 x 10924 Euro

Commercial diagnostic assays

Mertens M, Schmidt K, Ozkul A, Groschup MH (2013)The impact of Crimean-Congo hemorrhagic fever virus on public healthAntiviral Research 98 (2013) 248–260


CCHF: Diagnostic methods at FLI

Molecular biology

realtime RT-PCR(Woelfel et. al 2007)

Serology

IFAEuroimmun

adapted to animal sera

ELISA1) In-house ELISA

2) Vector Best ELISA(adapted to ruminants)

Western-blot assayin-house

based on recombinant

N-protein

http://www.hamburg-web.de/fotos/original/10111-kuh.jpg


^q


West Nile Virus FP7 projects:€14M

2011 2012 2013 2014 2015 20162010

EDENext

PREDEMICS

SILVER

EUROWESTNILE

EMPERIE

WINGS

VECTORIE

EVA

ARBO-ZOONET

© Christian Desaintes, DG Research, Health Infectious Diseases, European Commission


FP7 funded research infrastructures

EVA

Transvac

NADIR (BSL3 animal)

ERINHA (BSL4)


BSL3 BSL4


Jonas Schmidt-ChanasitPetra EmmerichStephan Günther

Ute Ziegler Marc MertensIsolde SchusterMartin EidenAnne Balkema-BuschmannAriel Vina-RodriguezJoke AngenvoortSusanne JäckelMarkus KellerMartin H. Groschup

Norbert BeckerHanna Jöst

Kommunale Aktionsgemeinschaftzur Bekämpfung der Schnakenplage

State veterinaryinvestigation laboratory

Kerstin Müller, FU BerlinMichael Lierz, JLU Giessen Dominik Fischer, JLU GiessenMonika Rinder, LMU Munich

Judith TyczkaJaqueline LambertzKlaus-Jürgen DannerMarkus ReuleClaudia SauerwaldHans-Peter HamannMartin Peters

Stephanie HillmersWulf-Iwo BockLothar HoffmannDirk SteinhauerKarl ZimmerRobert HövelerSylvia Blahak…..

Renaud LancelotNoël Tordo, Pasteur InstitutPhillipe Pourquier, ID-VetLoïc Comtet, ID-VetRobert Farkas, Budapest UniversityZati Vatansever, Kafkas University

Helge KampenDirk Höper

Doreeen Werner , ZALF Müncheberg

recent advances in the field of arbovirus diagnostics and ... viral diseases, may 23 rd /24 th, 2013...

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