1. ENIVD 06/06 02:19 First international proficiency study on West Nile virus molecular detection. Matthias Niedrig, Sonja Linke Christian Drosten Hervé Zeller Source: H. Gelderblom, RKI ROBERT KOCH INSTITUT West Nile

2. ENIVD 05/06 02:19 Distribution of disease vectors and WN virus in Europe source: Snow & Ramsdale, Biologist (2002) 49 (2) Hubalek & Halauzka; EID, (1999) 5 (5) Aedes albopictus Ochlerotatus atropalpus Ochlerotatus japonicus WN

3. ENIVD 05/06 02:19 Import of suspected or confirmed WN cases into Europe Period of time Imported from Imported toViral agent Number of cases/deat hs Type of travellerReference Nov 1994Ivory CoastSwitzerlandEbola virus1/0Repatriation7 Apr 1996BrazilSwitzerlandYellow Fever virus1/1Tourist8 Sep 1997GhanaGermanySuspected VHF 1 1/1Refugee9 Nov 1998The GambiaBelgiumSuspected VHF 2 1/1Tourist10 Aug 1999Ivory CoastGermanyYellow Fever virus1/1Business11 Jan 2000i.a. Ivory CoastGermanyLassa virus1/1Tourist12 Feb 2000Sierra LeoneUKLassa virus1/1Repatriation13 Mar 2000NigeriaGermanyLassa virus1/1Medical transport14 Jul 2000Sierra LeoneThe NetherlandsLassa virus1/1Business15 Dec 2000KenyaGermanySuspected VHF 3 1/1Tourist16 Mar 2001Sierra LeoneGermanySuspected VHF 2 1/0Repatriation17 Mar 2001i.a. ChileFranceHantavirus1/0Tourist18 Aug 2001BulgariaGermanyCCHF virus1/0TouristPers. comm. a) Sep 2001ChadFranceRVF virus2/0Business19 Nov 2001The GambiaBelgiumYellow Fever virus1/1Tourist20 Sep 2002NepalSpainSuspected VHF 1 1/0TouristPers. comm. b) Oct 2002CameroonRep.of IrelandSuspected VHF 2 1/0BusinessPers. comm. c) Feb 2003Sierra LeoneUKLassa virus1/0Business21 Feb 2003i.a Chinadifferent countriesSARS-CoV33/1Tourist/Business6 Mar 2003USAFranceWest Nile virus1/0TouristPers. comm. d) Aug 2003USAGermanyWest Nile virus2/0Tourist22 Jun 2004USAFranceHantavirus1/1TouristPers. comm. d) Sep 2004USAGermanyWest Nile virus1/0Tourist22 Oct 2004USAFranceWest Nile virus3/0TouristPers. comm. d) Oct 2004TunisiaFranceWest Nile virus1/0TouristPers. comm. d) Nov 2004SenegalFranceCCHF virus1/0RepatriationPers. comm. d) eight cases in total

4. ENIVD 05/06 02:19 cell culture supernatants of infected cells with the respective viruses heat treatment for 1h at 56°C and gamma irradiation of 30 kGy check for infectivity by cell cultivation dilute with human plasma, aliquots of 100 µl are frozen at –70°C before freeze-drying for 12 hours samples were distributed by normal mail service Preparation of PCR samples for the External Quality Assurance (EQA)

5. ENIVD 05/06 02:19 Phylogenetic tree of some tested WNV samples and reference strains based on the UPGMA method by using 216 nt sequence of 5'-UTR/C gene fragment Source: Alex Platonov

6. ENIVD 05/06 02:19 Evaluation of the WN-PCR EQA

7. ENIVD 05/06 02:19 Evaluation of WN PCR diagnostic by EQA (1) WNV-1 copy N° [ge / ml] N°of laboratories % 100.000 2896,6 10.000 2689,7 1.000 2275,9 100 1965,5 WNV-2 1034,5 YF, SLE, DEN 1 - 4, TBE, neg. 42,9

8. ENIVD 05/06 02:19 Evaluation of the WNV-PCR diagnostic by EQA (2)

9. ENIVD 05/06 02:19 Evaluation of WNV PCR diagnostic by EQA (3) Log RNA copies / ml Fraction of laboratories Probit analysis of the fractions of laboratories achieving positive results (y-axis) in relation to the virus RNA concentration in a given positive sample (x- axis). Data points represent individual samples in the proficiency test panel. The thick line is the regression line calculated on the basis of a probit model (dose- response curve), the thin lines are 95% confidence lineage 2 lineage 1

10. ENIVD 05/06 02:19 Results WNV PCR EQA Does the PCR method used has an influence on the quality of the results? n= 28; 15 real time PCR, 11 RT-PCR, 2 commercial Do other factors influence the PCR results ? genome preparation: n= 28, 19 Qiagen, 5 Roche, 4 other There is no significant correlation between the PCR method and the quality of the results. There is no significant correlation between the genome preparation method and the quality of the results.

11. ENIVD 05/06 02:19 - Conclusions - WNV PCR EQA using cell culture supernatants, diluting samples with human plasma and freeze-drying before shipping is useful for sample preparation - gamma-irradiation is a suitable method for inactivation of samples 7 laboratories showed excellent results (sensitivity of 100%), whereas 10 laboratories that were less sensitive need to improve their methods PCR methods for detection of WNV should also detect lineage 2 which is now present in Europe. 4 laboratories create false positive results

12. ENIVD 05/06 02:19 EQA studies performed by the ENIVD between 1999 and 2005

13. ENIVD 05/06 02:19 What‘s coming next

14. ENIVD 05/06 02:19 Acknowledgement: Parts of this project were supported by the European Network for diagnostics of "Imported" viral Diseases (ENIVD) presently funded by DG-SANCO of the European Community under the program „AIDS and other communicable diseases“ H. Bin, Central Virol. Laboratory, Sheba Medical Center, Tel Hashomer, Israel D. J. Gubler, CDC, Fort Collins, USA A. Platonov, Central Research Institute of Epidemiology, Moscow, Russia H. Zeller, Jean Mérieux-INSERM P4 laboratory, Institut Pasteur, Lyon, France C. Drosten, Bernhard Nocht Institut, Hamburg, Germany Stephan W. Aberle, Franz Heinz, Medical University of Vienna, Austria

15. ENIVD 06/06 02:19 European Network for diagnostics of "Imported" viral Diseases (ENIVD) for further information: www.enivd.de www.enivd.net www.enivd.org