1. Crimean-Congo Haemorrhagic Fever DIAGNOSIS Hervé Zeller National Reference Center - WHO Collaborating Centre for Arboviruses and Viral Haemorrhagic Fevers, Institut Pasteur, Lyon

2. Viral Haemorrhagic Fevers Flaviviridae (dengue, yellow fever, Groupe TBE)

3. Arenaviridae (Lassa, Junin, Machupo, Guanarito) Viral Haemorrhagic Fevers Flaviviridae (dengue, yellow fever, Groupe TBE)

4. Filoviridae (Ebola, Marburg) Arenaviridae (Lassa, Junin, Machupo, Guanarito) Viral Haemorrhagic Fevers Flaviviridae (dengue, yellow fever, Groupe TBE)

5. Filoviridae (Ebola, Marburg) Arenaviridae (Lassa, Junin, Machupo, Guanarito) Bunyaviridae (CCHF, RVF, Hantaviruses) Viral Haemorrhagic Fevers Flaviviridae (dengue, yellow fever, Groupe TBE) Envelopped RNA viruses

6. FamilyGenus VIRUS DISTRIBUTION FlaviviridaeFlavivirusYellow FeverAfrica South America Dengue 1,2,3,4. Tropical areas Omsk HFRussia AlkhurmaSaudi Arabia Kyasanur Forest HFIndia BunyaviridaePhlebovirusRift Valley FeverAfrica, Saudi Arabia Nairovirus Crimean-Congo HF Africa, Eurasia HantavirusHantan Dobrava Puumala Eurasia Sin Nombre, AndesAmericas

7. Hemorrhages are inconstant : Emerging part of the iceberg …Most frequently asymptomatic infections +++

8. Biosafety Issues Related to Haemorrhagic Fever Viruses Criteria for classification –Disease severity –Transmissibility to laboratory workers –Availability of treatment –Availability of vaccine Classification BSL 1 to BSL4

9. CCHF VIABILITY SENSITIVITY TO DESINFECTANTS: sodium hypochlorite 2%, glutaraldehyde 2%, formaldehyde SENSITIVE TO DESSICATION INACTIVATION : IRRADIATION UV TEMPERATURE : 1 hour 60°C not complete inactivation beta propiolactone 4°C not complete inactivation Triton X100

10. S M L N G1 G2 10 nm Nairovirus structure L

11. CCHF Nairovirus genome _____________________________________________ SegmentNucleotidesAmino acids Protein _____________________________________________ S1659-1712442-482 N M48881551 G1 G2 NSm? L122554036 L? _____________________________________________

12. VHF SUSPECT CASE

13. Malaria – Hepatitis – Typhoidis – Toxicosis Septicemia – Leptospirosis Rickettsiosis…

14. VHF SUSPECT CASE Epidemiological data, risk evaluation biological analysis, differential diagnostic Malaria – Hepatitis – Typhoidis – Toxicosis Septicemia – Leptospirosis Rickettsiosis…

15. VHF SUSPECT CASE Contact between clinicians and biologists Epidemiological data, risk evaluation biological analysis, differential diagnostic Malaria – Hepatitis – Typhoidis – Toxicosis Septicemia – Leptospirosis Rickettsiosis…

16. CCHF : laboratory data LEUCOPENIA, particularly neutropenia THROMBOCYTOPENIA Hematocrite increases early then falls down ASL, AST levels increases Proteinuria and hematuria Mild azotemia, bilirubine increase

17. Viral detection: blood specimen –RT-PCR (nested) – Cell culture (Vero E6 cells) CCHF : laboratory diagnosis

18. Viral detection: (blood specimen) –RT-PCR (nested) – Cell culture (Vero E6 cells) Antibody detection : (serum sample) -IFA -ELISA IgM (immuno-capture) IgG -NT CCHF : laboratory diagnosis

19. Viremia 10-12 days (although afebrile). Can be detected by PCR up to day 16 By day 9 all patients will have IgM or IgG antibody Information needed : DATE OF ONSET OF FEVER

20. viremia 5 IgM RT-PCR ELISA IgM IgG IFA CCHF : viral/antibody kinetics Viral isolation 010 IgG 16 IgM duration: 2-3 months up to 6 months…

21. Primers for RT-PCR on S segment From J. Smith, 1990

22. RT-PCR /Southern blot hybridization/antibody : retrospective study From Burt et al, J Virol Methods 1998, 70:129- 137 Day of illness PCR + Virus + PCR + Virus - PCR - Virus + PCR - virus -Ab + Total tested 31113 411 1 2 53235 65343815 75489 812477 95156 10333 11121155 121122 1314388 1441388 152133 16 2 133 18111 Total18346226580

23. RT-PCR /Southern blot hybridization/IFA antibody : 26 samples from 19 patients from day 3-12 of illness From : Burt et al, J Virol Methods 1998, 70:129-137 day of illness PCR + Virus + PCR + Virus - PCR - Virus + PCR - virus - IFA Ab + Total tested 311 411 511 64125 731155 861289 9222 10 11111 12111 Total141381926

24. Hyalomma sp. ticks RT-PCR Viral isolation

25. DUGBE AP 92 AnD 15786 ArD 8194 ArTeh 193-3 HD 49199 ArMg 951 C 68031 ArB 604 HD 38562 ArD 39554 ArD 97264 ArD 97268 HAZARA 3 2 1 1 1 1 1 3 1 1 2 3 2 2 4 2 2 3 2 2 2 1 3 1 Grèce Sénégal Mauritanie Iran Madagascar Chine Rép. Centrafr. Burkina Faso ORIGINEPROFIL RFLP (100) (96) (100) (99) (100) (84) (57) 536 pb amplicons of the S fragment of CCHF genome using primers CSDR3/CSDF2. RFLP with Hinf I, Hae III, and Alu I endonucleases Rapport IP Dakar 1993

26. Phylogenetic analysis of 46 partial sequences (219 bp) of the S segment of CCHF virus Turkey 2003

27. Phylogenetic analysis of 46 partial sequences (219 bp) of the S segment of CCHF virus. Seven major genetic groups. Strains from the Middle and Far East and from different African regions cluster in clearly separated groups. TURKEY 2003 Preliminary data: 96-98% homology with strains from the Balck Sea area and Kosovo KOSOVO AF404507; STAVROPOL AF481802 ; DROSDOV U88412 ; ROSTOV AY277672 Drostein et al, J Clin Microbiol 2002, 40 1122

28. National Reference Center - WHO Collaborating Centre for Arboviruses and Viral Haemorrhagic Fevers, Institut Pasteur, Lyon Marie-Claude Georges Isabelle Schuffenecker Ingrid Marendat Séverine Murri Hervé Zeller BSL 4 BSL 3