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)
Viral Haemorrhagic Fevers

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

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

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

6. Family Genus VIRUS DISTRIBUTION
Flaviviridae Flavivirus Yellow Fever Africa South America
Dengue 1,2,3,4. Tropical areas
Omsk HF Russia
Alkhurma Saudi Arabia
Kyasanur Forest HF India
Bunyaviridae Phlebovirus Rift Valley Fever Africa, Saudi Arabia
Nairovirus Crimean-Congo HF Africa, Eurasia
Hantavirus Hantan Dobrava Puumala Eurasia
Sin Nombre, Andes Americas

7. Hemorrhages are inconstant : Emerging part of the iceberg
…Most frequently
asymptomatic infections +++
.Among all the hemmorrhagic fevers, hemorrhages are the smaller part of the iceberg “  Symptoms ”
In fact, those diseases are very frequently asymptomatic :
either because we are faced with mild infections showing only non hemorrhagic symptoms,
or because it is a really asymptomatic infection.
I shall give you soon the example of what we have shown in Ebola 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
Triton X100

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

11. CCHF Nairovirus genome
_____________________________________________
Segment Nucleotides Amino acids Protein
S N
M G1 G2 NSm?
L L?

12. VHF SUSPECT CASE

13. Malaria – Hepatitis – Typhoidis – Toxicosis Septicemia – Leptospirosis
VHF SUSPECT CASE
Malaria – Hepatitis – Typhoidis – Toxicosis
Septicemia – Leptospirosis
Rickettsiosis…

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

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

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

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

19. CCHF : laboratory diagnosis
Viremia 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. CCHF : viral/antibody kinetics
IgM
IgG
viremia 5 10
RT-PCR 16
Viral isolation
ELISA IgM IgG
IFA
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
Day of illness
PCR + Virus +
PCR + Virus -
PCR - Virus +
PCR - virus -
Ab +
Total tested 3 1 4 2 5 6 8 15 7 9 10 11 12 13 14 16 18
Total 34 22 65 80
From Burt et al,
J Virol Methods 1998, 70:

23. RT-PCR /Southern blot hybridization/IFA antibody :
26 samples from 19 patients from day 3-12 of illness
day of illness
PCR + Virus +
PCR + Virus -
PCR - Virus +
PCR - virus -
IFA
Ab +
Total tested 3 1 4 5 6 2 7 8 9 10 11 12
Total 14 19 26
From :
Burt et al, J Virol Methods 1998, 70:

24. Hyalomma sp.
ticks
RT-PCR
Viral isolation

25. 536 pb amplicons of the S fragment of CCHF genome using primers CSDR3/CSDF2. RFLP with Hinf I, Hae III, and Alu I endonucleases
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
Grèce
Sénégal
Mauritanie
Iran
Madagascar
Chine
Rép. Centrafr.
Burkina Faso
ORIGINE
PROFIL
RFLP
(100)
(96)
(99)
(84)
(57)
Rapport IP Dakar 1993

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

27. Seven major genetic groups.
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 AF ; DROSDOV U88412 ; ROSTOV AY277672
Drostein et al, J Clin Microbiol 2002,

28. BSL 3 BSL 4 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 3
BSL 4