The Department of Emerging and Re-emerging Infections, Central Research Institute

1. The Department of Emerging and Re-emerging Infections, Central Research Institute of Epidemiology; Moscow, Russia 2000-2004 Contacts: platonov@pcr.ru

2. West Nile fever Crimean-Congo hemorrhagic fever Tick borne encephalitis Omsk hemorrhagic fever Japanese encephalitis other flavivirus infections hantaviral infection are subjects we deal with

3. Borreliosis Rickettsiosis Enteroviral infection Meningococcal infection Haemophilus influenzae type b Pneumococcal infection are subjects we deal with

4. treatment and control epidemiology and surveillance genodiagnostics and genotyping

5. Russia: Annual sum of effective temperatures (t >= 10oC) Moscow 200-500 500-1000 1000-1500 1500-2000 2000-2500 2500-3000 3000-3500

6. West Nile fever: classical and molecular epidemiology, diagnosis, pathogenesis, treatment

7. Molecular investigation of the simultaneous outbreaks of West Nile fever in the USA and Russia in 1999. (Bioterrorist's attack? No, the attack of nature) The beginning – last week of July, The peak of outbreak – first week of September ?

8. Genetic distance of West Nile virus isolates based on complete nucleotide sequence "New York group" – equine, flamingo, human, and mosquito isolates in 1999, as well as the USA strains from 2000-2002 and Israel strains from 1999-2001 Astrakhan strain, 1999 Italian strain, 1998 Kenyan strain, 1998 Mosquito isolate, Romania 1996 and human clinical isolate, Volgograd 1999, (as well as the Volgograd strains from humans, crows and C.pipiens, 1999-2002) Eg-101 Kunjin strains Genetic distance

9. The nearest relative of the Volgograd WN isolate was the mosquito isolate from Romania-1996. The strains differed from each other in 46 nucleotide positions, 43 of them were located in coding region. 35 mutations were silent so the strains differed in 8 amino acid only. So the homology of WN-Volgograd-4 and WN-Romania-1996M strains was 99.77% for aa sequence and 99.58% for nt sequence. Is the difference of 0.42% really small? Do these strains belong to the same clone?

10. Problem: the origin of "Volgograd" variant of West Nile virus. Is this variant endemic or are several variants of West Nile virus introduced in Volgograd region every year by migrating birds?

11. >500 Volgograd region Number of WNF cases >35 32 15 0 14 >5 West Nile fever in Russia in 1999-2003 WNF outbreak in 1999: Volgograd - 38 deaths; Astrakhan - 5 deaths; Krasnodar - 3 deaths. Astrakhan region Krasnodar Volgograd Astrakhan Number of WNF cases 95 49 ? 33 24 12 ?

12. Genetic tree based on envelope gene fragment, 512 nt, of WN virus isolates from Volgograd 1999-2002 Volgograd WN clone appear to be endemic in Volgograd region because the same isolates were found in 1999-2002 patient-1999 C.pipiens pool-2001 two patients in 2000, one in 2002 patient-1999 patient-1999 9 of 1999 two crows in 2001 0.002 p-distance (one substitution) crow-2001

13. Selected publications and presentations: Platonov AE. West Nile encephalitis in Russia 1999-2001: Were we ready? Are we ready? In: West Nile virus: Detection, surveillance, and control. D.J.White and D.L.Morse, eds. New York, Annals of the NYAS, 2001; V.951, pp.102-116. Platonov A.E., Shipulin G.A., Shipulina O.Yu., et al. Outbreak of West Nile infection - Volgograd region, Russia, 1999. Emer. Infect. Dis. 2001; 7: 128-132 Platonov A.E., Karan L.S., Yazyshina S.B., et al. Microheterogenicity of the Volgograd clone of West Nile virus. International Conference on Emerging Infectious Diseases. Atlanta, March 24-27, 2002, p. 112. Platonov A.E., Zhuravlev V.I., Lazorenko V.V. The weather and the mosquito-borne infections: What was wrong in 1999 in Southern Russia? International Conference on Emerging Infectious Diseases. Atlanta, February 29 - March 3, 2004, p.63-64.

14. Crimean-Congo hemorrhagic fever: early pathogen detection, viral load measurement, genotyping

15. Crimean hemorrhagic fever (CHF) was diagnosed in the Russian Federation since 1944. 334 human cases were registered in the Rostov region from 1963 to 1971 years, 51 cases (15%) were fatal. CHF outbreaks have been reported also in the Republics of Kalmykya and Dagestan, the Stavropol, Astrakhan and Krasnodar regions (colored in red) of the Southern Russia Crimean In 2000-2003 more than 200 CHF cases with 18 deaths were laboratory confirmed in Russia. Both old endemic regions and new region (Volgograd - colored in yellow) were affected. In the latter region there were 18 CHF cases in 2000, 9 cases in 2001, 4 cases in 2002-2003; the Crimean-Congo hemorrhagic fever virus (CCHFV) RNA was found in the ticks. The most affected region was Stavropol region, where we have done our studies on viral load at CCHF. Congo Volgograd Stavropol After the period of relative composure the CHF epidemic situation has become worse last years.

16. Crimean-Congo hemorrhagic fever in Stavropol Region, population 2,655,000

17. Laboratory diagnosis of Crimean-Congo hemorrhagic fever IgG diagnosis is possible IgM diagnosis is possible PCR diagnosis is possible Critical time for treat- ment and prevention -- diagnostic methods

18. The decrease of viral load in patients with Crimean-Congo hemorrhagic fever Viral load,arbitrary units, logarithmic scale

19. Viral load correlates with the severity of Crimean-Congo hemorrhagic fever (day 3 – day 5 of hospitalization) Viral load,arbitrary units, logarithmic scale

20. Phylogenetic tree based on partial sequences of the small S segment of CCHF isolates South Europe: Rostov, Volgograd, Stavropol, Astrakhan, Kosovo, Albania 0.05 p-distance Africa: South Africa, Senegal, Nigeria, Mauritania, Burkina Faso, C.A.R. U.A.E. South Africa, Uganda Greece U.A.E. - United Arab Emirates, Pakistan, Madagaskar Senegal, Maurinatia, Iran China, Kazakhstan

21. CCHF PCR-positive result in tick pools from Volgograd H.marginatum 962 ticks 155 pools 13 CHF-positive H.scupense 220 ticks 22 pools 2 CHF-positive D.marginatus 17 ticks 2 pools 0 CHF-positive R.rossicus 26 ticks 2 pools 0 CHF-positive

22. Selected publications and presentations. Platonov A.E., Karan L.S., Yazyshina S.B., et al. Molecular identification of Crimean-Congo hemorrhagic fever virus in human clinical cases in Southern Russia. International Conference on Emerging Infectious Diseases. Atlanta, March 24-27, 2002, p. 83. Karan L.S., Sannikova I.V., Platonov A.E., et al. Viral load at Crimean-Congo hemorrhagic fever and its clinical significance. International Conference on Emerging Infectious Diseases. Atlanta, February 29 - March 3, 2004, p.125. Karan L.S., Shipulin G.A., Platonov A.E. [PCR-based laboratory diagnosis of Crimean-Congo hemorrhagic fever]. Clinical Laboratory Diagnostics, 2003, No 10, p.50-54. PCR-based assay for CCHF RNA detection is approved by the State Institute for Biological Standards and Control

23. The advantages for physicians and epidomiologists: the diagnostics of unusual or new pathogen

24. Birds mortality was observed in 1997-2001 in Southern Europe. The WNF infection was suspected due to concurrent West Nile fever equine enzootic

25. Genodiagnostics of infectious diseases (approach 2) non-specific probes Possible infectious agents + + direct sequencing of specific fragment …CTTGGAGCACGGTATCTAGAGTTTGAAGCYYTGGGGTTC.. identification of pathogens: both usual, unusual, or new pathogens

26. Phylogenetic tree of flaviviruses based on NS5 Denge fever West Nile fever Yellow fever Japanese encephalitis Tick-borne encephalitis Negishi, LI Omsk hemor- rhagic fever Up to 100 flaviviruses may be identified using NS5 gene

27. Non-specific NS5-basedassay for the detection of genus Flavivirus RNA Tick-borne encephalitis strains West Nile fever strains К- К- К+ Omsk hemorrhagic fever strains Japanese encephalitis strains К- К+ К- К+ К+ Also positive results with Langat, Powassan, looping ill, Saint Louis encephalitis viruses

28. Specific E gene-basedassay for the detection of Japanese encephalitis virus RNA 1 2 3 4 5 6 7 8 9 NCC- C+ 1 2 3 4 5 6 7 8 9 1011121314151617 C-C+ Lines 1-17 – avian brain samples C+ - positive control 10 11 12 13 14 15 16 17 18 ок к- к+ Lines 1-5,10-15 – JE strains (positive) Line 6- SLE strains (negative) Line 7-9- TBE strains (negative) Line 16- Powassan strain (negative) Line 17– Langat strain (negative) Line 18– Looping ill strain (negative)

29. Genetic tree (E gene nucleotide sequences) of avian JE isolates and reference strains of JEV, WNFV, SLEV Japanese encephalitis West Nile fever Saint Louis encephalitis

30. Birds mortality in 1997-2001 in Southern Europe - Natural enzootic JE infection in Europe?

31. Ceccherelli R. Gubler D. Lanciotti R.S. Renzoni G. Rossi G. Tarantino C. Антонов В.А. Бочкова Н.Г. Бутенко А.М. Виноградова Е.Б. Вышемирский О.И. Галимзянов Х.М. Говорухина М.В. Громашевский В.И. Ефременко В.И. Еременко Е.И. Жуков.А.Н. Журавлев В.И. Ковалев Н.Г. Краснова Е.М. Литовкина Н.А. Лазоренко В.В. Лопатина Ю.В. Маленко Г.В. Миронов К.О. Обухов И.Л. Погодина В.В. Подколзин А.Т. Родионова Е.Н. Рыжков В.И. Смирнова С.М. Тялина Ю.Ю Фролочкина Т.И Швагер М.М. Шипулин Г.А. Шипулина О.Ю. Якименко В.В., Яцышина С.Б. Карань Л.С. Булгакова Т.А. Венгеров Ю.Я. Гаранина С.Б. Малеев В.В. Миронов К.О. Платонов А.Е. Платонова О.В. Рудникова Н.А. Федорова М.В.

32. West Nile Virus Shoot Thank you