The Department of Emerging and Re-emerging Infections Central

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

West Nile fever w it h Crimean-Congo hemorrhagic fever w ec ts su ar e Japanese encephalitis bj Omsk hemorrhagic fever e de al Tick borne encephalitis other flavivirusinfections hantaviral infection

Borreliosis e ar h it w al de Meningococcal infection e w Enteroviral infection s t ec bj su Rickettsiosis Haemophilus influenzae type b Pneumococcal infection

gy o l io em d nc e d pi an illa e e rv su nt e m at d ol tre an tr on c ics t os n ag d ng di n pi no a ty ge no ge

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

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

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

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

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-1996 M 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?

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?

Krasnodar Volgograd Astrakhan Number of WNF cases WNF outbreak in 1999: Volgograd - 38 deaths; Astrakhan - 5 deaths; Krasnodar - 3 deaths. Number of WNF cases West Nile fever in Russia in 1999 -2003 >500 >5 >35 Volgograd region 32 15 14 0 Astrakhan region ? ? 95 24 49 33 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 patient-1999 0. 002 p-distance (one substitution) crow-2001 9 of 1999 two patients in 2000, one in 2002 two crows in 2001

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.

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

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 Volgograd Stavropol After the period of relative composure the CHF epidemic situation has become worse last years. 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.

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

Laboratory diagnosis of Crimean-Congo hemorrhagic fever Ig. G diagnosis is possible Ig. M diagnosis is possible PCR diagnosis is possible Critical time for treatment and prevention -- diagnostic methods

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

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

Phylogenetic tree based on partial sequences of the small S segment of CCHF isolates 0. 05 Africa: South Africa, Senegal, Nigeria, Mauritania, Burkina Faso, C. A. R. U. A. E. United Arab Emirates, Pakistan, Madagaskar p-distance South Europe: Rostov, Volgograd, Stavropol, Astrakhan, Kosovo, Albania South Africa, Uganda Greece China, Kazakhstan Senegal, Maurinatia, Iran

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 CHFpositive D. marginatus 17 ticks R. rossicus 2 pools 26 ticks 0 CHFpositive 2 pools 0 CHFpositive

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

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

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

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

Phylogenetic tree of flaviviruses based on NS 5 West Nile fever Denge fever Yellow fever Japanese encephalitis Tick-borne encephalitis Negishi, LI Omsk hemorrhagic fever Up to 100 flaviviruses may be identified using NS 5 gene

Non-specific NS 5 -based assay for the detection of genus Flavivirus RNA Tick-borne encephalitis strains К- К- Japanese encephalitis strains К- West Nile fever strains К+ К+ Omsk hemorrhagic fever strains КК+ К+ Also positive results with Langat, Powassan, looping ill, Saint Louis encephalitis viruses

Specific E gene-based assay for the detection of Japanese encephalitis virus RNA 1 2 3 4 5 6 7 8 9 NC C- 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)

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

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

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. 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.

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