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

of Epidemiology; Moscow, Russia

2000-2004Contacts: platonov@pcr.ru

West Nile fever

Crimean-Congo hemorrhagic fever

Tick borne encephalitis

Omsk hemorrhagic fever

Japanese encephalitis

other flavivirus infections

hantaviral infectionar

e su

bjec

ts w

e de

al w

ith

Borreliosis

Rickettsiosis

Enteroviral infection

Meningococcal infection

Haemophilus influenzae type b

Pneumococcal infection

are subjects we deal w

ith

epidemiology

and

surv

eillance

treatm

ent

and

contro

l

genodiagnostics

and

genotyping

Russia: Annual sum of effective temperatures (t >= 10oC)

200-500

500-1000

1000-1500

1500-2000

2000-2500

2500-3000

3000-3500

Moscow

West Nile fever:classical and

molecular epidemiology, diagnosis,

pathogenesis, treatment

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

?

Genetic distance of West Nile virus isolatesbased on complete nucleotide sequence

Genetic distance

"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

Mosquito isolate, Romania 1996and human clinical isolate, Volgograd 1999,(as well as the Volgograd strains from humans, crows and C.pipiens, 1999-2002)

Italian strain, 1998Kenyan strain, 1998

0.01

Kunjin strains

Astrakhan strain, 1999

Eg-101

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?

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?

West Nile fever in Russia in 1999-2003

Astrakhan

VolgogradKrasnodar

0

100

200

300

400

500

1997 1998 1999 2000 2001 2002 2003N

um

ber

of

WN

F c

ases

>5>35

>500 Volgograd region

32 15 14 0

0

100

200

300

400

500

1997 1998 1999 2000 2001 2002 2003

Nu

mb

er o

f W

NF

cas

es

??

Astrakhanregion

24 49 33 12

95

WNF outbreak in 1999:Volgograd - 38 deaths; Astrakhan - 5 deaths;Krasnodar - 3 deaths.

0.001

Genetic tree based on envelope gene fragment, 512 nt,of WN virus isolates from Volgograd 1999-2002

0.002 p-distance(one substitution)

9of

1999

Volgograd WN clone appear to be endemic in Volgograd region because the same isolates were found in 1999-2002

twopatients in 2000,one in2002

twocrows in 2001crow-2001

patient-1999

patient-1999

patient-1999

C.pipienspool-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

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 yellowVolgograd - 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.

After the period of relative composure the CHF epidemic situation has become worse last

years.

Volgograd

Stavropol

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

0

10

20

30

40

50

60

1998 1999 2000 2001 2002 2003year

nu

mb

er

of

CC

HF

ca

se

s

lethal

all

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

0

20

40

60

80

100

days 1-4 days 5-7 week 2 week 3 week 4 week 5

days of disease

po

sit

ive

sa

mp

les

, %

PCR IgM IgG-- diagnostic methods

PCR diagnosis is possible

IgM diagnosis is possible

IgG diagnosis is possible

Critical time for treat-ment and prevention

Laboratory diagnosis of Crimean-Congo hemorrhagic fever

0

1

10

100

1000

10000

100000

1 2 3 4 5 6 7 8 9 10 11 12 13 14

day of hospitalization

Vir

al l

oad

, ar

bit

rary

un

its,

log

arit

hm

ic s

cale

The decrease of viral load in patients with Crimean-Congo hemorrhagic fever

Vir

al l

oad

, ar

bit

rary

un

its,

log

arit

hm

ic s

cale

Viral load correlates with the severity of Crimean-Congohemorrhagic fever (day 3 – day 5 of hospitalization)

0,1

1

10

100

1000

mild moderate severe

0,1

1

10

100

1000

without hemorrhagicsyndrome

with hemorrhagicsyndrome

0.05

Phylogenetic tree based on partial sequences of the small S segment of CCHF isolates

South Europe:Rostov, Volgograd, Stavropol,Astrakhan,Kosovo,Albania

China, Kazakhstan

Africa:

South Africa,Senegal,Nigeria,Mauritania,Burkina Faso,C.A.R.U.A.E.

South Africa,Uganda

Greece

0.05

p-distance

U.A.E. -United Arab Emirates,Pakistan,Madagaskar

Senegal,Maurinatia,Iran

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

CCHF PCR-positive result in tick pools from Volgograd

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

direct sequencing of specific fragment…CTTGGAGCACGGTATCTAGAGTTTGAAGCYYTGGGGTTC..

++

identification of pathogens: both usual, unusual, or new pathogens

Possibleinfectious agents

Genodiagnostics of infectious diseases (approach 2)

non-specific probes

Dengefever

Yellowfever

West Nilefever

Tick-borneencephalitis

Omsk hemor-rhagic fever

Japaneseencephalitis

Negishi, LI

Phylogenetic tree of flaviviruses based on NS5

Up to 100 flaviviruses may be identified using NS5 gene

Non-specific NS5-based assay for the detection of genus Flavivirus RNA

Tick-borne encephalitis strains West Nile fever strains

Also positive results with Langat, Powassan, looping ill, Saint Louis encephalitis viruses

К+

К+

К+

К-

К- К-

К-К+

Japanese encephalitis strains Omsk hemorrhagic fever strains

1 2 3 4 5 6 7 8 9 NC C- C+

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)

1 2 3 4 5 6 7 8 9 1011121314151617 C-C+

Lines 1-17 – avian brain samplesC+ - positive control

Specific E gene-based assay for the detection of Japanese encephalitis virus RNA

Genetic tree (E gene nucleotide sequences) of avian JE isolates and reference strains of JEV, WNFV, SLEV

JE-Tmer/s|AF501311| 25-JE-Italy2003-twotrace-need JE-Tmus/p|AF501312| JE-Svul/h|AF501313| JE-Tphil/h|AF501314| JE-Cliv/b|AF501315|

JE-NAKAYAMA/AF112298/ JE-NAKAYAMA-RFVL/S75726/ JE-Ling/L78128/ JE-YLG/AY243837/human brain-China Kunjin-MRM61C/D00246/ WN-Eg101/AF260968/ AY033390 ISR99-GULL Israel-1999 Seagull WN-VOLGOGRAD-1999 AF380670 Isr00-PigT Israel-2000 Pigeon SLE-PV0-620/AF205492/

0.05

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.ЮФролочкина Т.ИШвагер М.М.Шипулин Г.А.Шипулина О.Ю.Якименко В.В.,Яцышина С.Б.

AF3

4631

3 30

0062

2 N

Y20

00W

N-N

Y-M

-274

1W

N-N

Y99

-EQ

HS

WN

-HN

Y19

99

AY

0333

89 IS

98-S

T1

Isra

el-

1998

Sto

rk

AY0

3338

8 IS

R98-

Goo

1 Is

rael

-199

8 G

oose

AF3

4631

7 00

-824

NY2

000

AF346

316 3

0002

59 N

Y2000

AF346315 00-2741 NY2000

AF346314 3000352 NY2000

AF346312 3000271 NY2000AF346310 3000365 NY2000AF346309 3000017 NY2000

AF380664 Isr00-GooM Israel-2000 Goose

AF346318 00-3356 NY2000

AF346319 00-3282 NY2000

WN-NY99-FLA382-99

AF346311 3000548 NY2000

AF380669 Isr00-Eq1 Israel-2000 Horse

AF380663 Is

r97-Go

o1 Is

rael-1997 G

oo

se

AF380665 Is

r00-Go

oN

Israe

l-2000 Go

os

e

AY

033390 ISR

99-GU

LL

Israe

l-1999 Se

agu

llA

Y033391 Is

r99-Go

o Is

rael-1999 G

oo

se

AF3

8066

8 Is

r00-

Ro

s Is

rae

l-20

00 R

os

ella

WN

-As

trak

han

-199

9/A

ST

R98

6/

AF3

8066

6 Is

r00-

Go

oM

aV Is

rae

l-20

00 G

oo

se

AF3

8067

0 Is

r00-

Pig

T Is

rae

l-20

00 P

ige

on

AF3

8067

1 Is

r00-

Pig

C Is

rae

l-20

00 P

ige

on

AF3

8066

7 Is

r00-

Go

oM

aS Is

rae

l-20

00 G

oo

se

WN

-KEN

YA

-199

8 A

F146

082

727

AV

ERIN

A 3

WN

-RO

MA

NIA

-199

6M A

F260

969

715

TA

RASO

VA

11

bir

d-20

01 n

ew

428

ARCHAKOVA 7

435 PAC 5

445 DODIN 4(V)

499 PROSCHANOVA 1 506 KUKISHEVA 8 703 KRASOTCHENKO 9

707 DOBROVENSKI 10

719 ANDREEVA 12

723 KORSHUNO

VA

13

431 KN

YA

ZEV

A 6

WN

-VO

LG

OG

RA

D-1999 A

F317203

448 NO

VIK

OV

2 492 B

AR

AN

OV

14

WN

-EG101

KU

NJIN

-MR

M61C

WestNile

VirusShoot

Thank you