Infection by West Nile virus : epidemiology,

diagnosis and surveillance

UMR ANSES/INRA/ENVA
Stéphan Zientara

ONIRIS MANIMAL, 2016

Paris

Maisons-Alfort
 EU-RL on equine diseases
EIAV, EAV, EHV, WN, VSV, glanders,
dourine, CEM, EEV, EEW, EEV

OIE RL
FMDV
EHDV

NRL
FMDV
BTV
AHSV
HEV…
West Nile virus
Epidemiology
Vectors of the WN virus
WN in birds
WN in other animal species
WN in humans
WN in horses
WN diagnosis
WN in France
WN prevention
 World distribution of flaviviruses
Alfuy virus Jugra virus Rocio virus
Apoi virus Jutiapa virus Saboya virus
Aroa virus Kadam virus Sal Vieja virus
Bagaza virus Karshi virus San Perlita virus
Banzi virus Kedougou virus Saumarez Reef virus
Batu Cave virus Kokobera virus Sepik virus
Bouboui virus Koutango virus Sokoluk virus
Bukalasa bat virus Kunjin virus Spondweni virus
Bussuquara virus Kyasanur Forest disease virus St. Louis encephalitis virus
Cacipacore virus Langat virus Stratford virus
Carey Island virus Louping ill virus Tamana bat virus
Cell fusing agent virus Meaban virus Tembusu virus
Cowbone Ridge virus Modoc virus Tick-borne encephalitis virus
Dakar bat virus Montana myotis Tyuleniy virus
Dengue virus leukoencephalitis virus Uganda S virus
Edge Hill virus Murray Valley encephalitis virus Usutu virus
Entebbe bat virus Naranjal virus Wesselsbron virus
Gadgets Gully virus Negishi virus West Nile virus
Iguape virus Ntaya virus Yaounde virus
Ilheus virus Omsk hemorrhagic fever virus Yellow fever virus
Israel turkey Phnom Penh bat virus Yokose virus
meningoencephalomyelitis virus Potiskum virus Zika virus
Japanese encephalitis virus Powassan virus
Rio Bravo virus

World-wide: Courtesy Dr. Tamás Bakonyi

 Flavivirus : structure
Family : Flaviviridae
Genus : flavivirus >70 virus
Structure

Virus à ARNsb+, 11000nt

Structural protein :«E»
Highly immunogenic

50 nm

ssRNA+ 11kb

Kramer et al., 2007; http://viralzone.expasy.org

8
 Flavivirus

Pegivirus
 Mosquitoe Aedes

Unknown vector

Ticks, birds

TYUV DQ235148.1

Mosquitoes Culex

WNV-1a Ro96 AF260969.1

Ticks,
groupe encéphalite

HCV-3i JF735125.1

0.2
Beck et al, IJERPH, 2013
 Phylogeny of Flaviviruses

Serocomplex
of japanese
encephalitis

Modifié d’après Beck et al, 2013

 Phylogenetic tree of flaviviruses

NS5 gene (nt 9060-10 065)

12
Global distribution of the major Japanese encephalitis serological group members

SLEV
WNV
JEV
MVEV
 Molecular diagnostics of West Nile virus infection
Phylogeny of WNV
divergent strains
NS5-3’UTR (10102-10920)
(Bakonyi T et al., EID, 2006)
8 lineages

Lineage 1: worldwide distribution

West Africa, Middle East, East and Southern
Europe, America and Australia.

Lineage 2: Africa
Senegal, Ouganda, R.C.A, Kenya,
Madagascar.
Lineages 3 and 4 : central Europe

Lineage 5 : indian strains

15
Scanned images of West Nile virus isolated from
brain tissue from a crow found in New York.
The WN virus
WN epidemiology
Vectors of the WN virus
WN in birds
WN in other animal species
WN in humans
WN in horses
WN in birds
WN diagnosis and prevention
WN in France
 Transmission cycle

vectors

D. Furry, 2003.
 Distribution géographique du virus West Nile

1999

1937

19
 West Nile in the USA 1999 : 62 cases, 7 deaths

2000 : 21 cases, 2 deaths

2001 : 66 cases, 10 deaths

2002 : 4156 cases, 284 deaths

2003 : 9862 cases, 264 deaths

2004 : 2539 cases, 100 deaths

2005 : 3000 cases, 119 deaths

2006 : 4269 cases, 177 deaths

2007 : 3630 cases, 124 deaths

2008 : 1356 cases, 44 deaths

2009 : 720 cases, 32 deaths

1999-2015
2010 : 1 021 cases, 57 deaths

2011 : 702 cases, 486 neuroinvasie cases

1,911 deaths 43 deaths

2012: 5,674 cases! (2873 neuroinvasive cases),

286 deaths, 703 vireamic
donors
2013: 2,469 cases! (1,267 neuroinvasive cases),
111 deaths

2014 : 2,122 cases, 1,283 neuroinvasive cases,

669 encephalitis, 85 deaths

between 1999 and 2010, ~1.8 millions infections with ~360,000 2015: 1,312 neuroinvasive cases,
sick people; 12,852 cases of encephalitis/meningitis and 1,308 deaths 111 deaths
(Science, Kilpatrick AM, 2011)
West Nile Virus Neuroinvasive Disease Incidence by State
– United States, 2016 (as of November 15, 2016)
 1999 2000 2001

2002 2003 2004

2007
2005 2006

1999-2009 : >28 000 human cases ; 1 100 deaths

1999-2007 : >60 00 dead corvids
1999-2009 : >24 000 equine cases (in 2002 : about 15 000 cases and 4 500 deaths)
NS5-3’UTR (10102-10920)
(Bakonyi T et al., EID, 2006)

Lineage 1: worldwide distribution

West Africa, Middle East, East and Southern
Europe, America and Australia.

. Origin WNV
. Variant 2002 WN02

Lineage 2: Africa
Senegal, Ouganda, R.C.A, Kenya,
Madagascar.
Lineages 3 and 4 : central Europe

Lineage 5 : indian strains

26
26
NS5-3’UTR (10102-10920)
(Bakonyi T et al., EID, 2006)

Lineage 1: worldwide distribution

West Africa, Middle East, East and Southern
Europe, America and Australia.

Substitution T249 P in the helicase (NS3)

NS3 : increases the viremia and mortality in American crows

. Origine
WN 2002 induces a higher rate of infection WNV
in mosquitoes
. Variant 2002 WN02
WN 2002 transmitted more rapidly than NY99?

Lineage 2: Africa
Senegal, Ouganda, R.C.A, Kenya,
Madagascar.
Lineages 3 and 4 : central Europe

Lineage 5 : indian strains

27
27
Is98 is a Euro-Mediterranean strain with high neuro-invasive
properties (isolated from a dying stork in 1998 in Israel)
(Malkinson et al, EID, 2002)

Reverse genetics of the WNV Is98 infectious clone.

IS98 Infectious clone
(IC)
ssRNA+ genome
ssRNA
Marker

11000 nt
9000nt

Bahuon et al, PLos One, 2012

Production of NS3249P (parental) and NS3249T viruses in Vero cells.

Virulence studies in model mice and birds.
 H. Zeller, eCDC

30
30
West Nile
1994-2006
North America
1999
2000
2003
2004
2006 1999
2004

1994 2000
1996 1998
1996
2003
1997 1997
2003
2000-5
2003

2002
horses human birds D’après H. Zeller, 2007
 Emerging viruses in Europe…

1996

2004

1998 Lineage 2
Lineage 1
2010, Bulgaria, Greece, Portugal, Spain, Turkey
2011, Albania, Macedoina, Ukrain
2012, Croatia, Kosovo, Serbia, Monténégro eCDC
2013, Bosnie-Herzegovine
Europe:
WNV
re-
emergence
Human cases
•2010:
• 926
•2011:
• 388
•2012:
• 860
•2013 (20 sept):
• 436 Lineage 2

US*: Lineage 1
•2013 (17 sept):
• 890
(*) Source: CDC Courtesy M-A Jimenez Clavero
 As of 10 November 2016, 206 cases of West
Nile fever in humans have been reported in the
EU Member States and 263 cases in the
neighbouring countries, since the beginning of
the 2016 transmission season
 Europe vs. North America

• North America: • Europe:

– Single introduction.
– Rapid expansion.
– Many outbreaks.
– Many human and
veterinary cases.
– Birds largely affected.
– Multiple introductions.
– Sporadic, self-limited
outbreaks.
– Increased incidence in the
last years.
– Differences/territories.
– Birds seldom affected.
 Vectors
> 75 species of mosquitoes
 Species of mosquito positive for WN by year

Aedes vexans
1999 Culex pipiens 2001
Cx restuans
20 species

Aedes vexans
Anopheles punctipennis 2002
Culex pipiens pipiens
2000 Cx restans
Culiseta melanura 35 species
Ochlerotatus cantator
Oc japonicus
Oc triseriatus
Psorophora ferox
2003 43 species

Competence ; capacity
Culex genus most efficient
Europe Cx pipiens, Cx modestus,
Cx perexiguus

(10 genres : Ochlerotatus,

Aedes, Anopheles,
Coquillettidia, Aedomya,
Mansonia, Mimomyia,
Psorophora, Culiseta et
Uranoteania)
Asie Cx quinquefasciatus, Cx
tritaeniorhynchus et Cx
Vishnui
Afrique Cx univittatus, Cx
poicilipes, Cx neavei, Cx
decens, Aedes
albocephalus, Mimomyia
spp
Amérique du nord Cx pipiens, Cx restuans,
Cx nigripalpus, Cx
quinquefasciatus, Cx
tarsalis, Cx salinarius
Vector (Culex spp)
C Griot
Mosquito Habitat Elimination
Role of the birds?
Avian mortality (USA, Canada, Israel)

Crows and blue jays

 1999 – 2002

> 57 000 dead corvids

geese
 Bird reservoirs

Susceptible birds
(temporary amplificators):
• Corvidae (USA)
• Other passerines:
 Blackbirds (Usutu, Autriche, 2002)
 Sparrows (Israël)
• Geese (Israël)
• Raptors…
Resistant birds
(asymptomatic reservoirs):
• Ducks
• Galliformes (chicken, turkey)
• Pigeons
• Gruidae
• …
MARSHES

EDEN
 Hungary
2003 encephalitis
geese + human cases

Lineage 1

2004 and 2005

encephalites
goshawk
(Accipiter
gentilis) Lineage 2
Other species
« overwintering »
C Griot
Xenopus model

Experimental infection in vitro and in vivo

1- WNV infection of XTC-2 cells (Xenopus fibroblasts)

. CPE or not ?
. Serial passages : virus titration (vero celles) and rt-RT-
PCR quantification

Not predictive of in vivo replication

2- Infection of Xenopus laevis by

WNV injection
The WN virus
WN epidemiology
Vectors of the WN virus
WN in birds
WN in other animal species
WN in humans
WN in horses
WN diagnosis and prevention
WN in France
 West Nile
virus in horses
 Clinical signs in Horses

• Muscle fasciculations—60%
• Recumbency—35%
• Depression—51%
• Weakness—100%
• Ataxia—100%
• Fever—65%

• Mortality rate
• 30-38% overall
• 70% in recumbent horses
 Molecular diagnostics of West Nile virus infection

Hyperesthesia, Ataxia, Somnolence

Fort Dodge

63
 Treatment of WNV

Treatment is symptomatic.
IV fluids
IV DMSO, NSAIDs
Sling
Dexamethasone - controversial
• Low-dose
 0.02 mg/kg
 Anti-inflammatory without immune suppression
Interferon alpha
 Survival

If horse presents recumbent

• 30% survive
If horse first presented on his feet
• 97% survive
 Mortality may result from injuries
incurred during acute neurologic disease

Laceration
Head trauma
Fracture
Septic joints
The WN virus
WN epidemiology
Vectors of the WN virus
WN in birds
WN in other animal species
WN in humans
WN in horses
WN diagnosis
WN in France
 Molecular diagnostics of West Nile virus infection

4. Diagnostics

M
E
C

ARNss +
 Serology ELISA (IgM, IgG)
Seroneutralization tests (PRN T90)

 Virology Virus isolation

RT-PCR (classical or real-time)
Antigen detection (immunohistochemistry,…)
70
 Maps of the distributions of the main flaviviruses found in Europe (2003-2012)

West Nile (WNV) : human

cases
Tick-borne encephalitis
(TBEV) : human cases

Vector :
mosquitoes Vector: ticks
Beck et al., 2013

Usutu (USUV) : clinical cases
in wild birds (red)
Different flaviviruses
with overlapping areas:
interpretation of
serological diagnostic
with care
Multiplex immonassay technology : principle (I)

WNV sE JEV TBEV

EDIII EDIII EDIII

1. Incubation step
Beads : 4 plex 2. Wash step

Antigen coupled
bead

Serum antibodies
Biotinylated anti-horse
immunoglobulins

Strepavidin –R-
phycoerythrin (SPE)
3. Reading step
1250 beads/well
Multiplex immuassay technology : production of antigens (II)

Selection of immunodominant Ag

E glycoprotein : major
neutralizing antigen

• DI and DIII : specific epitopes

• DII : epitope recognized by broadly cross-
reactive antibodies
Glycoprotéine d’enveloppe E et domaine III

Expert Reviews in Molecular Medecine 2008

Beasley and Barett, 2002

MIA : reference sera production against the main flaviviruses (I)

Blood samples D8 D21 D35 D 58

inoculation
D0

Strain Titer TCID50

WNV 1 Israel 98 1.5 X107
WNV2 Austria 1.5 X107
strain
%S/N for reference sera
JEV Nakayama 6X105 (kit ID screen WNV competition, ID Vet)
strain 100
90
TBEV Hypr 1.1X107 80
- 70 D0
60
D8
% S/N

Threshold 50
D21
%S/N < 40% 40
D35
30
D58
20
10
0
WNV-1 WNV-2 JEV TBEV
Luminex technology with
reference sera
Luminex results with reference sera
A B
7 6
TBEV JEV
6
5

5
4
4 JEV.EDIII
JEV.EDIII
ratio

ratio
WNV.EDIII 3
WNV.EDIII
3 WNV.sE
WNV.sE
TBEV.EDIII
2 TBEV.EDIII
2

1 cut-off 1 cut-off

0
0
TBEV_D0 TBEV_D4 TBEV_D8 TBEV_D11 TBEV_D14 TBEV_D20 TBEV_D35 TBEV_D58
JEV_D0 JEV_D4 JEV_D8 JEV_D11 JEV_D14 JEV_D20 JEV_D35 JEV_D58
TBEV reference sera
JEV reference sera

C D
16 25
15 WNV lineage 1 24 WNV lineage 2
23
14 22
21
13 20
12 19
18
11 17
16
10 15
9 14
JEV.EDIII JEV.EDIII

ratio
13
ratio

8 12
WNV.EDIII WNV.EDIII
7 11
10 WNV.sE
6 WNV.sE 9
5 8 TBEV.EDIII
TBEV.EDIII 7
4 6
5
3 4
2 3
1 cut-off 2 cut-off
1
0 0
WNV1_D0 WNV1_D4 WNV1_D8 WNV1_D11 WNV1_D14 WNV1_D20 WNV1_D35 WNV1_D58 WNV2_D0 WNV2_D4 WNV2_D8 WNV2_D11 WNV2_D14 WNV2_D20 WNV2_D35 WNV2_D58

WNV lineage 1 reference sera WNV lineage 2 reference sera

Reference equine sera sampled from ponies experimentally infected with different
flaviviruses: TBEV (panel A), JEV (panel B), WNV lineage 1 (panel C) and WNV lineage 2
(panel D) collected on different days post infection and tested by flavivirus luminex with
four antigen-coupled beads (JEV.EDIII, WNV.EDIII, WNV.sE and TBEV.EDIII).
Application of MIA with field sera (III)
74 sera coming from Austria and positive or negative by the ID
screen WNV competition kit (ID Vet) were analysed by Luminex

ELISA positive

Luminex fluorescence results for Austrian sera

The WN virus
WN epidemiology
Vectors of the WN virus
WN in birds
WN in other animal species
WN in humans
WN in horses
WN diagnosis and prevention
WN in France
West Nile in France

2003
2006 1962-65
2000
2004
2015
 1962-1965 : Camargue

80 equine cases
25 deaths
A few human cases
 2000 : Petite Camargue

76 equine cases
21 deaths
No human cases

Serosurvey : 5133 sera 8% IgG+

3% IgM+
 WN-Italy-1998; AF205883
WN-Israel-2000; AF380669
WN-Morocco-1996; AF205884
WN-France-2000; AF418554
WN-Romania-1996M; AF260969
WN-Volgograd-1999; AF239988
WN-Senegal-1993; AF001570
WN-Kenya-1998; AF146082
WN-Israel-1998; AF205882
WN-New York-1999F; AF196835
WN-Tunisia-1997; AF418555
WN-CAR-1989; AF001558 Lineage I
WN-Algeria-1968; AF001567
WN-Senegal-1979; AF001569
WN-France-1965; AF001560
WN-Egypt-1951; AF001568
WN-South Africa; AF205880
WN-CAR-1967; AF001566
WN-Ivory Coast-1981; AF001561
Kunjin; AF001572
WN-India-1980; AF196526
WN-Uganda; AF001573
WN-Senegal-1990; AF001556
WN-Nigeria
WN-CAR-1972; AF001563
WN-CAR-1983; AF001557
WN-CAR-1972; AF001565
WN-Uganda; AF001562
Lineage II
WN-Madagascar-1986; AF001564
WN-Madagascar-1988; AF001574
WN-Kenya; AF001571
WN-Madagascar-1978; AF001559
JE SA 14; UO4522
0.1
2000 : 76 cases, 21 deaths
5133 sera ; 8% IgG and 3% IgM
 2003 : Var

7 equine cases (2 deaths)

7 human cases
2003 : Results ELISA IgG

305 IgG+ (n =906)

32 stables(n = 40)

Sero-
prévalence:
33,7% of horses
IC 95%: 30,6%–36,8%

80% of stables
IC 95%: 64%–91%
Results ELISA IgM

23 IgM+ (n = 305)

Seroprevalence
7,5% animaux /IgG+
Localisation vs seroprevalence IgM

Dry Zones +++

Wet Zones -
September - November 2004

1 case
Lunel
1 case
Arles
2 cases
Le Cailar
1 case
Saliers

1 case
Beauvoisin

2 cases
2 cases St Gilles
Vauvert
1 case 2 cases 18 cases 1 case
St Laurent Aigues-Mortes Saintes Maries Albaron
dAigouze de la Mer

 15/11/2004

- 32 equine cases;

13 avian seroconversions confirmed in Camargue in 4 surveillance sites

 2006 : Pyrénées orientales

5 equine cases ; 1 death

No human case
West Nile virus (October 30, 2015)
2006 1962-65
2000 2003
2004
2015
Au 30/10/2015 : 49 cases
-41 cases meningo-encéphalitis

-5 asymptomatic infections and 3

febrile forms

-+1 infection asymptomatic case

(RESPE suvey)

2st suspicions (RESPE)

1er case the 11/08/2015 (Albaron,
Fourques
Letality neuroinvasive forms : 14.6%
(6/41)
Evolution du nombre de chevaux détectés infectés
par le virus West-Nile et présentant des signes
cliniques, entre le 11 août et le 20 octobre 2015 par
rapport à la date d’apparition des signes cliniques
(n=41 chevaux, la date d’apparition des signes n’a
pas encore été renseignée pour 2 chevaux ayant
potentiellement présenté des signes dans les
semaines 38 à 42
Location

-32 cases in 24 stables in Bouches-du-Rhône (13)

-15 cases in 13 stables Gard (30)

-1 case in 1 stable (Hérault (34)

Source : plateforme ESA (19/10/15)

•Dr Philippe Garcia (clinique vétérinaire de la Crau)
•Dr Philippe Garcia (clinique vétérinaire de la Crau)
 One human case (WN)

Identified the 02/10/15 by CNR arbovirus

Febriel form since 27/09 (Dengue-Chikungunya
surveillance survey) (Nîmes).

Low mortality in wild birds

Pool of mosquitoes Culex pipiens (11/09)

Positive by PCR
(EID Méditerranée, CNR Arbovirus)
• 2 USUTU viruses isolated in birds (Haut-
Rhin, Rhône)

• Virus isolated in 1959 in South Africa

• 2 human cases in Africa in 1981 and 2004

• Identification in Austria in 2001 (avian mortality) and in Italy in 1996

• 2 human cases in Italy (encephalitis/ immunodepression)

West Nile virus
Epidemiology
Vectors of the WN virus
WN in birds
WN in other animal species
WN in humans
WN in horses
WN diagnosis
WN in France
WN prevention
 Prevention of WNV
No stagnant water
• 4 days required for mosquito breeding
• Old tires
• Gambusia fish eat larvae in tanks and ornamental pools
 Prevention of WNV
No stagnant water
• 4 days required for mosquito breeding
• Old tires
• Gambusia fish eat larvae in tanks and ornamental pools
Aerial spraying--may be of limited value
 Prevention of WNV

No stagnant water
• 4 days required for mosquito breeding
• Old tires
• Gambusia fish eat larvae in tanks and ornamental pools
Aerial spraying--may be of limited value
Topicals--permethrins
 Prevention of WNV
No stagnant water
• 4 days required for mosquito breeding
• Old tires
• Gambusia fish eat larvae in tanks and ornamental pools
Aerial spraying--may be of limited value
Topicals--permethrins
 Prevention of WNV
Vaccination
4 vaccines licensed in the USA

• Inactivated vaccine (Fort Dodge)(2001)

• Recombinant canarypox vaccine (Merial)(2004)

• DNA vaccine (2005)

• Recombinant yellow fever vaccine (YF 17D)(2006)

Vaccines in France

Pfizer Animal Health/Zoétis MERIAL (Proteq WN ND)

confirmed cases of WN infection in horses in the US,
1999-2008
vaccination
Number of cases

16000 15257

14000
12000
10000
8000
6000 5181

4000
733 1341 1072 1086
2000 65 504 218
25
0
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008
West Nile Vaccine
Widely Used
 2009 Equine U.S. State: West Nile Virus:
State: West Nile Virus: MT 14
AL 6 NE 3
AR 1 NJ 1
CA 15 NM 6
CO 20 NV 3
FL 3 PA 2
GA 0 SD 4
IA 2 TX 9
ID 8 66 unvaccinated
UT 4
IL 4 1 vaccinated
VA 3
KY 6 WA 67
LA 3 WI 1
MO 2 WV 1
MS
Surveillance
 WN Surveillance since 2000 in France

Surveillance « multi-species » :
• Humans
• Avian
• Mosquitoes
• Horses
Passive or active surveillance
 Objectives and means of surveillance

Horses : indicators
Passive surveillance
Birds
Mosquitoes ???

Estimated sensitivity of surveillance methods of WN in the American context (CDC).

 Follow-up of sentinel birds
In 2004:
30 sites in 6 departements
300 birds serologically analyzed from June to October
 Serological survey

duck

 IgG ELISA
seroneutralisation
on positive ELISA
magpie
Location of the seroconverted
birds and equine cases (2004)

viropositive
 Assessment of the surveillance system
technically good performance

• good participation and coordination of the partners

• Frequency and quality of the samples: 97% conformity
compared with the initial protocole

………….but difficult to manage

WEST NILE ENTOMOLOGICAL SURVEILLANCE 2001

EID

Unité « virus émergents , Marseille », Faculté de Médecine

20 sites of capture : 12 in 30 and 34, 8 in 13

997 tested pools (14 355 mosquitoes ; 12 species) : all negative

French surveillance system

Passive « clinical » surveillance

horses
•Activation of the surveillance system
•In the Mediterranean basin
• at the national level (RESPE)
 French surveillance system

Passive surveillance
Birds
* SAGIR system at the national level
* Abnormal mortality : viral analyses

•Targeted information campaign (hunters, professionnels, …)

Horses
•Targeted information equine vets
•Epidemiosurveillance system (RESPE)

If virus circulation, contacts with DGS, EFS, EFG, …

Risks of introduction of equine encephalitis viruses in Europe
(Durand B et al. PlosOne, 2013; EPIZONE)

Identification of « hotspots »
Remerciements

• Sylvie Lecollinet
• Cécile Beck
• Steeve Lowenski
• Céline Bahuon
• Josiane Maingault