21

Epidemiology and Diagnosis of West Nile Virus Infection
Ikuo TAKASHIMA1*, Hiroaki KARIWA1 and Kazuya SHIRATO2
1

Laboratory of Public Health, Department of Environmental Veterinary Sciences
Graduate School of Veterinary Medicine, Hokkaido University
Kita 18 Nishi 9, Kita-ku, Sapporo, 060-0818, Japan2
Department of Virology III, National Institute of Infectious Diseases, Murayama BranchMusashi-Murayama, Tokyo 208-0011, Japan*e-mail: takasima@vetmed.hokudai.ac.jp

Abstract
West Nile (WN) virus produces a mosquito-borne infection manifesting in fever and, in serious cases,
encephalitis. The virus that causes WN infections, WN virus, belongs to the family Flaviviridae, genus
Flavivirus. The WN virus emerged in New York City in August, 1999, as the first case on the American
continent, and seven human deaths were reported. Along with humans, deaths were observed among horses
and various birds, such as crows. The WN virus epidemic expanded to the southern United States in 2000
and most of the rest of the country by 2004, extending as far as Argentina in 2007. Since there is a possibility of the WN virus spreading to Japan, where the Japanese encephalitis virus is also prevalent, the TaqMan
assay was adapted to develop a sensitive, specific diagnostic test to differentiate WN virus from Japanese
encephalitis virus.
Key words: epidemiology, diagnosis, West Nile virus infection

1. Epidemiology
1.1 Global epidemiology
The WN virus (WNV) was first isolated in 1937
from the blood of a febrile patient in the West Nile district of Uganda. WNV has since been found endemic
over a wide range of areas in Africa, the Middle East,western Asia and Australia. Outbreaks of varying size
occurred in Israel in 1941 and 1951-1954, and in Africa
in 1974. After that, no large outbreaks were observed for
20 years, but from 1994 to 2000, WNV outbreaks
occurred among humans and horses in Algeria in 1994,
Morocco in 1996, Romania in 1996, Tunisia in 1997, theCzech Republic in 1997, the Congo in 1998, Italy in
1998, Israel in 1997-2000, Russia in 1999, France in2000 and the United States in 1999-2002 (Marfin &Gubler, 2001).
1.2 Epidemiology on the American Continent
During the five years following the first diagnosis of
WN encephalitis in August, 1999, in New York City, theWNV epidemic spread over 41 states. The total numberof diagnosed patients was 26,997 and the total numberof deaths was 1,008 (Table 1). (CDC (Centers for Disease Control and Prevention), 2008). Deaths in the
United States were seen most often among persons older
than 50 years and the average mortality rate was ca.
3.73%. Between 1999 and 2006, WNV was detected in
Global Environmental Research
12: 21-25 (2008)
printed in Japan

Table 1 Reported WNV disease cases inhumans, United States, 1999-2007.
(modified from CDC 2008)
Year

Total cases

Deaths

1999

62

7

2000

21

2

2001

66

9

2002

4,156

284

2003

9,862

264

2004

2,539

100

2005

3,000

119

2006

4,269

177

2007

3,022

76

Total

26,997

1,008

62 species of mosquitoes, with Culex species accounting
for more than 98% of the total reported. Over the same
period, 317 species of WNV-positive dead birds werereported, with American crows and blue jays accounting
for more than 62% of the these cases. WNV outbreaks
have also been reported among islands in the Caribbean
Sea and in Argentina. The means of importation of thevirus into the United States is unknown.

2. Causal Virus
WNV belongs to the family Flaviviridae, genus©2008 AIRIES22

I. TAKASHIMA et al.

Flavivirus and is included in the Japanese encephalitis(JE) virus serocomplex group. This group of viruses
includes the St. Louis encephalitis virus of the UnitedStates and the Murray Valley encephalitis virus ofAustralia. The viral genome consists of single-stranded
positive RNA and encodes three structural proteins (C,prM and E) and seven non-structural proteins.
WN virus isolates have been subjected to phylogenetic analysis, and can be subdivided into two major
lineages. Lineage 1 includes most of the virus isolated
since 1996, the American strains identified in 1999 and
2000, and the strains identified in Romania in 1996,
Israel in 1999, and Volgograd, Russia in 1999. Lineage 2
includes most of the strains prevalent in Africa(Lanciotti et al., 1999).

3. Transmission Cycle and Infection RouteThe major transmission cycle of WNV occurs
between mosquitoes and birds. The model of virus transmission proposed for the United States is shown in
Fig. 1. Birds are the virus-amplifying hosts, and mosquitoes of the Culex species are the major vectors in manyoutbreaks. Humans and horses are infected by bitesfrom virus-carrying mosquitoes, and while they show
clinical symptoms such as fever and encephalitis, they
are called ‘dead-end hosts’ as they do not serve as aninfection source for mosquitoes, because the viremia inhumans and horses is not high enough to infect the mosquitoes. Major vector species of mosquitoes in the
United States are Cx. pipiens, Cx. quinquefasciatus, Cx.
restuans, Cx. salinarius and Cx. tarsalis. Crows areoften used as sentinel animals to monitor virus activity
in the United States.
Routes of human infection other than mosquito bites
include fetal infection through the placenta, infantile
infection via breast feeding, infection due to bloodtransfusion or organ transplantation and laboratoryacquired infection during handling of the live virus orinfected materials.
Culex
mosquitoes
Birds

Primary
cycle

Birds

Culex
mosquitoes

Bridge vector
mosquitoes?

Humans&HorsesFig. 1 Transmission cycle of WN virus. (modified from
Campbell et al. 2002)

4. Clinical Symptoms
In humans, the incubation period of WNV is three tofifteen days following infection. Most cases are asymptomatic, but in some cases, fever and fatal encephalitis
may develop. Mild cases show a febrile period of three
to six days, headache, backache, myalgia, fatigue, rashand lymphadenopathy. Severe cases develop acute myelitis and encephalitis. The mortality rate of the outbreaks
recorded in the United States has been approximately
3.73%.
In horses, the incubation period is five to ten days.
Most infected horses are asymptomatic, but some show
clinical symptoms. In the United States, reported symptoms include ataxia, weak legs, lying down, astasia,
myospasm, fever, facial neuralgia, lip palsy, facial
spasm, gnashing and blindness. The mortality rate,
including euthanised cases, is 38%.
In birds, infection is asymptomatic, except in the
United States and Israel. In the cases recorded in the
United States, high mortality was observed among
American crows and blue jays. Symptoms included
depression, anorexia, weakness and loss of body weight.
Severe cases manifest neurological signs such as ataxia,
tremor, revolution and paresis. The clinical signs last forseven days.

5. Diagnosis
Diagnosis of WNV involves virus detection andserological examination. Virus isolation or virus genome
detection is performed using sera or cerebrospinal fluids.
IgM-captured ELISA, neutralization test, hemagglutination inhibition test and complement fixation test areused to detect WNV-specific antibodies. Acute phasesera, plasma, cerebrospinal fluids or post-mortem brain
materials can all be used for virus isolation. For
serological examination, paired sera from patients in theacute phase and convalescent phase should be collected
and tested. Differential diagnosis should be considered
for other flavivirus infections including JE, St. Louis
encephalitis, tick-borne encephalitis, Murray Valley
encephalitis and dengue. Other viral infections, such asalphavirus infection and herpesvirus infection, should bealso considered.
5.1 Development of a sensitive diagnostic test fordifferentiation of WNV and Japanese
encephalitis (JE) virus
Like WNV, JE virus is a mosquito-borne flavivirus
from the JE virus (JEV) serocomplex, and causes
encephalitis in humans and horses; it is widespreadthroughout most of Asia (Igarashi, 1992; Rosen, 1986).
In areas where JEV is endemic, such as Japan, distinguishing between WNV and JEV is critical for correct
identification of a WNV invasion. However, JE
serocomplex flaviviruses cross-react antigenically andare thus not readily differentiated by serological methods (Martin, et al., 2000). Molecular diagnostic methods

Epidemiology of West Nile Virus Infection

are therefore preferred, and the reverse transcriptase
polymerase chain reaction (RT-PCR) has been used todevelop sensitive and specific assays for the identification of WNV (Igarashi, et al., 1994; Porter, et al., 1993).
Recently, more sensitive assays, such as fluorogenic
real-time (TaqMan) PCR, SYBR Green-based real-time
PCR and loop-mediated isothermal amplification(LAMP), have been developed for diagnostic detectionof the WNV genome (Lanciotti, et al., 2000; Papin,
et al., 2004; Parida et al., 2004). In areas where JEV isendemic, it is necessary to perform specific assays forboth WNV and JEV to make a definitive diagnosis.
However, the diagnostic methods described above were
designed to detect only WNV or, more specifically, thestrain of WNV isolated in the United States. Here, we
describe the development of a sensitive moleculardiagnostic method that can detect and distinguishbetween WNV and JEV, using TaqMan RT-PCR analysis with a probe common to WNV and JEV strains(Sirato et al., 2005).

To determine the sensitivity of the TaqMan assay, theassay was performed with serially-diluted cDNA fromthe NY99-6922 strain of WNV (Fig. 2). It wasestablished that a cut-off (Ct) value of <40 and ∆Rnsignal of >0.5 indicated that WNV was present. The
specificity of the primer sets was tested using various
flaviviruses (Table 2). The TaqMan assay was performed using the indicated volume of cDNA
synthesized from total RNA. The results indicated thatthe primer set for WNV could detect only WNV strains,
including both lineage 1 and 2 viruses. The primer set
for JEV could detect only JEV strains, including
genotypes 1 (Ishikawa) and 3 (JaGAr01, Nakayama, andBeijing). The MVE virus, which is one of the JE
serocomplex viruses, was not detected by either primer
set. Flaviviruses of other serocomplexes, such as TBE
virus, Dengue virus, Langat virus, and Powassan virus,
were also not detected by either primer set.
To measure the sensitivity of the TaqMan assay forother WNV and JEV strains, the assay was performed
10000 PFU

3.5

1000 PFU

3

100 PFU

2.5
Delta Rn

23

10 PFU

2

1 PFU

1.5

0.1 PFU1

0.01 PFU

0.5

0.001 PFU

0
1

4

7 10 13 16 19 22 25 28 31 34 37 40 43
Cycle number
Cycle number

Fig. 2 Determination of the cut-off values for the TaqMan assay. The assay
was performed with serially diluted cDNA from the NY99-6922 strainof WNV. The dotted line is drawn at ∆Rn = 0.5. (Data from Shirato
et al. 2005.)

Table 2 Specificity of the multi-probe TaqMan assay. (modified from Shirato et al. 2005)
Primer set used
WNV primers
JEV primers
Virus
Amount of template Ct*
Result**Ct
ResultWNV
Lineage 1
NY99-6922
1 µg of total RNA
16.8 ± 0.18
Pos
> 40.0Neg
NY99-A301
1 µg of total RNA
17.7 ± 0.15
Pos
> 40.0Neg
BC787
1 µg of total RNA
17.6 ± 0.03
Pos
> 40.0
Neg
6-LP
1 PFU
33.6 ± 0.00
Pos
> 40.0
Neg
6-SP
1 PFU
31.2 ± 0.07
Pos
> 40.0Neg
Eg101
1 µg of total RNA
17.0 ± 0.04
Pos
> 40.0Neg
Kunjin (OP393)
100 PFU34.7 ± 0.20
Pos
> 40.0Neg
Lineage 2FCG
1 µg of total RNA
17.2 ± 0.03
Pos
> 40.0Neg
JEV
Genotype 1
Ishikawa
Genotype 3
Nakayama
Beijing
JaGAr011,000 PFU

> 40.0Neg

23.5 ± 0.03

Pos

1 µg of total RNA
1 µg of total RNA
1 µg of total RNA> 40.0> 40.0> 40.0

Neg
Neg
Neg

20.1 ± 0.19
15.8 ± 0.2
16.3 ± 0.11

Pos
Pos
Pos

Pos=positive; Neg=negative* Data are represented as the mean ± standard deviation.
** Ct values < 40 with ∆Rn signals of > 0.5 were considered positive.

24

I. TAKASHIMA et al.

using serially diluted cDNA samples from titrated virus
stocks (Table 3). At least 10-1 plaque-forming units(PFU) of virus was required for fluorescence detection
by the TaqMan assay with WNV or JEV primer sets.
However, the sensitivity for detection of Kunjin virus
was lower than that of other viruses; at least 10 PFU of
Kunjin virus were required for detection. Although the
TaqMan assay was first performed using a 50 µl reaction
volume, there was no change in sensitivity when theassay was performed using 25 µl or 12.5 µl reaction
volumes. The TaqMan assay could detect 10-1 PFU ormore of virus even in these smaller reaction volumes(data not shown). Therefore, all TaqMan assays were
performed using a 25 µl reaction volume thereafter.
We also examined whether the primer sets used inthis study could detect viruses in animal tissues. As
Japan is a WNV-free country, we were unable to obtainarchived clinical samples from animals naturally
infected with WNV. Therefore, the TaqMan assay was
performed using tissues from experimentally-infected
mice. BALB/c mice were infected with 106 PFU of the
NY99-6922 strain of WNV via the intraperitoneal route,and tissue samples were collected on the indicated days

Pos

Pos

Pos

Pos

Pos

Neg

Pos

Pos

Pos

Pos

Pos

Neg

Acknowledgements
This work is supported by Grants-in Aids for Scientific Research from the Ministry of Education, Culture,
Sports, Science and Technology, Japan.

Table 3 Sensitivity of the multi-probe the TaqMan assay.
(modified from Shirato et al. 2005)
Virus dose (PFU)
Virus
103 102 101 100 10-1 10-2
WNV
Lineage 1
NY99-6922
Pos* Pos Pos Pos Pos Neg
6-LP
Pos Pos Pos Pos Pos Neg
6-SP
Pos Pos Pos Pos Pos Neg
Eg101
Pos Pos Pos Pos Pos Neg
Kunjin (OP393) Pos Pos Pos Neg Neg Neg
Lineage 2
FCG
Pos Pos Pos Pos Pos Pos
JEVGenotype 1
Ishikawa
Genotype 3
JaGAr01

post-infection (p.i.). Total RNA was extracted, andcDNA was synthesized and tested using the TaqMan
assay. The assay detected viral RNA in blood, spleen
and brain samples (Table 4). Viruses were detected atboth the early (three days p.i.) and late (eight days p.i.)
stages of infection. Plaque assays were also performed
to detect viruses in infected animal tissues.
This paper describes a highly sensitive genetic
diagnostic method that can detect WNV and JEV using
TaqMan RT-PCR analysis with a single probe that iscommon to both WNV and JEV strains. WNV strains ofboth lineage 1 and 2 were successfully detected whenusing the primer set for WNV, and genotype 1 and3 JEV were detected when using the primer set for JEV.
Viral RNA was detected in experimentally-infected animal tissues. Therefore, it is considered likely that themethod described here is sufficiently sensitive and specific for detecting WNV and JEV strains in both humanand animal samples.

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Table 4 Detection of WNV and JEV in experimentally infected mouse tissues using the multi-probe
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The detection limits were 10 PFU/ml (blood) and 100 PFU/g (spleen and brain).
** Ct values < 40 with ∆Rn signals of > 0.5 were considered positive.

Epidemiology of West Nile Virus Infection
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25

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Ikuo TAKASHIMA
Ikuo TAKASHIMA is a Professor at theLaboratory of Public Health, Departmentof Environmental Veterinary Sciences,
Graduate School of Veterinary Medicine,
Hokkaido University. He obtained his
PhD from the Graduate School of
Veterinary Sciences at the University of
Wisconsin in 1979. He has been studying
the epidemiology of viral zoonoses.
Currently, he is working with epidemiology and diagnosis of West Nilevirus infections. There is a possibility of West Nile virus invading Japan;
therefore, preventive measures including diagnosis and early detection
systems are urgently needed. Prof. Takashima led the 21st Century COE
Program “Program of Excellence for Zoonosis Control” at Hokkaido
University from 2003 to 2007. He has been working with Russianscientists to monitor zoonosis such as West Nile virus infections andtick-borne encephalitis which are prevalent in Russia and may invade
Japan.

(Received 18 January 2008, Accepted 5 June 2008)