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West Nile virus

Laura D Kramer, Jun Li, and Pei-Yong Shi

West Nile virus is a mosquito-borne flavivirus originally isolated in 1937 from the blood of a febrile woman in the
West Nile province of Uganda. The virus is widely distributed in Africa, Europe, Australia, and Asia, and, since 1999,
it has spread rapidly throughout the western hemisphere, including the USA, Canada, Mexico, and the Caribbean and
into parts of Central and South America. Before 1994, outbreaks of West Nile virus were sporadic and occurred
primarily in the Mediterranean region, Africa, and east Europe. Since 1994, outbreaks have occurred with a higher
incidence of severe human disease, particularly aecting the nervous system. In North America, the virus has caused
meningitis, encephalitis, and poliomyelitis, resulting in significant morbidity and mortality. The goal of this Review
is to highlight recent advances in our understanding of West Nile virus virology, ecology, clinical disease, diagnosis,
and development of potential vaccines and antiviral therapies.

Many members of the genus Flavivirus within the family
Flaviviridae cause substantial human disease, including
West Nile, dengue, yellow fever virus, Japanese
encephalitis virus, and tick-borne encephalitis virus.1
Neuroinvasiveness is a common feature of flavivirus
infections where Culex mosquitoes are the predominant
mosquito vector. Since 1999, about 19 525 cases of West
Nile disease have been reported in the USA, of which
8 606 (44%) caused neuroinvasive disease with 771
fatalities (39% of all; 90 % of neuroinvasive disease;
table).2 The 200203 epidemics were the largest outbreaks
of meningitis or encephalitis ever reported in the western
hemisphere, making West Nile virus the dominant
vector-borne viral pathogen in North America.
Seroprevalence varied from 03% in Alberta, Canada, to
95% in Nebraska, with even higher rates in local areas
following the large scale outbreak in 2003.3,4 In North
Dakota, USA, in 2003, blood donor screening suggested
that 735 000 individuals were infected with West Nile
virus, with 04% of these presenting with neuroinvasive
disease.5 Interestingly, almost no morbidity and mortality
have been recognised in human beings, equines, and
birds in Latin America and South America, although two
equine cases were recently reported in Argentina.6 The
reasons for this are not clear but hypotheses include
protection by antigenically cross-reactive flaviviruses,7
decreased virulence of the circulating virus, high
biodiversity in tropical regions leading to a dilution
eect,8 and decreased intensity of surveillance and
diagnostic eorts.

West Nile virus ecology

West Nile virus is maintained worldwide in an enzootic
cycle, transmitted primarily between avian hosts and
mosquito vectors. Mosquitoes of the genus Culex are
implicated as the predominant vectors in the enzootic
cycle throughout the range of the virus distribution.9
The predominant mode of perpetuation of the virus in a
temperate environment over adverse seasons is likely to
be by vertically infected diapausing (ie, physiologically
enforced dormancy between periods of activity) adult
mosquitoes.1012 However, the transmission cycle may also
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be reinitiated through reintroduction of virus by

migratory birds from their winter breeding grounds or
from locations where the virus may be transmitted all
year round,13 or by recrudescence from low concentrations
of virus in avian tissue.14
Non-vector routes of transmission have been
describedeg, oral transmission in birds,14 cats, and
other vertebrates.1416 Although human beings are most
commonly infected by mosquito bites, transmission can
also occur through blood transfusion,17 transplantation,18
breast milk,19 and intrauterine transmission.20 Organ
recipients are at very high risk for neuroinvasive disease
after blood transfusion, donor transmission, or
community exposure.21 In 2002, transmission through
organ transplantation was recognised for the first time in
four patients who were recipients of organs from a
common donor.18 Risk of neuroinvasive disease in an
organ recipient infected with West Nile virus is estimated
as 40% (95% CI 1680%)22 compared with less than 1% in
the general population. Administration of Omr-IgG-am,
an intravenous immunoglobulin product with high-titre
neutralising antibodies to West Nile virus, after disease
onset did not ameliorate symptoms in patients infected
by organ transplantation.23

Lancet Neurol 2007; 6: 17181

Wadsworth Center, New York
State Department of Health,
Albany, New York, USA
(L D Kramer PhD, P Y Shi PhD);
and Department of Neurology,
Wayne State University School
of Medicine Detroit, MI 48201,
Correspondence to:
Pei-Yong Shi, 120 New Scotland
Avenue, Albany, New York
12208, USA

Molecular epidemiology
Phylogenetic analyses of global West Nile virus strains
have revealed two distinct lineages (I and II). Lineage I
strains are commonly involved in human and equine
outbreaks. Phylogenetic analyses of West Nile virus
isolated from the USA indicates that the virus remains
highly conserved genetically and was successfully
introduced only once into North America. A single
conserved aminoacid change in the envelope gene















Neuroinvasive disease (%)









Data from the US Centers for Disease Control and Prevention as of Jan 10, 2006.4

Table: Human illness from West Nile virus infections reported in the USA, 19992005



E and M
Lipid bilayer


frame encodes a polyprotein that is processed by viral and

cellular proteases during and after translation into three
structural proteins (C, premembrane or membrane, and
envelope) and seven non-structural proteins (NS1, NS2A,
NS2B, NS3, NS4A, NS4B, and NS5). Structural proteins
are mainly involved in viral particle formation, whereas
non-structural proteins function in viral replication, virion
assembly, and evasion of host innate immune response.

Host proteins and flavivirus resistance



C prM E


NS1 2A 2B NS3 4A 4B


Figure 1: West Nile virion and genome

The virus structure as reconstructed by cryo-electron microscopy. One asymmetric unit of the icosahedron is indicated
by the triangle on the surface shaded view. The central section of the reconstruction shows the concentric layers of
mass density. Reproduced with permission from the American Association for the Advancement of Science.27 NS3 is a
serine protease (with NS2B as a cofactor), 5-RNA triphosphatase (RTPase), nucleoside triphosphatase (NTPase), and
RNA helicase. NS5 is an RNA-dependent RNA polymerase (RdRp) and N-7 and 2O-methyltransferase (MTase) involved
in the methylation of the 5 RNA cap structure. E=envelope, m=membrane, UTR=untranslated region.

(V159A) is shared by most strains isolated since 2002

(North American dominant)24,25 and is also found in old
world strains of West Nile virus. This genotype first
emerged in 2002 when the activity of West Nile virus
increased expansively, and has eectively displaced the
genotype originally introduced to the USA. Riskassessment analyses suggest that human transport
(mosquitoes on aeroplanes) may be an important pathway
for introduction to new areas, including Hawaii9 and the
Galapagos Islands,26 in addition to transport by birds.

Basic virology
Cryoelectron microscopy showed that virions of West Nile
virus have icosahedral symmetry of 50 nm in diameter,
with no surface projections or spikes (figure 1).27 The
outermost layer contains the viral envelope and membrane
proteins embedded in a lipid bilayer, forming the envelope
of the virion. Inside the envelope is the nucleocapsid core,
which consists of multiple copies of the capsid protein and
genomic RNA. The West Nile virus genome is a singlestranded RNA of plus-sense polarity (ie, mRNA). The viral
genome is about 11 000 bp in length, consisting of a
5untranslated region (UTR), a single long open reading
frame, and a 3untranslated region. The open reading

Host proteins have important roles in West Nile virus

replication. Translation elongation factor alpha (EF-1)
binds specifically to the 3stem-loop of the plus-sense
genomic RNA,28 wherease host proteins TIAR and TIA-1
bind to the 3stem-loop of the minus-sense RNA.29 The
functions of such hostprotein binding to viral RNA are
unclear. The Src family kinase c-Yes was recently reported
to be important for maturation of West Nile virus
particles.30 Inbred mouse strains exhibit significant
dierence in their susceptibility to flavivirus infection.
Resistance to virally induced morbidity and mortality in
mice is flavivirus-specific and is inherited as a single
dominant allele. 2-5-oligoadenylate synthetase 1b,
OAS, was identified as a candidate for the flavivirusresistance phenotype.31 Compared with the resistant
mice, susceptible mice produce an OAS1b protein
lacking 30% of the C-terminal sequence, resulting in the
inactivation of the OAS/RNase L pathway. Consequently,
a large amount of virus is produced in the susceptible
mice. Sequence comparison of OAS and RNase L genes
between patients with West Nile disease and controls
showed multiple single-nucleotide polymorphisms, but
no insertion, deletion, or nonsense mutations.32 More
recently, Scherbick and colleagues found that, although
RNase L has a role in the antiviral response to West Nile
virus, the activation of RNase L is not a major component
of the OAS1b-mediated flavivirus-resistance phenotype.33

Evasion of host innate immune response

Flavivirus non-structural proteins suppress host antiviral
immune responses; however, the molecular details are
unknown. Expression of the dengue virus-2 NS4B and, to
a lesser extent, NS2A and NS4A proteins, results in
down-regulation of interferon--stimulated gene
expression.34 The inhibition could be mediated by the 125
N-terminal aminoacid residues of dengue virus-2 NS4B
protein.35 During West Nile virus infection, the host
response limits viral spread through the activation of the
interferon regulatory factor 3 pathway.36 Interferon-
signalling and STAT-2 translocation to the nucleus were
inhibited when Kunjin (a naturally attenuated subtype of
West Nile virus) NS2A, NS2B, NS3, NS4A, and NS4B,
but not NS1 and NS5 proteins were individually
expressed.37 Furthermore, West Nile virus replication
prevents the phosphorylation and activation of JAK1 and
Tyk2.38 Kunjin replicon accumulated an aminoacid
substitution (Ala30Pro) in NS2A that could reduce the
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NS2A-mediated inhibition of the interferon response.39

Dengue and langat viral NS5 protein could also block the
interferon-stimulated JAKSTAT pathway independently
of STAT1 activation and translocation into the nucleus.40

Clinical presentation
Most individuals infected with West Nile virus are
asymptomatic. Symptoms may develop in 2040% of
people with West Nile virus infection.41,42 The incubation
period is 214 days before symptom onset. Most patients
that are symptomatic present with flu-like symptoms
(West Nile fever). West Nile virus is characterised by fever,
headache, malaise, myalgia, fatigue, skin rash,
lymphadenopathy, vomiting, and diarrhoea.43 Less than
1% of infected individuals develop severe neuroinvasive
diseases, a majority of them can be primarily classified
into three clinical syndromes: West Nile meningitis, West
Nile encephalitis, and acute flaccid paralysis. Clinical
features of these syndromes may overlap in the same
patient. Whether these syndromes are dierent aspects of
a continuous clinical spectrum or distinct entities is
unknown. A recent study of 228 patients reported that
most patients with neuroinvasive disorders can be
classified as either having West Nile meningitis or West
Nile encephalitis and patients with the latter have a higher
mortality rate and more severe complications.44 Thus, the
distinction between these syndromes seems to be clinically
useful. In addition, other syndromes have also been
described, including rhabdomyolysis,45,46 chorioretinitis,47
myositis,48 and autonomic nerve involvement.49 Although
uncommon, additional neurological syndromes may
occur, such as hepatitis, pancreatitis, myocarditis, orchitis,
uveitis, and vitritis.43,50,51 Patients with West Nile virus may
present with two, three, or even more of these syndromes
at the same time. Here we focus on meningitis,
encephalitis, and acute flaccid paralysis.

West Nile meningitis and encephalitis

West Nile meningitis usually presents with fever and
signs of meningeal irritation, such as headache, sti
neck, nuchal rigidity, and photophobia.5053 Kernigs and
Brudzinskis signs may be found on physical
examination.54 When the infectious process involves the
brain parenchyma, West Nile encephalitis develops and
additional clinical features can appear. Patients may have
an altered level of consciousness, disorientation, and
focal neurological signs and symptoms (eg, dysarthria,
seizures, tremor, ataxia, involuntary movements, and
parkinsonism).5053 These variable clinical presentations
show the selectivity of neuroinvasion in West Nile virus
in certain cell populations, such as substantia nigra in
brainstem, basal ganglia,53 and cerebellum.49 All of these
clinical features have also been reported in patients with
other flavivirus infections; thus, these flaviviral infections
are dicult to dierentiate clinically in the acute
Most patients with West Nile fever completely recover
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within days to months.50,55,56 However, patients with West

Nile encephalitis have a poorer outcome than those with
West Nile mengitis. In a recent study of 221 patients with
West Nile virus infection, the 12 patients who died during
hospitalisation had West Nile encephalitis. 25% of
surviving patients with West Nile encephalitis returned
home without the need for increased care, compared
with 76% of patients with West Nile meningitis.50 Patients
with West Nile encephalitis with movement disorders
had a favourable outcome in an earlier study.53

Acute flaccid paralysis

Clinical features in patients with West Nile meningitis or
encephalitis are usually familiar to many physicians and
prompt them to search for a viral cause. However, acute
flaccid paralysis may not be familiar to some clinicians,
particularly when it occurs in the absence of meningitis
or encephalitic signs and symptoms,57 resulting in
diculties for an accurate diagnosis.
About 10% of the hospitalised patients in the 1999 New
York City outbreak had acute flaccid paralysis57. However,
the underlying cause (poliomyelitis) for this acute
paralysis was not recognised until 2002.5860 The selective
lesion of spinal anterior horns by West Nile virus was
documented decades ago.61 In fact, by inoculating West
Nile virus intramuscularly or intraperitoneally,
encephalomyelitis was induced in patients with cancer.
Autopsies from these patients with encephalomyelitis
induced by West Nile virus clearly showed all typical
pathological features that are now described in North
American patients with neuroinvasive diseases from
West Nile virus.62,63 These studies were the first clinical
and pathological documentations for West Nile virus
poliomyelitis. Unfortunately, these studies have been
largely ignored for many years because West Nile virus
was not an epidemic in North America. During the
summer of 2002 through to 2005, there a substantial
number of cases of West Nile virus infection with acute
flaccid paralysis.5759,6466 The main clinical feature was
acute asymmetric flaccid paralysis (dark limbs;
figure 2).57,66,67 Paralysis reached a plateau within hours in
most patients. There was minimal or no sensory
disturbance; most patients had substantial muscle ache
in the lower back; bowel and bladder functions were
disturbed in some patients.58,68
12 weeks before the onset of muscle weakness many
patients have mild flu-like symptoms including headache,
fever, malaise, gastrointestinal upset, skin rash, and
some patients have neck rigidity and changes in mental
status. However, half of our patients did not have viral
prodrome or any meningitis or encephalitic signs.57 Most
patients were healthy before the illness and had no
evidence of immunological suppression.57,65 On neurological examination of patients with acute flaccid
paralysis, most cranial nerves are usually normal.
However, there may be facial weakness in more than half
of these patients. Flaccid limb weakness is conspicuous.


since 1999.

Clinical laboratory features

<2 days
52, F

48, M

36, F

27, M

<7 days
69, M

63, M

39, M

<30 mins
36, M

<7 days
51, M

23 days
44, M

<14 days
50, M

Figure 2: Clinical features induced by West Nile virus paralysis

The main clinical features of our patients are illustrated. Weak limbs at the peak of paralysis are darkened. Degree
of darkness corresponds to the severity of weakness. Duration of weakness and characteristics (age [years] and sex
[F=female, M=male]) are listed below each patient. Reproduced with permission from Elsevier.70

Muscular atrophy develops in the late phase of the illness.

Sensory examination is normal or minimally aected.
Deep tendon reflex can be diminished in severely
paralysed limbs.51,52,57,65
Data on recovery from paralysis induced by West Nile
virus are scant. Among patients hospitalised in New York
and New Jersey in 2000, only a third regained the ability
to walk within a year.69 In another study, three patients
with acute flaccid paralysis needed to use a wheelchair
after an 8-month follow-up.53 Although no detailed
quantitative assessment of motor function was described
in these studies, they suggest an unfavourable outcome.
However, a study in 2002 showed that patients infected
with West Nile virus had a remarkable variation in
recovery.66 Some patients recovered completely within
weeks. A group of our patients with paralysis induced by
West Nile virus were followed up for about 2 years using
certain quantitative measures. Highly variable recoveries
were reported and it was noted that the initial severity of
the illness did not always predict a severe final outcome.67,70
Prediction of whether patients with paralysis will develop
a post-West Nile virus poliomyelitis syndrome similar to
postpolio syndrome is not yet possible.
Although there have been patients that were diagnosed
as having Guillain-Barre syndrome (GBS) or GBS-like
disease, 54,65,71 all cases with detailed electrophysiological
data do not support a demyelinating neuropathy.51,57,59,66,72,73
There was only one exception in which detailed nerve
conduction studies showed the development of a
neuropathy with conduction velocities below 35m/s in
one patient with West Nile virus infection.71 Therefore, it
is reasonable to state that demyelinating neuropathy has
been extremely unusual among patients in the USA

To diagnose West Nile virus infection, serum from

patients should be tested for IgM antibodies against the
virus (usually by ELISA), which are usually indicative of
a recent West Nile virus infection. Blood samples that
are collected between the eighth and 21st day after onset
likely to give the best yield. IgM antibodies are only
detectable 8 days post-symptom onset in some
patients.74,75 Thus, there may be a negative result from a
blood sample obtained before the eighth day after
symptom onset. After the 21st day, the titre of IgM could
Anti-West Nile virus IgM can persist for 1 year or
longer in some patients; a single positive test is not
necessarily associated with the patients current illness.
In addition, cross-reactivity with other flaviviruses may
occur. Thus, a definitive diagnosis requires testing two
serum samples taken at least 2 weeks apart. A fourfold
rise in antibody titre in acute and convalescent sera will
confirm the diagnosis. A definitive diagnosis of West
Nile meningitis, encephalitis, or poliomyelitis can also be
made by looking for IgM antibodies in the cerebrospinal
fluid from patients with pertinent neurological symptoms.
Alternatively, neurological syndromes of those with West
Nile virus can be examined by detection of West Nile
virus RNA with reverse transcriptase PCR of cerebrospinal
fluid, or by immunohistochemical demonstration of
West Nile antigens in neural tissues; however, these
alternative approaches are rarely used in clinical
Other abnormal findings in the cerebrospinal fluid
include raised concentrations of lymphocytes and
proteins. Neutrophils may contribute (at least 50%) in
the early phase of the illness. West Nile meningitis and
encephalitis have similar degrees of pleocytosis.
However, patients with West Nile encephalitis tend to
have higher concentrations of total protein in the
cerebrospinal fluid and usually experience a more
severe outcome.77
MRI is usually normal in most patients. However,
abnormalities have also been described,51,57 such as lesions
in the focal cerebral hemispheric white matter (figure 3),
pons,51,53 substantial nigra,53 and thalamus with increased
signal intensity on T2-weighted images. An important
MRI finding is the focal abnormal signal intensity within
the anterior horns.51,57 In this subgroup of patients, the
level of abnormal spinal MRI findings corresponds to the
paralysis. This observation provides strong evidence for
the selective damage of the anterior horn of the spinal
cord.57 Abnormal MRI findings in the spinal roots have
also been reported.51 A similar finding of root
enhancement was also reported in one of our patients
(figure 3). Even though this could represent a primary
spinal root involvement of West Nile virus (radiculopathy
or radiculitis),51 it is more likely that this MRI change is a
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without GAD

with GAD

Figure 3: Abnormal MRI findings in patients with West Nile virus

Axial FLAIR (fluid-attenuated inversion recovery) image of the brain from a 57-year-old woman with encephalitis (A) shows abnormal signals in bilateral thalamus
and other areas of basal ganglion. Focal white matter lesions are also seen (B). Sagittal T2-weighted MRI of the lumbar spinal cord (C); abnormal signal intensity
(arrows) is conspicuous within the cord. A transverse view of the cord at the mid-lumbar level (D); abnormal signal intensity (arrows) is confined to the anterior
horns; T1-weighted lumbar spine MRI from a patient with both meningitis and acute flaccid paralysis shows no discernable abnormality (E); however, after giving
GAD contrast, spinal roots are significantly enhanced (F). Parts C and D reproduced with permission from Wiley.57

result of axonal degeneration secondary to spinal motor

neuron loss. The second possibility is supported by our
autopsy study that showed pathological changes
consistent with an acute Wallerian degeneration in the
spinal roots.70
Electrophysiological studies are helpful for the
diagnosis of paralysis induced by West Nile virus.51,57,66,72
Motor-nerve conduction studies may reveal severely
reduced amplitudes of compound muscle action
potentials in symptomatic limbs. However, if the nerve
conduction study is done in the early phase of the illness,
compound muscle action potentials can be normal
because Wallerian degeneration can take 710 days to
complete. Nerve conduction velocities are usually
preserved, and sensory nerve conduction is typically
normal. Needle electromyography shows severe denervation in muscles of weak limbs and its corresponding
paraspinal muscles. Taken together, these abnormalities
in the paralysed limbs localise the lesions to the anterior
horn motor neurons or their ventral nerve roots. The
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localisation is typically consistent with the aforementioned

MRI findings.

Neuropathology and pathogenesis of clinical

West Nile virus is thought to initially replicate in
dendritic cells after being bitten by an infected
mosquito. The infection then spreads to regional lymph
nodes and into the bloodstream.2 The way in which the
virus invades the nervous system is still unknown;
retrograde transport along peripheral nerve axons has
been proposed.70,78 On the basis of animal models, a
Toll-like receptor-dependent inflammatory response
could be involved in brain penetration of the virus and
neuronal injury.79 CNS expression of the chemokine
receptor CCR5 and its ligand CCL5 are prominently
upregulated by the West Nile virus, associated with
CNS infiltration of CD4+, CD8+ T cells, NK1.1+ cells,
and macrophages that express CCR5.80 In mice, in
response to the West Nile virus infection, neurons


pathological findings, motor neuron damage at the

anterior horn of the spinal cord is probably the major
contributor to West Nile virus paralysis.
Recovery among patients that have been paralysed is
remarkably variable. The reason for this variation is still
unclear. Data suggest that the variation may be caused by
dierent degrees of motor neuron or motor unit loss.70


Figure 4: Pathology of the spinal cord and roots from an autopsy of a patient infected with West Nile virus
Haematoxylin and eosin-stained sections of the thoracic spinal cord from an autopsy (magnification 250X).
Motor neurons at the anterior horn are almost depleted (A). By contrast, most motor neurons (arrowheads) in
another segment of the thoracic spinal cord are well preserved (B). A few motor neurons seem swollen (arrows
in B). In addition, conspicuous inflammatory cells infiltrate diffusely in both segments of the cord.
Immunohistochemistry of the spinal root with antibodies against CD68 (brown-colour dots in C) and CD3
(brown-colour dots in D; magnification 250X). Abundant inflammatory cells and axonal ovoids are present in
the root (arrowheads and arrows). CD68 cells (brown-color in C) are numerous and distributed in linear
arrays along the degenerating axons (the array of arrowheads). These features are highly suggestive of a
secondary inflammatory reaction to an ongoing Wallerian degeneration in the root. Reproduced with
permission from Elsevier.70

secrete chemokine CXCL10, which recruits eector T

cells via the chemokine receptor CXCR3.81 A recently
identified receptor for West Nile virus, V3 integrin,
provides new insights into how the virus invades
dierent tissues.82
Histological CNS findings of West Nile virus infection
are usually characterised by perivascular lymphoplasmacytic infiltration, microglial nodules, reactive
proliferation of astrocytes, variable necrosis, and neuronal
loss with predilection to structures like the thalamus,
brainstem, and cerebellar Purkinje cells.48,6164,74 These
variable anatomical involvements explain dierent
clinical presentations.
The selective destruction of anterior horn motor
neurons seems particularly relevant to understanding
the pathogenesis of neuroinvasiveness. This selective
lesion with inflammatory infiltration has been confirmed
in several autopsy studies (figure 4)64,70 and West Nile
virus antigens were localised in the anterior horn.83 This
localisation is consistent with early pathological studies84,85
as well as the findings from people intramuscularly
inoculated with West Nile virus.62 Moreover, almost all
flaviviruses cause motor neuron damage at the anterior
horns; however, poliomyelitis is much less common in
patients with other non-West Nile flaviviruses.8589 On the
basis of clinical presentation, neuroimaging, and

The diagnosis of West Nile virus paralysis should be

considered whenever clinical presentation of viral
meningitis or encephalitic and acute asymmetric
paralysis with normal sensory examination occurs
during the seasons when mosquito-borne diseases
might occur. Absence of viral prodrome does not
exclude the diagnosis.57,65 Electrophysiological studies
and neuroimaging can be helpful. A definitive diagnosis
for neuroinvasive diseases usually requires a positive
IgM antibody test from the serum or cerebrospinal fluid
in an appropriate clinical setting. Confirmation of
recent infection is confounded by long-lasting IgM
antibodies; IgM can last up to 16 months.90 Thus,
caution should be taken when interpreting these
laboratory results.
All flaviviruses of the Japanese and tick-borne
encephalitis complex can cause meningitis and
encephalitis. Because meningitis and encephalitis with
these viruses are clinically similar, at least in the acute
phase, dierential diagnosis has to rely on serological
assays on sera or cerebrospinal fluid or on PCR testing.
In patients with acute flaccid paralysis, dierential
diagnosis can include GBS, myopathy, neuromuscular
junction disorders, and other virus-related motor neuron
diseases. Sensory disturbance and slow conduction
velocities from nerve conduction studies may be sucient
to dierentiate those with GBS and West Nile virus
paralysis. Acute motor axonal neuropathy, as a subtype of
GBS, can closely imitate West Nile virus paralysis, but is
less problematic in the USA since acute motor axonal
neuropathy is extremely rare in this country and it
symmetrically aects limbs.
In addition, all these flaviviruses aect the lower motor
neurons. Flaccid weakness and needle electromyography
abnormalities consistent with damage of the lower motor
neurons have been reported in St Louis encephalitis,88,90
Japanese encephalitis,87 Murray Valley encephalitis,91 and
Powassan encephalitis.92 Pathological studies have shown
inflammatory involvement in the anterior horns in
Japanese encephalitis, nearly identical to that seen in
encephalitis causes similar pathological abnormalities;93
however, its clinical presentation is unique and is
characterised by cervical cord motor neuron involvement
and conspicuous head-drop due to neck extensor
denervation. These infectious agents should be considered
when dealing with a patient with an acute lower-motorneuron disorder. Because it is challenging to clinically
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dierentiate among these diseases, epidemiological data

may be critical for the diagnosis, and specific serological
or PCR tests are commonly necessary.

Vaccine development
Human vaccines for flavivirus infections are currently
available only for yellow fever, Japanese encephalitis, and
tick born encephalitis.1 Because the premembrane and
envelope proteins are highly antigenic and elicit strong
and long-lasting immune responses, multiple approaches
have been explored to deliver these antigens into animals
for vaccine development. The first approach is based on
chimeric viruses delivering West Nile virus antigens. The
yellow fever 17D vaccine strain, which has been safely
used for large-scale human immunisation for over
60 years, is an excellent vector for delivering protective
antigens of flaviviruses. Chimeric vaccines were
constructed by replacing the yellow fever 17D
premembrane and envelope genes with the corresponding
genes of other flaviviruses, including the virus of Japanese
encephalitis,94 dengue,95 and West Nile.96 Similarly,
chimeric viruses of West Nile virus combined with
dengue virus 2 or 4 are immunogenic, and provide
complete protection against lethal West Nile virus
challenges.97,98 In mice, expression of West Nile virus
premembrane and envelope genes, through attenuated
measles virus, conferred protection from a lethal dose of
West Nile virus.99 Among these vaccine candidates,
chimeric viruses of West Nile virus and yellow fever virus
and with dengue virus are in phase I clinical trials. The
recent results of clinical trials showed that yellow fever
chimeric virus elicits strong immune responses without
apparent adverse events after a single dose.100 After
inoculation of 30 healthy adults with 105 plaque-forming
units or 15 people with 103 plaque-forming units (n = 15)
of West Nileyellow fever chimeric virus, all adults
developed neutralising antibodies to West Nile virus, and
most developed a specific T-cell response.
The second approach is based on immunisation of
animals with recombinant viral proteins, inactivated
West Nile virus, or DNA that expresses viral antigens.
Formalin-inactivated West Nile virus, a recombinant
canarypox virus vector, and a DNA plasmid expressing
West Nile virus premembrane and envelope proteins
have been developed and approved for equine use.101103
These results agreed with previous findings that
expression of tick-borne or Japanese encephalitis
premembrane and envelope proteins in mammalian
cells produced empty virus particles that contained
proteins embedded in the lipid bilayer without a
nucleocapsid. Mice immunised with such West Nile
virus particles were protected from infection.104
Furthermore, direct immunisation with recombinant
West Nile virus envelope protein alone protected mice
and horses against subsequent infections.105 In a hamster
model, animals immunised with 1 g of recombinant
envelope protein were completely protected from lethal
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challenge with West Nile virus for at least 6 months. No

viraemia or clinical disease were reported, and high
titres of viral neutralising antibodies were elicited.106
These promising results warrant further assessment of
recombinant envelope as a vaccine candidate in monkeys
and human beings.
The third approach is based on attenuated West Nile
virus isolates. Kunjin virus is a naturally attenuated West
Nile virus subtype that could provide protective immunity
in mice against the virulent New York West Nile virus
strain.107 An attenuated non-epidemic West Nile virus
strain (lineage II) was also shown to be an eective
vaccine against virulent epidemic strain (lineage I) in
mice.108 For other flaviviruses, introduction of deletions
into the capsid protein of TBEV has been reported as a
promising way to create attenuated, yet highly
immunogenic, flaviviruses.109 Deletions within the
3untranslated region were also shown to attenuate
dengue virus110 and tick-borne encephalitis virus.111 These
approaches could alternatively be used for development
of West Nile virus vaccines.

Antiviral therapy
There is no specific regime currently available for
treatment of flavivirus infections. Although several
compounds have been reported to inhibit recombinant
West Nile virus enzymes or to suppress virus in cell
culture, few of them have shown in vivo ecacy.
Antibody-based therapy has yielded the most promising
results. Passive administration of monoclonal antibodies
has previously shown prevention and alleviation of St
Louis encephalitis,112 Japanese encephalitis,113 and
encephalitis caused by yellow fever virus.114 Intravenous
immunoglobulin that contains high titers of West Nile
virus antibodies seemed eective in patients with West
Nile encephalitis in an open-label study; however, these
findings require further confirmation in controlled
studies.115 Humanised monoclonal antibodies against the
West Nile virus envelope protein have shown ecacy in
mice, even when given as a single dose at day 5 postinfection and when the virus has already infected the
CNS.116 The ecacy of these monoclonal antibodies
against the envelope protein still needs to be determined
in monkeys and human beings.
phosphorodiamidate-morpholinooligomers (PMOs) were reported to potently inhibit West
Nile virus infection in cell culture.117 A new Arg-rich
peptide was conjugated to the PMO for ecient cellular
delivery. Because one PMO targets a flavivirus-conserved
RNA sequence that is essential for viral RNA synthesis,
this PMO had a broad spectrum of anti-flavivirus activities
in cell culture.117 These PMOs could partially protect mice
from West Nile disease when given at day 5 post-infection
(Shi PY, unpublished). These results warrant further
improvement in anti-sense PMO-based therapies.
Interferon -2b is currently under clinical trial for
treatment of patients with West Nile virus-mediated


Cell lines containing persistently replicating RlucNeoRep



NS1 2A 2B NS3 4A 4B NS5 IRES Neo

Infection of cells with virus-like particles containing RlucNeoRep




Infection of cells with full-length WNV containing a luciferase reporter


C prM E

NS1 2A 2B NS3 4A 4B NS5 IRES Rluc

Figure 5: Cell-based, high-throughput assays for West Nile virus drug discovery
For each assay, a Renilla luciferase (Rluc) gene was engineered into a replicon (containing a deletion of three
structural genes) or into a full-length viral genome to monitor viral replication. Potential inhibitors could be
identified through suppression of luciferase signals upon compound incubation. A reporting cell-line assay (top):
the cell line has a persistently replicating replicon that contains dual reporter genes, luciferase and neomycin
phosphotransferase, resulting in RlucNeoRep; the assay allows screening of inhibitors of all targets involved in viral
replication. Virus-like particle infection assay (middle): Semliki forest virus vector containing viral non-structural
proteins (SFV NS14) was used to express West Nile virus (WNV) structural proteins C-prM-E under the control of
the SFV 26S subgenomic promoter (SFV-CprME). Transfection of RlucNeoRep-containing cells with SFV-CprME
packages RlucNeoRep RNA into VLPs. Infections of naive cells with such particles results in replication of
RlucNeoRep, yielding Rluc signals; this assay allows screening of inhibitors of viral entry and viral replication. A fulllength reporting WNV infection assay (bottom): a luciferase gene driven by an EMCV IRES was engineered at the
3-untranslated region of the genome, resulting in Rluc-WNV. Infection of cells with Rluc-WNV generates Rluc
signals; this assay allows screening of inhibitors of all steps of viral infection cycle, including entry, replication, and

meningoencephalitis. Interferon -2b has inhibited in

vivo West Nile virus replication.118 Mice with a defective
receptor for interferon or interferon (and therefore
defective in interferon response) showed higher mortality,
shorter survival time, and altered cellular tropism of
infection in comparison with wild-type mice inoculated
with the West Nile virus.119 Treatment of primary neurons
infected with West Nile virus with interferon increased
neuronal survival. There was substantial recovery in the
neurological function of five patients with West Nile
virus-CNS disease treated with interferon -2b soon after
symptom onset.120 However, one patient treated with
interferon -2b died, probably because of delayed
diagnosis and treatment, as well as other complications.121
More controlled studies are needed to demonstrate the
ecacy of the interferon treatment.
Three cell-based high-throughput assays have been
developed for the discovery of a drug for West Nile virus
(figure 5).122 A luciferase reporter was engineered into a
subgenomic replicon (containing a deletion of viral
structural genes) and into a full-length West Nile virus.
Replication of a reporting replicon or virus in cells results
in expression of luciferase, which can be used to monitor
antiviral activities of potential inhibitors. Alternatively, viral
structural proteins could package the luciferase-expressing
replicon into virus-like particles. Infection of cells with
such reporting virus-like particles could also be used to
screen potential inhibitors. Screening compound libraries
with these high-throughput assays has identified a new
triaryl pyrazoline compound that could inhibit flaviviruses
in cell culture, including West Nile, dengue, and yellow

fever virus.123 Mode-of-action analyses in West Nile virus

showed that the compound specifically suppressed viral
RNA synthesis. The in vivo ecacy and the specific target
of the triaryl pyrazoline inhibitor are unknown.

Improvement in our understanding of flavivirus virology,
ecology, and pathogenesis will substantially contribute to
the prevention and treatment of flavivirus infections. For
example, crystallographic studies have shown that
envelope proteins undergo a sequential structural change
during the fusion-activating transition.124 Small-molecule
inhibitors could be developed to block the structural
switch that is essential for viralhost membrane fusion.
The findings that flavivirus proteins antagonise host
innate immune response have provided opportunities to
develop novel antiviral therapy and vaccines for
flaviviruses. For antiviral therapy, inhibitors that block
the activity of interferon antagonism of viral proteins
would allow the innate antiviral response to eectively
suppress viral infection and, therefore, reduce viral
burden. For vaccine development, mutant flaviviruses
containing aminoacid changes in viral proteins to
suppress its function on inhibiting interferon response
could be generated through manipulation of infectious
cDNA clones. The mutant viruses are competent in
replication, but defective in interferon antagonism.
Inoculation of animals with such mutant viruses is
expected to elicit a strong immune response due to viral
replication. However, since the viruses are sensitive to
the hosts interferon inhibition, they will be eliminated
by the host without causing disease.
A rapid and virus-type-specific serological assay is
needed for clinical diagnosis of flavivirus infection. One
potential means to improve the current flavivirus
diagnosis is to use luciferase-reporting flaviviruses for
the standard antibody-mediated neutralising assay.
Similar to the luciferase-expressing West Nile virus
(figure 5), other flaviviruses containing the luciferase
reporter could be generated. Neutralisation of flaviviruses
will be indicated by luciferase signals within 24 h postinfection. The reporting virus-based assay will avoid the
time-consuming plaque assay (which requires about a
week depending on the type of flavivirus) and should
substantially shorten the time to diagnosis. In support of
this idea, reporting replicon-containing viruses have
recently been reported for measuring antibody-mediated
neutralisation of West Nile virus.125
It is important to understand the ecology of West Nile
virus, including the causes of spatial and temporal
variation in transmission, the roles of host reservoir
competence, acquired immunity in West Nile virus
amplification, and the impact of climate on vector ecology
and viral replication. Finally, despite extensive studies on
the phenotypic presentation of West Nile virus
neuroinvasive diseases, the pathogenic mechanism of
these diseases is still unclear. Unravelling the mechanism
http://neurology.thelancet.com Vol 6 February 2007


Search strategy and selection criteria

References for this Review were identified by searches of
PubMed using the terms West Nile virus and flavivirus
from 1950 to March 2006. Additional references and book
chapters that were cited in relevant papers were also used.
Abstracts from conferences were also included. Only
references in English were used. References were selected on
the bases of originality and relevance.

of selective invasion of West Nile virus into spinal motor

neurons may provide a therapeutic target for blocking of
West Nile disease. A combination of basic science and
translational research will greatly facilitate the prevention
and treatment of West Nile virus.
All authors contributed equally to the preparation of this Review.
LDK wrote sections on the ecology and epidemiology of West Nile virus.
JL wrote sections on clinical presentation and diagnosis. PYS wrote
sections on virology, evasion of innate immune response, development
of vaccines and antiviral therapy.
Conflicts of interest
We have no conflicts of interest.
We thank Dr Richard Kuhn for providing cryo-electron microscope
images in figure 1. We thank Dr Aashi Shah for his generous gift of
figure 3, parts A and B and 3B. LDKs group is supported by NIH
contract NO1AI254 90 and grant AI50758-01, and CDC grant U50/
CCU220532-01. JLs group is supported by grants NINDS K08NS048204
and MDA4029. PYSs group is supported by NIH grants AI061193 and
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