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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.
Introduction
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.
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
1999
2000
2001
2002
2003
2004
2005
Cases
62
21
66
4156
9862
2539
3000
952
905
970
Deaths
709
284
291
264
450
100
431
119
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
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E and M
5
Lipid bilayer
3
Core
5 UTR
3 UTR
Structural
C prM E
Non-structural
NS1 2A 2B NS3 4A 4B
Protease
Helicase
NTPase
RTPase
NS5
MTase
RdRp
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
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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.
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since 1999.
<2 days
52, F
<24h
48, M
<10h
36, F
<24h
27, M
<7 days
69, M
<10h
63, M
<3h
39, M
<30 mins
36, M
<7 days
51, M
23 days
44, M
<14 days
50, M
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without GAD
with GAD
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Diagnosis
C
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
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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
http://neurology.thelancet.com Vol 6 February 2007
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.
Two
antisense
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
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Rluc
SFV-CprME
SFV NS14
WNV cprME
C prM E
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
assembly.
Conclusions
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
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