Viruses 2012, 4, 3812-3830; doi:10.3390/v4123812
Immune Responses to West Nile Virus Infection in the Central
Hyelim Cho 1 and Michael S. Diamond 1,2,3,*
1 Departments of Molecular Microbiology, Washington University School of Medicine,
St. Louis, Missouri 63110, USA; E-Mail: firstname.lastname@example.org
2 Departments of Medicine, Washington University School of Medicine, St. Louis,
Missouri 63110, USA
3 Pathology and Immunology, Washington University School of Medicine, St. Louis,
Missouri 63110, USA
* Author to whom correspondence should be addressed; E-Mail: email@example.com;
Tel.: +1-314-362-2842; Fax: +1-314-362-9230.
Received: 20 November 2012; in revised form: 7 December 2012 / Accepted: 10 December 2012 /
Published: 17 December 2012
Abstract: West Nile virus (WNV) continues to cause outbreaks of severe neuroinvasive
disease in humans and other vertebrate animals in the United States, Europe, and other
regions of the world. This review discusses our understanding of the interactions between
virus and host that occur in the central nervous system (CNS), the outcome of which can be
protection, viral pathogenesis, or immunopathogenesis. We will focus on defining the
current state of knowledge of WNV entry, tropism, and host immune response in the CNS,
all of which affect the balance between injury and successful clearance.
immunopathogenesis; neuron; brain
flavivirus; innate immunity; adaptive immunity; pathogenesis;
West Nile virus (WNV) is a mosquito borne, neurotropic, positive-stranded, enveloped RNA virus
in the Flaviviridae family. WNV is related genetically to other viruses that cause severe visceral and
central nervous system (CNS) diseases in humans including dengue (DENV), yellow fever (YFV),
Japanese encephalitis (JEV), and tick-borne encephalitis (TBEV) viruses. WNV is maintained in an
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enzootic cycle between mosquitoes and birds, but also infects and causes disease in vertebrate animals
including horses and humans. WNV is transmitted primarily by Culex species mosquitoes and the virus
amplifies in bird reservoirs, with humans and horses largely considered as dead-end hosts .
Although human cases occur primarily after mosquito inoculation, infection after blood transfusion,
organ transplantation, and intrauterine transmission has been reported . At present, there are no
vaccines or therapeutic agents approved for humans against WNV.
WNV was first isolated in 1937 in Uganda from a woman with an undiagnosed febrile illness ,
and historically, has caused outbreaks of a relatively mild febrile illness in regions of Africa, the
Middle East, Asia, and Australia . In the 1990’s, the epidemiology of infection changed.
New outbreaks in Eastern Europe were associated with higher rates of severe neurological disease .
In 1999, WNV entered North America, and caused seven human fatalities in the New York City area
as well as a large number of avian and equine deaths. Since then, it has spread to all 48 of the lower
continental United States as well as to parts of Canada, Mexico, the Caribbean, and South America.
While the majority of human infections are asymptomatic, WNV can cause a severe febrile illness and
neuroinvasive syndrome characterized by meningitis, encephalitis, and/or acute flaccid paralysis [5–7].
Persistent movement disorders, cognitive dysfunction, and long-term disability all occur after West
Nile neuroinvasive disease. West Nile poliomyelitis-like disease results in limb weakness or paralysis.
Patients show markedly decreased motor responses in the paretic limbs, preserved sensory responses,
and widespread asymmetric muscle denervation without evidence of demyelination or myopathy .
Thus, the neurological and functional disability associated with WNV infection represents a
considerable source of morbidity in surviving patients long after the acute illness [9–13]. In the United
States alone between 1999 and 2012, ~36,000 cases and ~1,500 deaths have been confirmed.
The risk of severe WNV infection in humans is greatest in the elderly and
immunocompromised [14,15]. Two studies have estimated a 20-fold increased risk of neuroinvasive
disease and death in those over 50 years of age [14,16]. Beyond age, a limited number of host genetic
factors have been linked with susceptibility to WNV infection. A deficiency of the chemokine receptor
CCR5 increases the risk of symptomatic WNV infection, as a higher incidence (4.2%) of
loss-of-function CCR5Δ32 homozygotes was observed in symptomatic WNV infection cohorts
compared to that in the general population (1.0%) . A nonsense mutation in the gene encoding
2'-5'-oligoadenylate synthetase/L1 (OAS) isoform is associated with WNV susceptibility in laboratory
mice . Correspondingly, a hypomorphic allele of the human ortholog OAS1 is associated with both
symptomatic and asymptomatic WNV infection . Finally, an association of single nucleotide
polymorphisms (SNP) between symptomatic and asymptomatic WNV infections and IRF3 and Mx1
innate immune response and effector genes has been reported ; thus, genetic variation in the
interferon (IFN) response pathway appears to correlate with the risk of symptomatic WNV infection in
humans. In this review, we will summarize our understanding of the host-virus interface in the CNS
and how this determines WNV disease pathogenesis and clinical outcome.
2. Virology and Pathogenesis
Although cellular receptors have not yet been identified definitively, studies suggest that WNV
enters cells by endocytosis and fusion with the early endosome [21,22]. Following fusion between the
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viral and endosomal membranes, the nucleocapsid is released into the cytoplasm and 11 kilobase viral
genomic RNA associates with endoplasmic reticulum (ER) membranes. The single open reading frame
is translated into a polyprotein and enzymatically processed into three structural proteins (capsid (C),
pre-membrane (prM)/membrane (M), and envelope (E)) and seven non-structural proteins (NS1,
NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Negative strand viral RNA then is synthesized and
serves as a template for positive strand RNA synthesis . Positive strand RNA is packaged in
progeny virions, which bud into the ER to form enveloped immature virions. A maturation step,
cleavage of the prM protein to the membrane M protein, occurs in the trans Golgi network by
furin-like proteases [24–26] and results in a reorganization of E proteins on the virus surface into a
homodimeric array ; these virions are secreted into the extracellular space by exocytosis.
Following mosquito inoculation into the skin, it is believed that WNV replicates within epidermal
keratinocytes and Langerhans cells [28,29]. Migratory Langerhans dendritic cells enter afferent
lymphatics and travel to draining lymph nodes . Here, infection and the risk of dissemination are
countered by the rapid development of an early immune response including type I and II IFN
production and the effector functions of innate immune cells (
macrophages, and IgM-secreting B cells) [30–34]. Virus produced in the lymph node can enter
circulation via the efferent lymphatic system and thoracic duct, and viremia allows spread to secondary
lymphoid and visceral organs including the spleen and kidney [35,36]. In peripheral tissues, infection
is restricted by innate and adaptive immune responses including serum IgM , IFN-/ ,
IFN- [32,39], cytolytic CD8+ T cells [39–41], and cell-intrinsic IRF-3-dependent [30,42]
cells, NK cells, neutrophils,
3. WNV-Induced Pathology in the CNS
WNV causes encephalitis in several vertebrate species by virtue of its ability to infect and injure
neurons through direct (viral-induced) and indirect (immune response induced or bystander)
mechanisms . Pathologic observations in humans are limited by the small number of autopsy
studies on individuals succumbing to WNV infection. In these few reports, gross macroscopic
examination of the brain and spinal cord did not reveal any overt pathology .
Microscopic examination of the brain in humans and other animals reveals histological changes that
are consistent with the clinical disease [5,36]. This includes neuronal cell death, activation of resident
microglia and infiltrating macrophages, perivascular and parenchymal accumulation of CD4+ and
CD8+ T cells and CD138+ plasma cells, and formation of microglial nodules. These lesions, which can
be patchy in distribution, occur in the brainstem, cerebral cortex, the hippocampus, thalamus, and
cerebellum . Cellular infiltrates in the meninges also can be present. In some cases, destruction of
vascular structures with focal hemorrhage occurs, suggestive of a vasculitis; this may be associated
with local compromise of the blood-brain barrier (BBB) [44,45]. Immunohistochemical analysis
confirms that WNV antigen is present primarily in neurons from multiple regions of the brain,
although other cells (e.g., CD11b+ myeloid cells and possibly astrocytes) may be infected but to lesser
degrees [46,47]. In the spinal cord, an intense inflammatory infiltrate around large and small blood
vessels is observed with large numbers of microglia in the ventral horn. Anterior horn motor neurons
are targeted by WNV [8,48], and studies suggest that axonal transport from peripheral neurons can
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mediate WNV entry into the spinal cord and induce acute flaccid paralysis . Studies in hamsters
reveal that limb paralysis and tremors are directly associated with infection and injury of anterior horn
motor neurons in the lumbar section of the spinal cord .
To establish infection in neurons of the brain, WNV first must cross the BBB (Figure 1). The BBB
is composed of endothelial cells, astrocyte foot processes, and pericytes (PCs) and impedes the entry of
macromolecules and pathogens from the blood into the brain. The tight junctions between endothelial
cells form a diffusion barrier and pose obstacles for pathogens to enter the brain and to infect
vulnerable and largely non-renewable neurons . The mechanism by which WNV and other
encephalitic flaviviruses cross the BBB remains uncertain. Crossing of the BBB likely occurs through
a hematogenous route, as high levels of viremia correlate with greater and more rapid WNV entry into
the CNS [52,53]. Intravascular levels of pro-inflammatory cytokines, which are produced during
peripheral immune responses, also may modulate WNV entry into the CNS. WNV infection in
peripheral tissues induces Toll-like receptor (TLR)-3-mediated secretion of pro-inflammatory
cytokines, including IL-6 and TNF-α . Secreted TNF-α can modulate BBB permeability by
altering endothelial cell tight junctions, which may allow WNV to cross the BBB and infect
neurons [44,54,55]. Semaphorin 7A upregulation after WNV infection also is linked to increased
TNF-α production. Mice lacking Semaphorin 7A showed reduced TNF-α levels in serum, less BBB
permeability, and reduced viral entry into the brain. . Activation of matrix metalloproteinases also
may enhance the flux of WNV by degrading the extracellular matrix of the BBB . In BBB model
studies in vitro, treatment with inhibitors of matrix metalloproteinases prevented the disruption of tight
junction integrity associated with WNV infection .
Beyond compromise of the BBB, in some cases, WNV may penetrate into the CNS through
additional mechanisms. Peripheral neurons are susceptible to infection by WNV [59,60]; retrograde
axonal transport can bring WNV into the CNS, where transneuronal spread can occur. In contrast to
some viruses (e.g., rabies ), neuron-to-neuron spread of WNV requires axonal release of viral
particles . Other possible entry mechanisms for WNV include (i) infection or passive transport
through choroid plexus epithelial cells , (ii) a “Trojan horse” mechanism in which the virus is
transported by infected immune cells (e.g., neutrophils  or CD4+ or CD8+ T cells ) that cross
the BBB , (iii) infection of olfactory neurons and rostral spread from the olfactory bulb , or
(iv) direct infection of brain microvascular endothelial cells . The precise mechanism of WNV
entry into the CNS in humans requires further study, and may differ depending on the route of
infection and the pathogenicity of the WNV strain .
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Figure 1. Mechanism of neuroinvasion of West Nile virus (WNV). WNV may enter the
central nervous system (CNS) via multiple mechanisms including axonal retrograde
transport along peripheral neurons into the spinal cord or hematogenous transport across
the blood-brain barrier (BBB). Spinal cord entry is believed to result in interneuron spread
to motor neuron cell bodies within the anterior horn of the spinal cord and lead to flaccid
paralysis. The possible routes of virus entry across the BBB include (a) “Trojan horse”
model; intracellular transport within macrophages or neutrophils, (b) loss of integrity of the
BBB; cytokine-mediated (TNF-α, MIF) or matrix metalloproteinases disruption of tight
junctions and basement membranes; (c) direct infection of brain microvascular endothelial
cells with basolateral spread of the virus; (d) infection of choroid plexus epithelial cells; or
(e) direct infection of olfactory neurons adjacent to the cribriform plate.
5. Neuronal Injury
WNV infection of neurons can result in caspase 3-dependent apoptosis, which likely contributes to
CNS dysfunction and pathogenesis of severe disease. While no significant difference in peripheral or
CNS tissue viral burden was observed in WNV-infected caspase 3−/− mice, these animals were more
resistant to lethal WNV infection due to reduced neuronal cell death in the cerebral cortex, brain stem,
and cerebellum . Consistent with this, ectopic expression of WNV NS2B-NS3 non-structural
proteins activates caspase 3 and induces apoptosis in neuroblastoma cell lines , and primary
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neurons and neuroblastoma cells undergo apoptosis after WNV infection [48,68,70]. Cellular stress
pathways including cAMP response element-binding transcription factor homologous protein
(CHOP)-dependent apoptotic pathway also likely contribute to WNV-induced neuronal damage .
WNV infection may trigger apoptosis by activating non-caspase proteases, such as calpains and
cathepsins [72,73]. Finally, WNV infection can induce non-apoptotic pathways of cell death.
Cell necrosis can occur, as characterized by extensive cell swelling and loss of membrane integrity
likely due to the extensive budding of WNV progeny virions into the ER .
In addition to injury imposed directly by WNV infection, neurons may undergo cell death or injury
due to bystander damage caused by cytotoxic factors released by neuronal and non-neuronal cells.
Neurons that are dying secondary to viral infection or immune-targeted death may release
inflammatory molecules (e.g., Cxcl10L-1β, IL-6, IL-8, and TNF-α) [75,76] with potentially toxic
effects on uninfected neurons resulting in irreversible neuronal loss and atrophy. Analogously, glial
cells, which are not primary targets of direct WNV infection, can become activated and release
excitotoxic amino acids (e.g., glutamic and aspartic acids) and pro-inflammatory cytokines that
contribute to the pathogenesis of neurological diseases by virus infections [77,78]. For example,
TNF-α and IL1-β released by activated glial cells have direct roles in promoting bystander damage to
neurons . Elevated reactive oxygen species secreted by infected or activated microglial cells also
may result in oxidative damage to neurons .
6. CNS Immune Responses to WNV
Upon entry in the CNS, WNV spreads rapidly between different subtypes of neurons in distinct
regions . As neurons are largely non-renewable, controlled immune responses must limit spread
and eliminate virus while minimizing neuronal damage . A delay or absence of such responses in
genetically deficient mice or immunosuppressed humans results in rapid dissemination, neuronal
injury, with an increased risk of mortality. Recent work in animal models has shown that both innate
and cellular immune response in the CNS orchestrate control of WNV spread, which ultimately limits
the number of neurons targeted for infection or the amount of virus any given infected neurons
6.1. CNS Innate Immunity
Nucleic acid intermediates of RNA virus replication are recognized by pathogen recognition
receptors (PRR) such as TLR and RIG-I like receptors (RLR), which promote an antiviral state by
activating IRF-3 and IRF-7-mediated transcriptional programs and type I IFN responses. The
importance of these pathways for controlling WNV infection is highlighted by studies in mice that are
genetically deficient for key components
receptor-knockout mice (Ifnar−/−), Ifnb−/−, Mavs−/−, Tlr3−/−, Tlr7−/−, and Myd88−/− mice all show
enhanced viral replication in the CNS and mortality after WNV infection [38,47,83–85].
Irf3−/− neurons showed reduced induction of antiviral defense genes including Rig-I, Mda5, and Ifit1,
as well as blunted IFNα/β production . In Irf7−/− neurons, IFN-α production was blunted, which
resulted in increased WNV infection . Together, these studies suggest that IRF-3 and
IRF-7-dependent transcriptional programs are crucial for protective IFN response in neurons.
in this pathway: Type I IFN
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Stat1-dependent signaling pathways in part, determine the susceptibility of specific neuronal subtypes
to WNV infection in the brain. IFN-α/β and Stat1-dependent transcription of IFN-stimulated genes
(ISGs) inhibited WNV replication in neurons in vitro and in vivo. Rsad2 (also known as viperin), PKR,
and RNase L are induced in neurons of the CNS and restrict WNV infection in vivo [87,88].
6.2. Inflammatory Responses
Neurons in the CNS are immunologically active and initiate inflammatory responses by producing
chemokines that recruit immune cells (Figure 2). Infection of neurons by WNV induces expression of
the T cell chemoattractant Cxcl10, which promotes trafficking of WNV-specific CD8+ T cells via
binding to its cognate receptor Cxcr3 [89,90]. Enhanced expression of Ccl3 (MIP-1α), Ccl4 (MIP-1β),
Ccl5 (RANTES) by WNV infection leads to Ccr5-dependent trafficking of CD4+ and CD8+ T cells,
NK cells, and macrophages. Deletion or truncation of Ccr5 in mice leads to enhanced viral burden and
increased mortality [91,92], and appears to be associated with more severe disease in humans .
Trafficking of monocytes into the brain, as precursors of macrophages and possibly microglia, can
contribute to CNS injury  or survival after WNV infection , depending on the virulence of the
infecting WNV strain. In mice, deletion of Ccr2, a chemokine receptor on inflammatory monocytes,
leads to increased mortality after infection by virulent North American WNV strains, and this is
associated with reduced monocyte accumulation in the brain . Study with Il22−/− mice demonstrate
that reduced levels of Cxcr2, a chemokine receptor mediating neutrophil migration, correlate with
decreased viral loads in the CNS , suggesting that entry of WNV-infected neutrophils may
contribute to pathogenesis. In Tlr7−/− mice, CD45+ leukocytes and CD11b+ macrophages failed to
home to WNV-infected neurons due to blunted IL-23 responses, suggesting Tlr7 reduces WNV
infection in part, by enhancing IL-23-dependent immune cell infiltration and homing into the brain
6.3. Cellular Immunity
Studies in mice suggest that T cell-mediated immunity is an essential aspect of immune mediated
protection from virulent strains of WNV. The lack of a functional CD4+ and CD8+ T cell response
results in inefficient clearance of WNV infection from neurons of the brain [39,40,96]. Nonetheless, an
over-exuberant CD8+ T cell-mediated response can lead to injury and or death of infected or
uninfected neurons. In mice, within a few days of CNS infection, inflammatory cytokines and
chemokines produced by resident cells of the CNS attract antigen-specific CD8+ T cells into the CNS
[40,89]. In addition, CD40-CD40L and TNF-TNF-receptor interactions promote CD8+ T cell
migration across brain microvascular endothelial cells, likely by increasing expression of adhesion
molecules and modulating the integrity of tight junctions [97,98].
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Figure 2. Leukocyte trafficking into the CNS after WNV. Upon WNV infection of
neurons, virus-mediated upregulation of Cxcl10 recruits virus-specific CD8+ T cells via
interactions with Cxcr3. Expression of Ccl3, Ccl4, and Ccl5 by other neuronal cells recruits
Ccr5-expressing leukocytes. Monocytes and lymphocytes entering the perivascular spaces
may be retained initially via Cxcr4 binding Cxcl12 . Leukocyte egress from
perivascular spaces requires IL-1β, TNF-, and CD40 interactions, which likely
upregulates adhesion molecules including ICAM-1 and VCAM-1 [121,123,124].
CD8+ T cells control WNV infection in the CNS via multiple mechanisms (Figure 3) including the
production of antiviral cytokines (e.g., IFN-) or by triggering cell death of target cells through
perforin, Fas-Fas ligand, or TRAIL-dependent pathways. Infected neurons up-regulate MHC class I
molecules and thus, can be targeted by cytotoxic T cells . Perforin−/− mice showed higher viral
burden in CNS and increased mortality after WNV infection , as well as a failure to clear WNV
resulting in persistent CNS infection. Perforin-mediated control of infected neurons occurs through the
granzyme-dependent granule exocytosis pathway, which results in apoptosis of infected neurons
in vitro and in vivo [100–102]. Fas ligand (FasL) deficient mice also showed increased susceptibility to
lethal WNV infection . Interactions between Fas on infected neurons and FasL on CD8+ T cells
leads to programmed cell death of neurons through the activation of a death domain and a caspase
apoptosis cascade [102,104,105]. CD8+ T cells also use tumor necrosis factor-related
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apoptosis-inducing ligand (TRAIL; also known as CD253) to restrict WNV pathogenesis by
controlling infection in neurons. TRAIL binding to the death receptor DR5 on neurons activates a
caspase-dependent apoptosis cascade . Consistent with results establishing a protective effect of
effector CD8+ T cells in mice, humans with impaired T cell immunity have a greater risk of CNS
infection with WNV .
Although T cell responses are important for viral clearance, they can cause irrevocable damage to
the host. Under certain conditions, infection of mice lacking CD8+ T cells with an attenuated lineage 2
WNV (Sarafend) strain resulted in decreased morbidity and mortality compared to wild type
mice . Consistent with this, depletion of CD8+ T cells in mice infected with an attenuated genetic
variant of a North American WNV strain resulted in prolonged survival . Thus, depending on the
virological and immunological context, CD8+ T cells either can protect against or contribute to WNV
Figure 3. Mechanisms of CD8+ T cell clearance in the CNS. CD8+ T cells control WNV
infection in the CNS through multiple mechanisms. Infected neurons upregulate surface
expression of MHC class I molecules. Antigen-specific CD8+ T cells recognize infected
neurons via class I MHC and processed viral peptides and trigger cell death of target cells
through perforin, Fas-Fas ligand, or TRAIL-dependent pathways. Perforin-mediated
control of infected neurons occurs through the granzyme-dependent granule exocytosis
pathway, which results in apoptosis of infected neuron. Interactions between Fas on
infected neurons and FasL on CD8+ T cells leads to programmed cell death of neurons
through caspase-dependent pathways. CD8+ T cells also utilize TRAIL to restrict WNV
infection in neurons. TRAIL binds to DR5 on neurons, which can have a direct antiviral
effect against flaviviruses  or result in targeted apoptosis. Activated CD8+ T cell also
produce IFN-γ, which can induce genes with antiviral effect.
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7. Viral Persistence in the CNS
Although still controversial, persistent WNV infection and inflammation in the CNS of vertebrate
animals has been reported in mice, monkeys, and hamsters [109–113]. These results are consistent
with earlier studies in animals and humans showing flavivirus persistence after infection with Saint
Louis encephalitis, tick-borne encephalitis, and louping ill viruses [114–117]. In monkeys, the duration
of WNV persistence was at least 5.5 months, with infectious virus isolated from the cerebellum and
cerebral subcortical ganglia. Virus recovered more than two months after initial infection from these
monkeys retained neurovirulence . In hamsters, WNV persistence has been described up to
86 days after initial infection, and this was associated with long term neurological sequelae [112,113].
In mice, infectious WNV was detected in the brains up to 4 months in 12% of mice and viral RNA
persisted up to 6 months after infection . Consistent with this, virus-specific B and T cell immune
responses persisted in the brains of mice for at least 4 months after infection . Although viral
persistence in the CNS has not been documented in humans, chronic WNV infection in the kidney has
been reported in some patient cohorts [118,119].
8. Summary and Future Perspectives
WNV continues to spread and cause neurological disease and thus, remains a public health concern
in the United States and other countries. Research into the viral and host factors that determine the
pathogenesis and outcome of WNV infection is crucial for development of new therapeutic and
vaccines strategies. A more complete understanding of the mechanisms of immunopathogenesis in the
CNS could facilitate the development tailored anti-inflammatory agents that minimize neuronal
damage without preventing clearance. As examples, treatment with the Cxcr4 antagonist AMD3100
enhanced CD8+ T cell trafficking into the parenchyma of CNS and improved survival after WNV
encephalitis , whereas blockade of migration of nitric oxide-producing inflammatory macrophage
using anti-very late antigen (VLA)-4 integrin antibody prolonged survival after WNV encephalitis
. Combining such types of immunomodulatory agents with small molecule or antibody-based
antiviral molecules  that target viral replication or tropism might be a way to maximize viral
clearance and minimize neuropathogenesis after WNV infection.
Conflict of Interest
H. Cho declares no conflict of interest. M. Diamond is a consultant for MacroGenics and
NIH grants U54 AI081680 (Pacific Northwest Regional Center of Excellence for Biodefense and
Emerging Infectious Diseases Research), U19 AI083019, and R01 AI074973 supported this work.
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