West Nile Virus (Flaviviridae)☆

Laura D Kramer, NYSDOH, Slingerlands, NY, United States; State University of New York at Albany, Albany, NY, United States
Elizabeth Kauffman, NYSDOH, Slingerlands, NY, United States
ã 2017 Elsevier Inc. All rights reserved.

Classification 1
History and Geographic Distribution 1
Molecular Epidemiology 2
The Virion and the Viral Genome 4
Replication 5
Ecology 6
Vectors 6
Vertebrates: Birds 8
Vertebrates: Other 8
Other Factors 8
Vertebrate Pathogenesis 8
Clinical Disease 9
Diagnostics 9
Human 9
Surveillance Specimens 10
Prevention and Control 11
Vaccines and Therapeutics 11
Public Health Measures 11
Summary 11
Acknowledgment 11
References 12

Classification

West Nile virus (WNV) is a member of the family Flaviviridae, genus Flavivirus, which is composed of approximately 70 members
classified into 12 antigenic subgroups. The family Flaviviridae includes two additional genera, Pestivirus (including veterinary
pathogens such as bovine viral diarrhea viruses) and Hepacivirus (including the human pathogen hepatitis C virus). WNV belongs
to the Japanese encephalitis (JE) antigenic complex, which includes the human pathogens St. Louis encephalitis, Rocio, and Ilheus
viruses in the Americas, Japanese encephalitis virus in Asia, Murray Valley encephalitis virus in Australia, and other viruses, most of
them not associated with encephalitis (Brinton, 2002).

History and Geographic Distribution

West Nile virus was first isolated in 1937 from the blood of a febrile woman in the West Nile district of Uganda (Smithburn et al.,
1940). The next evidence of activity occurred when WNV was isolated from mosquitoes and birds and identified as the etiologic
agent in sick children in North Africa and the Middle East in the 1950s (Melnick et al., 1951; Nir et al., 1967; Nir et al., 1968). West
Nile neuroinvasive disease (WNND) was first recognized during an outbreak in elderly patients in Israel in 1957 (Spigland et al.,
1958). In the 1960s, WN encephalitis in equines was recognized in Egypt and France (Murgue et al., 2001; Schmidt and
Elmansoury, 1963). The largest outbreak of West Nile fever (WNF) occurred in 1974 in Cape Province, South Africa, leading to
approximately 10,000 cases (Jupp, 2001). Increasing frequency of severe outbreaks began occurring in 1996 in humans and horses,
largely in the Mediterranean Basin (Hubalek and Halouzka, 1999). The range of WNV expanded into the western hemisphere in
1999 with the introduction of the virus into the United States in the New York City area, although the mode of introduction
remains unknown. The virus became successfully established as it expanded its range westward to encompass the contiguous
United States, northward into Canada in 2001, and southward beginning in 2002 into Mexico, Central America, the Caribbean,
and South America (Gubler, 2007). In the United States, from 1999 to 2016, approximately 45,000 cases of WNV disease,
including >21,409 neuroinvasive (WNND) and 24,570 nonneuroinvasive (WNF) cases with 2005 deaths, were reported to
ArboNET, Arboviral Diseases Branch, Centers for Disease Control and Prevention (Fig. 1). In 2012, the United States experienced

☆
Change History: July 2017. Laura D Kramer and Elizabeth Kauffman updated all sectionswith in-text citations and updated material has been added to some
sections and updated Fig. 2 and Table 1 added new Fig. 1 and replaced old Fig. 2.

Reference Module in Biomedical Sciences https://doi.org/10.1016/B978-0-12-801238-3.02696-9 1

2 West Nile Virus (Flaviviridae)

Fig. 1 West Nile neuroinvasive disease (WNND) in the United States. From ArboNET, Arboviral Diseases Branch, Centers for Disease Control and
Prevention.

its second most severe outbreak since WNV was introduced and the largest since 2003, with 5,674 cases and 286 deaths, including
the first human case in Maine (CDC, 2013). The overall increase was largely due to the high number of cases in Dallas that year with
an incidence rate for WNND of 7.30 per 100,000 residents, compared with 2.91 per 100,000 in 2006 (Chung et al., 2013).
In contrast to North America, WNV outbreaks have not occurred in the Caribbean and Latin America; a single human
neurological case in Brazil (Vieira et al., 2015), a fatal case in Mexico in 2009 (Rios-Ibarra et al., 2010), and the equine cases in
Guatemala and Argentina (Morales-Betoulle et al., 2006; Morales et al., 2006) are the exceptions. Theories to explain the lack of
disease include preexisting antibodies against a heterotypical virus that confers cross protection, cross-reaction in diagnostic
serological tests, and attenuation or different origin of WNV strains circulating in Latin America (Elizondo-Quiroga and
Elizondo-Quiroga, 2013; Gubler, 2007; Guerrero-Sanchez et al., 2011; Komar and Clark, 2006). However, research indicates
that the virus strains isolated in Latin America are no less virulent and the avian hosts are no less competent than their North
American counterparts (Guerrero-Sanchez et al., 2011).

Molecular Epidemiology

WNV is the most widely distributed flavivirus in the world (Chancey et al., 2015; Kramer et al., 2008) (Fig. 2). Isolates comprise two
main lineages based on nucleotide sequence homology, although up to eight lineages have been reported (Donadieu et al., 2013;
Mackenzie and Williams, 2009; Prow et al., 2014) (Figs. 1 and 2). Lineage 1 includes two clades: (1a) isolates from Africa, Asia,
Europe, Middle East, North America, and Russia and (1b) isolates from Australia (Kunjin). It was proposed recently that the
original third clade (1c) that contains most Indian isolates should be considered a separate lineage, lineage 5 (Bondre et al., 2007;
Mann et al., 2013) (Fig. 3). Clade 1a that includes most of the lineage 1 isolates has been further subdivided into six clusters (Fig. 3)
(Mann et al., 2013; May et al., 2011). The strain of WNV introduced into the United States in 1999 has 99.7% nucleotide identity
with a 1998 Israeli goose isolate, both of which belong to cluster 4 (Lanciotti et al., 2002). Lineage 2 contains strains isolated in
sub-Saharan Africa and Madagascar and includes the prototype 1937 Ugandan isolate and the strain responsible for the South
African WN fever outbreak in 1974 (Jupp, 2001). Severe WNND in humans has been associated with lineage 1 strains predom-
inantly, although neuroinvasive lineage 2 virus has been isolated and showed to cocirculate with lineage I virus in Europe,
particularly Italy and Greece (Bakonyi et al., 2013; Hernandez-Triana et al., 2014). The lineage 2 strains belong to a monophyletic
clade derived from a single introduction into Europe of the prototype Hungarian strain as it dispersed in Europe and adapted to
local ecological niches (Barzon et al., 2015).
Comparison of isolates from lineage 1 indicates differences in virulence between North American and Afro-European strains,
but the degree of difference varies with the specific avian host. Enhanced virulence of the North American strains appears to be
partially correlated with envelope (E) protein glycosylation and in corvids (particularly American crow (Corvus brachyrhynchos))
 West Nile Virus (Flaviviridae) 3

Fig. 2 Worldwide distribution of WNV. Vector-virus interactions and transmission dynamics of West Nile virus. Note that Indian strains originally
lineage 1c now are considered lineage 5. Modified from Ciota, A.T. and Kramer, L.D. (2013). Vector-virus interactions and transmission dynamics of
West Nile virus. Viruses 5, 3021–3047, https://doi.org/10.3390/v5123021.

with a threonine to proline substitution in the NS3 (Brault et al., 2007). However, evaluation of pathogenicity of lineage 2 WNV
strains in SPF (specific pathogen-free) chickens found that NS3-249Pro mutation was not sufficient or necessary to confer virulence
(Dridi et al., 2015). Strains within both lineages 1 and 2 vary phenotypically with respect to virulence in animal models (Beasley
et al., 2002). Two new lineages have been identified recently; a lineage 3 virus was isolated from Culex pipiens in the Czech Republic
(Rabensburg virus) in 1997 and 1999 (Hubalek et al., 2010), and a lineage 4 virus isolated from Dermacentor marginatus ticks in
Russia in 1998 (LEIV-Krnd88–190) (Lvov et al., 2004) (Fig. 3). Rabensburg virus showed 77%–78% identity to lineage 1 and
2 WNV strains, 77% identity to strain LEIV-Krnd88–190, and 71%–76% identity to other representatives of the JE antigenic
complex. However, this virus differs biologically from other WNV strains in that it appears to be an intermediate between
mosquito-only flaviviruses and those that also infect vertebrates, as it is capable of infecting vertebrates only with in vivo passage
at decreased temperature of incubation (Aliota et al., 2012; Aliota and Kramer, 2012). Recently, it has been proposed that the
Sarawak strain of Kunjin virus, isolated in Malaysia, be placed in a new lineage 6 because its sequence and serological properties are
significantly different from other Kunjin viruses (Mackenzie and Williams, 2009; Scherret et al., 2001). Lineage 7 has been
proposed for Koutango virus, initially isolated in 1968 from a gerbil in the Koutango region of Senegal (Mackenzie and Williams,
2009; Scherret et al., 2001). Although no evidence for disease in humans or animals has been observed, Koutango virus was shown
recently to be highly virulent in a mouse model (Prow et al., 2014). New lineages have also been proposed for WNV isolates from
Spain (Vazquez et al., 2010) and Africa (Fall et al., 2014).
Phylogenetic analyses of WNV isolates from the United States indicate that the virus remains conserved genetically. The initial
strain of WNV introduced into the United States in 1999 has been designated as the “eastern US” strain or NY99 (Davis et al., 2005;
Lanciotti et al., 1999). In 2002, a single conserved amino-acid change in the E gene (V159A) was first detected and occurred with
increasing frequency from 2002 through 2004 throughout the United States, and it remains the dominant genetic variant
circulating throughout North America (Davis et al., 2005; Ebel et al., 2004). This genotype, “North American dominant” or
NA/WN02, probably resulted from a random mutation that became fixed in the viral RNA of the NY99. The dominant genotype
disseminates 2–4 days earlier following feeding, in two of the most important Culex spp. mosquito vectors, C. pipiens and C. tarsalis,
effectively reducing the extrinsic incubation period and thereby increasing the reproductive ratio or R0 of WNV (Ebel et al., 2004;
Moudy et al., 2007). This reproductive advantage and a higher fitness observed in some avian species (Brault et al., 2007) likely
contributed to displacement of the eastern US genotype. A third genotype (SW/WN03) that not only retains the V159A change but
also has changes in NS4A (A85T) and NS5 (K314 R) was first detected in the southern United States in 2003 (McMullen et al.,
2011). The mean nucleotide substitution rate in human isolates has been calculated to be 5.06
104 substitutions/site/year
(Anez et al., 2013). Temporal and spatial clustering of isolates suggests the occurrence in some cases of localized virus spread by
4 West Nile Virus (Flaviviridae)

Fig. 3 Neighbor-joining phylogenetic tree, using condensed, simplified branches that depicts the lineages 1–6 of West Nile virus. Indicated
phylogenetic groupings are defined based upon the genetic distance (i.e., percent nucleotide divergence) for isolates that cluster within lineages 1–6
(20%), clades 1a and 1b (12.7%–20.8%) of lineage 1, and clusters 1–6 (5.4%) of lineage 1a. Lineages 1 and 2 are the most geographically dispersed
and include both endemic and epidemic strains associated with outbreaks of neurological disease in humans, horses, and birds in the Americas, Europe,
Africa, and the Middle East. Strains circulating in the Americas belong to cluster 4 of lineage 1a, as defined by May et al. (2011). Figure taken from
Mann, B.R., McMullen, A.R., Swetnam, D.M. and Barrett, A.D. (2013). Molecular epidemiology and evolution of West Nile virus in North America.
International Journal of Environmental Research and Public Health 10, 5111–5129, used under terms and conditions of the Creative Commons
Attribution license (http://creativecommons.org/licenses/by/3.0/).

mosquitoes or resident birds and in other long-distance dispersal by migratory birds or possibly by human transport. Nonetheless,
it is clear from the phylogenetic evidence based on US isolates that there was a single introduction of WNV into North America
from the Old World (May et al., 2011). This is in contrast to WNV in Israel, where both lineage 1 and 2 strains are frequently
introduced, most likely during bird migration.

The Virion and the Viral Genome

The West Nile virion is spherical, enveloped, approximately 50 nm in diameter, with relatively smooth surfaces and icosahedral
symmetry (Fig. 4). It contains a host-derived lipid bilayer surrounding a nucleocapsid that consists of the viral RNA complexed with
multiple copies of the capsid protein. The viral genome is linear, positive-sense, single-stranded RNA, 11,029 bases in length. The 50
noncoding region of WNV has a methylated type 1 cap (m7GpppAmp); the 30 noncoding region lacks a polyadenylated tail and in
its place has a 50 -CUOH-30 . These two regions contain conserved secondary structures critical to viral replication (Brinton, 2014).
The viral genome encodes three structural and seven nonstructural proteins in a single long open reading frame of 10,299 nt
(97–10,395 nt), which is cleaved co- and posttranslationally (Brinton, 2002; Chancey et al., 2015). The structural proteins (capsid
(C), membrane (prM/M), and envelope (E)) are encoded at the 50 end of the genome and the nonstructural proteins (NS1, NS2a,
NS2b, NS3, NS4a, NS4b, and NS5) at the 30 end (Fig. 4C). The E protein, the predominant protein of the virion, plays a major role
in viral assembly, receptor binding, and membrane fusion and is the principal target for neutralizing antibody. A single mutation
has been identified at position 198 (T198F) of WNV E protein domain 1–II hinge that regulates virus “breathing” or conforma-
tional changes, and although this mutation does not have an effect on growth kinetics in vitro, it leads to attenuation in mice (Goo
et al., 2017). The NS proteins not only are responsible for viral RNA replication but also function in viral assembly and in evasion of
the host immune response. The putative function and nucleotide positions of the viral proteins are listed in Table 1.
 West Nile Virus (Flaviviridae) 5

Fig. 4 West Nile virion (A, B) and genome (C). WNV structure as reconstructed by cryo-EM. (A) A surface-shaded view with one asymmetrical unit of
the icosahedron indicated by the triangle. (B) Central section of the reconstruction, showing the concentric layers of mass density. (C) WNV genome,
single-stranded positive-sense RNA, approximately 11 kb in length, consisting of a 50 untranslated region (UTR), a single long open reading frame
(ORF), and a 30 UTR. The ORF encodes three structural and seven nonstructural proteins. (A, B) Modified from Mukhopadhyay, S., Kim, B.S.,
Chipman, P.R., Rossmann, M.G., and Kuhn, R.J. (2003). Structure of West Nile virus. Science 302, 248, as published in Kramer, L.D., Li, J., and Shi,
P.Y. (2007). West Nile virus: Highlights of recent advances. Lancet Neurology 6, 171–181, with permission from Science and Elsevier.

Table 1 Functions and nucleotide positions of West Nile virus proteinsa

Viral Function Position in

proteinb genome

Structural proteins
C Small, highly basic protein; binds to and encapsidates viral genomic RNA, regulation of viral replication and assembly 97–465
PrM/M PrM-E heterotrimers coat immature virion; cleavage to pr and M proteins directs E conformation and virion maturation (466–741)
742–966
E Three structural domains (DI, DII, DIII): receptor binding, cell entry, viral assembly; conformational changes needed 967–2469
for multiple stages of life cycle; targets for neutralizing Ab

Nonstructural proteinsc
NS1 Exists as monomer (soluble), dimer (membrane associated), and hexamer (secreted); essential for replication 2470–3525
(interacts with NS4A); virion assembly; immune response; neuroinvasion
NS2Ad Replication complex, interferon antagonist, virion assembly 3526–4218
NS2Bd Cofactor for viral protease, interferon antagonist, assembly and release of virions 4219–4611
NS3 Serine protease, RNA triphosphatase, NTPase and RNA helicase; interferon antagonist 4612–6468
NS4Ad Replication complex, cofactor for RNA helicase, interferon antagonist 6469–6915
NS4Bd Interferon antagonist, virion assembly 6916–7680
NS5 N-terminal methyl transferase methylates RNA and promotes translation, C-terminal RNA-dependent RNA polymerase 7681–10,395
RNA replication, potent interferon antagonist
a
Compiled from Brinton, M.A. (2002). The molecular biology of West Nile virus: A new invader of the Western Hemisphere. Annual Review of Microbiology 56, 371–402 and
Lindenbach, B. D., Thiel, H. J. and Rice, C. M. (2007). Flaviviridae: The viruses and their replication. In: Knipe, D. M. and Howley, P. M. (eds.) Fields virology. 5th edn. Philadelphia:
Lippincott-Raven Publishers.
b
C, core or capsid; PrM/M, premembrane/membrane; E, envelope; NS, nonstructural protein.
c
All nonstructural (NS) proteins are required for viral replication.
d
Functions not well characterized.

Replication

WNV is able to replicate in a great variety of vertebrate cells. Replication is a complex process mediated in a controlled fashion
through sequential interactions among viral RNA, proteins, and host factors (Fig. 5). The virion initially binds to the cell receptor
and then enters via receptor-mediated endocytosis. Virion and host cell factors mediating binding and entry are not well
understood, and WNV may utilize different cellular proteins in different cell types and host species (Brinton, 2014). Cell-surface
glycosaminoglycans can serve to initially attach and concentrate virus particles, and recent evidence suggests that subsequent
interaction with primary cell receptors leads to internalization (Perera-Lecoin et al., 2013). The best characterized receptors include
cellular avb3 integrin, C-type (calcium-dependent) lectin receptors (DC-SIGNR), and phosphatidylserine receptors (T-cell immu-
noglobulin and mucin domain (TIM) (Brinton, 2014; Perera-Lecoin et al., 2013). Virus is internalized via clathrin-coated pits and
6 West Nile Virus (Flaviviridae)

Fig. 5 Flavivirus replication cycle. Highlights of the flavivirus replication cycle are diagrammed. Modified from Lindenbach, B.D., Theil, H.-J., and Rice,
C.M. (2001). Flaviviridae: The viruses and their replication. In: Knipe D.M. and Howley P.M. (eds.) Fields Virology, 5th edn. Philadelphia: Lippincott-
Raven Publishers, with permission from Lippincott Williams and Wilkins.

delivered to endosomes, where acidification triggers E protein fusion with endosomal membranes, release into the cytosol, and
particle uncoating. Translation proceeds, forming a single polyprotein that is cleaved into the ten viral proteins. Genomic plus-
sense RNA is transcribed into complementary minus-sense RNA at the endoplasmic reticulum membranes, forming replication
complexes, which in turn serve as templates for the synthesis of new plus-sense RNA. The process is asymmetrical, yielding 10- to
100-fold excess plus strands. The plus-sense RNA is packaged by viral C protein to form the nucleocapsid that is enclosed in an
envelope consisting of a host-derived lipid bilayer and viral prM/M and E proteins. These immature noninfectious particles with a
spiky surface composed of prM-E heterotrimers bud into the lumen of the endoplasmic reticulum and then pass through the
secretory pathway. During passage through the Golgi, the prM proteins serve to prevent E from fusing with ER membranes. In the
more acidic environment of the trans-Golgi network, further conformation changes in E expose a cleavage site within prM, which is
recognized by cellular furin-like proteases. Cleavage of prM, probably immediately before exocytosis, results in release of the pr
peptide, retention of M in the envelope, and generation of the mature infectious viral particles. Mature virions are transported to the
plasma membrane in vesicles and released by exocytosis beginning 10–12 h after infection (Brinton, 2014; Lindenbach et al.,
2007). It is known that a percentage of virions released from the cell are still immature, but recent studies now have shown that
many virions released from the cell are partially mature virions containing clusters of uncleaved prM. These “mosaic” viruses are
infectious and may interact in a different manner with host cells and the host immune response (Goo et al., 2017; Pierson and
Diamond, 2012).

Ecology
Vectors
WNV is a zoonosis maintained in an enzootic cycle, transmitted primarily between avian hosts and ornithophilic mosquito vectors
(Fig. 6) (Kramer and Bernard, 2001). Mosquitoes of the genus Culex are the predominant vectors in the enzootic cycle throughout
the range of the virus’ distribution, although the particular species of Culex varies according to geographic location. In the northern
United States and Europe, Culex pipiens is the predominant vector, and in addition, in the United States, C. restuans may be an
important enzootic catalyst in the spring (Ciota et al., 2014). C. pipiens, an urban species that reaches high densities in midsummer,
also appears to be a bridge vector to humans in the northeastern and north central United States (Kilpatrick et al., 2005) and in
recent outbreaks in Europe and Israel. C. quinquefasciatus, a member of the C. pipiens complex, is the primary vector in the southern
United States and, presumably, Latin America and countries in the southern hemisphere. Distribution of the C. pipiens complex is
worldwide. C. tarsalis is the predominant vector in rural areas of western states in the United States (Bernard et al., 2001). Other
Culex spp., such as C. univitattus and C. antennatus (Europe and Africa), C. tritaeniorhynchus and C. vishnui complex (Asia), and
C. annulirostris (Australia), have also been implicated in the transmission cycle. Vector competence varies between species and
 West Nile Virus (Flaviviridae) 7

Fig. 6 West Nile virus transmission cycle. The enzootic cycle is illustrated, as well as epidemic and epizootic hosts. Solid arrows indicate confirmed
transmission pathways; dotted lines proposed pathways that have not been confirmed in nature.

within populations of individual species. The C. pipiens complex contains two genetically distinct forms, that is, C. pipiens form
“pipiens” and form “molestus.” The two forms differ in physiology and behavior with obvious implications for their epidemio-
logical importance (Farajollahi et al., 2011). Form “pipiens” is thought to be exclusively ornithophilic, while the urban form
“molestus” will feed on mammals. The two forms have been shown to not interbreed in northern Europe, in contrast to US
populations, which contain individuals with hybrid genetic signatures (pipiens
molestus), as does southern Europe (Fonseca et al.,
2004). Such hybridization may generate bridge vectors, disposed to feed on both birds and mammals. Indeed, US populations of
C. pipiens, C. nigripalpus (Edman and Taylor, 1968), and C. tarsalis (Tempelis et al., 1965) have been demonstrated to shift their
feeding from birds to mammals in the late summer and early fall and therefore may act as bridge vectors to infect equid and human
hosts (Kilpatrick et al., 2006b). It is likely that other species of bridge vectors also become involved since virus has been isolated
from mosquitoes of approximately 75 species of 10 genera worldwide (Petersen et al., 2013a), but the relative importance of each
species in transmission to birds or humans must take into account factors of vectorial capacity, including field infection rates and
population density (Kramer and Ciota, 2015). Many mosquito species actually may be incidental vectors of little epidemiological
significance, but further research is required to determine their importance in the ecology and epidemiology of WNV in North
America.
The mechanism(s) of WNV perpetuation over adverse seasons and years may vary by region and country, but possible
mechanisms include continuous low-level virus transmission, reinitiation after reintroduction of virus by migratory birds from
locations where virus is active year-round, vertical transmission to females about to enter reproductive diapause as adults in the fall,
and recrudescence of low levels of virus in chronically infected birds when mosquitoes are active. Viral RNA has been detected
occasionally in experimentally infected wild birds up to 18 weeks after inoculation, mainly in the spleen, kidney, brain, and skin,
but infectious virus has only been detected rarely (Wheeler et al., 2012). Virus has been isolated from diapausing adult C. pipiens in
the eastern United States in the winter (Nasci et al., 2001), from vertically infected male and female C. univitattus in Kenya (Miller
et al., 2000), and from C. quinquefasciatus larvae in California during the summer transmission season (Reisen et al., 2006). Vertical
transmission has been demonstrated in the field (Nelms et al., 2013; Unlu et al., 2010) and laboratory (Goddard et al., 2003) at
rates varying with the species and population of mosquitoes tested and method of infection.
Alternate modes of vector transmission have been observed in the laboratory and/or field. Nonviremic transmission has been
demonstrated in the laboratory, with low rates of infection in cofeeding mosquitoes (Higgs et al., 2005). This mode of transmission
may expand the enzootic transmission cycle to involve mammals and birds that generally mount viremias too low to infect feeding
mosquitoes. WNV has been isolated historically from soft ticks (Argasidae) in Egypt (Hoogstraal, 1972) and Russia (Lvov, 1980)
and recently from Israel (Mumcuoglu et al., 2005), but although they have been demonstrated to become infected in the
laboratory, their role in the transmission cycle is not clear. Ixodidae (hard ticks) allow the virus to pass transstadially but are
incompetent. Other arthropods have been suggested as alternative vectors, including dermanyssid mites, swallow bugs, and
hippoboscid flies.
8 West Nile Virus (Flaviviridae)

Vertebrates: Birds
Birds of more than 300 species in North America have been reported infected with WNV (Petersen et al., 2013a), as determined by
virus isolation or antibody, confirming their role as the primary vertebrate in the enzootic cycle. Blood meals by infected
mosquitoes are the most common route of infection, but transmission to birds also has been demonstrated by direct contact,
presumably via the fecal–oral route since there is significant shedding of virus, and by ingestion of infected mosquitoes or of carrion
by omnivorous birds such as corvids and raptors (Komar et al., 2003). Experimental and field studies indicate that birds vary
significantly in susceptibility and response to infection. The hallmark of WNV activity in North America is the significant morbidity
and mortality in a wide range of species, although bird deaths also have been noted in the Middle East and recently in Eastern
Europe. An Egyptian strain isolated in the 1950s from a sick pigeon (Columba livia) (Work et al., 1953) is virulent in experimentally
infected hooded crows (Corvus corone) (Work et al., 1953) and house sparrows (Passer domesticus). Corvids, especially crows,
magpies, and jays, appear to be among the most highly susceptible to disease, with 100% of American crows (Corvus brachyr-
hynchos) becoming sick and dying after infection with very low doses of WNV (Brault et al., 2004). Similarly, other avian species
have been demonstrated in the lab to be equally highly susceptible to disease, such as the greater sage-grouse (Centrocercus
urophasianus) (Walker et al., 2004) and tufted titmouse (Baeolophus bicolor) (Kilpatrick et al., 2013). Accurate mortality rates
from WNV in small birds in nature are difficult to ascertain because of rapid disappearance of bodies after death; some small
species, such as tufted titmouse (Baeolophus bicolor), have been demonstrated in the lab to be highly susceptible to disease
(Kilpatrick et al., 2013). In the northeastern and mid-Atlantic United States, the American robin (Turdus migratorius) appears to
be highly preferred by the predominant vector C. pipiens (Kilpatrick et al., 2006a). Disproportionate numbers of mosquito blood
meals are taken from robins, and they have high levels of IgG antibody to WNV, indicating their importance in the enzootic cycle.
In the laboratory, robins have average host competence determined by susceptibility, number of days infectious, and level of
viremia or mean infectiousness (Komar et al., 2001). High levels of WNV IgG have been detected in other birds as well, including
cardinals and wrens and, in some locations, house sparrows.

Vertebrates: Other
Thirty species of mammals and occasionally other vertebrates including reptiles and amphibians have been found infected with
WNV (Root, 2013). Their role in the transmission cycle is less significant than that of birds because of their generally low levels of
viremia, but some small mammals, such as rabbits and chipmunks, have been demonstrated in the laboratory to mount sufficiently
high levels of virus in the blood to infect a small proportion of feeding Culex spp. mosquitoes (Gomez et al., 2008; Platt et al., 2008;
Tiawsirisup et al., 2005). In the United States and Mexico, farmed alligators raised at high temperatures in crowded conditions
demonstrate significant mortality and mount high viremias (Miller et al., 2003). Transmission appears to occur directly between
alligators and through ingestion of uncooked infected horse meat. Enzootics in equines have occurred in the United States, France,
Italy, Morocco, and Israel. Unvaccinated equines develop infections ranging from asymptomatic to encephalitic disease and
demonstrate a case fatality rate of approximately 25% (Gardner et al., 2007). Because of their low viremias, they are incidental
hosts and do not contribute to the transmission cycle (Bunning et al., 2002).
Low levels of WNV antibody in domestic pets have been detected in serosurveys in the United States and in the highveld region
of South Africa. In the laboratory, dogs and cats are readily infected with WNV, but dogs do not develop detectable viremia, and
peak titers in cats are too low to infect most mosquitoes. Nonetheless, morbidity and occasional mortality have been observed in
very young dogs. Cats also may become infected by the oral route following feeding on dead infected mice. Sera from domestic
(Gaunt et al., 2015) and working dogs (Durand et al., 2016) demonstrated high seroprevalence to WNV. Wild-caught vertebrates
such as raccoons, Virginia opossums, fox squirrels, and eastern gray squirrels also demonstrated high levels of infection after
intensive activity in 2003 in the United States. It is unclear how much morbidity and mortality is associated with WNV in wild
mammals, but occasional cases of overt disease have been reported.

Other Factors
The relationships between land-use changes, climate, socioeconomic conditions, and occurrence of WNV are not clear. In a study in
Atlanta, Georgia, an array of risk factors was analyzed. Soil moisture and temperature were positively correlated with mosquito
abundance, as were proportion of low-income households, homes built before 1960, and housing density (Lockaby et al., 2016).
In another recent study, drought was the primary climatic driver of increased West Nile virus epidemics, rather than within-season
or winter temperatures or precipitation (Paull et al., 2017). Drought may increase epidemics via changes in mosquito infection
prevalence rather than mosquito abundance. These authors predict that in the next 30 years, increased drought severity from
climate change could triple WNV cases in regions with low human seroprevalence.

Vertebrate Pathogenesis
Infected mosquitoes expectorate virus with their saliva not only intradermally but also intravascularly while probing and feeding
(Styer et al., 2007a,b), and their salivary secretions contain potent pharmacological compounds that affect viral pathogenesis (Titus
et al., 2006). The initial site of viral replication in the vertebrate is thought to be subdural Langerhans’ dendritic cells at the site of
 West Nile Virus (Flaviviridae) 9

inoculation (Johnston et al., 2000; Lim et al., 2011). Activated dendritic cells migrate to draining lymph nodes where the virus
replicates further, antigen processing begins, and an early immune response may become evident (Quicke and Suthar, 2013). Virus
enters the blood by way of the efferent lymphatics and thoracic duct, resulting in a viremia that carries the virus to the visceral
organs of the body and possibly facilitates virus crossing the blood–brain barrier. However, the mechanism for the latter is
unknown and may occur via infected inflammatory cells (Bai et al., 2010), retrograde transport along peripheral nerve axons
(Samuel et al., 2007), replication in epithelial cells, or by another route (van Riel et al., 2015). Neurons, the main target cell in the
central nervous system, suffer pathology, as do bystander nerve cells. In addition, there is immune-mediated tissue damage.
Factors such as age, immune status, and genetic susceptibility of the host, strain of virus, dose of inoculum, and route of
infection, affect pathogenesis of WNV. In the mouse, variations in a single gene, Flv, on chromosome 5, determine the rodent’s
susceptibility to WN disease, but not infection (Brinton, 2001). Susceptibility has been mapped to the gene encoding the L1
isoform of the interferon-inducible, antiviral effector enzyme 20 –50 -oligoadenylate synthetase (Lim et al., 2009). An intact immune
system is critical to the prevention of disease, and both humoral and cellular factors play a role in protection against disease.
Neutralizing antibody is the principal means of viral clearance. Interferon-dependent innate immune responses are essential in
limiting infection of WNV, as is the adaptive immune response. Complement, dendritic cells, and chemokines probably also play
roles in viral clearance. The mechanism by which infection is cleared from neurons appears to involve CD8 + T cells in the perforin-
dependent class I major histocompatibility complex (MHC)-restricted system (Shrestha et al., 2006). Persistence of virus has been
noted to occur in some vertebrates following natural and experimental infection, even in the presence of neutralizing antibody
(Kuno, 2001). Hamsters shed WNV in their urine for  8 months, and virus can be isolated from their brains as long as 53 days after
infection (Tonry et al., 2005).

Clinical Disease

The range of symptoms associated with WNV infection extends from uncomplicated febrile illness to muscle weakness and WNND,
that is, meningitis, encephalitis, neuropathies, and acute flaccid paralysis (AFP, a poliomyelitis-like syndrome) (Gray and Webb,
2014). Symptoms generally become apparent 2–14 days after infection by mosquito bite (Petersen and Marfin, 2002), but 80% of
infected individuals are asymptomatic (Zou et al., 2010). The majority of symptomatic patients present with flu-like symptoms,
termed WN fever (Petersen et al., 2013b), which in the United States has been associated with substantial morbidity. The duration
of symptoms may be from days to months, and the patient may require hospitalization (Petersen et al., 2013a). Of the 45,975 cases
of WN disease reported in the United States from 1999 to 2016, 53.4% were WN fever, and 46.6% were WNND resulting in 2005
fatalities (4.3% of all, 9.3% of WNND) (Fig. 1). It has been estimated from WNND case reports that more than 3 million
individuals were infected with WNV from 1999 to 2010, and of these 780,000 developed WNF (Petersen et al., 2013b). Males are
more commonly infected than were females (Weatherhead et al., 2015). Movement disorders, that is, dyskinesias, frequently
characterized as parkinsonism are common (Gray and Webb, 2014). The constellation of fever, headache, and signs of meningeal
irritation cannot be easily discriminated from other flavivirus encephalitides. Patients with AFP experience loss of spinal anterior
horn cells and have accompanying asymmetrical weakness (Hart et al., 2014; Sejvar et al., 2003). Risk factors for WNND include
age, diabetes, and possibly a history of hypertension or cardiovascular disease. The median age of WNND cases in the United States
since 1999 is 57 years (46 years for WNF), but there have been reports of WNND in children and AFP in young adults (O’Leary
et al., 2004; Shaw et al., 2013; Trobaugh and Green, 2015). Infection may persist in the central nervous system of immunologically
compromised patients, leading to extended neurological illness and sequelae (Weatherhead et al., 2015). The mortality rate
following WN encephalitis is greater than following WN meningitis. Immunosuppression, chronic renal disease, and hepatitis
C infection have been recognized as risk factors for death after adjustment for age. Rare cases of hemorrhagic WNV disease have
been noted.
The vast majority of human cases are transmitted by the bite of an infected mosquito. However, human-to-human
transmissions after blood transfusions (Biggerstaff and Petersen, 2002; CDC, 2002c), organ transplantation, and through mothers’
milk have occurred in the United States (CDC, 2002a,b), and there has been one instance of intrauterine transplacental
transmission (CDC, 2002b). High-throughput nucleic acid detection assays were developed and implemented by blood banks
in response to transfusion transmission in order to protect the blood supply (Busch et al., 2005; CDC, 2004).

Diagnostics
Human
Studies of infected blood donors indicated the presence of WNV IgM and IgA at the earliest on day 3 and in all cases by day 8–9 after
RNA was detected; IgG antibody appeared about 1 day later (Fig. 7). The detection of IgM antibody in the cerebrospinal fluid (CSF)
by a monoclonal antibody (MAb) capture enzyme-linked immunosorbent assay (MAC ELISA) in conjunction with evidence of
neurological symptoms is considered diagnostic of WN disease (Prince et al., 2007). The presence of IgM antibody in the serum
alone is strongly suggestive of recent infection but not definitive due to persistence for at least 16 months (199 days in the CSF) in
patients with WNND (Roehrig et al., 2003) and to some cross-reactivity with antibody to other flaviviruses (Niedrig et al., 2007;
Petersen and Marfin, 2002). One long-term study detected IgM in 23% of study participants 8 years following infection (Murray
10 West Nile Virus (Flaviviridae)

Fig. 7 Schematic of virological and serological tests in West Nile virus encephalitis. Solid lines represent the more common results; broken lines
represent reported ranges. The shaded box is an example of a typical patient. Reproduced from Gea-Banacloche J., Johnson R.T., Bagic A., Butman J.A.,
Murray P.R., and Agrawal A.G. (2004). West Nile virus: Pathogenesis and therapeutic options. Annals of Internal Medicine 140, 545–553, with
permission from American College of Physicians.

et al., 2013; Roehrig et al., 2003). The plaque reduction neutralization test (PRNT) in Vero cell culture had been the gold standard
in serological diagnosis of flavivirus infections because of the extensive cross-reactions in other assays, until the expansion of Zika
virus activity in the Americas in 2015, complicating diagnosis. Nonetheless, a fourfold rise in neutralizing antibody titer between
paired acute-phase and convalescent-phase sera generally confirms WNV infection, as does a fourfold or greater titer to WNV
compared with related flaviviruses (Dauphin and Zientara, 2007). However, interpretation is complicated in individuals who have
had prior infection with another flavivirus. Microsphere immunoassay (MIA) using recombinant E protein has equivalent
sensitivity to ELISA (Wong et al., 2004). In primary flavivirus infection, the specificity of the MIA procedure is high (>90%)
relative to PRNT (Wong et al., 2004). However, as with PRNTs, the interpretation of WNV-E MIA is confounded when WNV is a
secondary flavivirus infection (Drebot et al., 2003). Sera from dengue (flavivirus) patients are highly likely to be positive in MIA
procedures using recombinant WN E protein. Addition of WNV NS5 to a multiplex test panel increases specificity, since sera from
patients with other past flavivirus infections are likely to be negative to WNV NS5 but reactive to WNV E (Wong et al., 2004).
Serological assays are more sensitive than detection of viral nucleic acid by reverse transcription polymerase chain reaction
(RT-PCR) once symptoms are evident because of the rapid clearance of virus by most individuals (Fig. 7); however, detection of
viral RNA is confirmatory (Sambri et al., 2013). Studies have shown that within 8 days of the appearance of symptoms, nucleic acid
assays in conjunction with the MAC ELISA enhance diagnosis (Tilley et al., 2006).
Clinical findings also aid diagnosis. Neurological symptoms indicate neuroinvasive disease (WNND). Electrophysiological tests
and magnetic resonance imaging (MRI) may be useful in cases of acute flaccid paralysis. Elevated lymphocyte counts and protein
levels may be present in CSF during WNV infection (Carson et al., 2006; Sejvar, 2014; Weatherhead et al., 2015). Epidemiological
data also may be critical for diagnostic consideration because other flavivirus infections cause similar symptoms.

Surveillance Specimens
Submission of dead birds, particularly American crows, originally provided an excellent sentinel system for WNV activity in the
United States, but now, it is used rarely because it depends upon participation by the public and can be confounded by level of
effort expended (Chuang et al., 2011; Eidson, 2005; Nemeth et al., 2009). More commonly, pooled mosquitoes are tested by real-
time RT-PCR for viral nucleic acid, by cell culture procedures that detect live virus followed by confirmation using specific assays
and/or by procedures that detect viral antigen. High-throughput virus-specific molecular assays, such as real-time RT-PCR, have
been developed to accommodate testing of large numbers of specimens in a timely manner (Grinev et al., 2017; Kauffman et al.,
2003; Shi et al., 2001; Zink et al., 2013). Rapid assays utilizing dip sticks to detect viral antigen also have been adopted for
surveillance but have low sensitivity (Burkhalter et al., 2006; Kesavaraju et al., 2012; Stone et al., 2005). Detection of antibody in
sera of live sentinel captive birds or wild-caught birds using an indirect or a competitive blocking ELISA, with confirmation by
 West Nile Virus (Flaviviridae) 11

PRNT, generally has proved less useful as an early warning indicator of viral activity but may be used in research efforts (Chiles and
Reisen, 1998; Ebel et al., 2002).

Prevention and Control

Vaccines and Therapeutics
Currently, treatment for WN disease is supportive; no therapeutic treatment has shown consistent clinical efficacy (Sejvar, 2014).
However, therapeutic antibodies acting on different stages of viral entry have shown the most promise. An example of such
interventions is passive administration of high-titer immune immunoglobulin (Ben-Nathan et al., 2009). Monoclonal antibody
(Mab), in particular humanized MAb against WNV E protein, has shown some benefit (Beigel et al., 2010; Oliphant et al., 2005).
Interferon offers promise based on animal models but may not be helpful in treating the late-stage central nervous system disease
(Kalil et al., 2005; Lewis and Amsden, 2007). Targets of intervention include viral RNA polymerase, RNA helicase, and other viral
replication enzymes (Geiss et al., 2009). WNV replicon-based high-throughput screening assays have been developed to aid in the
discovery of compounds with potential antiviral activity (Puig-Basagoiti et al., 2005). However, because most human infections
with WNV are typically asymptomatic, with approximately only 1% of cases developing severe neuroinvasive disease (encephalitis,
meningitis, or flaccid paralysis), there has been limited effort to develop antivirals. Although persistent infections have been
demonstrated, the disease is mostly transient followed by life-long immunity.
Veterinary WNV vaccines have been available for decades; protection has been demonstrated in equids following immunization
with inactivated cell-culture-derived virus, originally developed by Fort Dodge Animal Health (Ng et al., 2003). A recombinant
canarypox virus containing the prM and E genes of WNV also has been licensed for use in equids (El Garch et al., 2008). Two
recombinant live virus vaccines demonstrated initial efficacy in clinical trial in humans. One used a yellow fever 17D strain
infectious clone, with the original yellow fever virus prM and E genes replaced by attenuated WNV genes (Acambis’ ChimeriVax-
WN) (Monath et al., 2002), and another used attenuated dengue virus 4 with deletions in the 30 end as the backbone
(NIH/MacroGenics) (Pletnev et al., 2003). Both live attenuated and chimeric vaccines have the advantage of eliciting humoral
and cellular responses in the host after a single dose. Other approaches include DNA vaccines that encode prM, E, or C; subunit
vaccines of purified recombinant prM and E; and cross-reactive flaviviruses or WNV variants (Amanna et al., 2012; Amanna and
Slifka, 2014; Ledgerwood et al., 2011; Martin et al., 2007). Cost-effectiveness is considered a major obstacle for the human vaccine.
In addition, difficulty in conducting field case–control studies is another obstacle to human vaccine development because of
inconsistent disease incidence (Amanna and Slifka, 2014). It is expected that safe and effective human vaccines will be available
once the need for a human WNV vaccine is recognized. New arbovirus outbreaks, such as Zika virus in the Americas in 2015/2016,
stimulate fresh interest in flavivirus therapeutics and vaccines.

Public Health Measures

Reduction of WNV transmission currently relies on reduction of mosquito populations and minimizing contact between mosquito
vectors and humans (Abramides et al., 2013). Mosquito control agencies in the United States have undertaken extensive larviciding
to kill immature stages of Culex spp. vectors in the aquatic environment (Gray and Webb, 2014). Source reduction, that is, removal
of standing water, also has proved helpful. Personal protection measures, such as wearing loose clothing, using repellents,
particularly “DEET” (N,N-diethyl-m-toluamide or N,N-diethyl-3-methylbenzamide), or using window screens, reduce direct
contact with mosquitoes. Community action groups (citizen science) have worked to increase awareness and educate the public.

Summary

The introduction of WNV to the United States and its subsequent spread and successful establishment in the Western Hemisphere
alerted the world to the impact of globalization on pathogen dissemination. WNV was the first in a recent series of emerging
mosquito-borne diseases to be introduced into the naive environment of the Americas; it was followed closely by chikungunya
virus and Zika virus. Dengue virus continues to expand its range globally. The number of WNND cases in North America in
2002/2003 and 2012 represents the largest outbreak of neurological disease in the world. Future research undoubtedly will address
the development of efficacious therapeutics, effective vaccines, novel methods to control mosquitoes, and accurate risk prediction.
Each of these is dependent upon solid basic research on viral ecology, pathogenesis, and immunology.

Acknowledgment

The authors thank Mary Franke of the Wadsworth Center Arbovirus Laboratories for assistance with the update of this article.
12 West Nile Virus (Flaviviridae)

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