West Nile Virus

Authors: Wehbeh A. Wehbeh, M.D., James J. Rahal, M.D.

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Virology

West Nile Virus is an arthropod-transmitted RNA virus with a single-stranded, positive-polarity 11 kilo-base genome. The virion consists of a host-derived lipid bi-layer membrane surrounding an icosahedral core composed of multiple copies of the capsid protein. The West Nile virus RNA genome consists of a 5’ non-coding region (about 100 nucleotides), a single open reading frame coding for three viral structural proteins and seven non-structural (NS) proteins, followed by a 3’ non-coding region (about 600 nucleotides). The structural proteins include a capsid protein (C); an envelope protein (E) that functions in receptor binding, membrane fusion, and viral assembly; and a transmembrane protein (pr M) that mediates proper folding and function of the E protein. Late in virus maturation, the pr M protein is cleaved into M protein, which is incorporated into the mature virion. The E and M proteins determine host range, tissue tropism, replication, assembly, and stimulation of B and T cell immune responses. The E protein elicits most virus-neutralizing antibodies (9, 23, 25). The functions of the NS proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5) are not fully defined, but they make up the intracellular replication machinery of the virus. After the virus attaches to a cell surface receptor, it enters via receptor-mediated endocytosis. The West Nile virus RNA binds to ribosomes in the cytoplasm where it is translated as a single open reading frame. The resulting polyprotein is cleaved into structural and non-structural proteins by viral and host proteases. Positive RNA strands serve as a template for their replication. After assembly and packaging in the endoplasmic reticulum, viral particles are exocytosed via secretory vesicles (25).

Classification

The West Nile virus belongs to the family Flaviviridae and genus Flavivirus. The Flaviviridae family contains three genera: Hepacivirus (Hepatitis C virus), Flavivirus and Pestivirus (Bovine viral diarrhea virus, Classic swine fever virus and Border disease virus). The genus Flavivirus contains approximately 70 flaviviruses, several of which cause human diseases. Many species of flaviviruses have been grouped into 14 clones, which in turn are grouped into three clusters: the mosquito-borne cluster, the tick-borne cluster, and the no-vector cluster. All human flaviviruses belong to the first two clusters (53). The West Nile virus has been classified serologically within the Japanese encephalitis antigenic complex, which also includes Japanese encephalitis, St. Louis encephalitis, Kunjin virus (an Australian sub-type of the West Nile virus) and Murray Valley encephalitis (70). The close antigenic relationship between these flaviviruses explains the serologic cross-reactions observed in the laboratory. Two lineages of the West Nile virus exist, based on phytogenetic analyses of nucleic acid sequence data from a number of full-length genomes. Viruses belonging to lineage 1 are associated with human disease and have a worldwide distribution, ranging from West Africa to the Middle East, Eastern Europe, North America and Australia. Viruses belonging to lineage 2 consist of enzootic strains from Africa (70). The West Nile virus variant in the United States is included in lineage 1. The North American West Nile virus is closely related to that isolated from a dead goose in Israel, suggesting a Middle Eastern origin (52).

Epidemiology

The West Nile virus was first isolated from the blood of a febrile woman in the West Nile region of Uganda in 1937 (85) and again in 1950, from the blood of three febrile children in Egypt (38). In 1957, West Nile virus encephalitis occurred during an outbreak among elderly patients at a nursing home in Israel (38). In 1959, the West Nile virus encephalitis was reported in a 7-year old girl from Madras State in India (10). Until the early 1990’s, occasional outbreaks in humans, usually with mild febrile illness, were reported in Israel (1950’s) and South Africa (1974, 1983 and 1984) (70). In the 1990’s, the epidemiology of the West Nile virus infection expanded, with an increase in the severity of illness in humans and horses, and an associated increase in avian mortality (Romania, 393 human cases; Russia [Volgograd], 942 human cases, and U.S., 16,637 human cases between 1999 and 2004). The following outbreaks have been recorded: Romania 1996; Morocco 1996; Tunisia 1997; Italy 1998; Russia, the United States and Israel 1999; and Israel, France and the United States 2000 (72).

Infection due to the West Nile virus was first reported in the western hemisphere in the summer of 1999 when a cluster of patients with meningoencephalitis, frequently associated with muscle weakness, was noticed in New York City. The epicenter occurred in the borough of Queens. Initially, St. Louis encephalitis virus was considered as the causative agent, but specific serologic studies subsequently identified the West Nile virus as the etiology (64). The 62 patients diagnosed with West Nile virus infection lived within a 41.6 km² (16 mi ²) area in Northern Queens (64). Fifty-nine of the 62 patients were hospitalized during August and September of 1999, mostly with encephalitis, and seven patients died. During the next few years, West Nile virus infection in humans spread across North America. The discovery of West Nile virus in over-wintering Culex mosquitoes in New York City in 2000 indicated that the virus could survive in temperate climates, and heralded its spread westward (63). In 2000, 21 cases were diagnosed from three states, and in 2001, 66 cases from ten states. In 2002, a dramatic spread of the virus occurred and 4,156 cases were reported from 39 states. The virus reached Canada in the summer of 2002. Ontario was the first province affected, and 62 cases occurred with neurologic disease (69). In 2002-2003, West Nile virus infection was detected in resident and migrant birds in Yucatan State and Tamaulipas State in Mexico (30,56).

During 2003 and 2004, the number of reported cases in the U.S. was 9,862 and 2,470, respectively, from 40 states and the District of Columbia. The close genetic relationship between the West Nile virus isolated from New York and Israel suggests a Middle East origin of the North American outbreak, but the vehicle of its introduction (infected mosquito, bird or human) will remain unknown.

Interestingly, 1999 was not the first time that the West Nile virus caused human disease in New York. In the 1950’s, 103 patients were infected intramuscularly with the West Nile virus in an experiment to evaluate the antineoplastic effect of the virus. Some developed evidence of encephalitis and 15 patients died within four weeks after inoculation of the West Nile virus (87).

Early east coast isolates in the U.S. were similar by nucleotide sequence analysis (51). Isolates collected from New York between 2002 and 2003, during an increase in the intensity of the West Nile virus transmission, revealed the presence of a virus which segregated to a different clade (27). Strains from this new clade have a shorter extrinsic incubation period in mosquitoes, and became the dominant clade type in 2003. Other variant isolates collected in Texas in 2003 showed an attenuated phenotype in plaque morphology. In mice, neuro-invasiveness was attenuated, but not neurovirulence (20).

West Nile virus is maintained in a bird-mosquito-bird cycle. Ornithophilic, bird-feeding, mosquito species are the principal vectors of West Nile virus and wild birds are the principal, amplifying hosts of the virus (43). Though Culex mosquitoes have been the primary vectors in West Nile virus spread, the virus has been identified in 43 of 174 different species of mosquitoes in North America (33). Although the virus has also been isolated from other hematophagous arthropods (e.g. bird-feeding argasid [soft] or amblyommine [hard] ticks), their role in West Nile virus transmission is not known. In temperate regions, adult mosquitoes begin to emerge in the spring. After female mosquitoes ingest blood from infected birds, viral amplification occurs in the bird-mosquito-bird cycle until early fall when female mosquitoes begin diapause and bite less often. “Bridge-vector” mosquitoes bite both humans and birds and pose a threat of West Nile virus transmission to humans when they become infected (71).

It is not known which Culex species is most responsible for transmissions to humans. Data from Connecticut and New York State during early August 1999 indicated that Culex pipiens was present in high numbers and had high West Nile virus infection rates, coinciding with the subsequent peak in human disease in New York City. Culex salinarius is a species that feeds on both mammals and birds and readily bites humans. This species had high West Nile virus infection rates and was very abundant during an outbreak on Staten Island in 2000 (72). Human infection occurs mainly between June and November, with a peak between July and September, when the seasonal mosquito activity is highest.

Avian species develop high-level viremia when infected with West Nile virus, allowing transmission of the virus to feeding mosquitoes. Usually, infected birds survive and develop permanent immunity, but some species belonging to the family Corvidae (e.g. crows and jays) become ill and die. It is believed that West Nile virus is transported to different geographic areas by viremic birds (9). The West Nile virus became established permanently in the United States after introduction to the warmer south, where adult mosquitoes such as Culex quinquefasciatus feed throughout the year.

Although a broad range of mammals are susceptible to the West Nile virus infection, (8), recorded deaths occurred primarily in humans and horses after the West Nile virus arrived in North America. However, a few deaths have been documented in squirrels, a chipmunk, a big brown bat, a little brown bat, a skunk, a domestic rabbit, a domestic cat and three dogs (50). Some southern African isolates (Lineage 2) have been associated with deaths in a human, a dog and an ostrich chick (8).

Transmission

The most common route of West Nile virus infection to humans is through the bite of an infected mosquito. During the 2002 epidemic in the United States, five new routes of transmission were identified: Organ transplantation, blood product transfusion, breast feeding, transplacental transfer, laboratory acquisition.

Organ Transplantation

In August 2002, West Nile virus disease developed among four recipients of transplanted organs (2 kidneys, 1 liver, 1 heart) from a common donor. The donor had been admitted for injuries from unintentional trauma and had received blood transfusions from 63 donors. One of the donors had symptoms compatible with West Nile virus illness 2-3 weeks before donating blood, but was asymptomatic at the time of donation. He developed IgM antibody against West Nile virus at follow-up testing. The organ donor had no evidence of West Nile virus disease before and immediately after receiving blood transfusions, but West Nile virus was isolated from her serum and plasma samples obtained at the time of organ recovery. Three out of four organ recipients developed West Nile virus encephalitis and one died. The fourth organ recipient developed West Nile virus fever (45).

Blood Product Transfusion

The discovery of transfusion-associated transmission (TAT) resulted from the investigation of transplant-related West Nile virus infection. In 2002, 23 cases of West Nile virus infection related to various blood product transfusions were reported. Transmission of West Nile virus has been documented to occur by transfusion of blood cells, plasma and platelets. An estimated 80% of West Nile virus-infected persons remain asymptomatic, but can be viremic for a median of 6.5 days (17, 62). Thus, symptom screening at the time of donation is not sufficient to identify most viremic donors. During the summer of 2003, screening by nucleic-acid amplification tests (NAT) was implemented in the U.S. to test mini-pools. Individual samples are then tested from positive pools (17). There were six confirmed cases of West Nile virus TAT in 2003, even after implementing NAT screening. Virus loads in infectious donations were considerably lower in 2003 (0.06-0.5 pfu/ml—equivalent to 25-250 genome copies per ml.) than in 2002 (0.8-75 pfu/ml). A multivariate analysis revealed that fever, rash and eye pain were the most probable symptoms associated with infected donors, but the level of viremia did not differ between patients with and without symptoms (68). The lower limit of donor viremia that can lead to TAT of West Nile virus is unknown. Clinicians should consider theWest Nile virus disease in any patient with symptoms compatible with West Nile virus disease who has received a blood product transfusion during the 28 days preceding onset of symptoms.

Breast Feeding

Flaviviruses seem to have tropism to exocrine glands (salivary gland, mammary gland, mucus-secreting cells and pancreas) (50). In September 2002, a 40-year old woman in Michigan received West Nile virus-contaminated blood transfusions after childbirth and developed West Nile virus meningoencephalitis three days later. Her infant was breast-fed for the first 17 days and had little outdoor or other exposure to mosquitoes. West Nile virus nucleic acid and West Nile virus-specific IgM and IgG antibodies were detected in a breast milk sample obtained 16 days after delivery. The infant remained healthy, but West Nile virus-specific IgM antibody was present in the infant’s serum at 25 days of age. IgM antibodies might be excreted in human breast milk in low concentrations, but passive transfer of IgM antibody through breast milk is inefficient. The presence of West Nile virus-specific IgM in the infant’s serum suggests independent IgM production by the infant, i.e.West Nile virus infection (16). No other case of West Nile virus transmission through breast-feeding has been documented to date. Clinicians should report any West Nile virus infection in mothers or infants associated with breast-feeding.

Transplacental Transfer

Intrapartum infection with Japanese encephalitis virus, dengue virus and West Nile virus can be associated with an adverse outcome. The first reported case of congenital West Nile virus infection was in 2002, when a 20-year old healthy woman was diagnosed with West Nile virus meningoencephalitis in the 27th week of gestation. She delivered an infant five weeks later with evidence of West Nile virus infection and brain abnormalities (14). In 2003, the Centers for Disease Control and Prevention (CDC) established a registry to document pregnancy outcomes following maternal West Nile virus infection. Sixty-seven of 74 registered pregnancies yielded live-born infants, with one set of twins. There was no statistically significant increase in the frequency of spontaneous abortion, low birth weight, pre-term labor, or microcephaly compared to the general US population. Five infants had major congenital abnormalities (1 – lissencephaly; 1 – trisomy 21; 1 – cleft lip; 2 – microcephaly). The frequencies of trisomy 21 and lissencephaly were higher than expected. Three infants had possible congenital West Nile virus infection. One, with lissencephaly, died from neuroinvasive West Nile virus disease, though the authors postulated that maternal West Nile virus infection occurred late in the third trimester after lissencephaly had developed. One with a neonatal rash was well, and the third had West Nile virus neuroinvasive disease at nine days of age. In the last two cases, maternal West Nile virus infection occurred prior to delivery (66).

Laboratory Acquisition

In 2001, a suspected case of West Nile virus infection acquired in the laboratory was reported in New York. In 2002, two laboratory-acquired West Nile virus infections were documented in two microbiologists from exposure through percutaneous inoculation. Both had self-limited mild disease. These events were among factors leading to the designation of West Nile virus as a Bio-Safety Level 3 (BSL-3) agent (15).

Clinical Manifestations

Most cases of West Nile virus infection are asymptomatic. After infection, the incubation period in humans ranges from 2-14 days, but incubation periods up to 21 days have been observed among immunocompromised patients (9, 70, 71). Seroprevalence studies conducted during the 1999 New York City epidemic indicated that 1 in 5 (20%) infected persons will have a febrile illness (62). The rates of West Nile virus infection did not differ by age; however, the frequency and severity of illness did increase with age (62, 64).

Recognition of the clinical symptoms of West Nile virus disease continues to evolve. West Nile virus disease can present as West Nile virus fever, West Nile virus neuroinvasive disease (meningitis, encephalitis, meningoencephalitis, acute flaccidparalysis [AFP]), or various other clinical manifestations. In earlier outbreaks, West Nile virus fever predominated and typically presented with sudden onset of fever, headache, myalgia, lymphadenopathy, and a generalized roseolar or maculopapular rash that resolved without desquamation. The acute signs of West Nile virus fever usually resolved within a week, but fatigue and malaise lingered. In more recent outbreaks, West Nile virus meningoencephalitis has predominated among diagnosed cases, with rash and lymphadenopathy less common than in prior outbreaks (12, 18, 64, 96). However, it is important to note that the frequency of West Nile virus fever in recent outbreaks is poorly defined because surveillance has focused mostly on patients with neurologic disease.

Less than 1% of individuals infected with West Nile virus develop neuroinvasive disease. A serosurvey done in New York City in 1999 and 2000 showed that 1 in 150 infections result in meningitis or encephalitis. The same was noted in a 1996 Romanian survey where 1 in 140 to 320 infections led to neuroinvasive disease (31, 62, 89). Advanced age is the most important risk factor for neuroinvasive disease; risk is highest for individuals 50 years or older (9, 64). Other risk factors for neurologic disease are not well understood, but immunosupression and immune senescence could increase the magnitude and duration of viremia and neuroinvasive disease. Some studies have shown that diabetes was an independent risk factor for severe West Nile virus disease (33, 64).

The clinical features of West Nile virus meningitis are similar to that of other viral meningitides. Patients complain of fever, headache and nuchal rigidity. Those with alteration in mental status have encephalitis or meningoencephalitis (28). Tremors and myoclonus are seen in approximately 80% and 20% of cases, respectively (80). The most common cranial neuropathy is either unilateral or bilateral/peripheral facial paralysis. Patients with central nervous system involvement usually have a CSF pleocytosis, typically with mildly elevated protein and normal glucose levels. The presence of hyperkinetic movement disorders or focal neurologic deficits in patients with or without clinical evidence of meningitis during the appropriate season should suggest West Nile virus infection (94). The presence of plasmacytoid lymphocytes and mollaret-like cells in the CSF are clues to West Nile virus disease (11,73).

West Nile virus encephalitis should be classified as West Nile virus meningoencephalitis since the CSF abnormalities are similar to those seen in meningitis. As noted previously, age is an important risk factor for the development of West Nile virus meningoencephalitis. In the New York City epidemic, 88% of patients hospitalized were older than 50 years; their median age was 71 years (64). In another study, the median age of patients with meningoencephalitis was 70 years, compared with a median age of 35 years for patients with meningitis alone. The clinical presentation of meningoencephalitis due to West Nile virus is similar to that of other viral encephalitides, with fever, headache and altered mental status. The presence of tremors (postural or kinetic), rigidity, bradykinesia, postural instability and myoclonus should suggest West Nile virus infection since these signs are not common in non-flaviviral viral encephalitis (80). Seizures were reported in 1-4% of cases (28, 69, 89).

In the 2002 West Nile virus epidemic, numerous cases of isolated acute flaccid paralysis (AFP) were reported in immunocompromised, non-immunocompromised, elderly and young patients. This syndrome, unlike meningoencephalitis, does not have a predilection for the elderly, and has been reported in patients as young as 27 years (33, 55, 90). One report suggested that younger patients (under age 50) develop monoparesis and older patients (over age 65) develop paraparesis or quadriparesis (28). AFP can present in a pure form, with meningitis, or with meningoencephalitis. The incidence of AFP among patients with neuroinvasive disease, either in pure form, or in combination with meningoencephalitis, is not clear. AFP alone may occur in 10-20%, and combined with meningoencephalitis, in 35-50% of neuroinvasive cases (9, 94). Patients may present with weakness of one or more extremities, which may progress rapidly to asymmetric flaccid paresis. Hyporeflexia or areflexia occurs in the involved limbs. Bowel and bladder involvement occurs in approximately one-third of cases. Although many patients describe pain in the involved limbs, sensory loss is not observed. Fever and CSF pleocytosis are associated with most cases, but not in all. Radiologic, pathologic and electrodiagnostic tests indicate involvement of anterior horn cells. This may be seen as decreased attenuation on magnetic resonance imaging (33). Clinical reports in which both sensory and motor function were affected, and autopsy findings of more extensive spinal cord involvement support the occurrence of transverse myelitis, as well as AFP alone, due to West Nile virus (41, 90). Isolated AFP, as seen in poliovirus infection, may be due also to coxsackie virus and enterovirus (71). Thus, the term poliomyelitis should not be used to describe a single viral infection, but a syndrome caused by several viruses, including West Nile virus (81). This syndrome may involve the brain stem, as well as spinal cord, causing respiratory paralysis (“bulbar polio”) and requiring emergency ventilatory assistance.

Other clinical manifestations of West Nile virus disease include: ocular complications (optic neuritis, chorioretinitis, retinal hemorrhages, vitreous inflammation, visual loss), bladder dysfunction (urinary retention or incontinence), myocarditis, orchitis, myositis, pancreatitis and fulminant hepatitis (33, 70, 71). Few reports describe West Nile virus infection presenting as Guillain-Barre Syndrome (GBS) alone, a demyelinating neuropathy, rather than an axonal neuropathy that is seen in West Nile virus poliomyelitis syndrome (1, 94).

Most children infected with West Nile virus will be asymptomatic or have a mild febrile illness. However, rare cases of West Nile virus neuroinvasive disease occur in children. In 1968, a 7-year old girl developed West Nile virus encephalitis in India. She gradually recovered, but developed facial palsy three weeks after hospital admission (39). In 2002, an outbreak of West Nile virus disease occurred in Sudan. Thirty-one cases were diagnosed with encephalitis; four died. Median age was 36 months (21). In 2004, a report of West Nile virus infections described one child with acute flaccid paralysis, a case of encephalitis with fulminant hepatitis in an 11-year old, and a 9-year old child with ocular involvement (97).

Case-fatality rates among hospitalized patients have ranged from 4% to 18% (18, 28, 64, 69, 89, 96). Advanced age is the most important risk factor for death. Among patients older than 70 years, case-fatality rates were 15% in Romania, 29% in Israel, and 35% in Michigan (18, 28, 89). The case-fatality rates among patients with meningoencephalitis was 9% in the 2002 outbreak in the United States (70). Long-term morbidity among hospitalized patients can be substantial. In 2000, 37% of hospitalized patients in New York and New Jersey had recovered at time of discharge, and 53% had improved, but not to their previous level of function (96). At 12 months, only 37% achieved full recovery. Younger age at infection was the only significant predictor of recovery (49). Among patients with West Nile virus disease, but without West Nile virus neuroinvasive disease in Illinois, 63% continued to have symptoms at 30 days (93). Only 28% of patients in Canada who survived encephalitis or neuromuscular weakness, or both, were discharged without assistance. Most had persistent neurologic deficits at 30 days after discharge (69). In Louisiana, 16 patients with West Nile virus neuroinvasive disease were followed for 8 months. Fatigue, headache and myalgia persisted in 10 of 16, 2 of 18 and 3 of 16 patients, respectively. Gait and movement disturbances persisted in 6 of 16 patients (80).

One-year follow up of 18 persons with West Nile virus poliomyelitis in Colorado showed little or no improvement in seven, near-baseline strength in five, and significant improvement in six. Less profound initial weakness was associated with better outcome. Most of the recovery occurred within four months. Eleven other patients with poliomyelitis and respiratory paralysis were followed up at one year. Six had died; four deaths were related to voluntary withdrawal of ventilatory support. Of five survivors, two had persistent severe disability, and two (both younger than 45 years) recovered sufficiently to be employed and were functionally independent. One was lost to follow-up (79).

Laboratory Diagnosis

The diagnosis of West Nile virus infection depends primarily on serologic tests. In neuroinvasive infection, the peripheral white blood cell count can be elevated, normal or low, and lymphopenia is common. The cerebrospinal fluid (CSF) profile is that of any viral meningitis. A low to moderate pleocytosis occurs, usually with predominance of lymphocytes. The CSF contains moderately elevated protein and normal to slightly low glucose concentrations. Hyponatremia can be seen with meningoencephalitis (18, 64, 96). Magnetic resonance imaging scans may be normal or show focal lesions in the pons, basal ganglia, thalamus, brain stem or spinal cord. Enhancement of the leptomeninges, periventricular areas or both may occur (6, 9, 70, 80). The first step in diagnosis of West Nile virus infection is to test serum and CSF (if there is central nervous system involvement) for IgM and IgG antibody against West Nile virus by Enzyme Linked Immunosorbent Assay (ELISA). West Nile virus viremia precedes the onset of symptoms, is short lived and may have cleared by the time the patient becomes symptomatic. Thus, serum nucleic acid amplification tests and virus isolation are not useful in clinical diagnosis of West Nile virus infection.

The IgM antibody response to West Nile virus is rapid and should be detected in serum once clinical symptoms are present. IgM antibody capture enzyme immunoassay (MAC-ELISA) is typically performed to detect IgM levels. The FDA has approved a few commercially available MAC-ELISA and indirect IgG ELISA kits for use in the US. Goat antihuman IgM is coated onto 96-well flat bottom plates and is used as a capture antibody.

Diluted human sera are reacted with the anti-human IgM after blocking of the plates with nonfat dry milk. Viral antigens are then added to the plates. Flavivirus group-reactive monoclonal antibody (conjugated to horseradish peroxidase) is then reacted with the immobilized viral antigen. Reactions are measured using a microplate reader at an absorbance of 450 nm after the addition of benzidine substrate. The maximal dilution exhibiting positive signal is the serum titer. MAC-ELISA is more sensitive than complement fixation (CF) and hemagglutination inhibition (HI) in early diagnosis of acute infections by West Nile virus, and it has replaced CF and HI in most public laboratories. IgM antibody is large and does not cross the blood-brain barrier. Detection of IgM in spinal fluid implies synthesis by CNS lymphocytes and is indicative of recent infection (82). IgM antibody persistence in the sera of some patients has been identified for 500 days after onset of West Nile virus infection (76). Interpretation of serum anti- West Nile virus IgM results requires caution in areas where West Nile virus infection has occurred in prior years. In such circumstances, the diagnosis may be confirmed by a four-fold increase in serial IgG serum antibody. Such testing may also be necessary in the rare instance when serum IgM antibody is not yet present very early in the disease process.

Several limitations exist for MAC-ELISA. First, cross reactivity exists between West Nile virus and other JE serogroup viruses, dengue, and yellow fever. Thus, prior immunization, travel history, and history of previous encephalitis or meningitis are useful when interpreting diagnostic tests. Second, non- West Nile virus IgM molecules present in patient serum from infections other than West Nile virus can decrease the sensitivity of MAC-ELISA by competing for binding to anti-human IgM antibody on the ELISA plate. Third, rheumatoid factors can cause false-positive results of MAC-ELISA (82). Therefore, a 4-fold increase in neutralizing antibody titer may be necessary to provide a more specific diagnosis of West Nile virus infection. The plaque reduction neutralization test (PRNT) is not available from most commercial or public laboratories, and is selectively performed by the C.D.C. Division of Vector-Borne Infectious Disease (DVBID) laboratory and selected state public health laboratories. PRNT may resolve potential cross-reactions between West Nile virus, SLE and JE by ELISA, and provide specific diagnostic confirmation during the initial period of an outbreak.

Two highly sensitive new immunoassays, based on nonstructural protein 5 and a recombinant form of the envelope (E) protein reliably distinguish between different flavivirus infections (e.g., SLE, West Nile virus and dengue virus). They also differentiate between immunity due to vaccination or natural infection, and may reduce testing time to less than three hours (82). These two assays are not yet available commercially.

Currently, nucleic-acid-amplification tests are of limited clinical value. Their use is reserved primarily for surveillance of viremic blood donors. West Nile virus can be isolated from human blood and CSF, as well as from brain tissue obtained during biopsy or autopsy (5, 42, 85). However, West Nile virus recovery is rare and should be attempted in a biosafety level 3 facility experienced in flavivirus isolation.

Pathogenesis

The outcome of West Nile virus infection is influenced by many factors, including virus strain, inoculum, route of inoculation, age, genetic susceptibility and immune status of the host. Experiments with the related dengue virus indicate that initial viral replication after inoculation occurs in Langerhans dendritic cells of the skin (40, 46, 59). Infected dendritic cells migrate to regional lymph nodes and produce a primary viremia. Factors in insect saliva may alter local and systemic host immune responses, and facilitate West Nile virus transmission (98). The CNS and other organs can be seeded when a secondary viremia occurs. In healthy individuals, West Nile virus viremia rapidly disappears when symptoms appear, concomitantly with development of IgM, IgG and neutralizing antibodies. However, West Nile virus viremia may persist for longer periods in some hosts. West Nile virus has been recovered from the blood of an immunocompromised patient 28 days after infection (87). West Nile virus viremia persisted for 31 days in a transplant recipient, and was associated with delay in the development of IgM antibody and symptoms (45). Viremia may also occur in asymptomatic patients without subsequent illness (68).

The mechanism by which West Nile virus disseminates to the CNS is not clear. The synchronous appearance of virus at many sites in the brain, cranial nerves and spinal cord points to hematogenous spread (33). Four mechanisms have been suggested to explain entry of West Nile virus into the brain: a) virus enters through a neuronal route after infection of peripheral nerves; b) virus enters via axonal transport through olfactory neurons; c) virus crosses the blood-brain barrier via replication in vascular endothelial cells in brain capillaries, followed by transcytosis and release of virus into brain parenchyma; d) diffusion of virus from vascular endothelial cells in situations where the blood-brain barrier is permeable due to damage from related or unrelated trauma (3, 25). Age-related factors, such as immune senescence or changes in the blood-brain barrier (hypertension or cerebrovascular disease) may relate to more severe neuroinvasive disease and increased mortality.

In cases of severe fatal neuroinvasive West Nile virus disease, West Nile virus particles are found within neurons, associated with neuronal degeneration and necrosis, mostly in gray matter. The thalamus, brain stem, cranial nerves, spinal cord or leptomeninges can be severely involved, resulting in a multiplicity of neurologic abnormalities. Pathologic findings include perivascular inflammation and microglial nodules, composed mainly of lymphocytes and histiocytes. CD8-bearing T-lymphocytes predominate in these nodules and in the perivascular infiltrates (34, 67, 77, 83).

SUSCEPTIBILITY IN VITRO AND IN VIVO

Antiviral Agents

In vitro studies have demonstrated that nucleoside analogues (ribavirin, 6-azauridine, glycyrrhizin, cyclopentenylcytosine, mycophenolic acid pyrazofurin) can inhibit West Nile virus in cell culture (2, 19, 47, 61). Ribavirin is protective if applied prior to infecting Vero cells with West Nile virus, but it is not therapeutic. At high concentrations (600-1000 μ Μ), ribavirin is cytotoxic. Ribavirin treatment alone increases the mortality of West Nile virus infected hamsters (60) and did not show a synergistic antiviral effect when combined with interferon (alphacon-1). In animal models of other flavivirus infections, ribavirin has failed to reduce disease or viremia (44, 58). No improvement in survival was noted among 37 patients treated with enteral ribavirin during the West Nile virus outbreak in Israel in 2000 (18).

Cytokines and Cytokine Inducers

The innate (non-adaptive) immune system is an important part of the host response against viruses, and is an essential determinant in the outcome of West Nile virus infection (25). Interferon alpha-2b, when added 1.5-2 hours before or after infection of Vero cells with West Nile virus, is protective and therapeutic (2). Interferon alpha has also protected mouse neuroblastoma cells against West Nile virus infection (57). When tested against 11 pathogenic flaviviruses, interferon alpha was a potent inhibitor of viral replication in vitro (19). Interferon alpha and its inducers (Poly I:C and Ampligen) are effective against flaviviruses in-vivo, but their effectiveness decreases if given late after infection. Interferon α-2b and the two interferon inducers decreased morbidity and mortality in an animal model of flavivirus infections (54). Again, the efficacy of interferon alpha 2-b and Ampligen was decreased if treatment was delayed (60). Interferon alpha has shown a therapeutic effect in mice infected with St. Louis encephalitis virus. When interferon alpha was administered 24 and 48 hours after infectious challenge, 60% of animals survived. There was no benefit if interferon was given 5 days after challenge (7).

Few case reports suggest that interferon alpha may be beneficial against human flavivirus infections. Improvement in the outcome of dengue fever in children and adults was noted in Cuba in 1981, after treatment with interferon alpha (54). Among four patients with severe Japanese encephalitis, two recovered after they received interferon alpha, and two who did not receive such therapy died (37). Three case reports have described five patients with neuroinvasive West Nile virus infection who survived after treatment with interferon alpha 2-b (22, 48, 78).

A randomized, double-blinded, placebo-controlled study of interferon alpha therapy for Japanese encephalitis in children was conducted in Vietnam. No difference in mortality and functional outcome was demonstrated. However, 78% of treated children were in coma at enrollment (86). In an open-label study of interferon alpha-2b therapy of St. Louis viral meningoencephalitis, the neurologic status of 15 treated patients was compared to that of 17 previously untreated patients during the same outbreak. The results suggest that early initiation of therapy enhanced neurologic recovery at three weeks after enrollment (75). A randomized, non-blinded study evaluated the potential therapeutic benefit and safety of interferon alpha-2b in patients with West Nile virus meningoencephalitis during the summers of 2002 and 2003 in the U.S. Change in the National Institute of Health Stroke Scale (NIHSS) was used to measure neurologic function at enrollment and at three weeks. The outcome of fifteen treated patients was compared to eight untreated patients. A statistically significant improvement occurred in the treated group (95).

Neutralizing Antibodies

B-cell mediated humoral immunity is another essential component of the host immune response against West Nile virus infection. Neutralizing antibodies limit the dissemination of West Nile virus infection in animal models. Mice are protected from lethal West Nile virus challenge if they are treated with neutralizing antibody before or after viral exposure (4, 24, 29). However, the benefit of antibody decreases with time after inoculation with West Nile virus (4, 29). These results suggest that neutralizing antibody is important in the control of disseminated West Nile virus infection, but that such efficacy depends upon timely administration. Immunotherapy may not be sufficient to eliminate the virus from the host since immunocompromised mice succumbed to West Nile virus infection, even though they received antibody therapy (92). The roles of IgG and IgM antibody in West Nile virus immunity have been studied in a mouse model. When challenged with West Nile virus, mice deficient in secreted-IgM, but capable of expressing surface IgM and secreting other immunoglobulins, had higher levels of viremia and greater mortality than wild-type mice. Passive transfer of polyclonal anti- West Nile virus IgM or IgG antibody decreased the mortality in secreted-IgM deficient mice (26). The role of antibody to West Nile virus envelope (E) protein was also studied. Passive immunization with anti-E protein sera partially protected mice from infection with West Nile virus (91). This suggests that anti-E antibody may be protective if given before West Nile virus infection, while antibody produced during infection cannot eliminate the infection, but might limit its dissemination (92). Case reports describe the outcome of three patients with West Nile virus encephalitis treated with intravenous immunoglobulin containing a high titer of West Nile virus antibody. Two patients, one a lung-transplant recipient and the other with chronic lymphocytic leukemia, survived. A third patient with chronic lymphocytic leukemia died (35, 36, 84).

ANTIVIRAL THERAPY

Experimental Therapy

Three double-blinded, placebo-controlled, randomized clinical trials are in progress during 2005. These are testing different therapeutics among patients with, or at high risk of, neuroinvasive disease. One trial assesses the safety and efficacy of intravenous immunoglobulin G (Omr-IgG-am) containing a high concentration of anti- West Nile virus antibody. The second assesses the safety and efficacy of interferon alpha n3 (Alferon), and the third is an exploratory study of the safety, tolerability, pharmacokinetics and potential effectiveness of an antisense compound, AVI-4020 (www.cdc.gov/ncidod/dvbid/westnile/clnicalTrials.htm).

ADJUNCTIVE THERAPY

The potential value of systemic corticosteroids has not been tested. One report described the clinical course of a patient with West Nile virus meningoencephalitis and AFP who was treated with methylprednisone. The patient’s Glasgow coma scale improved from 8 (severe coma) to 15 (mild brain injury) within 24 hours of initiation of methylprednisone (74).

VACCINES

To date, no human West Nile virus vaccine is available, but a number are in development. One vaccine, Chimeri Vax (Acambi C, Cambridge, United Kingdom) is in clinical trials. This vaccine uses the live attenuated yellow fever (17D) vaccine virus as a backbone to develop a chimeric virus that contains premembrance (prM) and envelope (E) protein genes of West Nile virus and the non-structural protein genes of 17D virus (33, 92).

An inactivated vaccine is available for horses. It is given intramuscularly in two doses three weeks apart. Neutralizing antibody titers are low one year after immunization, so annual boosters are needed (33, 65).

PREVENTION

West Nile virus has found its niche in North America and it is unlikely that diseases associated with it will disappear. Therefore, public education and mosquito control are most important in prevention. Mosquito control is the most effective strategy for prevention of human infection. This should include surveillance for larval and adult mosquitoes, and determination of the prevalence of virus in the mosquito population. Control also involves source reduction; i.e. reduction in mosquito breeding grounds such as stagnant water in used tires, rain gutters, unused swimming pools, and other potential containers. Insecticides (larvicides or adulticides) can be used against immature or adult mosquitoes to enhance source reduction if necessary. Control programs should monitor the development of resistance to infecticides among the mosquito population (13).

The public should be educated about the modes of West Nile virus transmission and prevention of mosquito bites. Persons older than 50 or those who are immunocompromised are at highest risk for severe disease. Preventive measures should include use of mosquito repellent when outdoors. Spraying clothing with N,N-diethyl-meta-toluamide (DEET) or permethrin adds extra protection, since mosquitoes can bite through thin clothing. Wearing protective clothing (long sleeves, socks, and long pants) when outdoors should be encouraged. It is advisable to remain indoors or use protective clothing and repellent from dusk to dawn, the primary mosquito-biting hours. Intact window and door screens prevent mosquitoes from entering homes. Mosquito netting should be used over beds when camping (32).

Nosocomial transmission of West Nile virus can be prevented by screening blood products and donors or organs for transplantation. A high level of suspicion should be maintained between June and November, when human infection with West Nile virus predominates, coinciding with the seasonal activity of mosquitoes.

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