Updated: 23.07.2004
Manual of Diagnostic Tests
and Vaccines for Terrestrial Animals
Chapter 2.10.7.
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CHAPTER 2.10.7.




West Nile virus (WNV) is a member of the genus Flavivirus in the family Flaviviridae. The arbovirus is maintained in nature by cycling through birds and mosquitoes; numerous avian and mosquito species support virus replication. For many avian species, WNV infection causes no overt signs while other birds, such as American crows (Corvus brachrhynchos) and Blue Jays (Cyanocitta cristata), succumb to fatal systemic illness. Among mammals, clinical disease is primarily exhibited in horses and humans.
Clinical signs of WNV infection in horses arise from viral-induced encephalitis or encephalomyelitis. Infections are dependent on mosquito transmission and are seasonal in temperate climates, peaking in the early autumn in the Northern Hemisphere. Affected horses frequently demonstrate mild to severe ataxia. Signs can range from slight incoordination to recumbency. Some horses exhibit weakness, muscle fasciculation, and cranial nerve deficits. Fever is not a consistently recognised feature of the disease in horses.
Identification of the agent: Bird tissues generally contain higher concentrations of virus than equine tissues. Brain and spinal cord are the preferred tissues for virus isolation from horses. In birds, kidney, heart, brain or intestine can yield virus isolates. Cell cultures (using, for example, rabbit kidney or Vero cells) are used most commonly for virus isolation. WNV is cytopathic in susceptible culture systems. Viral nucleic acid and viral antigens can be demonstrated in tissues of infected animals by reverse-transcription polymerase chain reaction (RT-PCR) and immuno-histochemistry, respectively. The most sensitive method for identifying WNV in equine tissues is a nested format of the RT-PCR procedure.
Serological tests: Antibody can be identified in equine serum by IgM capture enzyme-linked immunosorbent assay (IgM capture ELISA), haemagglutination inhibition (HI), IgG ELISA or plaque reduction neutralisation (PRN). The HI and PRN methods are most commonly used for identifying WN antibody in avian serum. In some serological assays, antibody cross-reactions with related flaviviruses, such as St Louis encephalitis virus or Japanese encephalitis virus, may be encountered.
Requirements for vaccines and diagnostic biologicals: A formalin formalin-inactivated WNV vaccine derived from tissue culture and a WNV live canarypoxvirus vectored vaccine are is available for use in horses.


West Nile virus (WNV) is a zoonotic mosquito-transmitted arbovirus belonging to the genus Flavivirus in the family Flaviviridae. The genus Flavivirus also includes Japanese encephalitis virus (see Chapter 2.5.14.), St Louis encephalitis virus, and Kunjin virus, among others (5). WNV has a wide geographical range that includes portions of Europe, Asia, Africa, Australia and North America. Migratory birds are thought to be responsible for virus dispersal, including reintroduction of WNV from endemic areas into regions that experience sporadic outbreaks (5). WNV is maintained in a mosquito-bird-mosquito transmission cycle, whereas humans and horses are considered dead end hosts. Genetic analysis of WN isolates separates strains into two clades. Lineage 1 isolates are found in northern and central Africa, Israel, Europe, India, Australia (Kunjin virus) and North America. Lineage 2 strains are endemic in central and southern Africa and Madagascar, with co-circulation of both virus lineages in central Africa (2, 6). While recent human and equine outbreaks have been due to lineage 1 viruses, strains from each lineage have been implicated in human and animal disease.
WNV was recognised as a human pathogen in Africa during the first half of the 20th century . Although several WN fever epidemics were described, encephalitis as a consequence of human WN infection was rarely encountered prior to 1996, but since then, outbreaks of human West Nile encephalitis have been reported from Romania, Russia, Israel and North America (3, 10, 11). During the 1960s, West Nile encephalitis of horses was reported from Egypt and France (17, 19). Since 1998, outbreaks of equine WN encephalitis have been reported from Italy, France, and North America (7, 14, 15). In North America, from 1999 to 2002, the virus range dramatically expanded from a discrete region along the East Coast of New York State to include most of continental United States of America (USA), and Canada, with increasing numbers of horses and wild birds affected each year (15, 16, 22).
The incubation period for equine WN encephalitis following mosquito transmission is estimated to be 315 days. A fleeting viraemia of low virus titre precedes clinical onset (4, 19). WN encephalitis occurs in only a small per cent of infected horses; the majority of infected horses do not display clinical signs (15). The disease in horses is frequently characterised by mild to severe ataxia. Additionally, horses may exhibit weakness, muscle fasciculation and cranial nerve deficits (7, 15, 16, 20). Fever is an inconsistently recognised feature. Treatment is supportive and signs may resolve or progress to terminal recumbency. The mortality rate is approximately one in three clinically affected horses. Differential diagnoses include other arboviral encephalidites (e.g. eastern, western or Venezuelan equine encephalomyelitis, Japanese encephalitis), equine protozoal myelitis (Sarcocystis neurona), equine herpesvirus-1, Borna disease, and rabies.
Although many species of birds, including chickens, are resistant to disease, the outcome of infection is generally fatal in susceptible birds. Birds may exhibit neurological signs prior to death (21). WNV has been associated with sporadic disease in small numbers of other species, including squirrels, chipmunks, bats, dogs, cats, reindeer, sheep, alpacas, alligator and a harbour seal during intense periods of local viral activity. Most human infections occur by natural transmission from mosquitoes, but laboratory acquired infections have been reported. In clinically suspicious cases, diagnostic specimens from all animals, particularly birds, should be handled at containment level 3 following appropriate laboratory procedures (see Chapter I.1.6. Human safety in the veterinary microbiology laboratory) (18).
Due to the occurrence of inapparent WN infections, diagnostic criteria must include a combination of clinical assessment and laboratory tests.


1.   Identification of the agent
     a)   Culture
          Specimens for virus isolation include brain and spinal cord from encephalitic horses (15, 16); a variety of bird tissues including heart, brain or intestine may be used with success (21). In general, virus isolates are obtained more easily from avian specimens. Virus may be propagated in susceptible cell cultures, such as rabbit kidney (RK-13) and African green monkey kidney (Vero) cells, or embryonating chicken eggs. Intracerebral inoculations of newborn mice are less likely to yield virus isolates from mammalian tissues than cell culture methods. More than one cell culture passage may be required to observe cytopathic effect (CPE). Confirmation of WNV isolates is achieved by indirect fluorescent antibody staining of infected cultures or nucleic acid detection methods (see below).
     b)   Immunological methods
          Immunohistochemical (IHC) staining of formalin-fixed avian tissues is a reliable method for identification of WN infection in birds. The specificity of identification (e.g. flavivirus specific or WNV specific) depends on the selection of detector antibody. The brain and spinal cord tissues of horses with WN encephalitis are inconsistently positive in IHC tests; approximately 50% of equine WN encephalitis cases yield false-negative results. Failure to identify WNV antigen in equine central nervous system does not rule out infection.
     c)   Nucleic acid recognition methods
          Nucleic acid detection by reverse-transcription polymerase chain reaction (RT-PCR) significantly enhances the identification of WNV-infected tissues, particularly when a nested PCR approach is applied to fresh, unfixed, equine brain and spinal cord specimens (12). The RT-nested PCR method to detect WNV nucleic acid encoding a portion of the E gene is described below. This method was developed using a 1999 North American isolate and has been successful in detecting WNV RNA in animal tissues during recent North American outbreaks. St Louis encephalitis virus is not detected by this method. Lineage 1 West Nile viruses from China (Peoples Rep. of), France, Egypt, Israel, Italy, Kenya, Mexico and Russia demonstrate a highly conserved nucleotide sequence in the target region, regardless of species of origin. Analysis of sequence information for the Uganda 1937 Lineage 2 strain (GenBank M12294) in the region targeted by the PCR primers indicate that amplification of lineage 2 strains of WNV would not be expected. Other viruses from, the Japanese encephalitis serogroup have not been examined. Non-nested methods, including real-time PCR, pose less risk of laboratory cross-contamination and may be applied successfully to avian tissue samples (13). Tissues selected for PCR are the same as those selected for virus isolation attempts.
             Reverse-transcription nested polymerase chain reaction (RT-nPCR) procedure
          The RT-nPCR described here includes several procedures: extraction of RNA, reverse transcription to generate DNA from RNA and first stage PCR, second stage PCR using nested primers and, finally, detection of the appropriately sized amplicon by gel electrophoresis. WN E protein gene regions of 445 bp (base pairs) and 248 bp are amplified in the first-stage and nested procedures, respectively. The kits and reagents described below are provided as examples. Equivalent products may be available from other sources. Extreme care in handling all materials and inclusion of proper controls are essential to ensure accurate results. The precautions to be taken have been covered in Chapter I.1.4, Validation and Quality Control of Polymerase Chain Reaction Methods used for the Diagnosis of Infectious Diseases. Duplicate samples of each diagnostic specimen should be processed and tested to increase confidence in test results. Use appropriate precautions when handling hazardous reagents such as ethidium bromide.
             Extraction of viral RNA
          From 50 to 100 mg of tissue, extract total RNA using Trizol reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturers instructions. Also extract total RNA from WN control stock virus containing 10100 tissue culture infective dose (TCID50) per 100 l volume.
             Reverse transcription and first stage PCR
               First stage primers:
               1401: 5-ACC-AAC-TAC-TGT-GGA-GTC-3
               1845: 5-TTC-CAT-CTT-CAC-TCT-ACA-CT-3
          i)   Suspend extracted RNA samples in 12 l RNase-free water.
          ii)   Denature at 70C for 10 minutes.
          iii)   Add 2 l of each denatured RNA sample to 48 l of RT-PCR mixture containing a final composition of:
               10 mM Tris/HCl, pH 8.3
               50 mM KCl
               2.0 mM MgCl2
               0.8 mM deoxynucleoside triphosphate (dNTP) pool
               25 units M-MLV (Moloney murine leukaemia virus) RT
               1.25 units RNase inhibitor
               1.25 units AmpliTaq GoldTM (Applied Biosystems, Foster City, CA, USA)
               37.5 pmol each of the first stage primers.
               Include no RNA controls using 2 l RNase-free water in place of denatured RNA.
          iv)   Incubate reaction tubes at 45C for 45 minutes.
          v)   Incubate reactions tubes at 95C for 11 minutes.
          vi)   PCR amplification through 35 cycles:
               Denaturation at 95C for 30 seconds,
               Primer annealing at 55C for 45 seconds,
                Primer extension at 72C for 60 seconds (for the 35th cycle, primer extension at 72C for 5 minutes).
          vii)   Hold samples at 4C until second stage PCR.
             Second stage (nested) PCR
               Second stage primers:
               1485:    5-GCC-TTC-ATA-CAC-ACT-AAA-G-3
                1732: 5-CCA-ATG-C-TA-T-CA-C-AG-A-CT-3
          i)   For each sample and control, add 1.5 l of the first-stage amplification product to 48.5 l of PCR mixture with a final composition of:
               10 mM Tris/HCl, pH 8.3
               50 mM KCl
               2.0 mM MgCl2
               0.8 mM deoxynucleoside triphosphate (dNTP) pool
               1.25 units AmpliTaq GoldTM (Applied Biosystems, Foster City, CA, USA)
               37.5 pmol each of the nested primers.
          ii)   Incubate reactions tubes at 95C for 11 minutes.
          iii)   PCR amplification through 35 cycles:
               Denaturation at 95C for 30 seconds,
               Primer annealing at 55C for 45 seconds,
                Primer extension at 72C for 60 seconds (for the 35th cycle, primer extension at 72C for 5minutes).
          iv)   Hold samples at 4C or 20C until electrophoresis.
             Analysis of PCR products by gel electrophoresis
          i)   Prepare a 2.5% NuSieve 3/1 (FMC Bioproducts, Rockland, Maine, USA) agarose solution in 0.045 mM Tris/borate, pH 8.6, 1.5 mM EDTA (ethylene diamine tetra-acetic acid) (1 x TBE buffer). Boil the agarose on a hotplate or in a microwave oven until completely dissolved. Cool the agarose to 4555C. Add 5.0 l ethidium bromide solution (10 mg/ml) per 100 ml warm agarose and pour agarose gel with comb. Allow to solidify then remove comb.
          ii)   Add 30 l ethidium bromide solution (10 mg/ml) per 600 ml 1 x TBE tank buffer. Position gel in apparatus and fill buffer tanks.
          iii)   Mix 15 l of each sample and control with 5 l gel loading solution (e.g. Sigma product G-2526, St Louis, MO, USA) Include 100 bp DNA ladder (e.g. Life Technologies, Grand Island, NY, USA product 15268-019, range 1001500 bp) in at least one gel well. Load samples into agar wells and electrophorese at 6575 volts until the gel loading dye has travelled approximately two-thirds the length of the gel.
          iv)   Visualise and photograph gel under ultraviolet illumination.
             Interpretation of the test
          For the PCR test to be valid, the positive controls must show the appropriate size band (248 bp). The no RNA controls should have no bands. Samples are considered to be positive if there is a band of the same size as the positive control. Duplicate samples should both show the same reaction. If there is a disparity, the test should be repeated, starting with extraction from tissue. If further validation is required, the final nested PCR product can be sequenced and compared with the published sequences of WNV from GenBank.
2.   Serological tests
     Antibody can be identified in equine serum by IgM capture enzyme-linked immunosorbent assay (IgM capture ELISA), hemagglutination inhibition (HI), IgG ELISA or plaque reduction neutralisation (PRN) (1, 9). The IgM capture ELISA described below is particularly useful to detect antibodies resulting from recent natural exposure to WNV. Equine WN-specific IgM antibodies are usually detectable from 710 days post-infection to 12 months post-infection. Most horses with WN encephalitis test positive in the IgM capture ELISA at the time that clinical signs are first observed. WNV neutralising antibodies are detectable in equine serum by 2 weeks post-infection and can persist for more than 1 year. The HI and PRN methods are most commonly used for identifying WN antibody in avian serum. In some serological assays, antibody cross-reactions with related flaviviruses, such as St Louis encephalitis virus or Japanese encephalitis virus, may be encountered. The PRN test is the most specific among WN serological tests; when needed, serum antibody titres against related flaviviruses can be tested in parallel. Finally, WN vaccination history must be considered in interpretation of serology results, particularly in the PRN test and IgG ELISA. IgM capture ELISA may be used to test avian or other species provided that species-specific capture antibody is available (e.g. anti-chicken IgM). The PRN test is applicable to any species, including birds.
     a)   Equine IgM capture ELISA
          WNV and normal antigens for the IgM capture ELISA may be prepared from mouse brain (see Chapter 2.5.3), tissue culture or recombinant cell lines (8). Commercial sources of WNV testing reagents are available in North America. Characterised equine control serum, although not an international standard, can be obtained from the National Veterinary Services Laboratories, Ames, Iowa, USA. Virus and control antigens should be prepared in parallel for use in the ELISA. Antigen preparations must be titrated with control sera to optimise sensitivity and specificity of the assay. Equine serum samples are tested at a dilution of 1/400 and equine cerebrospinal fluid samples are tested at a dilution of 1/2 in the assay. To ensure specificity, each serum sample is tested for reactivity with both virus antigen and control antigen.
             Test procedure
          i)   Coat flat-bottom 96-well ELISA plates (e.g. Immulon 2HB, Dynex Technologies, Chantilly, VA, USA) with 100 l/well anti-equine IgM diluted in 0.5 M carbonate buffer, pH 9.6, according to the manufacturers instructionssuggested dilution for use as a capture antibody.
          ii)   Incubate plates overnight at 4C in a humid chamber. Coated plates may be stored for several weeks.
          iii)   Prior to use, wash plates twice with 200300 l/well 0.01 M phosphate buffered saline, pH 7.2, containing 0.05% Tween 20 (PBST).
          iv)   Block plates by adding 300 l/well freshly prepared 5% nonfat dry milk in PBST and incubate 60 minutes at room temperature. After incubation, remove blocking solution and wash plates three times with PBST.
          v)   Test and control sera are diluted 1/400 (cerebrospinal fluid is diluted 1/2) in PBST and 50 l/well of each sample is added to duplicate sets of wells (total of four4 wells per sample) on the plate. Include control positive and negative sera prepared in the same manner as samples.
          vi)   Cover the plates and incubate 75 minutes at 37C in a humid chamber.
          vii)   Remove serum and wash plates three times in PBST.
          viii)   Dilute virus and normal antigens in PBST and add 50 l of virus antigen to one set of wells per test and control sera and add 50 l normal antigen to the second set of wells per test and control sera.
          ix)   Cover the plates and incubate overnight at 4C in a humid chamber.
          x)   Remove antigens from the wells and wash the plates three times in PBST.
          xi)   Dilute horseradish peroxidase conjugated anti-Flavivirus monoclonal antibody (available from the Centers for Disease Control and Prevention, Biological Reference Reagents, 1600 Clifton Road NE, Mail Stop C21, Atlanta, Georgia, 30333, USA) in PBST according to manufacturers directions and add 50 l per well.
          xii)   Cover the plates and incubate at 37C for 60 minutes.
          xiii)   Remove conjugate and wash plates three six times in PBST.
          xiv)   Add 50 l/well freshly prepared ABTS (2,2-azino-di-[3-ethyl-benzthiazoline]-6-sulphonic acid) substrate + with hydrogen peroxide (0.1%) and incubate at room temperature for 30 minutes.
          xv)   Measure absorbance at 405 nm. A test sample is considered to be positive if the absorbance of the test sample in wells containing virus antigen is at least twice the absorbance of negative control serum in wells containing virus antigen and at least twice the absorbance of the sample tested in parallel in wells containing control antigen.
     b)   Plaque reduction neutralisation (applicable to serum from any species)
          The PRN test is performed in Vero cell cultures in either 25 cm2 flasks or 6-well plates. The sera can be screened at a 1/10 and 1/100 final dilution or may be titrated to establish an endpoint. A description of the test as performed in 25 cm2 flasks using 100 plaque-forming units (PFU) of virus is as follows.
          Prior to testing, serum is heat inactivated at 56C for 30 minutes and diluted (e.g. 1/5 and 1/50) in media. Virus (200 PFU per 0.1 ml) working dilution is prepared in media containing 10% guinea-pig complement. Equal volumes of virus and serum are mixed and incubated at 37C for 75 minutes before inoculation of 0.1 ml on to confluent cell culture monolayers. The inoculum is adsorbed for 1 hour at 37C, followed by the addition of 4.0 ml of primary overlay medium. The primary overlay medium consists of two solutions that are prepared separately. Solution I contains 2 x Earles Basic Salts Solution without phenol red, 4% fetal bovine serum, 100 g/ml gentamicin and 0.45% sodium bicarbonate. Solution II consists of 2% Noble agar that is sterilised and maintained at 47C. Equal volumes of solutions I and II are adjusted to 47C and mixed together just before use. The test is incubated for 72 hours at 37C. A second 4.0 ml overlay prepared as above, but also containing 0.003% neutral red is applied to each flask. Following a further overnight incubation at 37C, the number of virus plaques per flask is assessed. Endpoints titres are based on 90% reduction compared with the virus control flasks, which should have about 100 plaques.


In February 2003, the United States Department of Agriculture (USDA) issued a license for a formalin- inactivated WNV vaccine derived from tissue culture for use in horses. As recently as December 2003, the USDA licensed a live canarypoxvirus vectored WNV vaccine for use in horses. These vaccines have demonstrated sufficient efficacy and safety in adequately vaccinated horses. Vaccination may be helpful in preventing neurological signs associated with WN infection.
Guidelines for the production of veterinary vaccines are given in Chapter I.1.7. Principles of veterinary vaccine production. The guidelines given here and in Chapter I.1.7 are intended to be general in nature and may be supplemented by national and regional requirements.
1.   Seed management
     a)   Characteristics of the seed
          The isolate of WNV used for vaccine production must be accompanied by documentation describing its origin and passage history. The isolate must be safe in host animals at the intended age of vaccination and provide protection after challenge.
     b)   Method of culture
          The WNV should be propagated in cell lines known to support the growth of WNV. Cell lines should be free from extraneous viruses, bacteria, fungi, and mycoplasma. Viral propagation should not exceed five passages from the master seed virus (MSV), unless further passages prove to provide protection in the host animal.
     c)   Validation as a vaccine
          The MSV should be free from bacteria, fungi and mycoplasma. The MSV must be tested for and be free of extraneous viruses, including equine herpesvirus, equine adenovirus, equine viral arteritis virus, bovine viral diarrhoea virus, reovirus, and rabies virus by the fluorescent antibody technique. The MSV must be free from extraneous virus by CPE and haemadsorption on the Vero cell line and an embryonic equine cell type.
          In an immunogenicity trial, the MSV at the highest passage level intended for production must protect susceptible horses against a virulent challenge strain. A statistically significant number of vaccinated horses must be protected from viraemia when compared with the controls. Field trial studies should be conducted to determine the safety of the vaccine.
2.   Method of manufacture
     The susceptible cell line is seeded into suitable vessels. Minimal essential medium, supplemented with fetal bovine serum (FBS), is used as the medium for production. Incubation is at 37C.
     Cell cultures are inoculated directly with WN working virus stock, which is generally from 1 to 4 passages from the MSV. Inoculated cultures are incubated for 18 days before harvesting the culture medium. During incubation, the cultures are observed daily for CPE and bacterial contamination.
     Killed virus vaccines are chemically inactivated with either formalin or binary ethylenimine and mixed with a suitable adjuvant.
3.   In-process control
     Production lots of WN must be titrated in tissue culture for standardisation of the product. Low-titred lots may be concentrated or blended with higher-titred lots to achieve the correct titre.
4.   Batch control
     Final container samples are tested for purity, safety and potency.
     a)   Purity
           Samples are examined for bacterial and fungal contamination. To test for bacteria, ten vessels, each containing 120 ml of soybean casein digest medium, are inoculated with 0.2 ml from ten final-container samples. The ten vessels are incubated at 3035C for 14 days and observed for bacterial growth. To test for fungi, ten vessels, each containing 40 ml of soybean casein digest medium, are inoculated with 0.2 ml from ten final-container samples. The vessels are incubated at 2025C for 14 days and observed for fungal growth.
     b)   Safety
          Safety tests can be conducted in a combination of guinea-pigs, mice or horses. Field safety studies should be conducted before the vaccine receives final approval. Generally, two serials should be used, in three different geographical locations, and a minimum of 600 animals. About one-third of the animals should be at the minimum age recommended for vaccination (correlated to efficacy). If the final product is a modified-live vaccine, additional safety testing of the MSV is required to demonstrate a lack of virulence.
     c)   Potency
          Killed virus vaccines may use host animal or laboratory animal vaccination/serology tests or vaccination/challenge tests to determine potency of the final product. Parallel-line assays using ELISA antigen-quantifying techniques to compare a standard with the final product are acceptable in determining the relative potency of a product. The standard should be shown to be protective in the host animal (22). Live viral products are titred in cell cultures to determine the potency of the final product. The final release potency titre should include an additional 0.7 log10 for test variability and 0.5 log10 for end-of-dating stability than the minimum protective dose established in the immunogenicity trial.
     d)   Duration of immunity
          Duration of immunity studies are conducted before the vaccine receives final approval. The duration should be for the length of the mosquito season in the infected areas.
     e)   Stability
          All vaccines are initially given 24 months before expiry. Real-time stability studies are conducted to confirm the appropriateness of all expiration dating.
     f)   Preservatives
          Antibiotics are added during production, generally gentamicin sulphate or neomycin not to exceed 30 g/ml.
     g)   Precautions (hazards)
          Vaccination is only recommended for horses in WN-positive areas. Vaccinated horses may develop a serological titre that may interfere with the ability to export the horse.
5.   Tests on the final product
     a)   Safety
          See Section C.4.b.
     b)   Potency
          See Section C.4.c.


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23.   United States Department of Agriculture Animal and Plant Health Inspection Service, Veterinary Services Me morandum 800.90 (1998). Guidelines for Veterinary Biological Relative Potency Assays and Reference Preparations Based on ELISA Antigen Quantification. Available at: URL: http://www.aphis.usda.gov/vs/cvb/vsmemos.htm

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NB: There is an OIE Reference Laboratory for West Nile fever (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm).

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