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CHAPTER
2.2.19.
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WEST NILE ENCEPHALITIS
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SUMMARY
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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.
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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.
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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, liver 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.
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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.
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Requirements for vaccines and diagnostic biologicals: A formalin-inactivated WNV vaccine
derived from tissue culture, WNV live canarypoxvirus vectored vaccine, and a WNV DNA vaccine
are available for use in horses.
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A.
INTRODUCTION
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 (6). 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 (6). 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 (3, 7). 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.
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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, North America, France, and Tunisia (4, 11, 13, 15, 28). During the 1960s, West Nile
encephalitis of horses was reported from Egypt and France (21, 23). Since 1998, outbreaks of equine WN
encephalitis have been reported from Italy, France, North America, Morocco and Israel (8, 14, 18, 19). In the
Western Hemisphere, the virus range has dramatically expanded from a discrete region along the East Coast of
New York State to include the contiguous States of the United States of America (USA), Canada, Mexico, the
Caribbean islands, Central America, and Colombia (10, 19, 26).
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The incubation period for equine WN encephalitis following mosquito transmission is estimated to be 3–15 days.
A fleeting viraemia of low virus titre precedes clinical onset (5, 23). WN encephalitis occurs in only a small per
cent of infected horses; the majority of infected horses do not display clinical signs (19). The disease in horses is
frequently characterised by mild to severe ataxia. Additionally, horses may exhibit weakness, muscle
fasciculation and cranial nerve deficits (8, 19, 20, 24). 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.
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Although many species of birds, including chickens and turkeys, are resistant to disease, the outcome of
infection is generally fatal in susceptible birds. Outbreaks of fatal neurologic disease have been reported in zoo
birds in the USA and in domestic geese in Israel and Canada (1, 25, 28). WNV has been associated with
sporadic disease in small numbers of other species, including squirrels, chipmunks, bats, dogs, cats, white-tailed
deer, 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 1.1.6 Biosafety and
biosecurity in the veterinary microbiology laboratory and animal facilities) (22).
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Due to the occurrence of inapparent WN infections, diagnostic criteria must include a combination of clinical
assessment and laboratory tests.
B.
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DIAGNOSTIC TECHNIQUES
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1. Identification of the agent
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a) Culture
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Specimens for virus isolation include brain and spinal cord from encephalitic horses (19, 20); a variety of
bird tissues including kidney, brain, heart, liver, or intestine may be used with success (25). 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
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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 falsenegative results. Failure to identify WNV antigen in equine central nervous system does not rule out
infection.
c) Nucleic acid recognition methods
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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 (16). 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 (People’s 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
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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 (17). Tissues selected for PCR are the same as those selected for virus isolation attempts.
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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 1.1.4 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.
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From 50 to 100 mg of tissue, extract total RNA using Trizol ® reagent (Life Technologies, Grand Island, NY,
USA) according to the manufacturer’s instructions. Also extract total RNA from WN control stock virus
containing 10–100 tissue culture infective dose (TCID50) per 100 µl volume.
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Reverse-transcription nested polymerase chain reaction (RT-nPCR) procedure
Extraction of viral RNA
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’
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i)
Suspend extracted RNA samples in 12 µl RNase-free water.
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ii)
Incubate at 70°C for 10 minutes.
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iii)
Add 2 µl of each RNA sample to 48 µl 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 of the first stage primers.
Include ‘no RNA’ controls using 2 µl RNase-free water in place of denatured RNA.
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iv)
Incubate reaction tubes at 45°C for 45 minutes.
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v)
Incubate reactions tubes at 95°C for 11 minutes.
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vi)
PCR amplification through 35 cycles:
Denaturation at 95°C for 30 seconds,
Primer annealing at 55°C for 45 seconds,
Primer extension at 72°C for 60 seconds (for the 35th cycle, primer extension at 72°C for 5 minutes).
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Hold samples at 4°C until second stage PCR.
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Second stage (nested) PCR
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Second stage primers:
1485: 5’-GCC-TTC-ATA-CAC-ACT-AAA-G-3’
1732: 5’-CCA-ATG-CTA-TCA-CAG-ACT-3’
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i)
For each sample and control, add 1.5 µl of the first-stage amplification product to 48.5 µl 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.
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ii)
Incubate reactions tubes at 95°C for 11 minutes.
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iii)
PCR amplification through 35 cycles:
Denaturation at 95°C for 30 seconds,
Primer annealing at 55°C for 45 seconds,
Primer extension at 72°C for 60 seconds (for the 35th cycle, primer extension at 72°C for 5 minutes).
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iv)
Hold samples at 4°C or –20°C until electrophoresis.
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Analysis of PCR products by gel electrophoresis
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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 × TBE buffer). Boil
the agarose on a hotplate or in a microwave oven until completely dissolved. Cool the agarose to 45–
55°C. Add 5.0 µl ethidium bromide solution (10 mg/ml) per 100 ml warm agarose and pour agarose
gel with comb. Allow to solidify then remove comb.
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ii)
Add 30 µl ethidium bromide solution (10 mg/ml) per 600 ml 1 × TBE tank buffer. Position gel in
apparatus and fill buffer tanks.
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iii)
Mix 15 µl of each sample and control with 5 µl 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 100–1500 bp) in at least one gel well. Load samples into agar wells and
electrophorese at 65–75 volts until the gel loading dye has travelled approximately two-thirds the
length of the gel.
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iv)
Visualise and photograph gel under ultraviolet illumination.
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Interpretation of the test
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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.
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2. Serological tests
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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) (2, 12). 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 7–10 days post-infection to 1–2 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.
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a) Equine IgM capture ELISA
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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 (9). 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.
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Test procedure
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i)
Coat flat-bottom 96-well ELISA plates (e.g. Immulon 2HB, Dynex Technologies, Chantilly, VA, USA)
with 100 µl/well anti-equine IgM diluted in 0.5 M carbonate buffer, pH 9.6, according to the
manufacturer’s suggested dilution for use as a capture antibody.
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Incubate plates overnight at 4°C in a humid chamber. Coated plates may be stored for several weeks.
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iii)
Prior to use, wash plates twice with 200–300 µl/well 0.01 M phosphate buffered saline, pH 7.2,
containing 0.05% Tween 20 (PBST).
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iv)
Block plates by adding 300 µl/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.
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Test and control sera are diluted 1/400 (cerebrospinal fluid is diluted 1/2) in PBST and 50 µl/well of
each sample is added to duplicate sets of wells (total of four wells per sample) on the plate. Include
control positive and negative sera prepared in the same manner as samples.
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Cover the plates and incubate 75 minutes at 37°C in a humid chamber.
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Remove serum and wash plates three times in PBST.
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viii) Dilute virus and normal antigens in PBST and add 50 µl of virus antigen to one set of wells per test
and control sera and add 50 µl normal antigen to the second set of wells per test and control sera.
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Cover the plates and incubate overnight at 4°C in a humid chamber.
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Remove antigens from the wells and wash the plates three times in PBST.
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Dilute horseradish peroxidase conjugated anti-Flavivirus monoclonal antibody1 in PBST according to
manufacturer’s directions and add 50 µl per well.
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xii)
Cover the plates and incubate at 37°C for 60 minutes.
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xiii) Remove conjugate and wash plates six times in PBST.
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xiv) Add 50 µl/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.
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xv)
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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)
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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.
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Prior to testing, serum is heat inactivated at 56°C 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 37°C for 75 minutes before inoculation of
0.1 ml on to confluent cell culture monolayers. The inoculum is adsorbed for 1 hour at 37°C, 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 × Earle’s Basic Salts Solution without phenol red, 4% fetal bovine
serum, 100 µg/ml gentamicin and 0.45% sodium bicarbonate. Solution II consists of 2% Noble agar that is
sterilised and maintained at 47°C. Equal volumes of solutions I and II are adjusted to 47°C and mixed
together just before use. The test is incubated for 72 hours at 37°C. 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 37°C, the number of virus plaques per flask is assessed. Endpoint titres are based on 90%
reduction compared with the virus control flasks, which should have about 100 plaques.
C.
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REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
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. This was followed in December 2003, by a USDA
licensed live canarypoxvirus vectored WNV vaccine for use in horses. As recently as July 2005, the USDA
issued the first fully licensed WNV DNA vaccine for animals in the USA. The vaccine contains genes for two
WNV proteins, and therefore, does not contain any whole WNV, live or killed. These vaccines have
demonstrated sufficient efficacy and safety in adequately vaccinated horses. Vaccination may be helpful in
preventing neurological signs associated with WN infection.
1
Available from the Centers for Disease Control and Prevention, Biological Reference Reagents, 1600 Clifton Road NE,
Mail Stop C21, Atlanta, Georgia, 30333, USA.
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Guidelines for the production of veterinary vaccines are given in Chapter 1.1.7 Principles of veterinary vaccine
production. The guidelines given here and in Chapter 1.1.7 are intended to be general in nature and may be
supplemented by national and regional requirements.
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1. Seed management
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a) Characteristics of the seed
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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
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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
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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.
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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.
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2. Method of manufacture
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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 37°C.
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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 1–8 days before harvesting the culture medium. During
incubation, the cultures are observed daily for CPE and bacterial contamination.
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Killed virus vaccines are chemically inactivated with either formalin or binary ethylenimine and mixed with a
suitable adjuvant.
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The DNA for DNA vaccines is produced in an expression vector then extracted from the vector and purified for
formulation into a vaccine.
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3. In-process control
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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.
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Production lots of DNA are quantified by analytical methods and characterized before standardization and
blending at the correct DNA content.
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4. Batch control
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Final container samples are tested for purity, safety and potency.
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a) Purity
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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 30–35°C 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 20–25°C for 14 days and observed for
fungal growth.
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b) Safety
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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
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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 (27).
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.
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DNA vaccines are tested for bioactivity and DNA content using parallel-line direct antigen quantification
methods which compare a standard preparation to the final product.
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d) Duration of immunity
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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
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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
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Antibiotics are added during production, generally gentamicin sulphate or neomycin not to exceed 30 µg/ml.
g) Precautions (hazards)
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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.
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5. Tests on the final product
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a) Safety
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See Section C.4.b.
b) Potency
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See Section C.4.c.
REFERENCES
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1.
AUSTIN R.J., W HITING T.L., ANDERSON R.A. & DREBOT M.A. (2004). An outbreak of West Nile virus-associated
disease in domestic geese (Anser anser domseticus) upon initial introduction to a geographic region, with
evidence of bird to bird transmission. Can. Vet. J., 45, 117–123.
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2.
BEATY B.J., CALISHER C.H. & SHOPE R.E. (1989). Arboviruses. In: Diagnostic Procedures for Viral Rickettsial
and Chlamydial infections, Sixth Edition, Schmidt N.H. & Emmons R.W., eds. American Public Health
Association, Washington DC, USA, 797–856.
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*
* *
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NB: There is an OIE Reference Laboratory for West Nile fever (see Table in Part 3 of this Terrestrial Manual or
consult the OIE Web site for the most up-to-date list: www.oie.int).
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