Chapter 2.2.19. – West Nile encephalitis
Chapter 2.2.19.
west nileencephalitis
SUMMARY
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, 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.
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-inactivated WNV vaccine derived from tissue culture, WNV live canarypoxvirus vectored vaccine,and a WNV DNA vaccine are available for use in horses.
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.), StLouis 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.
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).
The incubation period for equine WN encephalitis following mosquito transmission is estimated to be 3–15days. 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.
Although many species of birds, including chickensand 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 atcontainment level3 following appropriatelaboratory procedures (see Chapter 1.1.6Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities)(22).
Due to the occurrence of inapparent WN infections, diagnostic criteria must include a combination of clinical assessment and laboratory tests.
b. DIAGNOSTIC TECHNIQUES
1.Identification of the agent
a)Culture
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
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. flavivirusspecific 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 (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 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.
•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 445bp (base pairs) and 248bp 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 1.1.4Validation 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 100mg 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 10–100tissue culture infective dose(TCID50) per 100µl 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 12µl RNase-free water.
ii)Incubate at 70°C for 10minutes.
iii)Add 2µl of each RNA sample to 48µl of RT-PCR mixture containing a final composition of:
10mM Tris/HCl, pH 8.3
50mM KCl
2.0mM MgCl2
0.8mM deoxynucleoside triphosphate (dNTP) pool
25units M-MLV (Moloney murine leukaemia virus) RT
1.25units RNase inhibitor
1.25units AmpliTaq GoldTM (Applied Biosystems, Foster City, CA, USA)
37.5pmol of the first stage primers.
Include ‘no RNA’ controls using 2µl RNase-free water in place of denatured RNA.
iv)Incubate reaction tubes at 45°C for 45minutes.
v)Incubate reactions tubes at 95°C for 11minutes.
vi)PCR amplification through 35cycles:
Denaturation at 95°C for 30seconds,
Primer annealing at 55°C for 45seconds,
Primer extension at 72°C for 60seconds (for the 35th cycle, primer extension at 72°C for 5minutes).
vii)Hold samples at 4°C 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-CTA-TCA-CAG-ACT-3’
i)For each sample and control, add 1.5µl of the first-stage amplification product to 48.5µl of PCR mixture with a final composition of:
10mM Tris/HCl,pH 8.3
50mM KCl
2.0mM MgCl2
0.8mM deoxynucleoside triphosphate (dNTP) pool
1.25units AmpliTaq GoldTM (Applied Biosystems, Foster City, CA, USA)
37.5pmol each of the nested primers.
ii)Incubate reactions tubes at 95°C for 11minutes.
iii)PCR amplification through 35cycles:
Denaturation at 95°C for 30seconds,
Primer annealing at 55°C for 45seconds,
Primer extension at 72°C for 60seconds (for the 35th cycle, primer extension at 72°C for 5minutes).
iv)Hold samples at 4°C or –20°C 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 0.045mM Tris/borate, pH 8.6, 1.5mM 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 5.0µl ethidium bromide solution (10mg/ml) per 100ml warm agarose and pour agarose gel with comb. Allow to solidify then remove comb.
ii)Add 30µl ethidium bromide solution (10mg/ml) per 600ml 1 × TBE tank buffer. Position gel in apparatus and fill buffer tanks.
iii)Mix 15µl of each sample and control with 5µl gel loading solution (e.g. Sigma product G-2526, St Louis, MO, USA) Include 100bp DNA ladder (e.g. Life Technologies, Grand Island, NY, USA product 15268-019, range 100–1500bp) in at least one gel well. Load samples into agar wells and electrophorese at 65–75volts 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 (248bp). 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) (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 7–10days post-infection to 1–2months 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 2weeks post-infection and can persist for more than 1year. 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 testand 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 (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 antigensshould 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 100µl/well anti-equine IgM diluted in 0.5M carbonate buffer, pH 9.6, according to the manufacturer’s suggested dilution for use as a capture antibody.
ii)Incubate plates overnight at 4°C in a humid chamber. Coated plates may be stored for several weeks.
iii)Prior to use, wash plates twice with 200–300µl/well 0.01M phosphate buffered saline, pH7.2, containing 0.05% Tween 20(PBST).
iv)Block plates by adding 300µl/well freshly prepared 5% nonfat dry milk in PBST and incubate 60minutes 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 PBSTand 50µl/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.
vi)Cover the plates and incubate 75minutes at 37°C in a humid chamber.
vii)Remove serum and wash plates three times in PBST.
viii)Dilute virus and normal antigens in PBSTand add 50µl of virus antigen to one set of wells per test and control sera and add 50µl normal antigen to the second set of wells per test and control sera.
ix)Cover the plates and incubate overnight at 4°C 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[1] in PBSTaccording to manufacturer’s directions and add 50µl per well.
xii)Cover the plates and incubate at 37°C for 60minutes.
xiii)Remove conjugate and wash plates six times in PBST.
xiv)Add 50µl/well freshly prepared ABTS (2,2’-azino-di-[3-ethyl-benzthiazoline]-6-sulphonic acid) substrate withhydrogen peroxide (0.1%) and incubate at room temperature for 30minutes.
xv)Measure absorbance at 405nm. 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 25cm2 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 25cm2 flasks using 100plaque-forming units (pfu) of virus is as follows.