OVERALL DETECTION AND IDENTIFICATION TECHNIQUES - Editorial Comment

SAMPLE COLLECTION & SHIPPING:

• The appropriate samples to be collected vary depending on whether diagnosis or virus detection is required from mosquitoes, dead wild birds, live sentinels, equines or humans.
• Accurate labelling of samples is important.
• Prior to collecting and shipping samples the laboratory to which the samples are to be sent should be consulted regarding the details of what samples are required, preservation required, shipping and labelling instructions. Detailed instructions are available from CDC.

(D67, D72, D147, P39.4.w9, P39.3.w15)

ANTIBODY DETECTION:

• Serological assays for arboviruses include IgM ELISA, IgG ELISA, PRNT, CF, HI, IFA (immunofluorescent antibody) and dipsticks. (P39.4.w12)
• Enzyme-linked Immunosorbent Assay (ELISA) techniques are in general used to detect antibodies and frequently replace the Haemagglutination Inhibition, Complement Fixation and Neutralisation Tests in many situations. The IgM capture ELISA (MAC-ELISA) is in current use for WN virus antibody confirmation. (B244.w1, B245.29.w29, D72)
  • An ELISA has been evaluated to detect antibody in wild birds, for use as a rapid screening test (the PRNT would be required for confirmation of results)  (J84.8.w9)
• Haemagglutination Inhibition (HI) is a serological test for WN virus antibody and, although it is still in use in many laboratories, there is an increasing tendency with time for sera to cross-react with other virus antigens within a given virus family; this test is gradually being replaced by ELISA techniques. (B245.29.w29)
  • HI may still be used for screening as it provides results within 24-48 hours. (J91.32.w1, V.w42)
  • This test has the advantage for surveillance of domestic and wild animals that a species-specific conjugate is not required. (P32.1.w1)
  • It has been noted that for flaviviruses HI tests on chicken sera are valid following an acetone extraction method. (J93.41.w2)
• The Plaque Reduction Neutralization Test (PRNT) is a serological test for WN virus antibody and, although expensive and problematic, is useful to differentiate between infection of two viruses which are closely related antigenically. (B245.29.w29)
  • The PRNT is the confirmatory test for identification of WN virus specific antibody.
• The Indirect Immunofluorescent Antibody (Indirect IFA) is a serological test for WN virus antibody. It uses an indirect format, therefore there is a problem with IgG competition for IgM binding. It is not a preferred test for serological diagnosis. (B245.29.w29). However it may be useful in laboratories without the time, funds or technical expertise for utilising ELISA. (J133.951.w43)
• The Complement Fixation Test (CF) can be used to detect viral antibodies. Whilst it is more difficult to maintain quality control for this test compared with the haemagglutination inhibition test, it may still be used to detect complement fixing antibodies that may be present after the IgM has waned. (B244.w1, B245.29.w29)
• An Immunoperoxidase Monolayer Assay (IPMA) has been described for testing human sera against several arboviruses, including WN virus, using a reaction readable visually; this may be of use for screening human sera in field situations. (J217.65.w1)

Comments on serological detection of antibody:

• "For most arboviruses, serological cross-reactivity with related viruses increases as the infection progresses." (B245.29.w29)
• Cross-reactions with related Flaviviruses occur in serological tests. (B244.w1)
• It is important to remember, in areas in which more than one flavivirus occurs, that in the event of infection by one flavivirus and later a second flavivirus, the antibody titre in serum taken at the time of the second infection may be higher for the virus which caused the first infection than for that causing the current infection.  (B244.w1)
• Paired serum samples (acute-phase and convalescent phase) are useful, allowing the detection of a rise in antibody titre and, for patients in whom the acute-phase sample was taken too early for IgM antibodies to be detected, both IgM and IgG antibodies are likely to be found in the convalescent-phase serum sample. (B245.29.w29)
• It is possible that if WNV Infection follows infection with another Flavivirus, IgM levels  measured by MAC-ELISA may give equivocal results. (J84.8.w1)
• In the USA cross-reaction may occur with St Louis Encephalitis virus (SLE) in particular, both being members of the Japanese Encephalitis group of flaviviruses. (J214.267.w11)

ANTIGEN DETECTION:

• Virus detection assays for arboviruses include virus isolation (in cell culture or mice), IFA, TaqMan RT-PCR, antigen-capture ELISA, RT-PCR/sequencing, dipsticks and NASBA. (P39.4.w12)
• Using Cell Culture techniques, WN virus can be grown on a variety of mosquito (Culicidae - Mosquitoes (Family)) cell lines, mammalian cell lines and in suckling mice. In  mosquito cell lines a cytopathic effect may not be seen and the culture needs to be screened by immunofluorescence [see section below] (B241.49.w49, B243.31.w1, B245.29.w29, J64.19.w1, J73.57.w1, J84.8.w7, J110.39.w1, D147)
• A variety of Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR) protocols, both standard and nested, have been described for detection of WN virus RNA. (J84.7.w25, J110.39.w1, J217.94.w1, B245.29.w29, D72)
  • PCR protocols can be used on a variety of sample types, including clinical samples such as CSF, post mortem tissue samples, and mosquito pools. 
  • PCR assays are highly sensitive but are susceptible to false positive results due to inadvertent contamination.
  • The TaqMan RT-PCR is a highly sensitive test for detection of virus RNA.
• A Nucleic Acid Sequence-Based Amplification (NASBA) assay has been developed with very high sensitivity and specificity, and allowing a rapid test result. It may be useful to complement virus isolation and TaqMan PCR testing or even as an alternative to TaqMan. (J93.40.w3)
• The Indirect Haemagglutination test was developed for rapid detection of WN virus antigen in a viraemic host, although the sensitivity is probably lower than virus isolation techniques. (J88.22.w1, B241.49.w49)
• An indirect IFA using IgM MAb specific for WN virus envelope protein followed by goat anti-mouse IgM fluorescein conjugate has been used for detection of virus in cell culture and tissues. It was found by the Arbovirus Laboratory, Wadsworth center, NYSDH, that the indirect IFA was a valuable confirmatory assay for use on bird tissues but that non-specific binding made it problematic for use on mammalian tissues. (J93.41.w4)
• Direct Immunofluorescence techniques are used for detecting WN virus antigen in cells, impression smears and cell culture. (J26.37.w1, J71.75.w1, J73.57.w1, J84.7.w29)
  • Immunohistochemistry (IHC) is extremely useful for histopathological studies in vertebrates and is particularly useful for use as a screening test in diagnostic laboratories which do not have BSL-3 facilities. (V.w42)
• An ELISA based on a monoclonal antibody has been developed for detection of WN virus antigen in samples of mosquitoes and bird tissues. 
  • The VecTest^TM dip stick test is an antigen-capture ELISA which may be used on mosquito homogenate, avian tissue, and oral or cloacal swabs from avian carcasses The test was shown to have a high specificity but was less sensitive than TaqMan or plaque assay. (P39.4.w10)

(J26.37.w1,J64.19.w1, J71.75.w1, J73.57.w1, J84.8.w1, J84.7.w29, J84.8.w9, J84.9.w18, J84.9.w21, J88.22.w1, J91.32.w1, J93.38.w4, J93.40.w3, J93.41.w4, J217.65.w1, B244.w1, B241.49.w49, B243.31.w1, B245.29.w29, D72, P39.4.w10, P39.4.w12, P51.49.w2, V.w42)

Further details of various tests, and their references, are provided in the literature reports below.

• --

Sample Collection & Shipping

• The appropriate samples to be collected vary depending on whether diagnosis or virus detection is required from mosquitoes, dead wild birds, live sentinels, equines or humans.
• Accurate labelling of samples is important.
• Prior to collecting and shipping samples the laboratory to which the samples are to be sent should be consulted regarding the details of what samples are required, preservation required, shipping and labelling instructions. Detailed instructions are available from CDC.

(D67, D72, D147, P39.4.w9, P39.3.w15)

CDC recommended tests for various samples are: (P39.4.w9, P39.3.w15)

• Human serum or CSF: In 2003 first choice tests ELISA and PRNT, other possibilities TaqMan, NASBA and virus isolation; TaqMan may detect in 57% of acute CSF but less than 10% of serum samples. (P39.4.w9)
  • In 2002 first choice tests were ELISA and PRNT, second choice tests HI and IFA; TaqMan may be useful in acute CSF samples.(P39.3.w15)
• Chicken serum: first choice tests ELISA and PRNT, second choice tests HI and IFA.[2002](P39.3.w15)
• Equine serum: first choice tests ELISA and PRNT, second choice tests HI and IFA.[2002](P39.3.w15)
• Human tissue: In 2003 first choice tests TaqMan and NASBA, other possibilities virus isolation and immunohistochemistry. In fatal cases approximately 100% of cases are positive by TaqMan and NASBA. (P39.4.w9)
  • In 2002 first choice tests were TaqMan, NASBA and virus isolation, second choice tests immunohistochemistry and standard RT-PCR (N.B. TaqMan and NASBA are more sensitive than virus isolation). 
• Avian tissue: In 2003 first choice tests TaqMan, NASBA and virus isolation, second choice tests VecTest, Antigen capture ELISA  RT-PCR; note that antigen-based tests require 100 plaque forming units. (P39.4.w9)
  • In 2002 first choice tests were TaqMan, NASBA and virus isolation, second choice tests antigen-capture ELISA and RT-PCR.(P39.3.w15)
• Equine/other species tissues: first choice tests TaqMan, NASBA, nested RT-PCR and virus isolation, second choice tests standard RT-PCR.[2002](P39.3.w15)
• Mosquito pool: In 2003 first choice tests TaqMan, NASBA or virus isolation, second choice tests VecTest, antigen-capture ELISA or RT-PCR. (P39.4.w9)
  • In 2002 first choice tests were TaqMan, NASBA and virus isolation, second choice tests antigen-capture ELISA and RT-PCR..(P39.3.w15)
• N.B. for serological assays sera (or human CSF) tested initially by IgG-ELISA or IgM-ELISA should be confirmed by PRNT against WN virus, St. Louis encephalitis virus and dengue. (P39.3.w15)

For humans, the following advice for the most appropriate specimens to test for viral encephalitides has been provided through the Emerging Infections Encephalitis Project, funded by the Centers for Disease Control and Prevention (CDC) [2001](D72):

(D72)

Appropriate samples for attempted viral isolation are prioritized by CDC as follows (D147):

"1) Clinically ill humans - CSF (serum samples may be useful early in infection)
2) Human (biopsy or postmortem) - brain tissue
3) Horses (postmortem) - brain tissue (including brainstem), spinal cord tissue
4) Birds - kidney, brain, heart
5) Other mammals - multiple tissues, especially kidney and brain"

(D147)

[NOTE: Research published in May 2004 suggests that, when available i.e. during the moult, samples of feather pulp from wing or tail flight feathers may be suitable for virus detection as this material contains relatively high titres of virus, at least in corvids (Corvidae - Crows, Birds-of-Paradise etc. (Family)) and was found to be more sensitive for virus detection than either spleen/kidney samples or cloacal swabs. (J84.10.w2)]

Labelling:

• The CDC Epidemic/Epizootic West Nile Virus in the United States: Revised Guidelines for Surveillance, Prevention and Control (D67) emphasizes that accurate taxonomic identification of specimens, a unique identification numbering system for specimens and durable tagging of filed specimens, as well as standardized forms for data collection and specimen submission, are particularly important when dealing with wild birds and mosquitoes. (D67)
• CDC requires, at a minimum, that "Tubes, cryovials, and other specimen containers should be clearly labeled with – at minimum – the specimen’s UID, patient’s name (human), state, date of onset, date of collection, and specimen type." (D147- Appendix A)

Shipping:

The following CDC "Special Collection, Shipping and Handling Instructions" are taken from CDC Epidemic/Epizootic West Nile Virus in the United States: Guidelines for Surveillance, Prevention and Control 3rd Revision (D147 - Appendix A) [Text copied directly]

Cell Culture Techniques

• WN virus can be grown on a variety of mosquito cell lines, mammalian cell lines and in suckling mice. In  mosquito cell lines a cytopathic effect may not be seen and the culture needs to be screened by immunofluorescence [see section below] (B241.49.w49, B243.31.w1, B245.29.w29, J64.19.w1, J73.57.w1, J84.8.w7, J110.39.w1, D147)

• "Virus isolates, amplified by cell culture or in suckling mice, are identified by virus neutralisation assays using specific antisera, by enzyme immunoassay, or by the detection of WNV-specific RNA sequences. Specificity of reagents must be validated against antigenically cross-reacting flaviviruses such as SLE [St Louis encephalitis virus] (in North America) and JE [Japanese encephalitis virus] (in Asia). " (J64.19.w1)
• "WN virus can infect and produce CPE in a variety of mammalian cell lines including VERO, BHK-21, chick embryo, and PS." (B241.49.w49) 
• WNV replication may be supported in a variety of mosquito cell lines (Aedes aegypti - Yellow fever mosquito, Aedes malayensis (Aedes (Genus)), Aedes albopictus - Asian tiger mosquito, Aedes pseudoscutellaris (Aedes (Genus))). In Aedes albopictus and Aedes pseudoscutellaris a cytopathic effect (CPE) has been reported. (B241.49.w49)
• "West Nile virus grows and produces CPE or plaques in a wide variety of cell cultures (including primary chick and duck embryo), as well as in continuous lines of human, primate, swine, rodent, and amphibian origin. It multiplies in Aedes aegypti and Drosophila cells and produces CPE in Aedes albopictus cells." (B243.31.w1)
• WN virus can be isolated in mammalian and mosquito cell lines; in mosquito cell lines a cytopathic effect may not be seen and the culture needs to be screened by immunofluorescence [see section below]. (D147)
• Virus isolation is still useful, despite the advent of highly sensitive assays such as PCR. (B245.29.w29)
• Virus isolation by cell culture allows detection of any virus growing in the cells. (J93.41.w4)
  • Time is required to allow the virus to grow. (J93.41.w4)
  • Time is required for identification, usually by immunofluorescent assay (IFA)  [see section below]. (J93.41.w4)
• Recent work has shown that, for pools of mosquitoes (Diptera: Culicidae) held for up to two weeks before testing by plaque assay in Vero cells, the temperature at which the pools were held affected the results: after storage for two days, at 20°C or 4°C only 10^1.5 or 10^3.6 PFU were recovered, compared to 10^5.5 and 10^5.7 for pools held at -20°C or -70°C respectively for the same time. Thus the temperature at which samples are held does affect the recovery of infectious virus. (J110.39.w1)
• Virus has been isolated by Vero plaque assay and detected by TaqMan RT-PCR from oral swabs and cloacal swabs taken from dead birds (Corvus brachyrhynchos - American Crow, Corvus ossifragus - Fish crow and Cyanocitta cristata - Blue jay) (died or euthanised following experimental WNV infection) as well as from the samples from the brains of the same birds. WN virus concentrations from all three sample types averaged greater than 10⁵ by both assays. It was suggested that dead birds could be tested for WN virus by taking oral and cloacal swabs and sending the swabs, frozen, to an appropriate virological laboratory for testing. (J84.8.w7)
• Virus was isolated from the brain and spinal cord of 3/5 horses necropsied following fatal WNV infection in Israel in 2000. (J73.57.w1)

Complement Fixation Techniques

• The complement fixation test can be used to detect viral antibodies. Whilst it is more difficult to maintain quality control for this test compared with the haemagglutination inhibition test, it may still be used to detect complement fixing antibodies that may be present after the IgM has waned. (B244.w1, B245.29.w29)

• "The CF test measures the ability of the antiviral antibody to fix complement in the presence of virus antigen." (B245.29.w29)
• Maintaining proper quality control is more difficult for the CF test than it is for the HI test. (B245.29.w29)
• CF antibodies appear later in infection than HI antibodies but have a shorter half life (around two years); therefore this test is sometimes used in attempts to determine the time after infection that a given single serum sample was taken. (B245.29.w29)
• The use of the CF test has decreased since the development of the IgM ELISA which allows direct measurement of IgM antibody. It may still be useful for indicating a recent infection in a single serum sample taken after IgM has waned. (B245.29.w29)
• A rising antibody titre may be demonstrated by CF test. (B244.w1)
• "Complement fixing antibodies appear by early in the second week of illness, titres peak by the third week, and begin to fall after 7 months after onset."  (B244.w1)
• CF tests were used historically for serosurveys (particularly until the 1950's) and are considered more specific than most other serological tests, although serological cross-reactions with other flaviviruses may still occur. (P32.1.w1)

ELISA Techniques

• Enzyme-linked Immunosorbent Assay (ELISA) techniques are in general use to detect antibodies and frequently replace the Haemagglutination Inhibition, Complement Fixation and Neutralisation Tests in many situations. The IgM capture ELISA (MAC-ELISA) is in current use for WN virus antibody confirmation. (B244.w1, B245.29.w29, D72)
  • An ELISA has been evaluated to detect antibody in wild birds, for use as a rapid screening test (the PRNT would be required for confirmation of results). (J84.8.w9)
  • Use of certain monoclonal antibodies can increase the sensitivity of the IgM-ELISA in horses. (J485.122.w1)
• An ELISA based on a monoclonal antibody has been developed for detection of WN virus antigen in samples of mosquitoes and bird tissues. 
  • The VecTest dip stick is an antigen-capture ELISA which may be used on mosquito homogenate, avian tissue, and oral/cloacal swabs from avian carcasses. The test was shown to have a high specificity but was less sensitive than TaqMan or plaque assay.  (P39.4.w10)

• For testing CSF samples of suspected human cases: "[IgM Capture] ELISA is more sensitive than  RT-PCR for WNV testing and should be considered when there is stronger suspicion of WNV than other etiologic agents". (D72)
• "Acute and convalescent sera can be tested by IgM Capture and IgG ELISA testing, with indeterminate and positive specimens confirmed by PRNT [Plaque Reduction Neutralisation Test]". (D72)
• ELISA (Enzyme-Linked Immunosorbent Assay) techniques are replacing other serological assays such as HI, CF and NT in many situations. (B245.29.w29)
• Indirect ELISAs for IgM and IgG were found to have good sensitivity, with all sera positive by other serological tests (HI, CF and neutralization test) also being positive by the ELISA. Screening for rheumatoid factor was required prior to use of the indirect IgM ELISA in order to avoid false positive reactions. It was recognised that this test could cross-react with antibodies to other flavivirus group viruses. (J117.17.w1)
• ELISAs, also known as EISs (enzyme immunoassays) allow quantification of antibody based on a single antibody dilution. (B216.12.w12)
• Both IgM and IgG ELISAs are being used (also HI) in Canada for initial serological testing in humans with suspected WNV infection. Testing is carried out on paired serum samples taken about three weeks apart. Test results are available within 48 hours. [2003] (W181.28Jan04.WNV3)
• IgM capture ELISA (MAC-ELISA): minimises the interference by IgG with binding of IgM to antigen and is therefore more sensitive than an indirect-ELISA for IgM. This test also allows the use of antigens from a variety of sources. (B245.29.w29)
  • A  96-well ELISA plate is used, pre-coated with commercial anti-human IgM antibody.
  • Human antiviral antibody is captured in the wells of this plate.
  • Individual wells are then reacted with different viral antigens to determine the specificity of the captured IgM.
  • Antiviral antibody is then used to detect the captured virus antigen.
  (B245.29.w29)
• Equivocal results can occur with IgM capture ELISA, requiring confirmation by an alternative serological assay. (B245.29.w29)
• IgM capture ELISA has been used to diagnose WNV encephalitis, by detection of WNV IgM in the CSF.  (B244.w1)
• Only a slight response may be seen if WNV infection follows infection with another Flavivirus. (J84.8.w1)
• Data from experimental infection of 12 horses indicated that the IgM capture ELISA was a "simple and efficient" method for detecting WN virus antibody and found that it could detect antibody at about the same time that antibody could be detected with the PRNT. (J84.8.w4)
• It has been suggested that this test alone, if giving a higher reactivity with WN virus than with St Louis Encephalitis virus (SLE) [Flaviviridae- St Louis Encephalitis virus], may be sufficient to distinguish between these two flavivirus infections in the USA. (J214.267.w11)
• Testing of the MAC-ELISA using a panel of sera positive for WN virus, St Louis encephalitis and Japanese encephalitis virus antibodies showed that positive-to-negative absorbance ratios (P/Ns) for WN virus antigen positive sera were generally much higher with WN virus antigen than with SLE virus antigen, allowing correct identification of WN virus infections 92% of the time. It was suggested that the MAC-ELISA provided a faster and less expensive way of identifying the virus involved in an infection, although PRNT may still be required for final confirmation. (J219.9.w1)
• In a typical human case of WNV infection the IgM ELISA P/N for WN virus is more than two times higher than the P/N to St Louis encephalitis virus. (P39.3.w15)
• The IgM ELISA is sensitive, relatively specific based on the P/N ration to distinguish between WN virus and SLE virus but cross-reactivity does occur between flaviviruses, it is of limited utility in secondary infections, it takes two days and IgM persistence may make interpretation of positive serum problematic. (P39.4.w9)
• The MAC-ELISA reduces the occurrence of false-negative reactions, since competition between IgM and IgG is minimised, and eliminated potential background due to extraneous antibody, reducing the frequency of nonspecific reactions and removing false-positive reactions due to rheumatoid factor. (J93.38.w6)
  • Broadly arbovirus group-reactive MAb conjugates can be used in combination with virus-specific antigens to give a system allowing concurrent screening for antibody to many arboviruses within a genus in one procedure. (J93.38.w6)
  • IgM capture ELISA may not detect antibody in early (day 0-7) acute-phase serum. (J93.38.w6)
  • Interpretation of results of IgM capture ELISA is based on the time at which the sample was taken and on confirmation of results with another test. For this test an acute-phase serum sample is one taken at least eight days and up to 45 days after the onset of symptoms. (J93.38.w6)
  • It is noted that acute-phase serum samples may be IgM-positive by MAC-ELISA but PRNT negative; this may be due to lack of neutralizing ability of early IgM rather than a false-positive result. (J93.38.w6)
  • For screening, serum samples should be tested at 1:400 dilution and CSF samples should be tested undiluted. Tests should be carried out in conjunction with IgG ELISA. (J93.38.w6)
• A disadvantage of the MAC-ELISA is the requirement for sustained supply of WN virus antigen, with Biosafety Level 3 facilities needed for WN virus antigen purification. (J283.119.w1) 
• MAC-ELISA IgM and direct IgG ELISA were used to test serum and CSF samples of patients infected during the outbreak of WNV infection in Bucharest, Romania, in 1996. IgM was detected in CSF and serum earlier than was IgG and in some individuals IgM could be detected in CSF prior to its being detected in serum. IgM antibody did not cross-react significantly with three flaviviruses in other antigenic groups. (J93.38.w5)
• A sandwich ELISA has been described using recombinant WN virus envelope (E) protein. In initial tests on a small number of serum samples IgG and/or IgM antibodies to the E protein were detected in 9/10 horses (IgG in 8/10 horses, IgM in 4/10 horses) and 6/6 humans (IgG in 5/6 humans and IgM in 2/6 humans, including one without detectable IgG) with confirmed WNV infection. (J279.2.w1)
• In horses: IgM MAC-ELISA can be used for serodiagnosis of WNV Infection. With a selected cut-off point ODr 2.0 and PN = 2.0, tested on 36 WNV infected horses and 383 horses from uninfected populations, the test had a sensitivity of 91.7%, specificity 99.2%. At this cut-off, a positive test reliably indicated infection and a negative test indicted probable non-infection; a small number of horses were false-negative with this test at this cut-off. (J275.20.w1)
• Using monoclonal antibodies (mAb) to equine IgM, the sensitivity of detection of WNV-specific IgM in horses using Ig-M ELISA was increased. Sensitivity was highest when anti-IgM I-22 mAb was used to capture IgM from equine serum. All test samples from the National Veterinary Service Laboratoy were correctly identified when this mAb was used. With another mAb, M2B-63, all positive and negative sera were correctly identified, but the P/N ratios were lower for two "weak positive" samples; reducing the P/N cut-off to >5 rather than >3 would give positive results. The established assay using polyclonal anti-IgM correctly identified 8/10 samples. (J485.122.w1)
• For 16 horses vaccinated against WN virus using Recombitek (Merial) (recombinant vaccine using canarypox virus vector) within the last year but at least three months prior to testing, all were negative for IgM (nine had detectable antibodies with PRNT). (J485.122.w1)
• In horses: MAC-ELISA can be used to detect IgM in both serum and CSF of WNV infected horses. The established cut-off for serum is 1:400; levels of 1:200 to 1:64,000 were detected in one study. In CSF, the P:N index is significantly greater than in serum. A few recently-vaccinated horses were positive with the MAC-ELISA on serum. (J275.18.w1)
• In horses: A competitive ELISA using neutralizing monoclonal antibody 5E8 (NT-ELISA) was developed and analysis of 246 WNV-negative horse sera was used to develop a cut-off percentage inhibition (mean PI plus three standard deviations). The NT-ELISA was positive in all 11 tested mice with experimental infection. In horses, it was positive in 173/180 (96.1%) of PRNT-positive horse sera and 1/32 (3.1%) of PRNT-negative sera; there was an overall agreement of 0.86 between the two tests. It was suggested that this test could be used for large-scale testing of sera for WNV-neutralising antibodies in multiple species. (J219.14.w1)
• In humans: Using a WN Virus preM/E recombinant antigen, an IgG ELISA had a sensitivity of 97.6% and specificity (not including sera positive for other flaviviridae) of 92.1%; it detected antibodies in 35% of sera from patients infected with or vaccinated against other flaviviridae. The IgM ELISA had sensitivity and specificity of 99.3% not including sera positive for other flaviviridae) and was positive for 12% of patients positive for other flaviviridae (31% for St Louis encephalitis patients). (J93.42.w1)
• In chickens: A study of different serological tests for the detection of antibodies in serum from chickens found that a micro-PRNT was more sensitive than IgM-capture ELISA or IgG indirect ELISA, although the ELISAs were more sensitive than HI test. Based on the findings of the study a combination of IgM and IgG ELISAs were recommended for screening of chickens, or of other species for which commercial conjugated secondary antibodies are available for use in these tests, as they are more sensitive than the HI and simpler and faster than either standard or microtitre PRNT.(J240.67.w1)
  • It was recommended that micro-PRNT should be used to confirm the specificity of serum found positive with IgM-capture ELISA or IgG indirect ELISA. (J240.67.w1)
• In birds: An IgM ELISA has been developed for use in detecting WN virus antibodies in chickens. The ELISA detected infection in experimentally-infected chickens as early as three to seven days postinfection, with peak P/N values usually between 10-13 dpi and then decreasing; IgM was detected for a variable time (19 to more than 61 days postinfection) indicating a need to bleed sentinel flocks at least once every two weeks if optimal results are to be obtained using the IgM ELISA for screening. Compared to HI and PRNT the ELISA detected WN virus antibody earlier and was considered to be the method of choice if early detection of WN virus antibodies in chickens is required. The test was also shown to detect IgM in serum samples from naturally infected chickens and in a blind-coded panel results were consistent except for one borderline result. The test was effective for detection of experimentally infected house sparrows (Passer domesticus - House sparrow) (serum samples collected at 13 dpi) but not for rock doves (Columba livia - Rock pigeon) (serum samples collected at 14 dpi). The test detected IgM in serum samples from experimentally infected Phasianus colchicus - Common pheasant, Corvus brachyrhynchos - American Crow, Quiscalus quiscula - Common grackle and Agelaius phoeniceus - Red-winged blackbird. When tested on 29 serum samples from naturally-infected birds, collected during September 1999 in New York,  IgM antibodies were detected in serum samples from two geese (one domestic goose (Anser - (Genus) spp.) and one Branta canadensis - Canada goose). Preliminary testing suggested that it may also be possible to develop a combined SLE/WN IgM-capture ELISA. (J93.41.w2)
• In birds: epitope-blocking ELISAs utilising various monoclonal antibodies (MAbs) have been developed and evaluated for detection of WN virus infection in multiple bird species. The assays using MAbs 2B2 and 3.1112G detected antibodies in all WN virus-infected birds; those using MAb 2B2 were also positive for most birds with St Louis encephalitis virus (SELV) infection, however binding of MAb 3.1112G was not blocked by anti-SELV serum samples, suggesting that this assay may be useful for differentiating between the two infections in birds in the North America. This assay is already used routinely in Australia to distinguish between Kunjin virus [now recognised as a subtype of WN virus - see: West Nile Virus - Viral Type Diversity (Viral Reports)] and other flaviviruses found in that region. Screening of more than 2,500 serum samples from various avian species in the USA has indicated that this assay specifically identifies WNV infections. Other MAbs either did not detect antibodies in some species of birds and/or did not distinguish between WN virus and SELV infections. (J93.41.w1)
• In domestic mammals: epitope-blocking ELISAs utilising various monoclonal antibodies (MAbs) have been developed and evaluated for detection of WN virus infection in domestic mammal species. The assays using MAbs 3.1112G and 6B6C-1 detected antibodies in all experimentally infected horses at 14 dpi or longer after infection; these assays were positive for all sera which were positive for antibodies by PRNT 90% neutralisation.  For experimentally infected cats, sera which were positive by PRNT at the 90% neutralisation level were also positive with blocking ELISA using MAb 3H6 or 6B6C-1; these ELISAs were also positive for two individuals which were PRNT₉₀ negative but gave low PRNT titres as an 80% plaque reduction level.  In the pigs, all four were positive with assays using MAbs 3.1112G and 6B6C-1 on serum samples at 28 dpi; lower numbers were detected as positive with other MAbs. The MAbs 3.1112G and 2B2, tested against sera from cows experimentally infected with St. Louis encephalitis virus (SELV) were negative while antibodies were detected by the flavivirus group-reactive MAbs 3H6 and 6B6C-1 and in three cows with MAb 6B5A-2 (SELV-specific). It was considered that the assays were reliable in detecting flavivirus antibodies in the various species and that they could provide a faster, less expensive alternative to PRNT for detection of infection with WN virus. (J93.41.w3)
• Indirect IgG ELISA: uses plates in which wells are coated with broadly cross-reactive murine monoclonal antibodies for each of the virus families.
  • The appropriate viral antigen is then added and captured.
  • Serum samples are then added to the wells.
  • Binding of antiviral antibody is detected using an anti-species antibody conjugated to enzyme.
  • (B245.29.w29)
• An Indirect IgG ELISA has been developed for the assay of antibodies to WN virus in birds. The ELISA, which used commercially available anti-wild bird horseradish peroxidase-conjugated antibodies generated from the sera of four species of bird from the orders Passeriformes - Wrens, Crows, Tits & Sparrows (Order), Columbiformes - Pigeons & Doves (Order), Galliformes - Pheasants, Quails & Turkeys (Order) and Anseriformes - Ducks, Geese & Swans (Order). It was shown able to detect anti-WN virus antibodies in 23 species of birds. All ELISA negative birds were also negative by PRNT, giving a predictive value of a negative test (PVN) 0f 100%. The predictive value of a positive test (PVP) was calculated as 70%. It was considered some ELISA-positive but PRNT-negative results might have been due to technical errors when carrying out the ELISA. The ELISA was considered to be a useful screening test, with positive results being checked by PRNT. (J84.8.w9)
• Capture ELISA based on monoclonal antibodies have been developed for detection of IgG to Alphavirus, Flavivirus and Bunyavirus arboviruses. The endpoint were shown to correlate with results of PRNT. A serum dilution of 1:400 was chosen as the screening dilution. For flaviviruses, 67/70 results (96%) showed agreement between the P/N value and endpoint titration, with the remaining thee samples being positive by screening P/N ration but negative by endpoint titration; thus endpoint titrations eliminated the occasional false-positive results. (J93.38.w7)
  • The tests should be used in tandem with the corresponding MAC-ELISAs for a complete serum antibody profile. (J93.38.w7)
  • The test allows rapid results and validation. (J93.38.w7)
  • It was noted that careful maintenance of the test schedule, particularly conjugate and substrate incubation times, were critical for consistency of results. Additionally, while capture MAb-coated microtitre plates could be stored for up to nine days if stored wet, covered, at 4°C, antigens, for optimum performance of the tests conjugates and substrates required preparation immediately prior to use. (J93.38.w7)
• The IgM capture ELISA is considered to be the test of choice for detection of recent exposure in the horse with IgM responses rising rapidly and declining usually by 30 days after the onset of clinical signs; in one study IgM responses lasted a variable time from about one to eight weeks. (P51.49.w2)
• Horses do not appear to develop an IgM response following vaccination with the USDA-licenced killed vaccine. (P51.49.w2)

Antigen detection:

• A monoclonal antibody-based antigen capture enzyme immunoassay (antigen-capture ELISA), with a confirmatory inhibition ELISA,  has been developed which can be used to detect WN virus antigen in mosquitoes and in avian tissues. The assay, using monoclonal antibody 3.91D, was found to be highly specific and was shown to be less sensitive than the TaqMan PCR assay but slightly more sensitive than that of a traditional RT-PCR assay. It was suggested that the test may be a useful, relatively easy to use and relatively inexpensive assay for detection of WN virus in mosquitoes and in avian tissues. (J93.40.w1)
• The VecTest^TM dip stick test is an antigen-capture ELISA which may be used on mosquito homogenate, avian tissue, and oral or cloacal swabs from avian carcasses. In crows and blue jays this test used on oral or cloacal swabs was found to give 100% agreement with brain tissue assay. The test was shown to have a very high specificity (no false positives) but was less sensitive than TaqMan or plaque assay. The WN virus VecTest assay required 3.7 log₁₀ PFU/ml compared to thresholds of 0.1  log₁₀ PFU/ml for TaqMan and 0.6-1.0  log₁₀ PFU/ml for plaque assay. (P39.4.w10)
• The VecTest dip-stick test is an immunochromatographic lateral flow wicking assay for detection of WN virus antigen and/or St Louis encephalitic virus (SLE virus) antigen, and may be used for differentiation between these. This test has the advantages that it produces results in less than 20 minutes, the kit can be stored at ambient temperatures, no specialised equipment or reagents are required and no specialised training is required to carry out the test. Evaluation of the individual WN virus and the combined WN/SLE assay found high specificity without cross reaction with the other antigen even at high concentration of the heterologous antigen. The sensitivity was found to be similar to that for ELISA tests for the viral antigens (WN virus detected at titres equal to or greater than 3.8 log₁₀ PFU/ml) and the assay detected the presence of WN virus in a pool of 50 mosquitoes in which a single WN virus-positive mosquito was present. Sensitivity was lower than that of RT-PCR or Vero cell plaque assay. There were no signs of cross-reaction nor false-positives from virus-negative mosquito pools. When tested against a small number of field-collected pools the test detected three of three pools which were positive by TaqMan RT-PCR and was negative for both negative pools. It was considered that the test could be useful for rapid assessment of medical threat and effective targeting of vector control measures but that test results should be verified with another test (e.g. RT-PCR) until more extensive valuations had been carried out. (J110.40.w1)
• Comparison of the VecTest™ with Vero cell plaque assay and TaqMan^® assay showed that the VecTest assay was less sensitive but highly specific. In 801 mosquito pools tested, TaqMan RT-PCR detected 34 WN virus-positive pools, plaque assay detected 25/24 (74%) of these pools and VecTest detected 18/34 (60%) of the TaqMan-positive pools (16/25 i.e. 67% of the plaque-assay positive pools and 2/9 pools that were TaqMan positive but plaque assay negative). All three assays indicated a high infection rate in the mosquito population. (J218.18.w3)
  • VecTest  is relatively inexpensive, does not require either expensive equipment or highly trained personnel and can test for WN virus and SLE virus on the same strip. As with TaqMan, but unlike plaque assay, it does not require live virus and can detect viral RNA in samples which have not been maintained in a cold chain. However it is less sensitive than TaqMan or plaque assay for detection of WN virus antigen in mosquito pools; this reduced sensitivity and increased risk that infection will not be detected early in the mosquito season may be overcome by testing larger numbers of specimens. (J218.18.w3)
• VecTest^TM was evaluated for use with field samples from dead crows. Samples of saliva, faeces, oral/cloacal tissue and in some cases associated maggots were used, from crows dead for an estimated time of less than 36 to less than 72 hours. Of 20 samples tested 19 showed a positive reaction for WNV. Five of the samples were cross-checked by IHC of internal tissues (brain, heart, kidney and spleen); all were positive by IHC. The remaining 15 samples were cross-checked against TaqMan RT-PCR. Three samples which were positive in the VecTest, as well as the one VecTest negative sample, were TaqMan negative. Taking the RT-PCR as the standard this gave a false-positive rate of 75% for the VecTest.  It was considered that further investigations were required, that the test should be considered experimental for use with samples from dead crows at this time, that all positives should be confirmed using another test but that the test may be useful "in early season screening, when rates of positives are typically low, or in nonpeak areas." It was noted that the VecTest detects the viral capsid while the RT-PCR detects WN virus genomic sequences. Two possible reasons for the discrepancy between tests were suggested, first that the VecTest may be detecting WNV capsid which may persist longer than viral RNA (due to abundant environmental RNAase) or second that the VecTest conjugate might react with a non-specific protein in the samples from the crows to give the false positives. (J84.9.w18)
• VecTest was evaluated for use with oropharyngeal or cloacal swabs from dead corvids without obvious signs of decay or decomposition. The TaqMan real-time RT-PCR was used on swabs or tissue samples to confirm the test results. Data from 222 Corvus brachyrhynchos - American Crow collected in Ontario in 2002 gave a sensitivity of 83.3% and specificity of 95.8% for oropharyngeal swabs; data from Manitoba gave a sensitivity of 83.9% and specificity of 93.6% for oropharyngeal swabs from 108 Corvus brachyrhynchos - American Crow . Data from birds (Corvus brachyrhynchos - American Crow and Cyanocitta cristata - Blue jay) collected in Ontario in 2001 and frozen for 10 months prior to testing suggested that sensitivity of cloacal swabs was lower than that of oropharyngeal swabs (58.3% versus 92.8%  for 33 birds and 40% versus 60% for 28 birds, respectively). Testing of other species in Manitoba gave lower sensitivities: for Cyanocitta cristata - Blue jay oropharyngeal swabs 71.4% sensitivity and 100% specificity (30 birds), cloacal swabs 57.1% sensitivity and 100% specificity (31 birds). It was considered that the test, used on swabs of oropharyngeal secretions, may be considered useful for surveillance particularly of dead Corvus brachyrhynchos - American Crow, particularly because the test procedure is simple without requiring expensive and technically demanding instruments  and provides rapid results (15 minutes). However the lower sensitivity might result in loss of information on early season WNV activity and the test may be less useful for other species, particularly non-corvid species, possibly related to species differences in virus titres in excretions./secretions at the time of death. (J84.9.w21)

Microsphere Immunoassay Techniques

• A fluorescent microsphere immunoassay incorporating recombinant WN Virus envelope protein (rE MIA) has been developed and may be useful for screening equine sera for WN Virus antibodies.

• A fluorescent microsphere immunoassay was developed incorporating either recombinant WN Virus envelope protein (rE MIA) or recombinant nonstructural proteins. The rE MIA, compared with PRNT on equine sera either recently vaccinated or naturally infected, had a diagnostic sensitivity of 99.3% and specificity of 97.4%. The MIA using non-structural proteins of WN Virus had much lower sensitivity, but one (rNS1 MIA) was able to distinguish horses vaccinated with the recombinant canarypox WNV vaccine (Recombitek, Merial Limited) from those either naturally infected or vaccinated with the inactivated vaccine (Innovator, Fort Dodge Laboratories). The re MIA "would appear to provide a rapid, convenient, inexpensive, and accurate test for the screening of equine sera for the presence of antibodies to WNV." (J212.18.w1)

PCR Techniques

• A variety of Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR) protocols, both standard and nested, have been described for detection of WN virus RNA.
• PCR protocols can be used on a variety of sample types, including clinical samples such as CSF, post mortem tissue samples, and mosquito pools. 
• PCR assays are highly sensitive but are susceptible to false positive results due to inadvertent contamination.
• The TaqMan RT-PCR is a highly sensitive test for detection of virus RNA.

(J84.7.w25, J110.39.w1, J217.94.w1, B245.29.w29, D72)

• Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR) tests are currently in use for viral testing of CSF and brain tissue. (D72)
• RT-PCR in particular and nucleic acid-based detection techniques in general have advantages of speed, specificity and sensitivity for detection of WN virus in samples. (J93.39.w1)
• For testing CSF samples of suspected human cases: "RT-PCR is less sensitive [than IgM Capture ELISA] for WNV, but provides testing for 13 different viruses. This test should be considered if suspicion of the etiologic agent is stronger for viruses other than WNV." (D72)
• PCR assays are extremely sensitive. However this sensitivity may lead to false positive reactions due to inadvertent introduction of nucleic acid. Therefore "a diagnosis should not be based solely on a positive PCR result but should be confirmed by a diagnostic serological assay." (B245.29.w29)
• The disadvantages of RT-PCR, whether standard or real-time, are the potentials for cross-contamination, occurrence of equivocal results requiring further confirmation and the fact that only the specific viral sequence determined by the primers will be detected; this last limitation has been reduced by the development of multiplex assays which can increase the number of viruses detectable in a single assay. (J93.41.w4)
• In addition to a traditional single stage RT-PCR which is suitable for detecting WN virus in avian tissue and in cell cultures but frequently may not be sufficiently sensitive to detect WN virus in equine brain, a RT-nested procedure (RT-nPCR) has been described which identifies directly in equine and bird tissues the strain of WN virus circulating in the USA. Tested on equine brain the RT-nPCR test detected WN virus in tissues from 13 WNV infection-positive horses and in none of 60 samples from horses not diagnosed as being infected with WN virus. The test also detected virus in plasma from two ponies following experimental infection with WN virus. The test also detected WN virus in the brains, and in some cases other tissues, of seven of 31 birds (the seven birds were also WN virus positive by virus isolation). The test was considerably more sensitive than the traditional single stage RT-PCR and much more sensitive than virus detection by cell culture. (J84.7.w25)
• A real-time 5'-nuclease RT-PCR has been developed, quantitative over a wide dynamic range and , because it is performed within a closed system, less sensitive than nested PCR to false-positive results. The real time RT-PCR had a detection sensitivity of 50-100 molecules (determined using synthetic RNA transcripts) and had a linear dynamic range of more than five orders of magnitude. Testing with CSF samples showed that the test did not detect WN virus in any of eight controls (persons with neurological disease not due to WNV infection) but detected WN virus sequences in 6/6 samples from patients with fatal illness in whom WNV infection had been confirmed serologically and in 1/4 samples from individuals with non-fatal WNV infection. (J115.13.w5)
• Recent work has shown that, for pools of mosquitoes (Diptera: Culicidae) held for up to two weeks before testing by RT-PCR, it did not matter whether the mosquitoes were stored at 20°C, 4°C, -20°C or -70°C. (J110.39.w1)
• A RT-PCR protocol has been described with a simple preparation method, involving boiling a mosquito homogenate at 99C for five minutes prior to adding the RT reaction mixture (removing the need for detergent and dilution in preparing the mosquito homogenate). The protocol was considered to be "highly sensitive" for various arbovirus and was shown able to detect one WN virus-infected mosquito in a pool of 100 mosquitoes and has been shown to be useful and effective in surveillance against arboviruses. (J217.94.w1)
• A rapid TaqMan RT-PCR assay has been developed, designed on the basis of the NY99 WN virus strain, which detects WN virus RNA in a variety of human clinical specimens (serum CSF and brain tissue) and in field-collected samples of mosquitoes and avian tissues. The TaqMan probes were labelled with FAM reporter dye at the 5' end and TAMRA quencher dye at the 3' end. The assay was highly specific for WN virus, not generating any fluorescent signal with any of the other arthropod-borne flaviviruses or other North American arboviruses tested. The assay was highly sensitive, 10-fold more sensitive than a traditional RT-PCR, detecting less than 1.0 PFU of virus, and was shown to detect WN virus in 100% of six WN virus-positive autopsy brain specimens, 16/28 CSF and 4/28 serum samples from patients with serologically-confirmed WN virus infection, 100% of culture-positive mosquito pools (detection of 1-10 PFU) and 98% of culture-positive avian tissue samples. (J93.38.w4)
• A variety of RT-PCR and RT-nested PCR protocols have been used for WN virus RNA detection. (J278.3.w1)
• The TaqMan (5' exonuclease real-time) RT-PCR combines the amplification and detection steps. In the opinion of the authors this currently (2003) represents the best throughput protocol for WN virus RNA detection. The test utilises a pair of RT-PCR primers and a TaqMan probe, which hybridises to the target sequence between the probes. The probe contains a reporter dye at its 5' end and a quencher dye at its 3' end; during the reaction the probe is cleaved, separating the reported and quencher dyes and resulting in increased fluorescence of the reporter dye. Accumulation of the PCR products is detected by monitoring the increase in fluorescence of the reporter dye.  (J278.3.w1)
  • Different primer/probe sets can be used, targeting different regions of viral DNA; different sets have different sensitivities.  (J278.3.w1)
  • The sensitivity of TaqMan assay is about 40-100 copies. (J278.3.w1)
  • This assay has been shown able to detect WN virus DNA from a variety of specimen types including from human clinical specimens which are negative by cell culture.  (J278.3.w1)
• A high-throughput real-time RT-PCR has been described, using a robotic system to set up samples allowing analysis of 96 samples in approximately 4.5 hours. The protocol had high sensitivity (estimated detection limit of approximately 40 copies of RNA and 0.08 PFU), with consistent RNA recovery and no cross-well contamination was detected in the 96-well plate during the robotic extraction. A standard RT-PCR assay has been optimized to give a similar sensitivity and was considered suitable for testing of small numbers of samples or for confirmation of previous test results. Using a nested RT-PCR protocol sensitivity was increased approximately 10-fold compared to the standard PCR, detecting WN virus at as low as 0.008 PFU per sample. (J93.39.w1)
• For the TaqMan RT-PCR the kidney appears to be the optimal tissue in corvids and processing of kidney is easier than processing of heart. (J5.47.w2)
• The TaqMan RT-PCR is the basis of one of the two nucleic acid-amplification tests being used to screen donated blood for evidence of viraemia. (N7.52.w1)
• A multiplex real-time RT-PCR, combining two sets of primers in one reaction, significantly saves time and money (in running the RT-PCR with one primer set then confirming by repeating the test with a different primer set) but gives a factor of 10 decrease in sensitivity, with the limit of detectability changing from 0.08 PFU (about 50 copy numbers of virus) to 0.8 PFU (about 500 copy numbers of virus). (J93.41.w4)
• In live equines, generally, detection of WNV RNA in blood by PCR is less likely than detection of IgM in serum, but on occasion (three of 140 clinically affected horses in one study), viral RNA may be detected in blood (using RT-nPCR) while no IgM is detected. (J212.16.w1)
• In dead equines, WN virus RNA is generally detectable in the CNS (particularly brainstem and medulla) by PCR (46 of 64 horses i.e. 71.9% in one study). Sensitivity was increased only slightly by nested RT-PCR rather than a single round of amplification. Quantitative (TaqMan) assay was found to have similar sensitivity and specificity to the nested RT-PCR. (J212.16.w1)

NASBA Techniques

• A Nucleic Acid Sequence-Based Amplification ( NASBA) assay has been developed with very high sensitivity and specificity, and allowing a rapid test result. It may be useful to complement virus isolation and TaqMan PCR testing or even as an alternative to TaqMan. (J93.40.w3)

• A nucleic acid sequence-based amplification (NASBA) assay for WN virus has been developed recently. Two forms of the assay were used, one using electrochemical luminescence (NASBA-ECL) and the other using a molecular beacon probe (NASBA-beacon assay) to detect the amplified RNA product. The assays particularly the NASBA-ECL version, were shown to have extremely high sensitivity and specificity. The NASBA-ECL assay detected 0.01 PFU of WN virus (compared to the NASBA-beacon assay, and the established TaqMan assay, which both detected 0.1 PFU of WN virus). Using the NASBA-beacon assay and real-time detection a result was available within less than one hour (detection sometimes by 14 minutes after the addition of enzyme and usually by 45 minutes). (J93.40.w3)
• NASBA is "a homogeneous isothermal process for amplification of specific RNA sequences through in vitro transcription." The technique uses three enzymes (reverse transcriptase, RNase and T7 RNA polymerase) and the final amplification product produced is a single-stranded RNA with polarity opposite to that of the target RNA. This product can be analysed on a standard agarose gel using ethidium bromide staining. Increased sensitivity and throughput may be achieved by two methods. In the NASBA-ECL assay the product is quantified using a target-specific capture probe bound to magnetic particles, in conjunction with a ruthenium-labeled detector probe and an instrument to measure electrochemiluminescence. In the NASBA-beacon assay molecular beacon probes are included in the amplification reaction. (J278.3.w1)
  • Clear cut positive and negative signals result from this method, particularly using the NASBA-ECL.  (J278.3.w1)
  • These assays are highly specific.  (J278.3.w1)

IPMA Techniques

• An immunoperoxidase monolayer assay (IPMA) has been described for testing human sera against several arboviruses, including WN virus, using a reaction readable visually; this may be of use for screening human sera in field situations. (J217.65.w1)

• An immunoperoxidase monolayer assay (IPMA) has been developed allowing detection of antibodies to six arboviruses, including WN virus, two other flaviviruses (dengue 2 and yellow fever viruses) and three phleboviruses (Rift Valley fever, sandfly fever Naples and sandfly fever Sicilian viruses) in human sera, the reaction being readable visually without any instrumentation required. The assay was shown to have 100% specificity while the sensitivity was considered "acceptable" (91% for WN virus, 96% for Rift Valley fever virus compared to an ELISA). It was suggested that the test may be useful for testing human sera against arboviruses particularly in the field). (J217.65.w1)

Indirect Haemagglutination Techniques

• The Indirect Haemagglutination test was developed for rapid detection of WN virus antigen in a viraemic host, although the sensitivity is probably lower than virus isolation techniques. (J88.22.w1, B241.49.w49)

• Glutaraldehyde-fixed, tanned erythrocytes were sensitized with gamma globulin from WN virus immune ascitic fluid. Blood from birds were tested and haemagglutination resulted with the blood from one bird, demonstrating the presence of WN virus in that bird. (J88.22.w1) The test was considered a rapid method for detection of WN virus in a viraemic host (J88.22.w1), "but the sensitivity is probably lower [compared to virus isolation techniques]."  (B241.49.w49)

Haemagglutination Inhibition (HI) Techniques

• Haemagglutination Inhibition (HI) is a serological test for WN virus antibody and, although it is still in use in many laboratories, there is an increasing tendency with time for sera to cross-react with other virus antigens within a given virus family; this test is gradually being replaced by ELISA techniques. (B245.29.w29) 
  • HI may still be used for screening as it provides results within 24-48 hours. (J91.32.w1, V.w42, W181.28Jan04.WNV3)
  • This test has the advantage for surveillance of domestic and wild animals that a species-specific conjugate is not required. (P32.1.w1)
  • It has been noted that for flaviviruses HI tests on chicken sera are valid following an acetone extraction method. (J93.41.w2)

• "The HI test utilizes the ability of antiviral antibody to block the capability of the virus to agglutinate erythrocytes." (B245.29.w29) 
  • Serial (usually two-fold dilutions) of serum are placed in the wells of a microtitre plate and a constant amount of virus (four to eight haemagglutinating units, generally) is added. The HI titre of the serum is taken as the highest dilution which inhibits the ability of the virus to haemagglutinate red blood cells.  (B216.12.w12)
• Requires the preparation of haemagglutination buffers, routine availability of gander erythrocytes and continual test standardization. There is an increasing tendency with time for sera to cross-react with other virus antigens within a given virus family, so that interpretation of a titre rise between acute-phase and convalescent-phase samples, even a four-fold rise, may be difficult to interpret. (B245.29.w29)
• HI assays are considered sensitive, specific, simple, reliable and inexpensive for detection and quantification of antibodies in animal serum samples. (B216.12.w12)
• The HI was used extensively from the 1950's onward for four decades. (P32.1.w1)
• A major advantage of the HI is that it does not require a species-specific conjugate. (P32.1.w1)
• This test is not excessively demanding in terms of skill required, resources or time. (P32.1.w1)
• This test is being replaced by ELISA, although it is still used in many laboratories. (B245.29.w29)
• A rising antibody titre may be demonstrated by HI test. (B242.w1)
• The haemagglutination inhibition test may be used for screening giving test results available within 24-48 hours (J91.32.w1, V.w42)
• A study of different serological tests for the detection of antibodies in serum from chickens found that the HI test was less sensitive than any of the other assays (IgM-capture ELISA, IgG indirect ELISA, PRNT and microtitre PRNT) tested. (J240.67.w1)
• It has been noted that for flaviviruses HI tests on chicken sera are valid following an acetone extraction method. (J93.41.w2)
• HI test is one of two types of serological tests being used in Canada for initial testing of humans with suspected WNV infection (the other being ELISA). Testing is carried out on paired serum samples taken about three weeks apart. Results are available within 48 hours. [2003](W181.28Jan04.WNV3)

Plaque Reduction Neutralization Techniques (PRNT)

• The Plaque Reduction Neutralization Test (PRNT) is a serological test for WN virus antibody and, although expensive and problematic, is useful to differentiate between infection of two viruses which are closely related antigenically. (B245.29.w29)
  • The PRNT is the confirmatory test for identification of WN virus specific antibody.

• Neutralization assays are generally the "gold standard" for detection and quantification of antibodies against a virus and neutralizing antibodies are considered to correlate directly with in vivo protective antibody. (B216.12.w12)
• PRNT, with a four-fold  or greater increase in WN virus-specific antibody titre between acute and convalescent serum samples, is required to unambiguously identify current WNV infection  in areas with year-to-year WN virus transmission. (J214.267.w11)
• "[PRNT] is by far the most expensive and problematic test [to diagnose arboviral infection] to perform, but it is still the only serological assay able to differentiate between infection by two closely antigenically related viruses." (B245.29.w29)
  • PRNT involves mixing a constant amount of virus with dilutions of the serum being tested, followed by plating of the mixture onto cells of an appropriate cell line for the individual virus. The vital dye neutral red is then added for visualization of the plaques and the number of plaques in an individual plate is divided by the original number of virions to calculate the percentage neutralization. Interpretation is typically based on 70% neutralization: the last dilution of serum capable of inhibiting 70% of the total plaques (virions). (B245.29.w29)
  • Different arboviruses require different cell lines and may form plaques of different size and morphology (depending on the cell type used); the time taken for plaque formation also varies between viruses and may be seven to ten days for flaviviruses. (B245.29.w29)
• Neutralizing antibodies appear slightly later than complement fixing antibodies but they remain at high titre for many years after infection. (B244.w1)
• Disadvantages of PRNT include the use of live virus in the test (therefore the requirement for biosafety level 3 containment conditions), the time required for completion of the test (six days for WN virus, 10 days for St Louis encephalitis virus) and the cost of the test (relatively expensive). (J219.9.w1)
• In a typical human case of WNV infection the PRNT for WN virus is four-fold higher than the PRNT to St Louis encephalitis virus (SLE) or dengue (DEN) and there is a four-fold rise in titre to WN virus between the acute and convalescent sera. (P39.3.w15)
• A study of different serological tests for the detection of antibodies in serum from chickens found that a micro-PRNT was most sensitive (more sensitive than HI, IgM-capture ELISA, IgG indirect ELISA and standard PRNT) and, unlike the standard PRNT tested, apparently was able to detect IgM antibodies on day seven after experimental infection. However it was noted that both the standard and micro PRNT assays are more complicated and slower to perform than are the ELISAs. (J240.67.w1)
  • The micro-PRNT may be the test of choice for assay of serum from species for which conjugated secondary antibodies required by ELISAs are not available. (J240.67.w1)
  • Micro-PRNT was recommended for confirming the specificity of serum found positive with IgM-capture ELISA or IgG indirect ELISA. (J240.67.w1)
• PRNT is more specific than IgG ELISA. It measures virus neutralising antibodies. (J89.22.w1)

Indirect Immunofluorescent Antibody Techniques (Indirect IFA)

• The Indirect Immunofluorescent Antibody (Indirect IFA) is a serological test for WN virus antibody. It uses an indirect format, therefore there is a problem with IgG competition for IgM binding. It is not a preferred test for serological diagnosis. (B245.29.w29) However it may be useful in laboratories without the time, funds or technical expertise for utilising ELISA. (J133.951.w43)
• An indirect IFA using IgM MAb specific for WN virus envelope protein followed by goat anti-mouse IgM fluorescein conjugate has been used for detection of virus in cell culture and tisues. It was found by the Arbovirus Laboratory, Wadsworth center, NYSDH, that the indirect IFA was a valuable confirmatory assay for use on bird tissues but that non-specific binding made it problematic for use on mammalian tissues. (J93.41.w4)

• Not a preferred test for serological diagnosis. (B245.29.w29)
• Uses endpoint titration. An indirect format, therefore there is the problem of IgG competition for IgM binding in the IgM IFA. (B245.29.w29)
• Rapid and may be useful for detection of antibodies which are difficult to demonstrate by other tests, but interpretation of results is frequently confused by the presence of nonspecific fluorescence. (B216.12.w12)
  • Virus substrate is usually infected cells fixed on glass microscope slides. The test serum is added and incubated, then the slide washed to remove unbound antibody, after which antiglobulin batched to the species from which the serum sample has been taken, and conjugated to fluorescein isothiocyanate is added and positive reactions are seen as apple-green fluorescence under a UV light.  (B216.12.w12)
• Results of tests using a commercially-available immunofluorescent assay (ARBOVIRUS IFA IgG^®, MRL Diagnostics, Cypress, California, USA), which uses Vero cells infected with a range of arboviruses including a strain of St. Louis Encephalitis (SLE) virus [Flaviviridae- St Louis Encephalitis virus], indicate that this assay may be useful for laboratories "in which cost, time and technical expertise may be prohibitory when considering incorporating an ELISA for arbovirus detection." The assay was  reasonable effective at detecting WN virus antibodies, although less sensitive for IgM detection than was a MAC-ELISA. (J133.951.w43)
• Testing of an IFA using commercially-available slides coated with WNV-infected Vero cells (PANBIO, Columbia, MD, USA) for detection of IgG and IgM found that the IgM was only slightly less sensitive than MAC-ELISA, with agreement (with MAC-ELISA) of 98%, clinical sensitivity of 96% and clinical specificity of 100%. The IgG IFA showed agreement (with  IgG ELISA) of 92%, clinical sensitivity of 100% and clinical specificity of 90%. The IgG IFA showed extensive cross-reaction with other flaviviruses (SLE and Dengue) while the IgM showed only minimal cross-reaction. It was noted that the IFA did not require in-house preparation of WNV antigen. It was suggested that the IFA was a cost-effective and reasonable alternative to IgG ELISA and MAC-ELISA for initial serological diagnosis of WNV infection. (J283.119.w1)
  • It was noted that attention is required to not only the presence but also the pattern of fluorescence for accurate interpretation, with care taken to compare equivocal slides with positive controls to confirm a similar pattern of fluorescence. (J283.119.w1)
• An indirect IFA has been used for identification of virus infected cells in culture and for detection of infected tissues. (J93.41.w4)
  • The indirect IFA used on both cell culture and tissues utilised an IgM MAb (H5.46) which is specific for WN virus envelope protein followed by staining with goat anti-mouse IgM fluorescein conjugate. IgG mAbs specific for WNV followed by goat anti-mouse IgG fluorescein conjugate were also used on tissues.(J93.41.w4)
  • The indirect IFA was found to be a very valuable confirmatory test for use on avian tissues. (J93.41.w4)
    • This assay is very sensitive and is rapid, however preparation of tissue sections may be too time consuming and potentially hazardous for it to be used for screening of large numbers of tissues. Care is required in handling tissues and results must be read individually. (J93.41.w4)
  • The indirect IFA was found to be problematic for use with mammalian tissues because of non-specific binding of the IgM MAb and also, with tissues of some mammal species, the second antibody. (J93.41.w4)

Direct Immunofluorescence (Immunohistochemistry) Techniques

• Direct Immunofluorescence techniques are used for detecting WN virus antigen in cells, impression smears and cell culture. (J26.37.w1, J71.75.w1, J73.57.w1, J84.7.w29)

• Virus specific antigen in cells may be detected by direct immunofluorescence on impression smears of organs. (J71.75.w1)
• Virus in cell culture may be identified using indirect fluorescent antibody test. (J84.7.w29) 
• Immunohistochemistry was used for detection of WN virus in prepared tissue sections and on tissue impression smears in the investigation of the WNV infection outbreak at Wildlife Conservation Society collections in New York City in 1999. (J26.37.w1)
• IHC was efficient and reliable for identification of infection from fixed tissues in birds; it was noted that polyclonal antibody cross-reacted with other flaviviruses including St Louis encephalitis virus.(P32.1.w15)
• Immunohistochemistry detected WN viral antigens associated with foci of inflammation in the spinal cords of two of five horses with fatal WN virus infection in Israel in 2000. (J73.57.w1)
• Monoclonal antibodies are now available for use in IHC which have high specificity for WN virus. However IHC is still not 100% sensitive. It is noted that some seronegative individuals, particularly immunosuppressed and diabetic patients, are IHC positive, while some seropositive individuals are IHC-negative. (P48.4.w11)
• In owls (Strigiformes - Owls, Frogmouths & Nightjars (Order)) heart and kidney appear to be the optimal tissues for immunohistochemical staining. (J5.47.w2)