West Nile Virus – Serology and PCR

Consistent with O. Reg. 671/92 of the French Language Services Act, laboratory testing information on this page is only available in English because it is scientific or technical in nature and is for use only by qualified health care providers and not by members of the public.

This page provides testing information for West Nile Virus (WNV) at Public Health Ontario (PHO). The WNV is a single-stranded RNA virus that is a member of the Flaviviridae family and can cause neuroinvasive disease in some individuals. The primary route of transmission for this virus is through the bite of infected mosquitos in endemic areas. Testing for WNV infection can be performed by serology or PCR depending on the specific clinical scenario.

WNV avidity test removed.  Test is not available both in PHO and NML.

Testing Indications

Testing for WNV infection is indicated when a patient displays clinically compatible signs/symptoms of infection and has a compatible exposure history (e.g. noted mosquito bites, travel to endemic area, among others). Serology is the primary testing modality for WNV infection.

Most WNV infections are asymptomatic. West Nile Virus disease should be considered in individuals with a febrile or acute neurological illness AND recent exposure to mosquitoes, a blood transfusion or organ transplantation. This is especially true during the summer months during active mosquito season. West Nile virus should be considered in the differential diagnosis in cases of suspected encephalitis and aseptic meningitis (e.g. herpes simplex virus, enteroviruses, among others) or infections caused by other arboviruses with a similar geographic distribution (e.g. Eastern equine encephalitis, Powassan virus, among others). The diagnosis should also be considered in any infant born to a mother infected with the virus during pregnancy or while breastfeeding.

Laboratory diagnosis of WNV infection can be accomplished by testing serum or CSF for virus-specific antibodies or nucleic acids. Serology testing is preferred for individuals suspected of WNV infection. Testing by PCR is not routinely recommended due to brief episodes of viremia and should be considered only for individuals that are immune compromised1. Testing of asymptomatic individuals is not recommended.

Acceptance/Rejection Criteria

Donor testing is not available through PHO. Specimens from patients being screened as potential donors (e.g. organ, tissue, cells, fertility, etc.) should be referred to a laboratory that performs donor screening assays. Specimens received for donor screening at PHO will be rejected.

Specimen Collection and Handling

Specimen Requirements

Test Requested Required Requisition(s) Specimen Type Minimum Volume Collection Kit

West Nile Virus Serology

Blood or serum

5.0 ml blood or 1.0 ml serum

Vacutainer tubes (serum separator tube, SST)

West Nile Virus Serology


400 µl

Sterile tube/container

West Nile Virus PCR1,4

EDTA blood, plasma (or serum)

5.0 ml blood or 1.0 ml serum

Non-heparin anticoagulant/
Lavender top tube

Submission and Collection Notes


Complete all fields of the General Test Requisition form, including:

  1. Test(s) requests and reasons for testing
  2. Patient setting, specimen type and site
  3. Clinical information, including any risk factors such as mosquito exposure, and any recent travel, must be provided.
  4. The Arbovirus (Non-Zika) Testing Intake Form is a mandatory requirement for West Nile virus PCR testing. PHO uses the information on the requisition and the mandatory intake form to assess testing criteria, assign appropriate tests, and provide mandatory information required by the National Microbiology Laboratory (NML) for relevant testing performed there.
    Note: Specimens submitted with mandatory information missing will not be tested until the information is provided.

Label the specimen container(s) with the patient’s first and last name, date of collection, and one other unique identifier such as the patient’s date of birth or Health Card Number. For additional information see: Criteria for Acceptance of Patient Specimens. Failure to provide this information may result in rejection or testing delay.


CSF Serology is the preferred method of testing for WNV. An accompanying serum sample is recommended when submitting a CSF for WNV serology.


Molecular testing (WNV PCR) must be preapproved by a PHO microbiologist. Contact PHO’s Laboratory Customer Service at 416-235-6556 or 1-877-604-4567 to request approval.

Timing of Specimen Collection

Acute and convalescent clotted blood or serum specimens for serology should be collected 2-3 weeks apart from patients with clinical illness.

Molecular (PCR):
Specimens for WNV PCR testing should be collected ASAP after the onset of symptoms. This should be discussed on a case-by-case basis with a PHO Microbiologist.


Haemolysed, icteric, lipemic or microbial contaminated sera or plasma are not recommended for testing.

PCR is not recommended for West Nile virus as it is not a sensitive test. It may be considered, after discussion with the Microbiologist, if the specimen is collected within 5 days of neurological onset during active mosquito season.

Storage and Transport

Centrifuge if using an SST. Place the specimen in a biohazard bag and seal. Specimens should be stored at 2 to 8°C following collection and shipped to PHO on ice packs as soon as possible.

Specimens for molecular testing should be frozen and shipped on dry ice.

All clinical specimens must be shipped in accordance with the Transportation of Dangerous Good Act/Regulations.

Special Instructions

For PCR requests, it is MANDATORY to provide all information requested on the Arbovirus (Non-Zika) Information Intake Form. The Arbovirus (Non-Zika) Information Intake Form may be exempted if all mandatory information is available on the PHO’s General Test Requisition. West Nile virus specimens for PCR requests that are submitted without the appropriate mandatory information will not be tested until this information is provided by the submitter.

Note: If only ordering West Nile virus serology, no arbovirus intake form is required.

If a patient has recently travelled to an area endemic for flaviviruses other than WNV, please document this on the laboratory requisition and contact the laboratory to request any follow up investigations if they are WNV IgM positive.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

West Nile virus IgG and IgM Enzyme Linked Immunosorbent Assay (ELISA) testing is performed daily Monday to Friday during peak season (summer months) and up to twice per week at other times. Turnaround time (TAT) for non-reactive ELISA IgG and IgM is up to 2 business days during peak season and up to 5 business days otherwise.

TAT for a confirmed reactive ELISA IgM is up to 5 business days from receipt at PHO.

TAT for results on specimens tested by Plaque Reduction Neutralization Test (PRNT) is up to 10 business days from receipt at PHO.

West Nile virus PCR tests are referred to NML. TAT is up to 21 calendar days from receipt at NML.

Test Methods

WNV IgM and IgG ELISAs are used as screening tests.  

Confirmatory PRNT testing of positives in health regions
All early season IgM reactive specimens will be further tested using the PRNT which is highly specific for WNV. However, PRNT testing is not necessary to make a diagnosis of WNV infection once WNV season is established within the local health region (3 positive PRNT results in an individual health region), at which time a reactive IgM ELISA test is sufficient for laboratory confirmation. In addition, repeat PRNT testing may not be performed if a patient has had a previous reactive PRNT test.

Note: During the summer season samples which are IgG positive and IgM negative will not be tested further by PRNT, as this indicates a past flavivirus infection, more than several months ago. If further investigation of these samples is required please contact the Customer Service Centre at 1-877-604-4567 and ask to speak to a PHO Microbiologist.

Molecular (PCR):
Although WNV PCR testing is available, it is not considered a first line test as it is less sensitive than CSF IgM ELISA due to the brief viremia experienced in WNV infection. PCR testing is not required to confirm the diagnosis, and usually is not necessary. However, under special circumstances, the National Microbiology Laboratory (NML) can perform WNV PCR testing on patients with suspected neuroinvasive disease with a reactive WNV IgM or immunocompromised individuals.

Recommended laboratory investigations and specimen requirements for cases of suspected West Nile Virus infection and cases with neurological involvement

It is recommended that a CSF specimen for WNV serology testing (WNV IgM) is submitted in addition to clotted blood or serum for acute and convalescent serology testing. The WNV CSF IgM is reactive in the majority of patients with WNV neuroinvasive disease.


Submitted specimens will first be analyzed by the IgG and IgM ELISA to determine if the patient has developed antibodies.

All specimens that are ELISA IgM reactive (early in the season) will be further analyzed for the presence of neutralizing antibodies by PRNT. Repeat PRNT testing may not be performed if a patient has had a previous reactive PRNT test.

Specimens that are ELISA non-reactive will not be tested by PRNT.

PCR testing will be considered on a case-by-case basis.



Table 1: Interpretation of WNV Laboratory Tests**

IgM ELISA IgG ELISA PRNT Interpretation of WNV Tests
Non-reactive Non-reactive Not tested No serological evidence of recent or past WNV infection.
Non-reactive REACTIVE Not tested Probable past WNV and/or other flavivirus infection or vaccination against a non-WNV flavivirus (e.g. yellow fever and Japanese encephalitis virus). PRNT testing is not warranted.
REACTIVE or Indeterminate REACTIVE REACTIVE ≥1:40 Consistent with recent or past WNV infection. IgM antibodies may persist for >1 year at low levels and may be indicative of a previous infection.*
REACTIVE Non-reactive or Indeterminate REACTIVE ≥1:40 Consistent with recent or acute WNV infection. A follow-up serum sample in two weeks is recommended to demonstrate the development of IgG antibodies. The failure to develop IgG antibodies suggests possible cross-reactive antibodies from another flavivirus infection.
REACTIVE Non-reactive or Indeterminate Non-reactive or Indeterminate 1:10 – 1:20 Probable recent or acute WNV infection. A follow-up serum sample in two weeks is recommended to demonstrate the development of IgG antibodies. The failure to develop IgG antibodies suggests a non-specific IgM reaction.
REACTIVE REACTIVE Non-reactive Probable recent or past WNV and/or other flavivirus infection or vaccination. A follow-up serum sample in two weeks is recommended, as well as consideration for testing for other flaviviruses, depending on the clinical history.*
Indeterminate REACTIVE Non-reactive or Indeterminate 1:10 – 1:20 Probable past flavivirus infection or vaccination. The IgG ELISA cannot differentiate between members of the Flavivirus genus. A follow-up serum sample in two weeks is recommended, as well as consideration for testing for other flaviviruses, depending on the clinical history.
Indeterminate Indeterminate Not tested West Nile Virus antibody status inconclusive. A follow-up serum sample in two weeks is recommended. Persistent indeterminate results for WNV IgM and IgG antibodies suggest a non-specific reaction.

Once WNV activity is established in a health region, PRNT testing will no longer be performed for the remainder of the season.  

A positive CSF WNV IgM is sufficient for laboratory confirmation of CNS WNV infection.  

Indeterminate results for any of the WNV assays may be due to the presence of low-level antibodies or non-specific reactions. Therefore, as with all laboratory tests, the results should be interpreted in the context of the clinical history.  

A reactive IgM antibody response using ELISA is specific for WNV and is rarely due to cross-reaction with other Flaviviruses. A secondary WNV IgM ELISA test confirms WNV IgM reactive results.  

A reactive IgG antibody response using ELISA may be due to infection with WNV or other flaviviruses, (e.g. Dengue, St. Louis encephalitis, Japanese encephalitis, Powassan, or Yellow fever virus) which may cross react.

A positive PCR result indicates that WNV virus nucleic acids were detected in the specimen and an acute/recent infection.

A negative PCR result indicates that WNV virus nucleic acids were not detected in the specimen. This does not exclude WNV virus infection.


Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

Specimens that are positive for West Nile virus are reported to the Medical Officer of Health as per the Health Protection and Promotion Act.

Data and Analysis

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Updated 31 May 2024