West Nile Virus – Serology and PCR

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Background
This page provides information on the testing available through Public Health Ontario (PHO) for West Nile Virus (WNV).

The WNV is a mosquito-borne RNA virus that is a member of the Flaviviridae family and can cause neuroinvasive disease in some individuals.
The WNV is transmitted through the bite of infected mosquitos in endemic areas, including Ontario and other parts of Canada^1-3. PHO performs surveillance on WNV in Ontario during active mosquito season (between July to October).

Testing for WNV infection can be performed by serology or PCR depending on the specific clinical scenario.

Updates
Effective July 2, 2025, submission of the new Vector-borne and Zoonotic Virus Testing Intake Form is mandatory, along with the General Test Requisition when requesting specific vector-borne or zoonotic virus tests. The new intake form replaces both the Arbovirus (Non-Zika) Testing Intake Form and the Mandatory Intake Form for Zika Virus Testing.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 13 juill. 2025.

Testing Indications

Testing for WNV infection is indicated in individuals with⁴:

clinically compatible signs/symptoms of infection and
relevant exposures (e.g. noted mosquito bites, travel to or residence in an endemic area with ongoing WNV transmission, among others) or
new onset of symptoms following an alternative exposure (e.g. blood transfusion, organ transplantation, infant born to a mother with a confirmed WNV infection during pregnancy or while breastfeeding, among others)

Most WNV infections are asymptomatic. Symptomatic individuals may develop a mild febrile illness up to 14 days following exposure (e.g. fever, headache, myalgias/arthralgias, etc). Some individuals may develop neuroinvasive disease accompanied by neurological symptoms (e.g. meningitis, encephalitis, paralysis).

Serology is the preferred method to detect WNV infection. Testing for WNV by PCR (serum or CSF) is not routinely recommended and should be considered only for individuals that are immune compromised¹. WNV PCR testing of other individuals may be considered on a case-by-case basis, after discussion with a PHO Microbiologist. Testing of asymptomatic individuals is not recommended.

Due to the overlap in geographic distribution of the corresponding vectors, infection with other vector-borne pathogens should be considered, following a clinical risk assessment, if suspecting WNV infection during active mosquito season.

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 will be rejected.

Specimens received without the appropriate forms (See: Submission and Collection Notes) are subject to cancellation.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
West Nile Virus Serology	General Test Requisition	Serum	5.0 ml blood or 1.0 ml serum	Red top or Serum Separator tubes (SST)
West Nile Virus Serology	General Test Requisition	CSF³	400 µl	Sterile tube/container
West Nile Virus PCR^1,4	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Serum	5.0 ml blood or 1.0 ml serum	Red top or Serum Separator tubes (SST)
West Nile Virus PCR^1,4	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Plasma	5.0 ml blood or 1.0 ml plasma	Non-heparin anticoagulant/ Lavender top/EDTA tube
West Nile Virus PCR^1,4	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	CSF	400 µl	Sterile tube/container

Submission and Collection Notes

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.

Each specimen submitted for testing must be accompanied by a separate PHO General Test Requisition, with all fields completed.

For West Nile virus PCR testing, it is MANDATORY to provide the clinical information, relevant travel(s), and relevant exposures for Vector-borne viruses requested on the Vector-borne and Zoonotic Virus Testing Intake Form. Test requests that are submitted without the appropriate mandatory information are subject to cancellation.

Testing for WNV PCR must be approved by a PHO Microbiologist. Submission of the mandatory Vectorborne and Zoonotic Virus Testing Intake Form will initiate the review process at PHO provided the form contains all necessary information. Requests received without the form, forms submitted with insufficient information or insufficient justification for testing are subject to cancellation.

CSF serology is the preferred method of testing for WNV in individuals with neuroinvasive disease. An accompanying whole blood or serum sample is required when submitting a CSF for WNV serology or PCR⁵.

Timing of Specimen Collection

Serology:
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 unless otherwise indicated via discussion with a PHO Microbiologist.

Limitations

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

Storage and Transport

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

For serum separator tubes: centrifuge sample prior to placing in biohazard bag.
Place each specimen type in an individual biohazard bag and seal. Insert the corresponding requisition in the pocket on the outside of each sealed biohazard bag.
Clotted blood/serum/plasma specimens should be stored at 2-8°C following collection and shipped to PHO on ice packs.

All specimens submitted for molecular testing should be stored at 2-8°C following collection and shipped to PHO on ice packs. If a delay in transport to PHO is anticipated (more than 72 hours), specimens should be frozen (at -80°C if possible) and shipped on dry ice.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Serology:
Peak season (early Spring to late Fall):

WNV serology testing is performed daily at PHO during peak season
The TAT for non-reactive results is up to 2 business days from receipt at PHO
The TAT for a reactive IgM result is up to 5 business days from receipt at PHO
The TAT for a confirmed reactive result, if PRNT is performed, is up to 10 business days from receipt at PHO

Off-peak season (all other times):

WNV serology testing may be performed up to twice per week at all other times as dictated by demand.
The TAT for non-reactive results is up to 5 business days from receipt at PHO
The TAT for a reactive IgM result is up to 5 business days from receipt at PHO
A final confirmatory result by PRNT is up to 10 business days from receipt at PHO

Molecular (Real-time RT-PCR):
WNV PCR tests are referred to NML. The TAT is up to 21 calendar days from receipt at NML.

Test Methods

Serology:
WNV serum and CSF serology are performed using an Enzyme Linked Immunosorbent Assay (ELISA).

Confirmatory testing of IgM reactive serum specimens is performed by Plaque Reduction Neutralization Testing (PRNT).

Molecular (Real-time RT-PCR):
WNV PCR testing is performed at the NML using a real-time reverse transcriptase PCR (RT-PCR).

Algorithm

Serology:
Serum: Serum will first be screened for IgG and IgM antibodies by ELISA. All Reactive IgM results regardless of IgG results, and Indeterminate IgM with Reactive IgG results will be confirmed by a Plaque Reduction Neutralization Test (PRNT). Indeterminate IgM with Non-Reactive/Indeterminate IgG results will not be tested for PRNT. Only serum specimens will be PRNT confirmed. Serum specimens that are ELISA non-reactive will not be tested by PRNT.

CSF: CSF will be tested for IgM antibodies only. No further PRNT testing is performed on IgM reactive specimens.

Molecular (PCR):
WNV PCR Testing will be considered on a case-by-case basis and must be approved by a PHO Microbiologist.

Additional WNV testing notes:

All early season IgM reactive sera will be confirmed by PRNT. Repeat PRNT testing may not be performed if a patient has had a previous reactive PRNT test.
Once WNV activity has been established within a health region (3 confirmed PRNT results from different individuals), PRNT will no longer be performed for the remainder of the season, unless by special request. 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.
Specimens which are IgM non-reactive and IgG reactive will not be confirmed by PRNT.

Interpretation

All results should be interpreted in the context of the specific clinical scenario. Given the overlap in the distribution of disease vectors, testing for other potential co-pathogens should be considered, where applicable.

Serology:

Table 1: Interpretation of WNV Serology Tests

IgM ELISA	IgG ELISA	PRNT	Possible interpretation of WNV Tests and Recommendations
Non-reactive	Non-reactive	Not tested	No serological evidence of infection. Advise a follow-up specimen in 2 to 3 weeks if clinically indicated.
Non-reactive	REACTIVE	Not tested	Previous WNV or other Flavivirus infection, or prior arboviral vaccination likely. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation and/or investigate other possible etiologic agents.
REACTIVE	Not tested	Not tested	Recent or past WNV infection possible. If specimen submitted was a CSF, only IgM testing is performed.
REACTIVE	REACTIVE	REACTIVE (Titre ≥1:40)	Recent or past WNV infection possible. Anti-WNV IgM antibodies may persist for >1 year after a previous infection7. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation if an acute or recent infection is suspected.
REACTIVE	REACTIVE	Not tested	Recent or past WNV infection possible. PRNT not performed as health unit has demonstrated ≥3 PRNT-confirmed cases during active mosquito season.
Indeterminate	REACTIVE	REACTIVE (Titre ≥1:40)	Recent or past WNV infection possible. Anti-WNV IgM antibodies may persist for >1 year after a previous infection7. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation if an acute or recent infection is suspected.
REACTIVE	Non-reactive or Indeterminate	REACTIVE (Titre ≥1:40)	Recent or acute WNV infection possible. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation if an acute or recent infection is suspected.
REACTIVE	Non-reactive or Indeterminate	Non-reactive or Indeterminate (Titre 1:10 – 1:20)	Recent or acute WNV infection possible. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation if an acute or recent infection is suspected. If result was obtained on second serum specimen, the result suggests a non-specific IgM reaction and other etiologic agents should be investigated.
REACTIVE	REACTIVE	Non-reactive	Recent or past WNV infection or other Flavivirus infection, or prior arboviral vaccination likely. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation if an acute or recent infection is suspected. Other etiologic agents should be investigated if the second PRNT is non-reactive.
Indeterminate	Non-reactive	Not tested	WNV antibody status inconclusive. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation if an acute or recent infection is suspected. Persistent indeterminate results for WNV IgM and IgG antibodies suggest a non-specific reaction.
Indeterminate	REACTIVE	Non-reactive or Indeterminate (Titre 1:10 – 1:20)	Recent or past WNV infection or other Flavivirus infection, or prior arboviral vaccination likely. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation if an acute or recent infection is suspected. Other etiologic agents should be investigated if the second PRNT is non-reactive.
Indeterminate	Indeterminate	Not tested	WNV antibody status inconclusive. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation if an acute or recent infection is suspected. Persistent indeterminate results for WNV IgM and IgG antibodies suggest a non-specific reaction.

Additional notes on WNV serology testing:

A ≥4-fold change in antibody titre between acute and convalescent specimens is indicative of an acute or recent infection.
A reactive CSF WNV IgM is sufficient for laboratory confirmation of CNS WNV infection.
Confirmatory testing of IgM/IgG reactive sera is necessary, unless there is sufficient serologic evidence to indicate WNV is established within a health region during active mosquito season (as described above).
Serologic cross-reactions in IgM or IgG may occur due to the persistence of antibodies to WNV from a previous infection, a recent or past infection with a related Flavivirus (e.g. Dengue virus, Japanese Encephalitis virus, among others), or other causes. This may manifest as a reactive or indeterminate serology result in IgM and/or IgG antibodies. Submission of a second serum specimen and IgM reactive by ELISA (and PRNT, as appropriate) can assist with interpretation.

Molecular (PCR):
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.

Additional notes on WNV PCR testing:
PCR is not a sensitive test for the detection of WNV and is not routinely recommended^5,6.

Reporting

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.

References

World Health Organization. 2017. West Nile virus. Available from: https://www.who.int/news-room/fact-sheets/detail/west-nile-virus
Public Health Ontario. 2024. Ontario Vector-Borne Disease Tool. Available from: https://www.publichealthontario.ca/en/Data-and-Analysis/Infectious-Disease/West-Nile-Virus
Public Health Agency of Canada. 2025. Surveillance of West Nile virus. Available from: https://www.canada.ca/en/public-health/services/diseases/west-nile-virus/surveillance-west-nile-virus.html
Ontario Ministry of Health. 2022. Appendix 1: Case Definitions and Disease-Specific Information Disease: West Nile Virus Illness. Available from: https://files.ontario.ca/moh-west-nile-virus-illness-en-2022.pdf
Kasule S, Fernholz E, Grant L, Kole A, Grys TE, Kaleta E, Theel ES, et al. 2024. Whole-Blood PCR Preferred for Timely Diagnosis of Neuroinvasive West Nile Virus Infections : Lessions From the 2021 Arizona Outbreak. Open Forum Infect. Dis. 11(5) : ofae188.
Lanciotti RS, Jerst AJ, Nasci RS, Godsey MS, Mitchell CJ, Savage HM, et al. 2000. Rapid Detection of West Nile Virus from Human Clinical Specimens, Field-Collected Mosquitoes and Avian Samples by a TaqMan Reverse Transcriptase-PCR Assay. J. Clin. Microbiol. 38(11): 4066-4071.
Centers for Disease Control and Prevention. 2024. Clinical Testing and Diagnosis for West Nile Virus Disease. Available from: https://www.cdc.gov/west-nile-virus/hcp/diagnosis-testing/index.html

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 13 juill. 2025.

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Mis à jour le 2 juill. 2025