Western Equine Encephalitis 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.

Background

This page provides information on the testing available through Public Health Ontario (PHO) for the Western Equine Encephalitis Virus (WEEV).

WEEV is a rare mosquito-borne RNA virus that is a member of the Togaviridae family.
Transmission of WEEV to humans is through the bite of an infected mosquito in specific geographic regions. The WEEV is endemic to South America^1,2. Sporadic human cases are rare in North America, with the most recent being identified in Canada and the US in the 1980s³.

Testing for WEEV infection can be performed by serology and/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.

*Uncontrolled print copy. Valid only on day of print: 04 July 2025.

Testing Indications

Testing for WEEV may be indicated in patients with²:

clinically compatible signs/symptoms of infection and
relevant exposures (e.g. travel to or residence in an area with active local WEEV transmission or a risk of WEEV transmission in animals or humans)

Most WEEV infections are asymptomatic4. Individuals may develop a mild febrile illness that can progress to neuroinvasive disease^1,4. Serology is the preferred test method for WEEV, particularly ≥4 days after symptom onset². Molecular testing via PCR is not routinely recommended and should only be considered for individuals that are immune compromised. 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 if suspecting a WEEV infection.

Acceptance/Rejection Criteria

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
Western Equine Encephalitis (WEE) serology	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)
Western Equine Encephalitis (WEE) PCR	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)
Western Equine Encephalitis (WEE) PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	CSF	400 µl	Sterile 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.

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 WEEV PCR is not routinely performed and must be approved by a PHO Microbiologist. Submission of the mandatory Vector-borne 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.

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 WEEV PCR testing should be collected as soon as possible after the onset of symptoms. This should be discussed on a case-by-case basis 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 /CSF 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:
Serology is performed once a week at PHO. Turnaround time is up to 8 days from receipt at PHO.

Molecular (real-time RT-PCR):
Molecular testing is not performed routinely by the NML and requires approval by a PHO Microbiologist. The TAT will be determined at the time of submission.

Test Methods

Serology:
Specimens submitted for WEEV serology are tested using a laboratory-developed Hemagglutination Inhibition (HI) Assay. There is no confirmatory test available.

Molecular (Real-time RT-PCR):
Molecular testing is performed by reverse transcriptase PCR (RT-PCR) at the NML upon special request. PCR test has not been fully validated and for research purposes only.

Algorithm

Serology:
Serum specimens will first be analyzed by HI Assay (in PHO) to determine if the patient has developed antibodies.

Molecular (Real-time RT-PCR):
PCR testing is not performed routinely (in NML) and will be considered on a case-by-case basis.

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:
Consult the table below for interpretations of WEEV serologic testing. Results should be interpreted with caution.

Table 1. Interpretation of WEEV Serologic tests

HI Result	Possible Interpretation and Recommendations
Non-Reactive (<1:10)	No serological evidence of WEEV infection. Advise a follow-up specimen in 2 to 3 weeks if clinically indicated.
Reactive (≥1:10)	May indicate acute or recent WEEV infection. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation. If result does not re-confirm on the follow-up HI, a non-specific reaction is likely.

Additional notes on WEEV serology:

WEEV HI testing of a single serum specimen is insufficient to establish the diagnosis of a WEEV infection if a reactive result is obtained. Submission of both acute and convalescent specimens is recommended to assist with interpretation.
The HI test does not distinguish the presence of WEEV IgM or IgG antibodies. Low HI reactive specimens (titres of ≤1:20) may be due to the persistence of antibodies to WEEV or a related vector-borne virus from a previous infection, cross-reactivity with antibodies to other vector-borne viruses (e.g. Chikungunya virus or Eastern Equine Encephalitis virus), or other causes.

Molecular (Real-time RT-PCR):
A positive PCR result indicates that WEEV nucleic acids were detected in the specimen and are suggestive of an acute/recent infection.

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

Reporting

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

Positive results from patients with encephalitis are also reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.³

References

Pan American Health Organization (PAHO). 2023. Epidemiological alert: Risk to human health associated with infection with Western Equine Encephalitis Virus in equines.
Pan American Health Organization (PAHO). 2024. Laboratory Guidelines for the Detection and Diagnosis of Western Equine Encephalitis Virus Human Infection. Available from: https://www.paho.org/sites/default/files/2025-03/eng-weefebruary-2024.pdf
Centers for Disease Control and Prevention. 2024. Data and Maps for Western Equine Encephalitis. Available from: https://www.cdc.gov/wee/data-maps/index.html
World Health Organization. 2023. Western equine encephalitis – Argentina. Available from: https://www.who.int/emergencies/disease-outbreak-news/item/2023-DON499