Powassan Virus – Serology and PCR

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This page provides serology and molecular testing information for Powassan virus (POWV) at Public Health Ontario (PHO).

POWV is a single-stranded RNA virus that is a member of the Flaviviridae family.
It is transmitted via a tick bite¹ and has been detected in some ticks in Ontario, Québec, and other parts of Canada^2,3, as well as the Northeastern United States⁴.

Testing for POWV infection involves serology and/or PCR depending on the clinical scenario.

Updates

Serum serology is the preferred method for POWV testing.
The “Specimen Requirements” table has been reorganized and updated to provide clearer guidance on the appropriate specimen type based on the specific test requested.
Under “Submission and Collection Notes,” the comments were revised to clarify the arbovirus testing approval process and address ongoing issues—such as clients continuing to call CSC for approvals and faxing intake forms to WRA prior to specimen submission to PHO.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 16 déc. 2025.

Testing Indications

Testing for POWV infection is indicated for individuals with:

clinically compatible signs/symptoms of infection AND
a relevant exposure history (e.g., outdoor activity in an endemic area, tick bite(s))

Most individuals exposed to POWV remain asymptomatic. Others may develop an acute febrile illness within 4 weeks of a tick bite, that is characterized by fever, chills, headache, gastrointestinal symptoms, myalgias, confusion, weakness, among others⁵. This may progress to a neuroinvasive disease, with meningitis and/or encephalitis accompanied by neurological deterioration or other focal neurologic signs/symptoms⁵.

Serum serology is the preferred method for POWV testing. PCR testing is not routinely recommended and should only be considered for immunocompromised individuals^6,7. Testing should not be requested for asymptomatic individuals.

Due to the overlap in geographic distribution of the corresponding vectors, infection with other vector-borne diseases, such as Lyme Disease should also be considered, following a clinical risk assessment, if suspecting POWV infection during active tick season.

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
Powassan virus serology	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Clotted blood or serum	5.0 ml Clotted blood or 1.0 ml serum	Serum Separation Tubes (SST)
Powassan virus PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Clotted blood or serum	5.0 ml Clotted blood or 1.0 ml serum	Serum Separation Tubes (SST)
Powassan virus PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Plasma	1.0 ml	EDTA blood, plasma with non-heparin anticoagulant
Powassan virus PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	CSF	600 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 to PHO for testing must be accompanied by a separate, fully completed:

PHO’s General Test Requisition AND
Vector-borne and Zoonotic Virus Testing Intake Form.

The submitted forms will be reviewed by a PHO Microbiologist, who will determine whether to approve or decline testing based on the information provided.

Approvals require that the Vector-borne and Zoonotic Virus Testing Intake Form to include all necessary information in applicable fields; fields indicated with a star are mandatory. If a testing method is indicated on the General Test Requisition (e.g., “Powassan virus serology” vs “Powassan virus”) , the request must meet the criteria outlined in the Testing Indications section above, or the request may be subject to cancellation.
Failure to submit the Vector-borne and Zoonotic Virus Testing Intake Form, to provide all required information, or submission of an inappropriate specimen type (as listed above) may result in the test request being declined.
If testing is declined, a cancellation report will be issued.
If testing is approved, the test will be performed, and a report will be issued within the established turnaround time.

Note the following:

Do not contact PHO’s Customer Service Centre for approvals. Approvals are based solely on the information provided on the submitted requisition and intake form.
Always send the completed Intake Form with the specimen at the time of initial submission.
Do not fax the Intake Form to the number on the form. Only fax if PHO has issued a cancellation report requesting additional mandatory information.

If submitting CSF, an accompanying serum sample must also be provided for POWV serology.

Timing of Specimen Collection

Serology:
Acute and convalescent sera should be collected for serologic testing, where applicable. The convalescent serum specimen should be collected at least 2 to 3 weeks after the initial acute specimen.

Molecular (Real-time RT-PCR):
Specimens for PCR testing should be collected as soon as possible after symptom onset.

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 specimens should be stored at 2-8°C following collection and shipped to PHO on ice packs.

All specimens submitted for molecular testing may 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:
POWV serology screening is performed once per week at PHO.

TAT is up to 8 business days from receipt at PHO.

Specimens reactive on PHO’s screening assay are referred to the National Microbiology Laboratory (NML) for additional confirmatory testing within 7 business days of the reactive result.

TAT for confirmatory POWV serology testing by PRNT is determined by the NML and may require up to an additional 21 business days following receipt of the specimen at the NML.

Molecular (Real-time RT-PCR):
Specimens are referred out to the NML. TAT will be determined by the NML at the time of submission as the test is not performed routinely.

Test Methods

Serology:
PHO uses a hemagglutination inhibition (HI) assay to screen for POWV antibodies. Confirmatory POWV serology testing is performed by plaque reduction neutralization test (PRNT) at the NML. A secondary screen for POWV IgM/IgG may also be performed at the NML.

Molecular (Real-time RT-PCR):
Molecular detection of POWV targets is performed by RT-PCR at the NML. This is not a sensitive test and is not routinely recommended.

Algorithm

Serum specimens submitted to PHO for POWV serology will be tested by the HI assay. This assay detects total POWV antibodies. Sera that are HI reactive will be referred to the NML for confirmatory testing. No further testing will be performed on HI non-reactive specimens.

POWV PCR requests are forwarded to the NML. These requests will be reviewed by a PHO Microbiologist. If a CSF specimen has been submitted for POWV PCR, an accompanying serum for POWV serology testing is required or testing may be declined.

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 may be considered where applicable.

Serology:
Consult the table below for interpretations of POWV serology. Results should be interpreted with caution.

Table 1. Interpretation of POWV Serologic tests

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

Additional notes on POWV serology:

It is difficult to establish diagnosis of a POWV infection by HI testing of a single serum specimen if a reactive result is obtained. Submission of both acute and convalescent specimens is recommended to assist with interpretation and confirmation.
Confirmatory testing of HI reactive specimens by PRNT is required to verify the presence of POWV antibodies. Low HI reactive specimens may be due to the persistence of antibodies to POWV from a previous infection, cross-reactivity with antibodies to other vector-borne viruses (e.g., St. Louis Encephalitis, Dengue, West Nile, Japanese B Encephalitis, and Yellow Fever), or other causes. HI reactive test results should not be interpreted in isolation without the corresponding PRNT confirmation, unless non-reactive.
A ≥4-fold change in neutralizing antibody titre between acute and convalescent sera collected 2-3 weeks apart is considered indicative of acute or recent infection.

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

A negative PCR result indicates that POWV nucleic acids were not detected in the specimen. This does not exclude POWV infection. Serology testing should also be performed.

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.

Positive results are reported to the Medical Officer of Health as per Health Protection and Promotion Act.

References

Government of Canada. 2024. Ticks and tick-borne diseases. Available from: https://www.canada.ca/en/public-health/services/diseases/ticks-tick-borne-diseases.html
Smith K, Oesterle PT, Jardine CM, Dibernardo A, Huynh C, Lindsay R, et al. 2018. Powassan virus and other arthropod-borne viruses in wildlife and ticks in Ontario, Canada. Am. J. Trop. Med. Hyg. 99(2): 458-465.
Public Health Agency of Canada. 2024. Tick surveillance in Canada: infographic 2022. Available from: https://www.canada.ca/content/dam/phac-aspc/documents/services/publications/diseases-conditions/surveillance-ticks-canada-infographic-2022/surveillance-ticks-canada-infographic-2022.pdf
Centers for Disease Control and Prevention. 2025. Powassan Virus Historic Data (2004-2023). Available from: https://www.cdc.gov/powassan/data-maps/historic-data.html
Government of Canada. 2024. Powassan virus disease. Available from: https://www.canada.ca/en/public-health/services/diseases/powassan-virus.html
National Microbiology Laboratory. 2025. Molecular Detection of Powassan Virus by Reverse Transcriptase PCR (RT-PCR). Available from: https://cnphi.canada.ca/gts/reference-diagnostic-test/5195?alphaReturn=pathogenByLetter&alphaChar=P
Centers for Disease Control and Prevention. 2024. Clinical Testing and Diagnosis for Powassan Virus Disease. Available from: https://www.cdc.gov/powassan/hcp/diagnosis-testing/index.html

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 16 déc. 2025.

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Mis à jour le 11 déc. 2025