Powassan Virus – Serology and PCR

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

The POWV is a single-stranded RNA virus that is a member of the Flaviviridae family.
The primary route of transmission for this virus is through a tick bite¹. Powassan virus is known to be present in some ticks in Ontario, Québec, and other parts of Canada^2,3, as well as the Northeastern United States⁴.

Testing for Powassan virus infection involves 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.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 06 juill. 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), if known.

Most individuals exposed to POWV are asymptomatically infected or develop an acute febrile illness within 4 weeks of a tick bite⁵. An initial prodrome of fever, chills, headache, gastrointestinal symptoms, myalgias, confusion, weakness, or nuchal rigidity, among others may occur⁵. Progression to neuroinvasive disease is possible and may present as meningitis and/or encephalitis accompanied by neurological deterioration or other focal neurologic signs/symptoms⁵.

Serology is the preferred method for POWV testing. Testing for POWV by PCR is not routinely recommended and should only be considered for individuals that are immune compromised^6,7. Testing should not be performed on 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	Blood or serum	5.0 ml 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 or serum	1.0 ml	EDTA blood, plasma with non-heparin anticoagulant Or Serum Separator Tubes (SST)
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 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.

If submitting CSF, a paired serum sample must also be provided.

Testing for POWV 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 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 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 days of the reactive result. TAT for confirmatory Powassan serology testing by PRNT is determined by the NML and may require up to an additional 21 calendar 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 (IgM and IgG). Confirmatory POWV serologic testing is performed using a plaque reduction neutralization test (PRNT) 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 for the detection of Powassan virus and is not routinely recommended.

Algorithm

Serum specimens submitted for POWV serology will first be analyzed by the HI assay to test for the presence of POWV antibodies. Specimens that are HI reactive will be referred to the NML for confirmatory testing for neutralizing antibodies by PRNT. HI non-reactive samples will not be tested by PRNT.

PCR testing by RT-PCR will be considered on a case-by-case basis as it is not a sensitive test and is not performed routinely. If CSF or other PCR testing is approved, submission of a paired serum for serology testing is required.

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 serologic testing. 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 result does not re-confirm on the follow-up HI, a non-specific reaction is likely.

Additional notes on POWV serology:

POWV HI testing of a single serum specimen is insufficient to establish the diagnosis of a POWV infection 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 (titres ≥1:20) 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 and tested by PRNT 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.

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 : 06 juill. 2025.

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Mis à jour le 2 juill. 2025