Oropouche Virus – PCR and Serology

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Background
This page provides information regarding the molecular and serologic testing available for Oropouche virus (OROV) at Public Health Ontario (PHO).

The OROV is an RNA virus (Peribunyaviridae family) that can be transmitted to humans by the bite of an infected midge or mosquito in parts of Central and South America and the Caribbean.^1,2
The virus has a similar clinical presentation to other arboviruses, including Dengue virus, Chikungunya virus and Zika virus. Additional information can be found in PHO’s Focus On Oropouche Virus in the Americas and PHAC’s Mosquitos and mosquito-borne diseases page. ^2,3

Updates
Effective May 4, 2026, the completion of Zika virus serology testing is no longer a pre-requisite for Oropouche virus testing.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 06 mai 2026.

Testing Indications

A clinical risk assessment is recommended for OROV, with specific reference to travel and exposure histories prior to symptom onset and possible alternative diagnoses, including Dengue, Chikungunya and Zika viruses.^2-4

Testing for OROV may be considered for individuals who meet the following criteria:

Presence of signs/symptoms compatible with OROV infection AND
Travel to, or residence in, areas with documented or suspected OROV transmission within the previous 14 days AND
Testing for other relevant vector-borne diseases has been completed, with either:
- No detection of Dengue, Chikungunya and Zika viral nucleic acids by PCR in specimens collected within 10 days of symptom onset OR
- No detection of antibodies raised against Dengue or Chikungunya viruses in specimens collected at least 7-10 days after symptom onset.

Molecular testing by PCR is the preferred diagnostic method. Serologic testing for OROV is not routinely recommended and is indicated only for individuals that meet the above criteria who are beyond the typical PCR testing window (≥7-10 days from symptom onset). Because of the overlapping geographic distribution of disease and clinical presentation, other more common vector-borne viral infections should be excluded. PHO offers diagnostic testing for Dengue, Chikungunya and Zika viruses. Refer to PHO’s Test Menu for specific information.

Asymptomatic individuals should not be tested.

Acceptance/Rejection Criteria

Specimens received without the appropriate forms (See: Submission and Collection Notes) and those that do not meet the pre-defined eligibility criteria are subject to cancellation.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Oropouche virus PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Serum	1 mL	Serum separator tube (SST)
Oropouche virus PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Other specimen type (e.g. CSF, tissues or urine)	1 mL	Sterile container
Oropouche virus serology (PRNT)	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Serum	1 mL	Serum separator tubes (SST)

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.

Clinical information and relevant travel and exposure histories for vector-borne viruses must be provided on the Vector-borne and Zoonotic Virus Testing Intake Form. This information will be reviewed by a PHO Microbiologist to determine eligibility. Failure to provide this information may result in rejection or testing delays.

If OROV is suspected, testing for other relevant vector-borne diseases (e.g. Dengue, Chikungunya viruses) must be ordered concurrently. Ensure that the most appropriate testing method is selected, depending on the time interval between symptom onset and specimen collection. Consult PHO’s Test Menu for additional information on these viruses.

For eligible OROV serology requests, submission of both acute and convalescent specimens is necessary to assist with result interpretation. If only a single specimen is received, PHO will perform PCR testing only, provided eligibility criteria are met.

Timing of Specimen Collection

Molecular (Real-Time PCR):
Specimens should be collected as soon as possible following symptom onset, ideally within 10 days⁶. Testing of other specimen types (e.g. CSF, urine, tissues) may be considered acceptable beyond 10 days, given limited data on the duration of OROV detection in these specimen types.

Serology - Plaque Reduction Neutralization Test (PRNT):
Submission of both an acute specimen (collected ≥7 days after symptom onset) and a convalescent specimen (collected 2-3 weeks after the acute specimen) is required to complete laboratory testing.

Limitations

Hemolysed, icteric, lipemic or microbially 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. Specimens can be shipped as Category B (UN3373).
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 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)

Molecular (Real-Time PCR)
PHO performs PCR screening for OROV with a TAT of up to 12 business days following completion of initial Dengue, Chikungunya or Zika virus testing.

Confirmatory testing for specimens with a “Detected” or “Indeterminate” result in PHO screening PCR is performed by the NML. An additional 21 business days may be required.

Serology:
Eligible specimens will be forwarded to the NML for serologic testing by PRNT. Testing is not performed routinely and a minimum of 21 business days may be required after referral by PHO.

Consult PHO’s Test Menu for Dengue virus, Chikungunya virus and Zika virus virus testing TAT.

Test Methods

Molecular (real-time RT-PCR)
PHO performs PCR screening for OROV.⁵ Both “Detected” and “Indeterminate” PCR results require confirmation at the NML.

Serology
Serology testing for OROV is performed by plaque reduction neutralization testing (PRNT) at the NML.

Interpretation

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

Molecular (real-time RT-PCR)

Result	Interpretation	Comment
Not Detected	Oropouche virus RNA Not Detected by real-time PCR	Indicates that Oropouche virus (and related Oropouche-like reassortant virus) nucleic acids were not detected in the specimen. This does not exclude Oropouche virus infection.
Detected	Oropouche virus RNA Detected by real-time PCR	A “Detected” PCR result (Oropouche PCR Positive) is considered presumptive and indicates that Oropouche virus or Oropouche-like reassortant virus nucleic acids may be present in the specimen and an acute/recent infection. Additional confirmation may be required.
Indeterminate	Oropouche virus RNA Indeterminate by real-time PCR	An “Indeterminate” result may be due to low viral target quantity in the clinical specimen approaching the limit of detection of the assay, or may represent nonspecific reactivity (false signal) in the specimen
Invalid	Oropouche virus PCR test Invalid	Tests results are invalid due to the failed amplification of the extraction control. Amplification failure may be due to inadequate specimen content, extraction failure, or PCR inhibition.

Depending on the time elapsed between symptom onset and specimen collection (e.g. >7-10 days), a “Not Detected” PCR result may not exclude prior infection.

Serology (PRNT):
Detection of OROV antibodies in serum may reflect a recent or prior infection. Seroconversion is indicated by a ≥ 4-fold increase in neutralizing antibody titre between paired acute and convalescent sera collected 2-3 weeks apart. Depending on the timing of specimen collection relative to symptom onset, a “Non-reactive” serologic result may not exclude an acute OROV 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.

References

Government of Canada. 2024. Oropouche virus disease in the Americas [Internet]. Available from: https://travel.gc.ca/travelling/health-safety/travel-health-notices/534.
Ontario Agency for Health Protection and Promotion (Public Health Ontario). Oropouche Virus in the Americas [Internet]. Toronto, ON: King’s Printer for Ontario; Available from: Focus On: Oropouche Virus in the Americas
Government of Canada. 2024. Oropouche virus disease: For health professionals [Internet]. Available from: https://www.canada.ca/en/public-health/services/diseases/mosquitoes/oropouche-virus/health-professionals.html.
Pan American Health Organization. 2023. Guidelines for the Detection and Surveillance of Emerging Arboviruses in the Context of the Circulation of Other Arboviruses.
CDC. 2024. Interim Guidance for Health Departments on Testing and Reporting for Oropouche Virus Disease. Available online at: https://www.cdc.gov/oropouche/php/reporting/index.html.
Naveca FG et al. 2017. Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses. Mem. Inst. Oswaldo Cruz. 112:7. 510-513.