Oropouche Virus – Serology and PCR

Conformément au Règlement de l’Ontario 671/92 de la Loi sur les services en français, les renseignements d’analyses de laboratoire liés à la présente page ne sont offerts qu’en anglais parce qu’ils sont de nature scientifique ou technique et destinés uniquement à l’usage des fournisseurs de soins de santé qualifiés et non aux membres du public.

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

The OROV is an RNA virus (Peribunyaviridae family) that is transmitted by the bite of an infected midge or mosquito in specific geographic regions¹.
The virus has a similar geographic distribution (endemic to some parts of Central and South America and the Caribbean^1,2,4) and clinical presentation as other arboviruses, including Dengue virus, Chikungunya virus and Zika virus.
Transmission of OROV from mother-to-fetus (vertical) may be possible, but a clear relationship has not yet been established^2,3.
Recommendations for laboratory testing have been described by the Pan American Health Organization (PAHO) and the Centers for Disease Control and Prevention (CDC)^5,6.

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 : 04 juill. 2025.

Testing Indications

Testing for OROV requires approval by a PHO Microbiologist.

A clinical risk assessment should be performed that includes a review of the individual’s clinical status, travel and exposure history and a consideration of alternative diagnoses, including infections by Dengue virus, Chikungunya virus or Zika virus.

Individuals that may be considered for OROV testing include:

Returned travellers OR individuals who reside in areas of risk that meet the following criteria^4-6:

Compatible symptoms⁴
Symptoms of Oropouche fever includes but are not limited to: fever, headache, chills, myalgia and/or arthralgia, nausea, vomiting, abdominal pain, photophobia or retro-orbital pain, and/or a maculopapular rash (starts on the trunk and spreads to extremities). Severe cases may display symptoms consistent with neuroinvasive disease.
Travel to an area with documented or suspected transmission of OROV⁴
Testing for other relevant arboviruses has been performed^4-6

Dengue virus, Chikungunya virus, and Zika virus targets are not detected by PCR^5,6 when testing is performed on a specimen collected within 10 days of symptom onset.

Dengue virus, Chikungunya virus or Zika virus serology (as appropriate) is non-reactive when testing is performed on a serum specimen that was collected more than 7 days from symptom onset.

Due to the overlap in arbovirus disease distribution, other more common arboviral infections must be ruled out first. PHO offers PCR and serology testing for Dengue virus, Chikungunya virus and Zika virus. Refer to PHO’s test information index for specific information on the appropriate tests to order for those viruses.

OROV testing may impact patient management
Testing of individuals that are critically ill or at risk of severe disease or its complications and who have compatible symptoms, and a relevant travel history may be considered.

Molecular testing by polymerase chain reaction (PCR) is the preferred test method. Testing of asymptomatic individuals is not recommended.

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
Oropouche virus PCR	General Test Requisition	Serum	1 mL	Serum separator tube (SST)
Oropouche virus PCR	General Test Requisition	Other specimen type (e.g. CSF, tissues or urine)	1 mL	Sterile container
Oropouche virus serology (PRNT)	General Test Requisition	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.

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 Oropouche virus 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.

Testing for Dengue virus, Chikungunya virus and Zika virus should be ordered at the same time as any request for OROV. The appropriate testing method is dependent on the time between symptom onset and specimen collection. Refer to PHO’s test information index for specific information on the appropriate tests to order for those viruses.

Serology testing for OROV is not routinely recommended and requires both an acute and convalescent specimen to be submitted before testing will be performed. All OROV serology requests will not proceed until both specimens have been received. If only a single specimen is received, the laboratory will only perform PCR, if approved.

Timing of Specimen Collection

Molecular (Real-Time PCR):
Collect specimen as soon as possible after symptom onset, ideally within 10 days⁶. Other specimen types (e.g. CSF, urine, tissues) may be considered acceptable beyond 10 days due to a lack of available information on viral detection in these specimen types.

Serology - Plaque Reduction Neutralization Test (PRNT):
Both an acute specimen (collected at least 7 days after of the onset of symptoms) and a convalescent specimen (collected 2-3 weeks after the acute specimen) are required to complete laboratory investigations.

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.

Special Instructions

Testing for Dengue virus, Chikungunya virus and Zika virus is required. The appropriate testing method should be selected based on the time from symptom onset, as described above. Requests should be submitted at the same time as any request for Oropouche virus testing.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Molecular (Real-Time PCR)
PCR screening for OROV is performed at PHO. The TAT is estimated to be up to 12 days after initial testing for Dengue, Chikungunya and/or Zika viruses is completed.

The TAT for other requests (and confirmatory testing) will be determined by the NML at the time of submission and may be up to 21 business days upon being referred out by PHO.

Serology:
Testing is not performed routinely and is available only by special request.

Specimens will be forwarded to the CDC by the NML only if the acceptance criteria indicated above are met. The TAT will be determined by the NML at the time of submission and may be a minimum of 21 business days upon being referred out by PHO.

Consult the specific PHO test information sheets for Dengue virus, Chikungunya virus and Zika virus testing TAT.

Test Methods

Molecular (real-time RT-PCR)
Molecular detection of OROV is performed by PCR at PHO. This test is for investigational use only.

Serology
Serology testing for OROV is performed by plaque reduction neutralization testing (PRNT) at the Centers for Disease Control and Prevention (CDC) in the US.

Algorithm

Requests for OROV testing are reviewed by a Microbiologist at PHO.

If the individual meets the criteria defined above (Testing Indications), testing for OROV will be approved and performed. It is a requirement that all specimens submitted for OROV testing must be tested for Dengue virus, Chikungunya virus or Zika virus by the appropriate method based on the time between symptom onset and specimen collection. If an appropriate method is not indicated, testing will be assigned based on the time from symptom onset indicated on the requisition.

Testing for OROV will not be performed if one or more assays for Dengue virus, Chikungunya virus or Zika virus are positive.

Specimens with OROV nucleic acids detected will be forwarded to NML for additional confirmation.

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 positive PCR result (Oropouche PCR Positive) 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.

Serology (PRNT):
The presence of anti-OROV antibodies in serum may indicate a recent or prior infection. A ≥ 4-fold increase in neutralizing antibody titre between acute and convalescent sera collected 2 to 3 weeks apart is considered indicative of recent seroconversion6. Depending on the time elapsed between symptom onset and specimen collection, a non-reactive serology 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

WHO. 2024. Disease Outbreak News - Oropouche virus disease - Region of the Americas. Available online at: https://www.who.int/emergencies/disease-outbreak-news/item/2024-DON530
PAHO. 2024. Epidemiological Alert Oropouche in the Region of the Americas: vertical transmission event under investigation in Brazil - 17 July 2024. Available online at: https://www.paho.org/en/documents/epidemiological-alert-oropouche-region-americas-vertical-transmission-event-under
Pan American Health Organization. 2024. Guidelines for Detection and Surveillance of Oropouche in cases of vertical infection, congenital malformation, or fetal deaths.
Government of Canada. 2024. Travel health notice – Oropouche fever in the Americas. Available online at: https://travel.gc.ca/travelling/health-safety/travel-health-notices/534
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.