Venezuelan 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 Venezuelan Equine Encephalitis virus (VEEV)

The VEEV is a mosquito-borne RNA virus that belongs to the Togaviridae family (genus Alphavirus)¹.
The virus is transmitted to humans via the bite of an infected mosquito in areas with ongoing transmission. Human infections are rare but have been documented primarily in Central and South America².

Testing for VEEV involves PCR or serology depending on the clinical scenario. Testing for the VEEV is performed at the Centers for Disease Control and Prevention (CDC) in Fort Collins, USA and requires approval by a PHO Microbiologist.

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: 03 July 2025.

Testing Indications

Testing for VEEV infection is indicated for individuals with:

Clinically compatible signs/symptoms of infection AND
Relevant exposure history (e.g. returned from travel to- or residence in- a VEEV endemic area, noted mosquito bites, among others).

Individuals expossed to VEEV can be asymptomatically infected or develop an acute febrile illness within 2 to 5 days after exposure¹. Initial symptoms of infection can include (but are not limited to): fever, headache, myalgias/arthralgias, nausea, vomiting or a rash³. Neuroinvasive disease may develop in more severe cases.

Due to the overlap in geographic distribution of the corresponding vectors, infection with other vector-borne pathogens, such as Dengue virus, should be considered if suspecting VEEV 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
Venezuelan Equine Encephalitis (VEE) 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)
Venezuelan Equine Encephalitis (VEE) serology	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	CSF	400 µl	Sterile container
Venezuelan Equine Encephalitis (VEE) 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)
Venezuelan Equine Encephalitis (VEE) 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.

Either an acute and convalescent serum or a CSF paired with serum specimens must be submitted. Note: CSF specimen alone will not be accepted.

Testing can only be performed based on relevant clinical information and must be approved by the PHO microbiologist and may be referred to the CDC in Fort Collins through National Microbiology Laboratory (NML). 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) :
Collect specimens for molecular testing early in the course of illness.

Note: Refer to the CDC-Submitting Specimens for Arboviral Tests for further information.

Limitations

Haemolysed, 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.
Clotted blood/serum specimens should be stored at 2-8°C following collection and shipped to PHO on ice packs.
Ship refrigerated specimens (e.g., clotted blood, serum, CSF) on ice packs, and frozen specimens (e.g., serum, CSF,) on dry ice. Do not ship clotted blood, EDTA/ non-heparin/citrated whole blood on dry ice.

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)

The TAT for VEEV testing will be determined at the time of submission as testing is not performed routinely.

Test Methods

Both VEEV serology and molecular testing by real-time PCR will be performed at CDC if all requirements and criteria for testing are met.

Reporting

CDC laboratory will send the reports to NML and will be faxed to PHO to be released.

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 specimens are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

References

Public Health Agency of Canada. 2021. Pathogen safety data sheets: infectious substances, Venezuelan equine encephalitis virus. Available from: https://www.canada.ca/en/public-health/services/laboratory-biosafety-biosecurity/pathogen-safety-data-sheets-risk-assessment/venezuelan-equine-encephalitis-virus.html
Aguilar PV, Estrada-Franco JG, Navarro-Lopez R, Ferro C, Haddow AD, Weaver DC. 2011. Endemic Venezuelan equine encephalitis in the Americas: hidden under the dengue umbrella. Future Virol. 6(6): 721-740.
Matthews R, Michael BC, Rodriguez-Morales, Ferreira L. 2025. Venezuelan equine encephalitis virus infection. BMJ Best Practice.

Additional Information

All molecular testing must be preapproved by the PHOL microbiologist. Contact PHOL customer service at 416-235-6556 or 1-877-604-4567 to request approval.

*Uncontrolled print copy. Valid only on day of print: 03 July 2025.

Contact Laboratory Customer Service

Microbiology and Laboratory Services

customerservicecentre@oahpp.ca

Updated 2 July 2025