Eastern Equine Encephalitis Virus – Serology and PCR

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Background
This page provides serology and PCR testing information for the Eastern Equine Encephalitis virus (EEEV) at Public Health Ontario (PHO).

The EEEV is a mosquito-borne RNA virus in the Togaviridae family (genus Alphavirus) that can cause severe neuroinvasive disease.
The virus is transmitted primarily through the bite of infected mosquitoes in endemic areas (e.g. North America)^{1, 2}.

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 : 05 juill. 2025.

Testing Indications

Testing for EEEV infection may be considered for individuals with:

clinically compatible signs/symptoms of infection and
relevant exposures (e.g. mosquito bites, travel to or residence in endemic areas, outdoor activities, among others)^1-3.

Many individuals exposed to EEEV remain asymptomatic, while others may develop a mild febrile illness that can progress to neuroinvasive disease^1-3. Serology is the preferred method to detect an EEEV infection^{1, 4-5}. Testing for EEEV by PCR is not routinely recommended and should only be considered for individuals that are immune compromised¹. Testing of asymptomatic individuals is not recommended.

Due to the overlap in geographic distribution of the corresponding vectors, infection with other vector-borne pathogens, such as West Nile Virus, should be considered, following a clinical risk assessment, if suspecting EEEV infection during active mosquito 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
Eastern Equine Encephalitis 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)
Eastern Equine Encephalitis PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Serum or plasma³	1.0 mL	Red top or Serum separator tubes (SST) Or Plasma collected in non-heparin anticoagulant
Eastern Equine Encephalitis PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Other specimens (e.g. CSF)³	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. All fields on each requisition must be completed.

It is MANDATORY to provide the clinical information, relevant travel(s), and relevant exposures for Vector-borne viruses requested on Vector-borne and Zoonotic Virus Testing Intake Form. Test requests that are submitted without the appropriate mandatory information may be subject to cancellation.

Testing for EEEV PCR is not routinely performed 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.

Note: If CSF or other PCR testing is approved, submission of a paired serum for serology testing is required.

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 (PCR):
Specimens submitted for molecular testing (PCR) should be collected as soon as possible after the onset of symptoms, unless otherwise indicated via discussion with a PHO Microbiologist.

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/plasma 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)

Serology:
Serology screening for EEEV antibodies 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 screen result. TAT for confirmatory EEV 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 (PCR):
Molecular testing (PCR) is performed at the NML if the request is approved by a PHO Microbiologist. This is not a routine test, and TAT will be determined in consultation with the NML at the time of submission.

Test Methods

Serology:
EEEV serology screening is performed using Hemagglutination Inhibition (HI) Assay. The HI assay detects total antibodies to EEEV (IgM/IgG). Confirmatory testing is performed at the Centers for Disease Control and Prevention (CDC) in Fort Collins using a plaque reduction neutralization test (PRNT).

Molecular (PCR):
Molecular testing is performed at the NML using a reverse transcriptase polymerase chain reaction (RT-PCR)

Algorithm

Serology:
Serum is first screened for EEEV antibodies (IgM/IgG) by HI. Specimens that are HI reactive will be further analyzed for the presence of neutralizing antibodies by PRNT. No further testing will be performed on specimens that are HI non-reactive.

Molecular (PCR):
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.

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 should be considered where applicable.

Serology:
Consult the table below for interpretations of EEEV serologic testing. Results should be interpreted with caution.

Table 1. Interpretation of EEEV Serologic tests

HI Result	Possible Interpretation and Recommendations
Non-Reactive (<1:10)	No serological evidence of EEEV infection. Advise a follow-up specimen in 2 to 3 weeks if clinically indicated.
Reactive (≥1:10)	May indicate acute or recent EEEV 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 EEEV serology:

EEEV HI testing of a single serum specimen is insufficient to establish the diagnosis of an EEEV infection if a reactive result is obtained⁵. Submission of both acute and convalescent specimens is recommended to assist with interpretation and confirmation.
Confirmatory testing of HI reactive specimens (HI titres ≥1:10) is required to verify the presence of antibodies to EEEV. Low HI reactive specimens (titres of ≤1:20) may be due to the persistence of antibodies to EEEV from a previous infection, cross-reactivity with antibodies to other arboviruses (e.g. Dengue virus, West Nile virus, Japanese Encephalitis virus or Yellow Fever virus), or other causes. HI reactive tests results should not be interpreted in isolation without the corresponding PRNT confirmation, unless non-reactive.
A ≥ 4-fold change in antibody titre between acute and convalescent specimens is indicative of an acute or recent infection.

Molecular (PCR):
A positive PCR result (POS) indicates that EEEV nucleic acids were detected in the specimen and indicates an acute/recent infection.

A negative PCR result (NEG) indicates that EEEV nucleic acids were not detected in the specimen. This does not exclude EEEV 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 from patients with encephalitis are also reported to the Medical Officer of Health as per Health Protection and Promotion Act.

References

Centers for Disease Control and Prevention. Eastern Equine Encephalitis Virus. 2024. Available from: https://www.cdc.gov/eastern-equine-encephalitis/site.html.
Government of Canada. Mosquito-borne disease surveillance: seasonal update. 2024. Available from: https://health-infobase.canada.ca/zoonoses/mosquito/
Government of Canada. Eastern and Western Equine Encephalitis virus: Infectious substances pathogen safety data sheet. 2024. Available from: https://www.canada.ca/en/public-health/services/laboratory-biosafety-biosecurity/pathogen-safety-data-sheets-risk-assessment/eastern-equine-encephalitis.html
National Microbiology Laboratory. Arbovirus, Rabies, Rickettsia and Related Zoonotic Diseases. Available from: https://cnphi.canada.ca/gts/laboratory/1020
Vetter SM. Eastern Equine Encephalitis Virus. In: Leber AL and Burnham CAD. Clinical Microbiology Procedures Handbook (5th ed). 2023. ASM Press. Washington DC, USA. 18.13.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 05 juill. 2025.

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Mis à jour le 2 juill. 2025