Japanese 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 Serology and PCR testing information available through Public Health Ontario (PHO) for the Japanese encephalitis virus (JEV).

The JEV is a mosquito-borne RNA virus that is a member of the Flaviviridae family and is genetically related to other common arboviruses, including Dengue virus and West Nile virus.
JEV transmission occurs primarily through the bite of an infected mosquito in an area that is considered endemic (e.g., Southeast Asia).

Testing for JEV is performed at the National Microbiology Laboratory (NML) in Winnipeg. Testing is not performed at PHO’s laboratory.

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: 05 July 2025.

Testing Indications

Testing for JEV is indicated for individuals with:

compatible clinical symptoms and
appropriate exposures (e.g., mosquito bites, travel to or residence in a JEV endemic area or an area with ongoing JEV transmission, among others)^1-5and
no history of vaccination against JEV

Most individuals infected with JEV remain asymptomatic. Symptomatic individuals may develop an acute febrile illness within 14 days of exposure, which may progress to a severe neuroinvasive disease¹.

Serology testing is the preferred method to detect infections caused by JEV^1-5. Molecular testing for JEV is not routinely recommended. It may be used to assist with the identification of an acute infection in some individuals (e.g., immunocompromised). 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
Japanese 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)
Japanese Encephalitis PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Serum	400 µl	Red top or Serum Separator Tubes (SST)
Japanese Encephalitis PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Other specimen types (e.g. plasma, CSF, tissues)	400 µl	EDTA/lavender top tube (plasma) Sterile container (other specimens)

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.

If submitting CSF, a paired serum specimen must also be submitted.

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 may be subject to cancellation.

Testing for JEV PCR must be approved by a PHO Microbiologist. Submission of the mandatory Vector-borne and Zoonotic Virus Testing Intake Formwill 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 clotted blood or serum specimens for serology should be collected 2-3 weeks apart from patients with clinical illness^{1, 3-5}.

Molecular (PCR):
Specimens for JEV PCR testing should be collected as soon as possible after the onset of symptoms^{3, 4} unless otherwise indicated after 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 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)

This is a Referred Out Test. Specimens are referred to the NML in Winnipeg.

TAT for Serology and molecular (PCR) testing is up to 28 days from receipt at PHO.

Test Methods

All JEV testing is performed at the NML.

Serology:
Screening testing for total antibodies to JEV (IgM/IgG) is performed using a Hemagglutination Inhibition Assay (HI). Confirmatory serology testing is performed by Plaque Reduction Neutralization Testing (PRNT)¹.

Molecular (PCR):
Molecular testing is performed by reverse transcriptase polymerase reaction (RT-PCR) on a case-by-case basis¹ if approved by a PHO Microbiologist. The NML indicates that their laboratory-developed test has not been fully validated/verified¹.

Algorithm

Serology:
Serum specimens submitted for JEV serology are first screened for antibodies to JEV by HI. Specimens that are negative by HI are not tested further. Specimens that are HI positive are reflexed to PRNT for confirmation.

Note: Due to the cross-reactivity of anti-Flaviviridae antibodies, PRNT for other arboviruses may also be performed by the NML on JEV HI reactive specimens to assist with result interpretation.

Molecular (PCR):
Molecular testing is performed on a case-by-case basis if approved by a PHO Microbiologist.

Interpretation

Serology:
An HI reactive/positive (POS) result from the NML indicates that anti-JEV antibodies (IgM/IgG) may be present in the specimen and requires further confirmation^{1, 5}. A corresponding positive PRNT result indicates that the patient’s serum contains neutralizing antibodies to JEV.

An HI negative (NEG) result from the NML indicates that antibodies to JEV were not detected¹. PRNT is not performed in these situations. Depending on the time elapsed between symptom onset and collection of the specimen, this may be due to the delayed development of an antibody response.

Additional notes on JEV serology:

Reactive serology results may represent recent or prior exposure to JEV or a related Flavivirus due to the cross-reactivity of anti-Flaviviridae antibodies. Reactive serology results may also occur due to a previous vaccination against a relevant Flavivirus.

Molecular (Real-Time PCR):
A positive (POS) RT-PCR result from the NML indicates that JEV nucleic acids were detected in the specimen and is suggestive of an acute/recent infection. Additional confirmation may be required¹.

A negative (NEG) RT-PCR result from the NML indicates that JEV nucleic acids were not detected in the specimen. This should not be used to rule out an acute infection due to the lack of sensitivity of this assay due to the characteristics of JEV viremia⁵.

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.

Specimens that are positive for Japanese encephalitis virus are reported to the Medical Officer of Health as per the Health Protection and Promotion Act.

References

National Microbiology Laboratory (NML). Arbovirus, Rabies, Rickettsia and Related Zoonotic Diseases. 2024. Available from: https://cnphi.canada.ca/gts/laboratory/1020
Public Health Agency of Canada (PHAC). Japanese encephalitis vaccine: Canadian Immunization Guide for Health Professionals. 2024. Available from: https://www.canada.ca/en/public-health/services/publications/healthy-living/canadian-immunization-guide-part-4-active-vaccines/page-11-japanese-encephalitis-vaccine.html
World Health Organization (WHO). Manual for Laboratory Diagnosis of Japanese Encephalitis Virus Infection. 2007. Available from: https://cdn.who.int/media/docs/default-source/immunization/vpd_surveillance/lab_networks/manual-lab-diagnosis-je.pdf?sfvrsn=e2b62a35_4
Centers for Disease Control and Prevention (CDC). Japanese Encephalitis Virus. 2024. Available from: https://www.cdc.gov/japanese-encephalitis/index.html
Hills S, Lindsey N, and Fischer M. Japanese Encephalitis. In CDC Yellow Book 2024. 2023. Available from: https://wwwnc.cdc.gov/travel/yellowbook/2024/infections-diseases/japanese-encephalitis#diagnosis

*Uncontrolled print copy. Valid only on day of print: 05 July 2025.

Contact Laboratory Customer Service

Microbiology and Laboratory Services

customerservicecentre@oahpp.ca

Updated 2 July 2025