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Detection of Antibodies Directed Towards West Nile Virus by PRNT

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Serological detection of neutralizing antibodies directed towards West Nile Virus (WNV) by Plaque Reduction Neutralization Test (PRNT).

Test Category:
West Nile virus (WNV)
Illnesses and Diseases:
  • West Nile encephalitis

Serum. Minimum volume of 200 µl required.

Collection Method:

2 mL screw cap tubes.

Specimen Processing, Storage and Shipping:

Store samples refrigerated or frozen until shipped for testing. Ship frozen samples on dry ice, and refrigerated samples on wet ice

Transportation of Dangerous Goods:

Shipping of specimens shall be done by a TDG certified individual in accordance with TDG regulations. For additional information regarding classification of specimens for the purposes of shipping, consult either Part 2 Appendix 3 of the TDG Regulations or section 3.6.2 of the IATA Dangerous Goods Regulations as applicable.

For additional guidance on the transport of infectious substances in other languages, please click on the link below.


Patient Criteria:

Suspected WNV infection. Submitted samples will first be analysed by the Viral Zoonosis Diagnostic Laboratory by either WNV HAI and/or WNV IgM/IgG ELISA to determine if the patient has developed anti-flavivirus antibodies. Samples that are reactive can be further analysed for the presence of neutralizing antibodies. Samples that are HAI and/or ELISA non reactive (negative) will not be tested by PRNT. This is not a routine test. Please contact the Viral Zoonosis Diagnostic Laboratory before sending specimens.

Accompanying Documentation:

Completed Viral Zoonoses requisition including sender laboratory name, address and telephone number. Patient name and / or identifier (specimen reference number), date of birth, test(s) requested, date of on-set of symptoms, and clinical and travel history of patient.


Initial screening serology such as WNV ELISA must be reactive before samples will be considered for PRNT testing. 

Methods and Interpretation of Results:

Antibodies directed towards WNV can cross react significantly in some serological assays with other flaviviruses (dengue, Powassan etc). The PRNT is a more specific assay and can be used to document the presence of serum antibodies specific for a particular flavivirus.  Serum samples are incubated with virus and if viral neutralizing antibodies are present, they will bind to WNV and prevent viral infection of cultural cells, and hence a reduction in the number of plaques detected.  The neutralizing titre of a sample is expressed as the reciprocal of the serum dilution at which there is a 90% reduction in the number of plaques detected.  If the patient has experienced more than one flavivirus infection, cross reactive results may yield uninterpretable results with this assay despite increased specificity.

Due to the cross-reactive nature of anti-flavivirus antibody, the detection of anti-flavivirus IgG (Eg. JE, DEN, etc.) in a single sera is indicative of past or present exposure to this agent, or a related agent from the same virus genus. The presence of WNV- specific IgM in a single serum sample is consistent with an acute infection with this virus (or a related flavivirus, note that anti-flavivirus IgM serological procedures are more specific than IgG serology for these viruses) and meets the criteria for a "probable case". However, a 4-fold rise or greater in neutralizing antibody titre, or an IgG or IgM seroconversion in paired sera, is required to document a "confirmed case" of infection with associated illness.   

There is increasing evidence for IgM persistence in blood/serum for up to a year or more after arbovirus (i.e. members of the flavivirus, alphavirus, and bunyavirus arthropod-borne virus groups) exposure.  Thus, detection of IgM by itself may not always be a confirmation of acute infection.

Isolation of an arbovirus, detection of specific antibody by plaque reduction neutralization assay or detection of nucleic acid by Real Time RT-PCR in a clinical specimen would constitute firm evidence of viral association with illness and provide "confirmed case" status.

Turnaround Time:

14 calendar days after the completion of ELISA testing.

Phone #: (204) 789-6097 or (204) 789-2106
Fax: (204) 789-2082
  1. WHO. 2007. Guidelines for plaque reduction neutralization testing of human antibodies to dengue viruses.