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West Nile Virus Diagnostic Testing

West Nile virus (WNV) is a member of the Flaviviridae family in the genus Flavivirus. Other members of the genus include St. Louis encephalitis virus, dengue virus, Zika virus and yellow fever virus. The hallmark of this group of viruses is their transmission by various species of mosquitoes. Up until 1999, WNV was considered an "Old World" virus, but in that year its introduction into the US in the New York City area triggered an epizootic that resulted in the spread of the virus to all 48 contiguous states. Currently, the virus is enzootic in much of North and South America. The virus is maintained in a mosquito – bird – mosquito cycle with spill over into other animals such as reptiles and mammals being incidental to the life cycle of the virus. The virus persists in passerines but infection does occur in other bird species such as raptors and corvids. Over 300 species of birds have been documented as having been infected with WNV. A recent publication indicates that WNV continues to exert negative impacts on the populations of certain bird species such purple finches, wrentits, and white-crowned sparrows.

The most significant impact of WNV in veterinary medicine excluding wildlife is the incidental infection of equines which can lead to viral encephalitis. Even though a viremia develops early in the infection of the horse, the amount of virus in blood is too low to infect mosquitoes. WNV can infect other animals such as dogs and cats, but clinical signs do not usually develop.

Testing strategies

The type of tests and timing of the sampling for diagnosing WNV infections depends on the species of the test subject:

Avian

Agent detection

RT-PCR testing
  • Whole blood – EDTA preferred (DO NOT use heparin for anti-coagulant as heparin is inhibitory in PCR assays).
  • Oropharyngeal or cloacal swabs preferably in small volume of transport medium or saline (oral swabs may not be appropriate for all bird species such as raptors).
  • Fresh tissue samples: brain, heart, kidney, liver, spleen, bone marrow.
  • Formalin-fixed tissue: same sample set as fresh tissue (a surcharge will be applied for the additional costs related to extraction of formalin-fixed tissues).
Virus isolation
  • Virus isolation can be performed on all samples suitable for PCR EXCEPT formalin- fixed tissues.

Antibody detection

  • Preferred sample is serum. Can be done with plasma, but toxicity due to the presence of anticoagulant may prevent accurate antibody titer determination. As with most serological assays, paired samples are needed for an accurate assessment of a recent infection (or vaccination).
  • Plaque-reduction neutralization assay (PRNT). Traditional test used for flavivirus antibody detection where cross- reactivity with other viruses may occur, e.g. St Louis encephalitis virus.

Mammals, Reptiles, and Amphibians

Agent detection

RT-PCR testing
  • Fresh tissues: brain, spinal cord, CSF.
  • Formalin-fixed tissue: same sample set as fresh tissue (a surcharge will be applied for the additional costs related to extraction of formalin-fixed tissues).
  • Whole blood from equine species can be used for WNV detection, however, the levels and duration of viremia are low, which may affect the diagnostic sensitivity of this specimen. Therefore, timing of collection is critical and the virus may be undetectable by the time clinical signs are noted. 
  • Whole blood from other mammals, reptiles, and amphibians is NOT a standard sample for agent detection due to the low levels of viremia produced during infection.
Virus isolation
  • Virus isolation can be performed on all samples suitable for PCR EXCEPT formalin fixed tissues.

Antibody detection

  • Preferred sample is serum. Can be done with plasma but toxicity due to the presence of anticoagulant may prevent accurate antibody titer determination. As with most serological assays, paired samples are needed for an accurate assessment of a recent infection (or vaccination).
  • Plaque-reduction neutralization assay (PRNT) (ALL MAMMALS, REPTILES, AMPHIBIANS)
    • Traditional test used for flavivirus antibody detection where cross-reactivity with other viruses may occur, e.g. St Louis encephalitis virus.
  • Microplate serum (virus) neutralization assay (SN) (EQUINE AND CANINE ONLY)
    • Microplate test gives titer values 2-4 fold higher than the plaque reduction assay and is more economical. It is recommended that samples with low microplate virus neutralization assay titers (=<16) should be tested by the PRNT assay.
  • Antibody capture ELISA tests (EQUINE ONLY)
    • IgM ELISA test. (serum, CSF, and plasma by special request). Test is used to detect an early immune response due to infection. Test interpretation must take into account vaccination status of the animal.
    • IgM and IgG ELISA testing panel. (serum, CSF, and plasma by special request). Test is used to define the infection or vaccination status of the horse. Paired serum samples may be necessary to define the infection status of a vaccinated horse.

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