Pennsylvania Animal Diagnostic Laboratory System
PADLS Conferences Spring 2001 16. Detection of West Nile flavivirus
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The Flaviviridae family includes arboviruses able to induce fatal encephalomyelitides in several species of vertebrates. These viruses are primarily transmitted and amplified between avian reservoir hosts by many species of mosquitoes in enzootic transmission cycles. West Nile flavivirus (WNV) has caused sporadic cases and outbreaks in humans, horses and birds with fatalities.^1,2,3 We developed an indirect immunohistochemical technique to detect flavivirus antigen in formalin fixed paraffin embedded tissues for diagnosis and pathogenesis studies.

We examined CNS tissues from 4 horses, 2 crows and 1 goose with natural WNV infection and brain from mice inoculated intracerebrally with WNV. Avian myocardium was also examined. WNV infection was previously confirmed by virus isolation, identification with RT-PCR and serological examination. Representative tissue samples of major organs and the whole CNS were fixed in 10% neutral buffered formalin solution and processed for histology and immunohistochemistry. Tissue sections were stained with hematoxylin and eosin, periodic acid-Schiff, Luxol fast blue and crystal violet for Nissl substance and were processed with indirect immunoperoxidase staining using an automated slide processing unit (Autostainer Immunostaining System S3400, Carpinteria, DAKO Corp., CA, USA). Tissue sections were mounted on Probe-On (Sigma Diagnostics, Missouri, USA) charged (+) glass slides and deparaffinized using Hemo-De (Fisher Scientific, New Jersey, USA). Endogenous peroxidases were inactivated with H₂O₂. Tissues were incubated in Proteinase K (DAKO) for 5 min at room temperature. The primary antibody was a murine polyclonal recognizing specific epitopes of West Nile flavivirus, 1:50 in 4% DAKO diluent and was incubated for 30 minutes at room temperature. The secondary antibody was a universal goat anti-mouse immunoglobulin conjugated to a peroxidase labeled polymer (EnVision+™, DAKO), which was incubated for 20 minutes at room temperature. Chromogen 3,3-diaminobenzidine-4HCl, was incubated for 3 min at room temperature. The sections were counterstained using Mayer's hematoxylin (DAKO), dehydrated and coverslipped. West Nile virus free avian and mammalian tissues were used as negative controls, as were equine nervous tissues containing rabies virus, encephalitic togaviruses and equine herpesvirus 1. Paired tissue samples were also stained using a primary mouse antibody nonspecific for WNV against other equine viruses including equine herpesviruses and eastern equine encephalitis. The absence of nonspecific binding of the secondary antibody was also evaluated by omitting the primary antibody.

Histological changes in horses included mild to severe, multifocal lymphocytic polioencephalomyelitis associated with moderate to severe hemorrhage in some cases, scattered foci of microgliosis, neuronal necrosis and occasional neuronophagia. In the avian tissues, moderate meningoencephalitis with hemorrhage and severe myocarditis with necrosis were observed. In mice there was mild encephalitis. In the horses, in sites of inflammatory lesions, WNV was mainly localized within the gray matter and had a finely granular appearance within the cytoplasm of a few morphologically normal and degenerate neurons. WNV was also present in a large number of morphologically normal nerve fibers, axonal hillocks, glial cells, in addition to spheroids, of the medulla oblongata and spinal cord in all horses. In the glial foci and areas of neutrophilic infiltration, small amounts of WNV were contained within the cytoplasm of glial cells, macrophages, and occasional neutrophils. Occasionally, morphologically normal neurons diffusely harbored intracytoplasmic WNV and viral antigen-positive glial cells also surrounded some neuronophagic neurons. In avian tissues WNV had a similar localization but was present in large quantities, especially within the cardiac myocytes and macrophages. The mouse brains contained moderate amounts of WNV within neuron cells dendrites and axons.

In horse CNS, the quantity of WNV antigen was scant when compared to the extent of the inflammatory lesions and sometimes we needed to examine multiple sections to detect positive signal. Conversely, in birds and mice the inflammatory lesions were very mild, whereas peroxidase immunohistochemistry detected large amount of WNV. This indirect immunohistochemistry technique on formalin-fixed paraffin-embedded tissues is rapid and inexpensive and can be used to detect flavivirus infection in the species we examined, especially when no fresh tissues for virus isolation and RT-PCR are available or when virus isolation and identification attempts on fresh tissues are unrewarding.

We still need to investigate the cross-reactivity of this antibody to other flaviviruses. WNV infected avian tissues contain large quantities of WNV and are the most suitable samples for positive controls.

References:

1. Cantile C., Di Guardo G., Eleni C., Arispici M.. Equine Veterinary Journal 32:31-35, 2000
2. Steele K.E., Linn M.J., Schoepp et. al.. Veterinary Pathology 37:208-224, 2000
3. Cantile C., Del Piero F., Di Guardo G. Arispici M. Veterinary Pathology 38 (4) 2001 (in press).

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