JPC SYSTEMIC PATHOLOGY
NERVOUS SYSTEM

April 2020
N-V19

 

Signalment (JPC #2839005):  13 year-old female thoroughbred, equine

 

HISTORY:  The mare was vaccinated for West Nile Virus when 4-months pregnant. She developed sudden onset of neurologic disease with flaccid paralysis of the tongue, lips, and jaws, and was unable to swallow. Due to poor prognosis, the horse was euthanized.

 

HISTOPATHOLOGIC DESCRIPTION:  Brainstem: Multifocally affecting the meninges, the gray matter, and to a lesser extent the white matter, there is perivascular cuffing with low to moderate numbers of lymphocytes, plasma cells, macrophages, and fewer neutrophils that expand Virchow-Robin space up to 10 times normal and often extend into the adjacent neuroparenchyma. Within gray matter, neurons are often markedly swollen with eosinophilic cytoplasm (degeneration), have clearing of the cytoplasm with peripheralization of Nissl substance (central chromatolysis), or are shrunken and angular with hypereosinophilic cytoplasm and nuclear pyknosis (necrosis). Affected neurons are occasionally surrounded by glial cells (satellitosis), with multifocal neuronophagia.  Multifocally, there are increased numbers of microglia and astrocytes (gliosis) and glial nodules with mild necrosis and spongiosis, mild perivascular edema, multiple small areas of hemorrhage, and occasional swollen axons (spheroids) within the white matter. 

 

MORPHOLOGIC DIAGNOSIS:   Brainstem:  Meningoencephalitis, perivascular, lymphohistiocytic, multifocal, moderate, with neuronal degeneration and necrosis, spheroids, mild hemorrhage, and gliosis, thoroughbred, equine.

 

ETIOLOGIC DIAGNOSIS:  Flaviviral encephalitis

 

CAUSE:  West Nile virus

 

GENERAL DISCUSSION:

·    West Nile virus (WNV), an arbovirus of the family Flaviviridae, genus Flavivirus, is an increasingly widespread cause of fatal disease in humans, horses, and birds

·    Originally discovered in 1937 in Uganda; first appearance in New York in 1999

·    Transmitted primarily via mosquito bites; also by ingesting infected prey (bird), and direct contact through open cuts (zoonotic potential)

·    Mosquitoes are considered the primary vectors in the US; thus, infections are seasonal in summer and fall

·      The virus is disseminated among animal populations by a variety of  Culex mosquitoes

·      Ticks are capable of harboring the virus

·    Several bird species, including corvids (crows, magpies, blue jays), other passerine species (house sparrows, grackles), shorebirds (gulls), raptors (hawks, owls), and flamingoes are highly susceptible to infection; domestic poultry and psittacines are generally resistant to fatal infections

·    Crows are the amplifying host; American robins also propagate and disseminate the virus

·    Birds are the reservoir for sylvatic transmission (bird – mosquito – bird cycle), with humans and horses becoming infected during the urban transmission cycle,  and are considered dead-end hosts

·      Birds: Virus distributed to call organs

·      Horses: Highly susceptible; virus mainly limited to CNS (dead-end)

 

PATHOGENESIS:

·      Mammalian / dead-end hosts: Virus injected by mosquito > propagates in keratinocytes, cutaneous dendritic cells/Langerhans cells, regional endothelial cells, and fibroblasts > spreads to lymph nodes, then viscera > viremia > virus enters brain hematogenously (crosses the BBB), or potentially through retrograde axonal transport > encephalitis with targeting of neurons and microglial cells (endothelial targeting not reported in horses, although it occurs in birds)

·      Monocytes and macrophages are important cellular targets and contribute to systemic spread

·      Macrophage dysfunction and infection of connective tissues may contribute to triggering mediators of the coagulation system, resulting in hemorrhages; there is no apparent targeting of endothelial cells by the virus   

·      Viral envelope contains two membrane-anchored glycoproteins, E2 used to attach to target cell, and E1 used to enter cell via endocytosis (glycoprotein E may confer invasiveness)

·      Toll-like receptor 3 (TLR3)-dependent inflammatory response to WNV infection is involved in BBB breakdown and subsequent viral infection of the brain and neuronal necrosis

·      Caspase 3-dependent apoptosis of target cells may play a role in pathogenesis in some species (i.e. experimentally infected mice and hamsters)

 

TYPICAL CLINICAL FINDINGS:

·    Weakness, recumbency, ataxia, anorexia, tremors, abnormal head posture, circling

·    Severity of clinical signs does not correlate with histologic findings

 

TYPICAL GROSS FINDINGS:

·      Usually absent

·      Acute infection: Occasionally, areas of hyperemia, hemorrhage, or malacia of thoracic and/or lumbar spinal cord

·      Horse: Lesions predominantly affect the lower brainstem and ventral horns of thoracolumbar spine; few other gross lesions

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:

·    Lesions primarily in brainstem and thoracolumbar spinal cord, and less often in the cerebral cortex and cervical spinal cord; extraneural lesions do not occur in horses

·    Nonsuppurative encephalomyelitis, with gliosis, glial nodule formation, neuronal degeneration and necrosis, ring hemorrhages, axonal degeneration and spheroid formation

·      Lesions more pronounced in gray matter

·      Lesion severity variable and may only consist of mild perivascular cuffing in the brainstem

 

ULTRASTRUCTURAL FINDINGS: 

·    35-45 nm diameter virions, with dense core surrounded by thin, diffuse outer layer

·    Virions within cytoplasmic vacuoles, less frequently in rough endoplasmic reticulum (rER)

·    100 nm smooth membrane vesicles (SMV) in dilated rER and vacuoles

·    Marked vesiculation and vacuolation of the cytoplasm, with disorganization of the rER and Golgi apparatus

 

ADDITIONAL DIAGNOSTIC TESTS:

·      In horses, antigen is scarce in CNS lesions (unlike birds); no extraneural antigen

·      Immunohistochemistry: Labeling may be sparse, found in cytoplasm of neurons, nerve fibers, and/or glial cells

·      In situ hybridization

·      PCR, brainstem: Preferred method of diagnosis in combination with histo findings

·      IgM capture ELISA (MAC-ELISA)

·      Virus isolation (requires Biosafety level 3 lab)

 

DIFFERENTIAL DIAGNOSIS:

Microscopic differentials for brain and spinal cord lesions:

·      Eastern equine encephalitis (EEE) (Togaviridae; Alphavirus; N-V09)

·      Rabies (N-V06)

·      Equine protozoal meningoencephalomyelitis (N-P01)

·      Equine herpesvirus-1 (alphaherpesvirus)

 

COMPARATIVE PATHOLOGY: 

WNV in Birds:

·      Affects many species, including water fowl, raptors, passerines, psittacines; lesions/susceptibility variable between species and age groups

·      American singer canaries: a recent study (experimental infection) determined that these are highly susceptible to WNV, even moreso than crows; the most common gross lesions were liver pallor and splenomegaly; histologic lesions were most severe in the liver, spleen and kidney (Hofmeister, Vet Pathol 2018)

·      Ruffed grouse: a recent study (experimental infection) determined that these birds are susceptible; they developed multifocal epicardial to diffuse myocardial pallor; histologic findings included severe, multifocal to coalescing, often perivascular, lymphoplasmacytic myocarditis with degeneration, necrosis, and mineralization of myofibers; all surviving birds seroconverted (Nemeth, Vet Pathol 2017)

·      Clinical signs:  Sudden death without premonitory signs, especially in crows 

·      Gross lesions:

·      Multiorgan hemorrhage:  myocardium (along with pale streaks/necrosis), intraosseous within the calvaria, intestinal mucosa (along with enterocolitis), kidney (or congestion), lungs (or congestion), one report of hemorrhagic tracheitis

·      Cerebral necrosis with hydrocephalus ex vacuo

·      Splenitis/splenomegaly, pancreatitis, adrenalitis, nephritis, hepatitis, Endophthalmitis and optic neuritis

·      Diffuse lymphoid necrosis

·      Microscopic findings:

·      Hemorrhage of the brain and meninges, especially cerebellum and lower brain stem, and ventral horns of the thoracolumbar spinal cord

·      Lymphoplasmacytic encephalitis with gliosis and astrocytosis, especially in the cerebrum

·      Lymphoplasmcytic pectenitis and choroiditis 

·      Necrotizing myocarditis

·      Other sites: Lymphoplasmacytic hepatitis and lymphoplasmacytic and histiocytic inflammation

·      Splenic and bursal lymphoid depletion

·      Pulmonary perivascular edema and hemorrhage

·      Red-tailed hawks and related species: Ocular lesions have been reported and include:  Pectenitis, choroidal/retinal lymphoplasmacytic inflammation, retinal degeneration and necrosis; more pronounced choroiditis with retinal collapse, atrophy and scarring (chronic) (Wunschmann A, Vet Pathol. 2017)

·      In the bird, the kidney, heart, brain, and spleen most consistently harbor WNV antigen but there is species variability; another study reported the heart, brain and spinal cord as being the most effected tissues (in the bird, the heart is one of the main target organs)

·      Neurons, glial cells AND endothelium may contain viral antigen in the CNS (also endothelium in heart)

·      Differentials for WNV include: Exotic Newcastle’s disease (Paramyxoviridae; Rubulavirus, N-V10); highly pathogenic avian influenza (Orthomyxoviridae; Influenza virus A, D-V25)

·      WNV reported in zoo / exotic species (Pathology of Wildlife and Zoo Animals, 2018):

·      Penguins: High rates of morbidity and mortality in free-range and zoo penguins during WNV outbreaks; gross lesions are not typically observed; histologic findings include cerebellar lymphoplasmacytic meningoencephalitis, mild gliosis, and satellitosis; frequent IHC-positive neurons, Purkinje cells, granular and glial cells

·      Cormorants and pelicans: WNV-related die-offs have been reported; histologic lesions in cormorants include lymphohistiocytic myocarditis, mineralization and hemorrhage; neuronal vacuolation, meningeal or neuropil hemorrhage; splenic lymphoid depletion with splenomegaly; hepatocyte and intestinal crypt necrosis

·      Flamingoes: WNV causes significant disease with widespread mortality; gross lesions (if the bird does not die before changes develop) include  pallor in heart, liver, spleen, and congestion in the brain and leptomeninges; histologic lesions are often absent, or may include necrotizing myocarditis with nonsuppurative inflammation, with similar lesions in the spleen and liver; neural changes are variable and range from lymphoplasmacytic perivascular cuffing to regions of malacia and parenchymal collapse

·      Several groups of raptors are susceptible to WNV, including hawks, eagles, barn owls, great horned owls, barred owls, falcons (though rare); disease varies between the groups; gross lesions are often absent; histologic changes most often occur in the heart, brain, spinal cord, liver, and spleen

 

WNV in Other Species:

·      Ruminants, dogs, cats, and pigs:  Less susceptible to WNV disease than horses; typically develop subclinical disease and brief viremia; if present, histologic findings are similar to the horse

·      Eastern fox squirrels: Lymphoplasmacytic inflammation in kidney, brain, heart, and liver

·      Arctic wolf: Severe renal lymphoplasmacytic vasculitis and multifocal cerebral cortical necrosis and gliosis

·      American alligators: Heterophilic to lymphoplasmacytic meningoencephalitis, necrotizing hepatitis, splenitis, pancreatitis, myocarditis, stomatitis, and glossitis

·      May serve as an amplifying host; develop high viremia and shed virus in feces

·      Natural infections with subsequent encephalitis have also been reported in bats, a chipmunk, a skunk, a domestic rabbit, reindeer, a white-tailed deer, gray squirrels, an alpaca, a Suffolk ewe, a Barbary macaque, a cat, and several dogs

·      Sheep: Lymphoplasmacytic meningoencephalitis with severe perivascular cuffing, and myelitis, with brain tissue positive for WNV in neurons and axons and less in glial cells; was the most common cause of viral encephalitis in sheep at CAHFS lab between 2002-2014

·      High viral antigenic load in some cases (unlike horses)

·      Differential diagnosis for viral lymphocytic encephalitis in sheep includes: bluetongue, SRLV, rabies, pseudorabies, Borna disease and Nipah virus infection

·      Mice, hamsters, and Rhesus macaques have been infected experimentally

·      Gastric and small intestinal lesions (distension) reported in mice, in addition to CNS – neuron lesions

 

Arboviruses:

·      Alphavirus (Togaviridae): EEE virus, Western equine encephalitis virus, Venezuelan equine encephalitis virus

·      Flavivirus (Flaviviridae): WNV, Japanese encephalitis virus, St. Louis encephalitis virus, Dengue, Yellow fever, Tick-borne encephalitis virus

·      Bunyavirus (Bunyaviridae): California encephalitis virus, LaCrosse virus

·      Phlebovirus (Bunyaviridae): Rift Valley fever virus, Sandfly fever virus

 

References:

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