217Chapter 21 Flavivirus Encephalitides

21 

Flavivirus EncephalitidesGretchen Henry Delcambre and Maureen T. Long

C H A P T E R 

Infections caused by Flaviviridae family viruses have made an impact on human health throughout history. Yellow fever virus (YFV), now eradicated from North America, is a centuries old arthropod-borne virus (arbovirus) that in Africa and South America annually causes morbidity in 200,000 people with an estimated 30,000 mortalities.1 Seventy-five percent of people who contract hepatitis C virus (HCV) from contact with

infected blood will develop severe chronic liver disease.2 This family of viruses had little documented recent impact on the domestic equine until the debut of West Nile virus (WNV) in the United States in 1999. With significant morbidity and mor-tality associated with the disease in human, avian, and equine populations, the Flavivirus genus has had a large spotlight of interest cast on it in both the clinical and research fields.

218 Section 2 Viral Diseases

Table 21-1 PartialTaxonomicStructure,PrimaryVector,Hosts,GeographicDistribution,andVeterinarySignificanceofGenusFlavivirus,FamilyFlaviviridae*

Serologic, Virus Group Virus Species Primary Vector Amplifying Hosts Location Veterinary Diseases

Tick-Borne VirusesTick-borne

encephalitis†Kyasanur forest disease Haemaphysalis spinigera Blanford’s rat, striped

forest squirrel, house shrew

India Mortality in nonhuman primates

Langat Haemaphysalis spp., Ixodes spp.

Rats Russia, Southeast Asia

Louping Ill Ixodes ricinus Sheep, grouse Iberian peninsula, United Kingdom

Ovine encephalitis and mortality, equine encephalomyelitis, canine central nervous system signs

Omsk hemorrhagic fever Dermacentor spp. Muskrats, voles Russia Mortality in birdsPowassan Ixodes spp., Dermacentor

spp., Haemaphysalis spp.

Lagomorphs, rodents, mice, skunks, dogs, birds

North America, Russia Experimental equine, canine encephalitis

Royal Farm Family Argasidae Small mammals, likely small ruminants

Afghanistan

Tick-borne encephalitis Ixodes ricinus, Ix. persulcatus

Small rodents Asia, Europe, Finland, Russia

Neurologic equine, canine, and primate cases described

Seabird tick-borne

Meaban, Saumarez Reef, Tyuleniy

Ixodes spp., Family Argasidae

Sea birds Africa, Australia, France, Norway, Russia, United States

Mosquito-Borne VirusesAroa Aroa Rodents VenezuelaDengue Dengue Aedes spp. Humans, nonhuman

primatesAsia; Africa; Caribbean;

North, Central, and South America

Japanese encephalitis

Japanese encephalitis Culex tritaeniorhynchus Birds, swine Asia, India, Russia, Western Pacific

Equine encephalitis, Abortion in swine

Koutango Culex spp. Senegal, Central African Republic

Murray Valley fever Culex annulirostris, Aedes normanensis

Birds, horses, cattle, marsupials, and foxes

Australia, Papua New Guinea

Equine encephalitis‡

St. Louis encephalitis Culex spp. Birds North, Central, and South America

No overt clinical signs

West Nile Culex spp. Passerine birds (crows, sparrows, robins)

Africa; Middle East; Europe; North, Central, and South America; Australia

Equine, Camelid, Ovine encephalitis

Kokobera Kokobera Culex annulirostris Macropods, horses?§

Mosquito-borne Ilheus, Sepik Psorophora ferox, Aedes aegypti, Ae. serratus

Birds Central and South America

Yaoundé Yaoundé Culex spp. BirdsNtaya Braganza, Israel turkey

meningoencephalitis, Ntaya, Tembusu, Yokose

Culex spp. Birds, small ruminants Central and South Africa

Spondweni Zika Aedes. spp., Mansonii spp.

Unknown; nonhuman primates

Africa, India, South Asia No overt clinical signs

Etiology

The Flaviviridae family consists of three distinct genera: Flavi-virus, Hepacivirus, and Pestivirus.3 The type species of these genera are YFV, HCV, and bovine viral diarrhea virus 1, respec-tively. Among the 53 species of Flavivirus, there are a number of historically significant and pathologically active viruses responsible for diseases including Japanese encephalitis (JE), dengue fever, tick-borne encephalitis (TBE), and West Nile encephalitis (WNE).4 Although some of these viruses are

transmitted directly or have an unidentified vector, the majority of them have a known tick or mosquito vector. About one-fourth of all flaviviruses are of veterinary importance, and about half of all members of Flaviviridae are zoonotic.

The members of the JE serogroup that are most likely to cause overt disease in horses are JE virus (JEV) and WNV. Disease in horses caused by Murray Valley fever (MVF) virus is geographically restricted to the South Pacific and is sporadic in occurrence.5-7 Several other members of the Flavivirus genus have been detected serologically in horses, but with limited reports of clinical disease (Table 21-1). Experimental

219Chapter 21 Flavivirus Encephalitides

Serologic, Virus Group Virus Species Primary Vector Amplifying Hosts Location Veterinary Diseases

Yellow fever Yellow fever, Banzi, Bouboui, Edge Hill, Uganda S, Wesselsbron

Aedes spp., Haemagogus spp., Sabethes spp.

Humans, nonhuman primates

Africa, Central and South America

Clinical disease in nonhuman primates

No Known VectorModoc Cowbone Ridge, Jutiapa,

Modoc, Sal Vieja, San Perlita

Unknown Rodents Western United States

Rio Bravo Apoi, Bukalasa, Carey Island, Dakar, Entebbe bat, Rio Bravo, Saboya

Unknown Bats United States, Mexico

Data checked with http://www.publichealthassociate.com. Accessed through the veterinary information network (vin.com).

*There are many classified and unclassified Flaviviruses omitted from this table for simplicity. Refer to www.ncbi.nlm.nih.gov/taxonomy to explore the most up-to-date information on taxonomic structure and virus subspecies of the Flavivirus genus.†Data from Dobler G: Vet Microbiol 140(3-4):221–228, 2010.‡Data from VET Watch, April 2011. Department of Primary Industries, Victoria, Australia. Accessed at http://www.dpi.vic.gov.au/agriculture/pests-diseases-and-weeds/animal-diseases/vetsource/vetwatch/vet-watch-april-2011.§Serologic evidence only.

Table 21-1 PartialTaxonomicStructure,PrimaryVector,Hosts,GeographicDistribution,andVeterinarySignificanceofGenusFlavivirus,FamilyFlaviviridae—cont'd

Figure 21-1 Idealized genomic organization of members of the genus Flavivirus. This is a positive-sense, single-stranded RNA virus consisting of two structural and seven nonstructural proteins.

5′UTR 3′UTRSTRUCTURAL NONSTRUCTURAL

C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5

pr M

reproduction of disease in horses must still be established for many of these viruses. The following discussion emphasizes WNV and JEV.

As with other Flaviviridae members, WNV and JEV are positive-sense, single-stranded ribonucleic acid (RNA) viruses8 measuring approximately 50 nm in external diameter.9 The virions are spheric and enveloped and contain a nucleocapsid composed of a capsid protein approximately 120 amino acids in length.10 Electron microscopy reveals an icosahedral sym-metry of the envelope and capsid. An approximately 11-kb genome contains a single open-reading frame (ORF) that is translated in its entirety and cleaved into 10 viral proteins by both cell and viral proteases11,12 (Fig. 21-1). There are three structural proteins, capsid (C), premembrane (prM)/membrane (M), and envelope (E), and seven nonstructural (NS) proteins. The NS proteins are cleaved after translation into NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 and are required for viral replication and assembly.11

The C, M, and E proteins are important in virulence for the JE group.13-18 Although not understood entirely, C protein is essential in virion assembly,19 and large deletions in its sequence renders the WNV virus nonvirulent.20 The M protein is formed from a precursor protein (prM protein), which is modified as immature virions are secreted through the Golgi network of the cell, leaving the C-terminal portion of the protein inserted in the envelope of the mature virion. Among other things, M protein may play a role in viral replication.21 The E protein is only secreted in its native conformation through association

with the prM protein. This immunodominant viral protein exists in the virion as a β-pleated sheet arranged head to tail, with the distal ends anchored in the membrane. This protein is dimeric, held together with intermolecular disulfide bonds, and lies flat against the lipid bilayer. The large E protein is important in receptor ligand binding and fusion to host cells, the latter being pH dependent. There are three major domains of this protein in WNV. Domain II contains a region important to virus binding in the brain. Domain III is important for vector and host virulence.22 In WNV, glycolysis of the E protein is strain dependent and associated with virulence. Viral binding to gly-cosaminoglycan on cells changes virulence in both JEV and WNV.

The NS proteins in flaviviruses are structurally and function-ally similar and are involved in synthesis of viral RNA.23,24 The glycoprotein NS1 is essential for virus function and appears to be important for cell activation as part of viral synthesis.25-27 The NS1 protein is found on host cell membranes and must interact with NS4A in this process. The proteins NS2A and NS2B are formed by cleavage of full-length NS2. Changes in the C-terminal of this protein results in loss of viral replicative ability. The NS3 protein is highly conserved between flavivi-ruses and, at the N-terminal, encodes a serine protease. The C-terminal of this protein has sequences typical of RNA heli-cases and triphosphatases.28 The NS4b protein appears to block antiviral cytokines.29 The NS5 protein is essential for viral rep-lication by forming the “cap” at the 5′ end of a genome. Viruses, as opposed to eukaryotic cells, have a type I cap at the end of

220 Section 2 Viral Diseases

Ardeidae (herons and egrets) and swine are also important amplifying reservoir hosts. Horses and humans are “dead-end” hosts and do not amplify the virus in quantities sufficient to infect mosquitoes.

Additional modes of transmission have been identified in the North American WNV encroachment. First, transmission through oral ingestion has been proven in both avian and mam-malian hosts, and oral and cloacal shedding has been demon-strated in birds.36-40 Second, WNV may be transmitted through contaminated blood transfusion or organ transplantation if donors are viremic.41-44 Third, vertical transmission through pla-centa and milk has been demonstrated in humans.42,45 This last feature is important in JE and several JE serogroup virus infections.

Vectors

The spread and yearly incidence of JE serogroup viruses coin-cides with the availability of vectors and reservoir hosts with transmission potential. Thus, outbreaks are seasonal and reflect mosquito activity. Culex spp. mosquitoes are the primary vector for the JE serogroup.40,44,46-48 WNV has been detected in 64 species of North American mosquito, but the overall vector efficiency (moderate to high) and wide feeding activity range of Culex spp. indicate that North American WNV outbreaks are propelled mainly by this genus.49 Most of the data support-ing this conclusion is based on vector efficiency studies under laboratory conditions, experimental feeding studies, and fre-quency of identification of WNV in mosquito pools.49,50 In the northeastern United States, more than half of WNV-positive mosquito pools are Culex pipiens.51-55 In the western United States, populations of the highly efficient Cx. tarsalis consti-tute the majority of positive pools, with Cx. pipiens the next most frequent.49,56-58 In the southeastern United States, Cx. quinquefasciatus and Cx. nigripalpus have the highest WNV infection rates.59-63 In the southwestern United States, epidem-ics are most often associated with positive pools of Cx. quin-quefasciatus, Cx. tarsalis, and Cx. pipiens.55,64-67 Culex restuans, frequently identified as part of the “Cx. pipiens” complex, is often one of the top five positive species.49,67 Positive vector species incidence is driven by environmental habitat, which may cause variation from these reported common species.68

Although Culex is important in the epidemiology and spread of WNV, relatively little is known regarding the actual vector of transmission to the horse. Blood meal analysis suggests that

Figure 21-2 Flavivirus replication cycle that demonstrates a first round of virus translation to produce proteins that are cleaved by host and viral proteases. Production of a positive-strand virus by viral proteins results in progeny pack-aged in mature virions. After rounds of translation and replication occur, virus is released by cell lysis or, more often, budding from the infected cell.

5′

5′

5′

3′

3′

3′

5′

3′

Structural

Translation

Cleavage

Cellproteases

RdRP(NS5)

Transcription

Replication

Packaging

Nonstructural

Figure 21-3 West Nile virus (WNV) and Japanese encephalitis (JE) virus life cycle in which the primary transmission cycle is between avian or porcine reservoirs and mosquitoes. Horses and humans are aberrant hosts.

Japaneseencephalitis virus

West Nile virus

VerticalOccupationalOrgan Blood

the genome, which in a cytoplasmic virus, such as WNV, must be formed solely by viral proteins. In addition, this is the site of the viral RNA-dependent RNA polymerase (RdRp), an essen-tial protein for formation of negative-strand RNA from the genome of the positive-strand “parent” RNA virus.29,30

JE serogroup viruses are thought to infect the cell through glycoprotein receptors that are likely highly conserved by hosts.30-32 After receptor-mediated endocytosis, there is fusion of the viral membrane with membranes of the endosomal vesicle, and the nucleoprotein is released into the cytoplasm. After translation, the serine protease NS2B-NS3A, along with cell proteases, cleaves the polyprotein at multiple sites to gener-ate viral proteins (Fig. 21-2). The RdRp copies the negative-strand RNA from the genomic RNA template. These negative-strand RNAs become templates for the synthesis of new genomic RNAs. There are likely alternating periods of replication and translation until a sufficient pool of structural proteins has accumulated. Once there is a pool of genomic RNA, virion assembly occurs in the rough endoplasmic reticu-lum (ER) membranes. Immature virions with the prM protein accumulate in vesicles and are transported through the host secretory pathway, where the E and prM proteins are modified. Virions are transported to the plasma membrane in vesicles and released by exocytosis. Mammalian cells will release progeny virus within 10 to 12 hours after infection.

Epidemiology

Life Cycle and Transmission

Japanese encephalitis serogroup viruses are vector-borne dis-eases, with transmission occurring to avian and mammalian hosts from blood meal–seeking mosquitoes33 (Fig. 21-3). Virus is maintained within vector populations by horizontal or verti-cal transmission. Climate-limited amplification of the virus within a vector occurs in warmer seasons but is minimal during colder temperatures34,35; therefore, reservoir hosts are required for amplification and maintenance of the virus year-round. Typical reservoirs include birds such as passerines. For JEV,

221Chapter 21 Flavivirus Encephalitides

in domestic dogs and cats.36,37 Neurologic disease has been confirmed as natural WNV infection in gray and fox squirrels but has been difficult to reproduce in experimental infection.91 New world camelids and sheep develop neurologic disease with natural exposure to WNV.92-94 Alligators can have an extremely high viremia and may be an important reservoir for WNV in the southeastern United States.90 There are reports of both farmed and free-ranging alligators with neurologic signs from which WNV has been isolated. In farm-raised alligators, cloacal shedding of virus has been demonstrated, with oral infection likely.

Geographic and Seasonal Distribution

The largest documented outbreak of equine neurologic disease caused by a flavivirus began in 1999 with WNV encroachment into the United States, emerging in New York City. In 2002, an epizootic resulted in 14,571 cases of equine WNE and accom-panied an epidemic of 4,156 human cases with 284 resulting deaths.95 Between 1999 and 2006, nearly 25,000 cases of equine WNE were reported in the United States, with an estimated average 30% to 40% case-fatality rate. Positive equine case reporting has decreased to an average of 300 a year from 2007 to 2010.

By 2005, WNV had been identified in all 48 continental U.S. states96 (Fig. 21-4). Canadian provinces reporting disease included Quebec, Ontario, Manitoba, Saskatchewan, and Alberta, with New Brunswick and Nova Scotia reporting evi-dence of WNV-positive birds.97 Serologic evidence of WNV has been reported in the Latin American and Caribbean countries of the Dominican Republic, Mexico, Guadeloupe, El Salvador, Puerto Rico, Cayman Islands, Jamaica, Belize, Cuba, and the Bahamas.40,98-103 As early as 2005, the presence of WNVreached as far south as Argentina. From 2006 through 2010, WNV in general appeared to wane in the United States; however, in 2011 an increase in reporting of human cases occurred, with horse cases remaining between 100 to 300 per year. With 5387 human, 654 equine, and 2436 avian cases, all groups had increased reporting during the 2012 season.

Japanese encephalitis virus annually causes 30,000 to 50,000 human encephalitis cases worldwide, with endemic areas including China, the southeast region of the Russian Federation, South and Southeast Asia, and Australia. Exact numbers of horses with clinical JE are difficult to ascertain; however, there are reports of JE isolation from horses in Taiwan, China, Paki-stan, and Australia in the literature since the 1980s. Outbreaks in horses have also been reported in India, Nepal, the Philip-pines, Sri Lanka, and Northern Thailand with the most recent outbreak in Spain in 2010.104

Occurrence of disease caused by JEV and WNV in horses and humans reflects vector activity, seasonally in temperate regions and year-round in subtropical and tropical regions. Intense virus activity in the United States begins in July, with a peak incidence in September and October.59,105-107 Temperature-dependent spatial modeling supports these disease dynamics, with risk increasing from 25% in late August to greater than 75% by the second week of September.108-110 A drop in ambient temperature with soft frost usually results in a rapid decrease in reporting activity.111,112 The appearance of disease from JE is highly variable, depending on the locale. Seasonal occurrence of disease in specific locales should be considered to facilitate timing of equine athletic events and to tailor vaccination regi-mens appropriately.

Intrinsic Risk Factors

Geriatric humans appear more susceptible to neuroinvasive disease from both JEV and WNV. This age bias in reporting

Cx. pipiens mosquitoes are primarily avian feeders. Mammalian feeders include predominantly Anopheles quadrimaculatus, Coquillettidia perturbans, and Aedes albopictus.69,70 Culex salina-rius, Cx. (Melanoconion) spp., Aedes vexans, Ae. albopictus, Pso-rophora ferox, Ps. columbiae, Coquillettidia perturbans, Anopheles quadrimaculatus, An. crucians, and Ochlerotatus atlanticus were reported engorged with equine blood in a 2-year surveillance study in south Louisiana.71 Culex pipiens, Cx. salinarius, and Ae. vexans are likely the most important bridge vectors, which feed from both reservoir and host.72-74

Experimental infection of horses through mosquito trans-mission studies is accomplished with Ae. albopictus, a common mammalian feeder and moderately efficient vector of WNV.75,76 In studies thus far involving low numbers of horses, 9 of 10 horses become viremic, and all seroconvert to the virus. One out of 11 horses develop neuroinvasive disease. Members of the Cx. tritaeniorhynchus group of mosquitoes are the most important vectors for JE and were used in the experimental transmission and reproduction of disease in horses.

Several species of ticks have been investigated for potential to transmit WNV. Transtadial transmission was demonstrated in one study of Ixodes ticks but failed to occur in a second study.77,78 Carios capensis can transmit WNV under experimental condi-tions to ducklings, and Ornithodoros moubata can transmit WNV under experimentally to mice.78,79

Hosts and Reservoirs

A reservoir host is one in which a pathogen is amplified in vivo so that it can be transmitted to a vector species.53 A blood meal taken from a mammal containing 105 to 107 plaque-forming units per milliliter (PFU/mL) of WNV results in infection of 30% to 100% of feeding mosquitoes, respectively. Humans vol-untarily infected with the Egyptian strain of WNV developed virus titers of 103 to 105 PFU/mL.80 Horses develop titers of 101 to 103 PFU/mL after experimental infection via exposure to WNV-positive mosquitoes.81 Dogs and cats experimentally exposed to WNV produce viremias lower than 104 PFU/mL, but individual cats can develop titers that would permit virus transmission.82 Cats can become infected after oral exposure.

Viral titers capable of transmitting JEV are similar to those for WNV transmission. Swine are a notable reservoir host for JE. Primary clinical manifestations include abortion, birth of weak pigs, and limited neonatal survival. Semen from infected boars contains infectious virus and has decreased sperm count and motility.

To date, more than 300 species of WNV-positive birds have been reported to the WNV Avian Mortality Database from the Centers for Disease Control and Prevention (CDC). High levels of viral amplification occur in many bird species, especially Pas-seriformes (e.g., songbirds) and Charadriiformes (e.g., shore-birds).39 Modeling of WNV outbreaks has identified some species of birds as “super-spreaders” including of the American robin, followed by the house sparrow, and, where present, the fish crow.83,84 Corvids (e.g., crows) are known to develop a high viremia (1010 PFU/mL) but with high mortality, which limits their potential as efficient reservoirs or their role in spread of the virus as compared to robins and house sparrows.36,38,39,85,86

Although corvid susceptibility has been described as unique to the North American outbreak, early studies with the Egyp-tian WNV strain produced high mortality in crows.87,88 The remarkably explosive North American outbreak of WNV has introduced new potential hosts for the virus. Seropositive, free-ranging mammals include the big brown bat, little brown bat, eastern chipmunk, eastern gray squirrel, eastern striped skunk, white-tailed deer, brown bear, rabbit, and raccoon.40,53,89,90 Sero-logic evidence of natural infection has been demonstrated

222 Section 2 Viral Diseases

reflection of brain and spinal cord disease through direct infec-tion of the spinal cord, interruption of motor tracts in the hindbrain, and loss of fine motor control through infection of the large nuclei of the thalamus and the basal ganglia. Ataxia can be attributable to interruption of general proprioception. Although ataxia is commonly detected and can be profound, many horses have difficulty standing, primarily because of pro-found weakness. These clinical signs are attributable to infection of the gray matter within the midbrain and hindbrain. In the spinal cord, lesions consisting of perivascular cuffing and gliosis tend to increase in frequency and severity caudally, with the most severe lesions appearing in the lumbar cord compared with the cervical cord.130 Lower motor neuron disease charac-terized by weakness would be a common clinical sign associated with these spinal cord lesions.

Involuntary skin and muscle fasciculations, tremors, and hyperesthesia, extremely common in WNV disease, likely result from loss of fine motor control, which is regulated mainly by the basal ganglia.137,138 Movement disorders are observed with flavivirus infection in a long-term Parkinson-like syndrome in rats and experimental infection in monkeys.138 Infection in the pons and medulla oblongata can explain clinical deficits of cranial nerves VII, XII, and IX.139

Like humans, horses also develop signs of flaccid paralysis, which in the quadripedal movement of the horse reflects lower motor neuron disease. These clinical signs likely reflect focal invasion of the lower motor neuron.

Two routes of neuroinvasion are proposed for WNV infec-tion. In the first, WNV causes a low-level viremia, followed by replication in the lymph nodes and entry into the central nervous system (CNS) across the blood-brain barrier.22 The second proposes transaxonal transmission.140 In the first theory, it is hypothesized that systemic viral infection results in local cytokine responses that increase the permeability of the blood-brain barrier to viral invasion. In particular, tumor necrosis factor alpha (TNF-α) increases vascular permeability and allows

appears true for WNV in horses.40,113-117 Although human men are more frequently affected with neuroinvasive diseases, there is conflicting evidence as to whether stallions, geldings, or mares are more likely to die as a result of WNV infection.118-121 Other factors that contribute to mortality risk in horses include light coat color, nonvaccinated status, pasture-only management, and solid stall walls.120,121 Additionally, the use of stable fans is posi-tively correlated with WNV infection because the weak air flow does not impede mosquito movement yet can disperse chemical odors emitted from the horse to attract mosquitos.120

Pathogenesis

In general, mammalian disease caused by infection with the JE serogroup viruses reflects a predilection for nervous tissue. Neu-rologic disease in the horse consists of changes in mentation, signs consistent with spinal cord abnormalities, and defects in cranial nerves of the hindbrain.122-132 Change in behavior likely results from viral infection and pathology induced in the neurons of the thalamus, medulla, and pons, with limited viral load in the cerebrum.125,126,133 Although the thalamus integrates all sensory input to higher centers, lesions within the midbrain and rostral pons may affect the reticular formation, which has an important role in regulation of consciousness.132,134 The retic-ular formation projects to the thalamus, which in turn sends diffuse projections to the entire cortex.132 This formation also travels directly to the base of the forebrain, which is the source of cholinergic stimulation to the entire cerebral cortex. Distur-bances of the reticular formation and the midbrain may induce behavioral changes ranging from severe aggression to somno-lence and even coma.

West Nile virus–induced motor deficits are multifocal, asymmetric, and primarily characterized by weakness and ataxia.124-126,130,133,135,136 These two clinical signs are likely a

Figure 21-4 A, Serial maps of the United States with green states reporting positive veterinary cases of West Nile virus (WNV). These maps demonstrate the spread of the virus from coast to coast. Veterinary cases are defined as nonavian, nonhuman cases, with horses composing 96.9% of these cases. B, Serial compellation of recent WNV-positive veterinary case maps. West Nile virus is an endemic virus.

2001 2002 2003

2004 2005

2009 2010 2011

A

B

223Chapter 21 Flavivirus Encephalitides

transport from peripheral nerves and anterograde spread to other parts of the CNS,154 or a combination of these.

Chemokine expression by infected neurons is integral in the recruitment of circulating leukocytes into the CNS. These include, CXCR2 for early neutrophil migration, CCR2 for monocyte recruitment; and CXCR3, CCR5, and CXCL10 for CD4+ and CD8+ T cell accumulation.169-171 Interferon gamma produced by NK T cells, Th1 cells,171 and CD8+ T cells (positive feedback) also recruits CD8+ T cells.

Although cell lysis occurs with viral replication, WNV also induces apoptosis in neurons, as demonstrated in cell culture and in vivo.147 In equine and human disease, virus load in neu-ronal tissues is low, indicating the possibility that global bystander injury leads to severe neurologic signs and neuron death. Some suggested mechanisms for neuronal injury and apoptosis include immune responses driven by activated CD8+ T cells,172 excitotoxicity via glutamate signaling,162,173-175 and mitochondria-mediated caspase activation.176 Although CD8+ T cells may be important in long-term protective immune responses, lesions in brains of mice with fatal WNV are pre-dominantly composed of CD8+ T cells.177,178 Activated CD8+ T cells are responsible for clearance of virally-infected cells by apoptosis-activating mechanisms and are subsequently respon-sible for pathology, particularly, the destruction of motor neurons of the brain and spinal cord. CD8+ T cells may also play a dominant role in inducing BBB permeability.179

Clinical Findings

Japanese encephalitis and WNV produce similar clinical signs, except that fatal JEV infection in horses usually results in blind-ness, coma, and death, whereas these signs are relatively limited in WNE horses.180,181 For both these infections, there is evidence of widespread subclinical infection in both humans and horses. Both exhibit the same disease course based on lineage of virus; clinical neurologic disease develops with neurotropic lineage type I WNV infection, whereas infection with the African lineage type II viruses is predominantly subclinical in nature.182,183 Kunjin virus, a WNV subtype causes milder clinical disease in horses. Infection with JEV may result in severe clinical disease in naïve horses, but great variation in virulence is seen in JE viruses.

When clinically apparent disease occurs, both systemic and neurologic abnormalities are observed in horses with WNV. A mild to moderate increase in rectal temperature (38.6° C-39.4° C [102° F-103° F]), anorexia, and depression are the most com-mon initial systemic signs.123 Abdominal pain, which also occurs in people, or a colic episode may be the first clinical presenta-tion.119,123,125,184 Gait abnormalities, including overt lameness or dragging of a limb, before development of an obvious neuro-logic syndrome have also been reported. Both spinal cord dis-ease and moderate mental aberrations occur. Onset of neurologic signs is frequently sudden and progressive, and the exact course of disease in any one animal is unpredictable.

Irrespective of onset, the literature is rich in descriptions of clinical disease in both humans and horses. While there is ample evidence for a polioencephalomyelitis, one of the initial signs of motor abnormality is a short, slow, stilted gait, described by observers as “lameness” with laminitis being a frequent differ-ential diagnosis at this stage. In human patients, however, bra-dykinesia or slow, deliberate movement is frequently described, and this may be the equine corollary.116 Spinal abnormalities are characterized by ataxia and paresis that can be asymmetric or involve one or two forelimbs or hindlimbs or by a flaccid paresis that is localized to one or more limbs. This state may be of short duration, or horses may become suddenly recumbent

infection of peripheral nerves.141 Evidence also indicates toll-like receptor 3 (TLR3) plays a role in the entry of WNV into the CNS.141,142

Experimental rodent models demonstrate that WNV has a primary predilection for neural tissues of the vertebrate host.22,143-145 Intraperitoneal injection of WNV (102 PFU) into 8- to 12-week-old mice results in dissemination into the CNS by 4 to 6 days after inoculation. The time course of infection in the hamster is similar. Experimental infection of horses results in viremia at days 3 to 5 and clinical signs in 7 to 10 days.75,146 WNV inoculation into the CNS results in direct infec-tion of nerve cell bodies. In rodent models, initial replication occurs in the basal ganglia, with subsequent dissemination to the cortex, cerebellum, and hippocampus.147-151 The large neurons of the ventral or anterior horns are infected later in the course of disease. Recent studies in a hamster model supports the transaxonal route of infection when injection of WNV into the sciatic nerve results in transmission of WNV through the peripheral nerve to the cell body of a motor neuron in the CNS.152-154 In this study, WNV was transported both antero-grade and retrograde, acute flaccid paralysis was induced, and clinical signs were ameliorated by treatment with monoclonal antibody to WNV.

Immune Responses

West Nile virus invades the hosts’ innate immune cells like macrophages and myeloid dendritic cells where it is recognized by cytosolic retinoic acid-inducible gene (RIG)-I-like and mela-noma differentiation antigen 5 (MDA5) receptors and endo-somal TLR3 and TLR7.155,156 Activation of these pattern recognition receptors lead to the downstream production of interferon gamma (IFN-γ), a type I-IFN.157 Interferons upregu-late major histocompatibility complex class 1 (MHC-1) expres-sion, CD8+ T-cell recruitment, and natural killer (NK) cell activity. Interferon-stimulated genes (ISGs) may hinder RNA translation, viral entry, or viral degredation.158-160 Mice lacking type I-IFN receptors suffer 100% mortality rates after subcuta-neous WNV infection with increased viral dissemination into typically nonaffected tissues and cell types.161 Increase in IFN signaling is supported by microarray analysis of gene transcrip-tion in gravely-ill, WNV infected horses.162 This analysis also demonstrated an increase in genes correlated to interleukin (IL)-15, IL-22, and IL-9 signaling pathways which promote antiviral immune responses.163-165

Antigen-presenting cells, including dendritic cells, macro-phages, and NK cells, initiate IFN production, which has down-stream antiviral effects or directly induce cytotoxic effects on infected cells. Macrophage and dendritic cell production of IL-12 induces the differentiation of T-helper cells into Th1 cells, which secrete IL-2 and IFN-γ. Interleukin-4, IL-5, and IL-10 expression by macrophages and dendritic cells drives differen-tiation of T-helper cells into Th2 cells. Th2 cells stimulate B-cell responses and inhibit Th1-cell response, in turn decreasing CD8+ T-cell activity.

The B-cell response is initiated shortly after 1 week postin-fection in horses. These cells produce T-cell dependent immu-noglobulin M (IgM) antibodies early in the disease but will isotype switch to a dominant production of IgG.166 Antibodies clear viral infection via neutralization, antibody-dependent-cell-mediated cytotoxicity, and complement activation.

Neuronal Injury

Viral invasion into the CNS occurs by disruption of the blood-brain barrier (BBB) via proinflammatory cytokine effects on endothelial cells,167 direct infection of circulating peripheral mononuclear cells that migrate into the CNS,168 retrograde

224 Section 2 Viral Diseases

should be pursued. Infectious CNS diseases that should be considered as differential diagnoses include alphavirus encepha-litis, rabies, equine protozoal myeloencephalitis (EPM), equine herpesvirus type 1 (EHV-1), botulism, and verminous menin-goencephalomyelitis (e.g., Halicephalobus gingivalis, Setaria, Strongylus vulgaris). Noninfectious causes to consider include hypocalcemia, tremorogenic toxicities, hepatoencephalopathy, and leukoencephalomalacia. In alphavirus encephalitis and rabies, signs of cerebral involvement are characterized by behav-ioral alterations, depression, seizure, and coma. The appearance of seizure and coma is rare in horses with WNV. Motor function is frequently abnormal in eastern equine encephalitis (EEE) and western equine encephalitis (WEE). In WNV suspects, circling and propulsive walking may occur, but head pressing is rare. Cranial nerve signs common in EEE and WEE are also common in WNV and include head tilt, pharyngeal/laryngeal dysfunc-tion, and paresis of the tongue. Other clinical signs of alphavirus encephalitis that may be observed in horses with WNV infec-tion are muscle fasciculations, hyperesthesia, excitability, blind-ness, somnolence, and progression to recumbency. Mortality in nonvaccinated horses with EEE is high, approximately 80% to 100% (as in rabies). The incidence of WEE in horses is fairly low in the United States, but mortality and severity of clinical signs would be similar to WNV. Spinal disease caused by EPM is a more difficult differential diagnosis if horses with WNV are not febrile and do not exhibit excessive muscle fasciculations. Both diseases demonstrate hindbrain disease with diffuse spinal cord abnormalities.

Confirmation of WNV infection with encephalitis in horses begins with assessment of (1) whether the horse meets the case definition based on clinical signs and (2) whether the horse resides in an area in which WNV has been confirmed in the current calendar year in mosquito, bird, human, or horse.135 Serologic testing developed by the National Veterinary Services Laboratory (NVSL) is based on detection of the IgM antibody response that uniformly occurs in acutely infected horses. The preferred test is an IgM antibody-capture enzyme-linked immu-nosorbent assay (MAC-ELISA).146 Horses develop a very intense IgM response on exposure to WNV that lasts approxi-mately 6 weeks. This immunologic reaction is much more reli-able than in human infection, in which a more persistent IgM response is common. Most diagnostic laboratories utilize the WNV MAC-ELISA for actual confirmation of disease (increases in IgM rarely occur after vaccination). The sensitivity and speci-ficity of this test are 81% and 100%, respectively.191

In the nonvaccinated horse, a fourfold change in paired neutralizing antibody titers confirms a diagnosis of WNV infec-tion. The most common neutralizing antibody test formats are the classic plaque reduction neutralization test (PRNT) (Fig. 21-5) for detecting antibody response and a more recently developed microwell format.122,135,146,192 Vaccination induces formation of neutralizing antibody to the E protein of the virus,193 which likely confounds interpretation of the PRNT. Since the marketing of equine WNV vaccines in 2001, reliance on the PRNT for serologic confirmatory diagnosis of WNV in horses has diminished. Microsphere immunoassay utilizing recombinant NS1 protein may be useful in detecting antibody response due to natural infection of vaccination.194

Other methods for confirmation of a diagnosis of WNV include postmortem detection of WNV by polymerase chain reaction (PCR), culture, and immunohistochemistry (IHC) in CNS tissues. Several methods for detection of WNV nucleic acids in equine tissue have been described. One method is nested PCR targeting the E protein and has demonstrated sen-sitivity for relatively low viral loads in equine tissues.195,196 Real-time PCR methodology has been used to detect WNV in equine tissues.196 Because of the limited viral load, the equine clinician must insist that ancillary testing be performed in several

and either die or require prolonged treatment. Horses that become recumbent often need aggressive supportive care.

The major hallmarks of equine WNV encephalomyelitis are muscle fasciculations and changes in personality. Fine and coarse fasciculations of the muscles of the face and neck are common. Fasciculations can be severe and can involve all four limbs and trunk, affecting normal activities such as walking, eating, and interactions with handlers and other horses. Muzzle and eyelid fasciculations are notable. Eyelid activity during this period is enhanced with light, and at times, horses appear pho-tophobic. Many horses have periods of hyperexcitability and apprehension, sometimes to the point of aggression. Frequently, a quiet horse will become hyperexcitable, and an abnormally aggressive horse will become compliant. Interspersed during periods of hyperexcitability, some horses appear to have abnor-malities of sudden sleeplike activity resembling narcolepsy. This can occur to the point of cataplexy, and horses may partially or completely collapse for a short period. Some horses show a persistent change of mentation, and a state of nonresponsive-ness, resembling coma, results.

Cranial nerves are frequently abnormal for short periods; weakness of the tongue, muzzle deviation, and head tilt are the most common abnormalities reported. Dysphagia has been reported, with esophageal obstruction a possible sequela. Auto-nomic nervous system dysfunction likely accounts for the respi-ratory failure and gastrointestinal disturbances.185 A cauda equina syndrome consisting of stranguria and rectal impaction is infrequently reported.

Overall, the combination, severity, and duration of clinical signs can be highly variable. After initial clinical signs abate, about 30% of horses experience a recrudescence in signs within the first 7 to 10 days of apparent recovery. Overall, about 30% of affected horses progress to complete paralysis of one or more limbs overall. Most of these horses are euthanized for humane reasons or die spontaneously.

Many horses will begin improvement within 3 to 7 days of displaying clinical signs. If the horse demonstrates significant improvement, full recovery within 1 to 6 months can be expected in most of these patients. However, residual weakness and ataxia are common, with long-term loss of the use of one or more limbs infrequently described. Changes in personality have also been reported to persist. Mild to moderate, persistent fatigue on exercise has been observed.186

Diagnosis

West Nile Virus

Ancillary diagnostic testing for horses with suspected WNV infection should include complete blood count (CBC), serum biochemistry analysis, and cerebrospinal fluid (CSF) analy-sis.187,188 CBC and serum biochemistry profiles of WNV-infected horses are usually normal, but basic blood work can rule out systemic causes of CNS abnormalities such as liver failure. Horses infected with WNV may have a mild absolute lympho-penia. Additionally, they may have elevated muscle enzymes secondary to trauma and prolonged periods of recumbency. A frequent finding is hyponatremia, which has also been described in humans with encephalitis, potentially caused by inappropri-ate release of antidiuretic hormone.189,190 Cerebrospinal fluid cell counts and protein concentration may be elevated. Dif-ferential cell counts in CSF of WNV-infected horses primarily have increased mononuclear cell populations. Protein concen-trations are frequently elevated (N < 70 mg/dL), and the color of the fluid can be mildly xanthochromic.188

No pathognomonic signs distinguish WNV infection in horses from other CNS diseases, and a full diagnostic evaluation

225Chapter 21 Flavivirus Encephalitides

limited in the cortex and cerebellum in the horse. Neuronal damage includes chromatolytic neurons and neuronophagia. Horses with long-standing disease may have areas of neuronal dropout. The spinal cord also has perivascular cuffing, glial nodule formation, and damage to neurons.124 It is still unknown if these cellular changes are attributable to viral damage or resulting inflammation.

Therapy

No known marketed antiviral medications demonstrate reliable activity against flaviviruses, and thus treatment of disease is supportive.123,125,202-204 In horses, the survival rate for WNV encephalitis is high compared with other infectious encepha-litides. Most horses appear to begin recovery 3 to 5 days after onset of signs, which makes it difficult to assess any pharmaco-logic intervention accurately, when a feature of analysis is resolving clinical disease. Flunixin meglumine (1.1 mg/kg every 12 hours [q12h] intravenous [IV] given early in the course of the disease) appears to decrease the severity of muscle tremors and fasciculations within a few hours of administration.

To date, much of the mortality in WNV horses results from euthanasia of recumbent horses for humane reasons. Recum-bent horses are mentally alert and frequently thrash, sustaining many self-inflicted wounds and posing risk to personnel. Therapy of recumbent horses is generally more aggressive and may include dexamethasone sodium (0.05-0.1 mg/kg q24h IV) and mannitol (0.25-2 g/kg q24h IV). Controversy remains as to whether corticosteroids enhance peripheral and CNS viral load.205-207 Detomidine hydrochloride (0.02-0.04 mg/kg IV or intramuscular [IM]) is effective for prolonged tranquilization. Low doses of acepromazine (0.02 mg/kg IV or 0.05 mg/kg IM) provide excellent relief from anxiety in both recumbent and standing horses. Until EPM is ruled out or WNV is confirmed, prophylactic therapy with antiprotozoal medications is recom-mended for horses in geographic areas where Sarcocystis neurona infection is prevalent. Other supportive measures may include oral and IV fluids and antibiotics for treatment of infections that frequently occur in recumbent horses (wounds, cellulitis, and pneumonia).

A variety of treatments have been recommended for horses with WNV; however, there is limited evidence at this time to support their efficacy.* The recommendation for IFN-α therapy is based on anecdotal reports in the human and veterinary lit-erature, yet limited information regarding efficacy in the horse

sections of brain, especially those with lesions observed by light microscopy.197

Serum titers should be evaluated for recent exposure to other encephalitides, including EEE, WEE, and EHV-1. Mea-surement of titers from paired sera is necessary in vaccinated horses for detecting recent exposure to these diseases. Because WNV can present with asymmetric weakness and ataxia, Western blot testing for EPM should also be performed on serum and CSF. The integrity of the BBB during acute infection is unknown. Initial work indicates little leakage, with most WNV-specific IgM within CSF considered to be of intrathecal origin.187

Japanese Encephalitis

Japanese encephalitis should be suspected in horses with com-patible clinical signs that reside in an area of virus activity. Diagnostic confirmatory tests include serologic assays such as neutralizing, complement fixation, hemagglutination inhibition, and ELISA tests.198-200 All single sera testing, including IgM assays, must be interpreted with caution in horses from areas with other endemic flaviviruses such as WNV.201 In fatal JE cases, viral isolation, PCR assays, and IHC for detection of virus in CNS tissues is confirmatory.

Pathologic Findings

Flaviviruses cause polioencephalomyelitis (inflammation of the gray matter) with lesions that increase in number from the diencephalon through the hindbrain and frequently increase in severity caudally through the spinal cord. The histologic changes within the brain, including inflammatory foci and detectable virus in the thalamus, medulla, and pons, are consistent with changes in behavior.

Gross pathologic findings are limited in WNV infection in the horse. The meninges may be congested. Small to moderately sized foci of hemorrhagic discoloration may be observed in the brain and spinal cord. These areas occur most often in the basal ganglia, rostral colliculus, pons, medulla, and lumbar spinal cord. Edema and softening of tissues are also common findings.

Histopathologic changes secondary to WNV infection are consistent with viral infection and neural cell death.124 The basal ganglia, thalamus, pons, and medulla have the highest numbers of lesions, with two to several cell layers of predominantly mononuclear perivascular cuffing. Recent IHC has revealed a minor presence of granulocytes (Delcambre, Long, et al, unpub-lished data). Predominantly confined to the gray matter, glial nodules are present in the brain parenchyma. These lesions are

Figure 21-5 Six-well plate exhibiting clearing of monolayer (plaques) of Vero cells as cytopathic effect of West Nile virus (WNV) infection. These plaques form the basis of the “gold standard,” the plaque reduction neutralization test (PRNT), for detection of neutralizing antibody to flaviviruses.

*References 97, 116, 199, 203, 208, 209.

Where these viruses are endemic, vaccination schedules must be maintained even with a decrease in the incidence of overt disease. Foals must receive three injections of vaccine with time 30 days between with the first and second injections and 60 days time between the second and third injections. Foals should not receive early vaccination if the mare has been vaccinated and should receive the first vaccine at 5 to 6 months of age. Horses that have recovered from clinical disease have long-term immunity and do not require annual boosters.

Public Health Considerations

West Nile virus is considered a zoonotic disease. A bird reservoir maintains the virus in an endemic life cycle in the environment, allowing for transmission by mosquitoes to humans. There is little risk of disease by direct contact with an infected horse, except during postmortem examination with inappropriate handling of infected tissues. Postmortem handling of tissues should be performed with personal protection similar to that for rabies suspect cases (see Chapter 19). The ecology of horse pastures and stables with standing water, a high degree of bio-logic debris, and “bridge” vectors between birds and mammals likely increase the risk of exposure in that environment. The same types of management tactics for prevention of disease in horses are important for people, except that there is no vaccine. Personal mosquito protection with a DEET-based product is recommended in areas with endemic disease.

The North American epidemic of WNV has demonstrated new modes of transmission, including blood-borne and occu-pational risks. Blood-borne transmission can occur between viremic hosts. In addition, occupational infection has occurred through necropsy of avian hosts. Veterinarians and horse owners should institute personal protection with appropriate clothing, gloves, and eye protection when coming into contact with animal tissues during the arbovirus season.

The complete reference list is available online at www .expertconsult.com.

is available. In a blinded study in which children with encepha-litis caused by JEV were treated with IFN-α, survival was not enhanced. In fact, length of hospitalization was increased in the IFN-α–treated group.

Therapy with WNV-specific recombinant immunoglobulin has also been recommended. In a blinded placebo-controlled trial with low numbers of animals, the risk for development of recumbency was less in horses receiving plasma from horses immunized against WNV (Long, unpublished data). However, plasma treatment did not change outcome and severity of WNV disease. In human patients, high-dose glutamate therapy has been suggested to prevent neuronal cell death. Another experimental therapy in mice is administration of beta-lactam inhibitors, which stimulate GLT1, a chemical that activates glutamate.

Prevention

Epidemiologic and anecdotal evidence exists regarding the effectiveness of vaccination against flaviviruses. Initial epide-miologic studies performed in 2000 established a point source for infection of WNV, demonstrating that outbreaks in horses may be controlled best by vaccination.146,210,211 This finding was consistent with prior experience with JE, in which vaccines were advocated for horses before the WNV epizootic. The dramatic decrease in WNV cases in the horse likely reflects the success of vaccination.

Currently, three killed vaccines and a recombinant vaccine are licensed for prevention of WNV viremia in the United States, and an inactivated virus vaccine is readily available against JE.201 Vaccination before the mosquito season is critical. The manufacturer’s labeling instructions must be followed for induction of immunity with initial immunization. More fre-quent vaccination in areas with year-round mosquito seasons is highly recommended. Limited information is available regard-ing long-term immunity after vaccination especially in the killed vaccines. However, it is not expected that the initial vaccine series will provide long-term protection beyond 1 year.

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226.e6 Section 2 Viral Diseases