**3.1.3 Polar bear**

136 Non-Flavivirus Encephalitis

An outbreak of acute encephalitis occurred in a herd of Thomson's gazelle in a Japanese zoo. Seven of 9 gazelle died with or without neurological symptoms within a 3-week period as described in the section 1.1 (Fig. 5A). All animals had nonsuppurative encephalitis characterized by necrosis and degeneration of neurons (Fig. 5B), gliosis and perivascular aggregates of lymphocytes (Fig. 5C) in the cerebrum (Yanai et al., 1998). Five cases had intranuclear inclusion bodies in neurons compatible with those of herpesvirus. Immunohistochemically, a positive reaction to EHV-1 antigen was demonstrated in neurons in the necrotic areas of the cortex in all cases. The neuropathology of EHV-9 infection clearly differed from EHV-1-induced encephalitis in the horse, which is characterized by vasculitis, thrombosis, ischemia, and lack of intranuclear inclusion in neurons (Wada et al., 1991).

Fig. 5. A: A dead Thomson's gazelle. B: Neuronal necrosis with frequent nuclear inclusion

Hoenerhoff et al. (2006) described as follows. The giraffe was in good body condition. There was a traumatic wound on the dorsal aspect of the tail base. There was a focal area of malacia within the cerebrum on cut section. There were no other lesions noted on gross

Histopathological examination included sections of brain; cervical, thoracic, and lumbar spinal cord; heart; lungs; liver; spleen; kidneys; adrenal glands; rumen; abomasum; jejunum; ileum; and colon. There was severe multifocal nonsuppurative meningoencephalitis within the white and gray matter of the cerebral cortex, hippocampus, thalamus, midbrain, and medulla

formation. HE. C: Perivascular aggregates of lymphocytes in affected brain. HE.

A

B C

**3.1.1 Thomson's gazelles** 

**3.1.2 Giraffe** 

necropsy examination.

Donovan et al. (2009) described as follows. The polar bear was in good body condition The meninges of the brain and spinal cord were diffusely congested. The caudodorsal lung lobes were edematous.

Histopathological findings were severe nonsuppurative meningoencephalitis, predominantly in the grey matter of the cerebrum. The most significantly affected region of the brain was the rostral telencephalon, particularly the rhinencephalon. Scattered regions of inflammation involved the olfactory bulbs, cerebrum, thalamus, midbrain, and rostral medulla. Inflammatory cells consisting of lymphocytes, plasma cells, macrophages, and fewer eosinophils formed perivascular cuffs within the meninges overlying the brain and Virchow-Robin spaces within the parenchyma, as well as more poorly delineated inflammatory cell infiltrates within the subjacent meninges, neuropil, and neuroparenchyma. Neuronal degeneration was characterized by peripheral localization or complete loss of Nissl substance, and neuronal necrosis consisted of cytoplasmic hyperosinophilia, loss of cytoplasmic detail, nuclear pyknosis, karyolysis, and karyorrhexis. Glial proliferation accompanied the inflammation, consisting of microgliosis and astrogliosis.

Within the nuclei of neurons and astrocytes, there were diffuse type and Cowdry type A inclusions. White matter tracts had random areas of vacuolation, axonal degeneration characterized by formation of axonal spheroids, demyelination, edema, and microg- lial and astrocytic gliosis. Additional histologic findings included mild, multifocal neutrophilic bronchiolitis, mild neutrophilic rhinitis, moderate pulmonary edema, mild lymphocytic myocarditis, and mild membranoproliferative glomerulonephritis.

#### **3.1.4 Goats**

Two young goats were inoculated intranasally with 10 ml of virus solution containing 5.25 x 106 PFU (Taniguchi et al., 2000a). These animals showed sudden neurological symptoms consisting of marked salivation, teeth grinding, convulsion, tremor and ataxia 8 days IP. One animal died 30 minutes after the onset of clinical signs and another was sacrificed 3 hours after the onset of teeth griding and foamy salvation. Both animals had fulminant encephalitis characterized by neuronal degeneration and necrosis with intranuclear inclusion bodies (Fig. 6).

#### **3.1.5 Carnivors**

In carnivore cats and dogs got fulminant encephalitis via nasal rout of inoculation with EHV-9, which suggested these animals to be fully susceptible to EHV-9 and that EHV-9 could cause fulminant encephalitis with high mortality in dogs and cats, as in gazelles and goats.

An acute lethal infection of EHV-9 was induced in cats by intranasal inoculation of 106 pfu (Yanai et al., 2003a). Four cats killed at 4, 5, 6 or 10 days after inoculation showed neurological signs consisting of hyper-excitability and aggressiveness, followed by tremors, occasional

Virology and Pathology of Encephalitis in Alien Hosts by Neurotropic Equine Herpesvirus 9 139

with intranuclear inclusions, glial reactions (Fig. 8A), perivascular aggregates of lymphocytes, and multi-focal perivascular hemorrhages (Fig. 8B). The olfactory bulb and the frontal and temporal lobes were predominantly affected. Immunohistochemistry revealed

Fig. 8. A: There were frequent positive reaction to EHV-9 antigen in the neuronal cells in the olfactory bulb in a dog inoculated with EHV-9 intra-nasally. IHC. B: There were prominent glial reactions in the brain. HE stain. C: There were occasional perivascular hemorrhages in

In suckling mice and rats inoculated intra-cerebrally with 102 to 105 plaque-forming units (PFU) of cell-free viral suspension, growth deterioration (Fig. 9) and neurological symptoms, including depression and seizures were observed, and inoculated animals died within 8 days of inoculation (Fukushi et al., 1997). The brain of dead animal had severe neuronal degeneration and necrosis accompanied by numerous intranuclear inclusion bodies

In mice, rats and hamsters inoculated with 102 to 106 PFU via intranasal route (Fukushi et al., 1997). The animals had neurological signs, including depression or convulsion, and died of acute encephalitis characterized by neuronal degeneration and necrosis with intranuclear inclusion bodies (Fig. 10A and B). Mortality rates were 100% with 104 PFU and 33% with 102

reactivity for EHV-9 antigens in neurons (Fig. 8C).

C

the affected brain. HE stain.

characteristic of herpesviruses.

**3.1.6 Rodents** 

A B

Fig. 6. Frequent intra-nuclear inclusion formation in the neuronal cells. HE stain.

convulsions, and depression. Histologically, the cats affected showed severe encephalitis characterized by neuronal degeneration and loss, intranuclear inclusions, perivascular aggregates of lymphocytes and gliosis in the cerebrum. The olfactory bulb and rhinencephalon were severely affected and collapsed, and eventually softened by complete destruction of both neuronal cells and glial cells. Using Double Label immunohistochemistry all cats showed proliferating astrocytes with double reactivities to both EHV-9 and GFAP, as well as EHV-9 antigen bearing neurons (Fig. 7A and B). These findings were most frequently observed in the intermediate stage of the infection (6th day post-inoculation by intranasal route) while in early stage of the infection (4th day post-inoculation by intranasal route), and 15th day of postinoculation by intraperitoneal route, only small number of astrocytes showed reactivity to EHV-9. At the later stage (10th day post-ionculation), malacia was observed in the cortex together with marked decrease in the number of astrocytic cells showing GFAP reactivity as well as neuronal number decreased by collapse.

Fig. 7. A: Affected cat had encephalitis with EHV-9 antigen in the brain. IHC. ABC methods. B: Double Label immunohistochemistry showed proliferating astrocytes with double reactivities to both EHV-9 and GFAP, as well as EHV-9 antigen bearing neurons. ABC method.

EHV-9 was inoculated intranasally at 107 PFU in five dogs to assess its pathogenicity (Yanai et al., 2003b). Dogs affected showed weight loss, pyrexia, anorexia, and neurologic signs on the fourth day. The EHV-9 virus was recovered from the examined brain. Histologically, dogs had a fulminant encephalitis characterized by severe neuronal degeneration and loss, with intranuclear inclusions, glial reactions (Fig. 8A), perivascular aggregates of lymphocytes, and multi-focal perivascular hemorrhages (Fig. 8B). The olfactory bulb and the frontal and temporal lobes were predominantly affected. Immunohistochemistry revealed reactivity for EHV-9 antigens in neurons (Fig. 8C).

Fig. 8. A: There were frequent positive reaction to EHV-9 antigen in the neuronal cells in the olfactory bulb in a dog inoculated with EHV-9 intra-nasally. IHC. B: There were prominent glial reactions in the brain. HE stain. C: There were occasional perivascular hemorrhages in the affected brain. HE stain.

### **3.1.6 Rodents**

138 Non-Flavivirus Encephalitis

Fig. 6. Frequent intra-nuclear inclusion formation in the neuronal cells. HE stain.

well as neuronal number decreased by collapse.

method.

A B

convulsions, and depression. Histologically, the cats affected showed severe encephalitis characterized by neuronal degeneration and loss, intranuclear inclusions, perivascular aggregates of lymphocytes and gliosis in the cerebrum. The olfactory bulb and rhinencephalon were severely affected and collapsed, and eventually softened by complete destruction of both neuronal cells and glial cells. Using Double Label immunohistochemistry all cats showed proliferating astrocytes with double reactivities to both EHV-9 and GFAP, as well as EHV-9 antigen bearing neurons (Fig. 7A and B). These findings were most frequently observed in the intermediate stage of the infection (6th day post-inoculation by intranasal route) while in early stage of the infection (4th day post-inoculation by intranasal route), and 15th day of postinoculation by intraperitoneal route, only small number of astrocytes showed reactivity to EHV-9. At the later stage (10th day post-ionculation), malacia was observed in the cortex together with marked decrease in the number of astrocytic cells showing GFAP reactivity as

Fig. 7. A: Affected cat had encephalitis with EHV-9 antigen in the brain. IHC. ABC methods. B: Double Label immunohistochemistry showed proliferating astrocytes with double reactivities to both EHV-9 and GFAP, as well as EHV-9 antigen bearing neurons. ABC

EHV-9 was inoculated intranasally at 107 PFU in five dogs to assess its pathogenicity (Yanai et al., 2003b). Dogs affected showed weight loss, pyrexia, anorexia, and neurologic signs on the fourth day. The EHV-9 virus was recovered from the examined brain. Histologically, dogs had a fulminant encephalitis characterized by severe neuronal degeneration and loss, In suckling mice and rats inoculated intra-cerebrally with 102 to 105 plaque-forming units (PFU) of cell-free viral suspension, growth deterioration (Fig. 9) and neurological symptoms, including depression and seizures were observed, and inoculated animals died within 8 days of inoculation (Fukushi et al., 1997). The brain of dead animal had severe neuronal degeneration and necrosis accompanied by numerous intranuclear inclusion bodies characteristic of herpesviruses.

In mice, rats and hamsters inoculated with 102 to 106 PFU via intranasal route (Fukushi et al., 1997). The animals had neurological signs, including depression or convulsion, and died of acute encephalitis characterized by neuronal degeneration and necrosis with intranuclear inclusion bodies (Fig. 10A and B). Mortality rates were 100% with 104 PFU and 33% with 102

Virology and Pathology of Encephalitis in Alien Hosts by Neurotropic Equine Herpesvirus 9 141

perivascular aggregates of lymphocytes (Fig. 11A) and gliosis (Fig. 11B). Neither neuronal

Fig. 11. A: Perivascular aggregates of lymphocytes in the affected brain. HE stain. B: Glial

In cattle seven calves were inoculated intranasally with 105 and 107 PFU of the EHV-9 (El-Habashi et al., 2011). Three animals showed brain lesions consisting of glial reactions and perivascular aggregates of lymphocytes in the olfactory bulb and the frontal and temporal lobes. Additionally, the animal inoculated with 107 PFU showed neuronal degeneration and loss, as well as nuclear inclusions compatible with herpesvirus. EHV-9 was isolated from the brain of a calf and the lungs of two calves. The results suggested that cattle are susceptible to experimental infection with EHV-9 and at risk from natural infection from reservoir hosts.

The infectivity and pathology of various routes including nasal, ocular, oral, intravenous (IV), or peritoneal routes of experimental infection were studied in hamsters (El-Habashi et al., 2010a). Clinically, all animals inoculated by the nasal route and ~25% inoculated by the oral and peritoneal routes showed neurological signs on days 3, 6 and 9 post-inoculation (PI), respectively. Neurological signs were not observed in animals administered EHV-9 by the IV and ocular routes. With the exception of animals administered EHV-9 by the IV route, all infected animals had lymphocytic meningoencephalitis. Although there were a number of differences in the severity and distribution of the lesions depending on the route of inoculation, the basic features of lymphocytic meningoencephalitis caused by EHV-9 were common. Lesions consisted of neuronal necrosis, perivascular aggregates of lymphocytes, plasma cells, and neutrophils, gliosis, intranuclear inclusion bodies, and diffuse lymphocytic infiltrates in the meninges. Viral antigen was detected in degenerated neurons in infected animals inoculated by the nasal, ocular, oral and peritoneal routes. The distribution of EHV-9 antigen was somewhat dependent upon inoculation route. There were no microscopic abnormalities nor viral antigen in animals treated by the IV route. This study provides new data about experimental EHV-9 infection in hamsters through routes other than the IV route. These results suggest that in the animals infected by the oral, ocular and peritoneal routes, EHV-9 might travel to the brain through nerves, other than by the olfactory route,

necrosis nor intranuclear inclusions were observed in affected horses.

**3.3 Neuropathogenesis of EHV-9 by experimental infections**

after initial propagation at the site of viral entry.

A B

cells reaction was observed. HE stain.

**3.2.2 Cattle** 

PFU in young mice (Fukushi et al., 1997). Although the morphological features of E HV9 induced encephalitis was common in rodents, the hamsters were thought to more susceptible to EHV-9 via nasal route (Taniguchi et al., 2000a).

Fig. 9. A: Inoculate suckling rats (arrow heads) showed growth deterioration.

Fig. 10. A: There were frequent intranuclear inclusions in degenerating neuronal cells. A mouse inoculated with EHV-9 intra-nasally. HE stain. B: Most neuronal cells with inclusion body had positive reaction for EHV-9 antigen. A mouse inoculated with EHV-9 intranasally. IHC.
