**3. Pathology**

134 Non-Flavivirus Encephalitis

ORF Codons Identity (%) HSV-1 Predicted or confirmed functions 64 1475 92 RS1 Transcriptional regulator, ICP4

67 269 94 – Virulence determinant virion protein

72 402 97 US6 Envelope glycoprotein (gD); role in cell

74 550 99 US8 Envelope glycoprotein (gE); complexes

EHV-9 can be propagated by fetal equine kidney cells (FEK), Madine-Darby bovine kidney cells (MDBK), rabbit kidney cells (RK-13), murine fibroblast L929 cells, and human HeLa 229 cells. Cytopathic effects varied in each cell line. Lytic CPE is observed in FEK, RK-13 and HeLa 229, while syncitium formation is observed in MDBK (Fig.). EHV-9 can be also propagated in neural cells derived from a fetal equine brain as well as a fetal murine brain. EHV-9 has been isolated from Thomson's gazelles, zebras (Borchers et al., 2008; Schrenzel et al., 2008), giraffes (Samy et al., 2009), polar bears (Schrenzel et al., 2008; Donovan et al., 2009) and onager (Schrenzel et al., 2008) as described in Section 1. Epizootiologically all of the cases in zoo animals associated with the presence of zebras. These data indicates that

EHV-9 can infect several animals experimentally. The experimental hosts include horse, goat, pig, cattle, hamster, mouse, rat, guineapig, dog, cat, and marmosette as described in

The phylogenic tree constructed by using a part of glycoprotein B gene sequence indicates

EHV-1 derived from zoo animals might be considered as another type of equid herpesvirus. Unfortunately equine herpesvirus 8 glycoprotein G sequence is not available at present.

Burchell's zebra and other zebras might be a natural host of EHV-9 in nature.

three groups of EHV-9, EHV-1 in horses and EHV-1 in zoo animals (Fig. 4).

**2.3 Phylogenic relatedness to other related herpesviruses** 

73 425 94 US7 Envelope glycoprotein (gI); complexes with

entry

gE in Fc receptor

with gI in Fc receptor

69 383 95 US3 Serine–threonine protein kinase 70 411 92 US4 Envelope glycoprotein (gG) 71 830 91 US5 Envelope glycoprotein (gp300)

65 291 94 US1 Regulatory protein ICP22

66 234 94 US10 Virion protein

68 296 93 US2 Virion protein

75 130 97 US8A Unknown

Identity was evaluated by protein-protein BLAST analysis. -: there is no homologue in herpes simplex virus 1 (HSV-1) genome

Table 2. Characteristics of EHV-9 proteins

**2.2 Host range in vitro and in nature** 

the section 3.

76 219 89 US9 Tegument protein

#### **3.1 Lethal Encephalitis in zoo, domestic and companion animals, and experimental small animals**

EHV-9 caused lethal encephalitis in several animals such as Thomson's gazelles, giraffes, and polar bears naturally and goats, cats, dogs, mice, rats, hamster and marmosets experimentally. All of these infections can be regarded as encephalitis with neuronal degeneration, perivascular cuffing and gliosis. Histopathological characteristics will be described.

Virology and Pathology of Encephalitis in Alien Hosts by Neurotropic Equine Herpesvirus 9 137

oblongata, and was most severe in the thalamus and ventrolateral portions of the cerebral cortex. Lesions were characterized by multifocal perivascular cuffs composed of moderate numbers of lymphocytes and histiocytes. The endothelium of affected blood vessels was swollen, and there were numerous microglial cell foci distributed diffusely in the gray and white matter. Multifocal neuronal necrosis was most evident in the ventral portion of the cerebral hemispheres, and areas of neuronal necrosis were occasionally associated with

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

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

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

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

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

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

fulminant encephalitis with high mortality in dogs and cats, as in gazelles and goats.

characteristic herpetic eosinophilic intranuclear inclusions in neurons and astrocytes.

inflammation, consisting of microgliosis and astrogliosis.

myocarditis, and mild membranoproliferative glomerulonephritis.

**3.1.3 Polar bear** 

were edematous.

**3.1.4 Goats** 

inclusion bodies (Fig. 6).

**3.1.5 Carnivors** 

### **3.1.1 Thomson's gazelles**

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

#### **3.1.2 Giraffe**

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 necropsy examination.

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 oblongata, and was most severe in the thalamus and ventrolateral portions of the cerebral cortex. Lesions were characterized by multifocal perivascular cuffs composed of moderate numbers of lymphocytes and histiocytes. The endothelium of affected blood vessels was swollen, and there were numerous microglial cell foci distributed diffusely in the gray and white matter. Multifocal neuronal necrosis was most evident in the ventral portion of the cerebral hemispheres, and areas of neuronal necrosis were occasionally associated with characteristic herpetic eosinophilic intranuclear inclusions in neurons and astrocytes.
