**1. Introduction**

126 Non-Flavivirus Encephalitis

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320

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simplex virus type 1 DNA in cells of cerebrospinal fluid of patients with herpes

Herpesviruses occasionally cause lethal infection in alien hosts by interspecies transmission such as B virus infection in humans from Macaques and pseudorabies virus infection in various animals from pigs. Most of the infection can be characterized as lethal encephalitis. One of typical examples is equine herpesvirus 9 (EHV-9), also called as gazelle herpesvirus 1 (GHV-1), which was isolated from enzootic encephalitis of Thomson's gazelles (*Gazella thomsoni*) in 1993 (Fukushi et al., 1996, Yanai et al., 1998). EHV-9 infection has been reported in non-equid species such as Thomson's gazelles, a reticulated giraffe (*Giraffa camelopardalis reticulata*) (Hoenerhoff et al., 2006), and a polar bear (*Ursus maritimus*) (Donovan et al., 2006). Experimental infections have also been investigated including domestic horses, pigs, cattle, and goats, companion animals including cats and dogs and common marmosets (*Callithrix jacchus*) (Section 3). These interspecies or cross-species infections can be characterized as viral lethal and inapparent encephalitis (Table 1).


Table 1. A list of animals infected with EHV-9

#### **1.1 Epizootiology of encephalitis in a herd of Thomson's gazelles**

Epizootic encephalitis occurred in a herd of Thomson's gazelles kept in a zoo in 1993. Twelve Thomson's gazelles were first introduced into the zoo in 1992. Then eight normal newborns and one malformed newborn have been delivered. Ten gazelles died of injury by various causes. The herd consisted of ten Thomson's gazelles at the outbreak. A first

Virology and Pathology of Encephalitis in Alien Hosts by Neurotropic Equine Herpesvirus 9 129

Sequences of gB and gG homologue genes of GHV-1 are closer to EHV-1 than to other equine herpesviruses including EHV-8. Therefore GHV-1 was recognized as a new member

It should be noted that a zebra kept in the same field with the gazelles had neutralizing antibody to GHV-1 but not to EHV-1 in neutralization tests examined several months after

of equine herpesvirus, equine herpesvirus 9 (EHV-9).

Fig. 2. CPE observed in MDBK cells inoculated with brain homogenate

An 18-month-old male reticulated giraffe (*Giraffa camelopardalis reticulate*) housed in a zoo presented with a one and a half day history of incoordination, stumbling, and abdominal pain and died (Hoenerhoff et al., 2006). Nonsuppurative encephalitis was histopathologically found in the brain of giraffe. Several zebras (*Equus burchelli*), which were apparently healthy, were housed in a pen with the giraffe. Herpesvirus was isolated from the reticulated giraffe. The isolated virus was identified as EHV-1 by PCR and monoclonal antibody assay at first. The giraffe had been housed with a group of zebras that were serologically positive for EHV-1. Considering the situation and histopathological findings, there was a possibility that the isolate could be EHV-9. Several gene sequences of the giraffe virus were almost identical to those of EHV-9 (Samy et al., 2008). Therefore the giraffe virus

A 12-year-old female polar bear (*Ursus maritimus*) developed a sudden onset of neurological symptoms in 2007 (Schrenzel et al., 2008; Donovan et al., 2009). Nonsuppurative pleocytosis of cerebrospinal fluid was observed. The bear was euthanized. Multifocal, random nonsuppurative meningoencephalitis was microscopically found involving most prominently the rostral cerebral cortex, as well as the thalamus, midbrain, and rostral

**1.2 A sporadic encephalitis in a giraffe** 

**1.3 Meningoencephalitis in a polar bear** 

the episode.

was EHV-9.

occurrence was sudden death of one gazelle, and then others showed neurological symptoms in turn over 2-week period (Fig. 1). The neurological symptoms in the gazelles were depression, incoordination, nystagmus, and convulsion. Laboratory tests showed occult blood and albumin in the urine samples. Seven out of the nine gazelles died. No evidence of infection was found in this foal. The other two gazelles recovered. The remaining one was a newborn that died of malnutrition, because the sick mother gazelle refused nursing. One of the recovered gazelles delivered a healthy neonate two months later. All dead gazelles did not have apparent changes at necropsy. Microscopically, all dead gazelles except the foal of malnutrition had nonsuppurative encephalitis, which characterized by neuronal degeneration and intracellular inclusion bodies (Fukushi et al., 1996, Yanai et al., 1998).

Fig. 1. Outbreak of lethal enzootic encephalitis in Thomson's gazelles

Homogenates of the brain, spinal cord, kidney, liver, lung and spleen were inoculated into Madin-Darby bovine kidney (MDBK) cells. Cells inoculated with the brain homogenate showed cytopathic effect characterized as syncitium formation accompanying nuclear inclusion bodies and detachment (Fig. 2), indicating the isolation of a virus. Physicochemical characterization of the isolate showed that the isolate was chloroform sensitive DNA virus of about 150 to 200 nm diameter. Considering the characteristics, the isolate was identified as a herpesvirus. Serum neutralization tests using antisera against pseudorabies virus, bovine herpesvirus 1, malignant catarrahl fever virus, and equine herpesvirus 1 (EHV-1) indicated that the isolated virus should be EHV-1 or EHV-1 related virus. Comparing DNA fingerprints and nucleotide sequences of a glycoprotein B gene and a glycoprotein G gene, the isolate should be regarded as a new herpesvirus, gazelles herpesvirus 1 (GHV-1) at first (Fukushi et al., 1996). DNA fingerprints of GHV-1 were different from those of EHV- 1, EHV-4, and EHV-8, although GHV-1 cross-reacted with EHV-1 and EHV-4 in neutralization tests. Southern analysis indicated that GHV-1 shared sequence homology with EHV-1. Sequences of gB and gG homologue genes of GHV-1 are closer to EHV-1 than to other equine herpesviruses including EHV-8. Therefore GHV-1 was recognized as a new member of equine herpesvirus, equine herpesvirus 9 (EHV-9).

It should be noted that a zebra kept in the same field with the gazelles had neutralizing antibody to GHV-1 but not to EHV-1 in neutralization tests examined several months after the episode.

Fig. 2. CPE observed in MDBK cells inoculated with brain homogenate
