**5. References**

144 Non-Flavivirus Encephalitis

induced disease. However, in more recent study, possible infection by different routes of inoculation including, nasal, ocular, peritoneal and oral routes were evident. There may be discrepancies in EHV-9 infection of the brain based on the route of inoculation when

Based on previous experimental studies in different animals inoculated via the nasal route, the olfactory pathway (i.e. through the olfactory nerves) is the major route of transmission of EHV-9 into the CNS. However, recent study that compared different routes of inoculation clearly indicates that virus can enter the CNS after administration of EHV-9 via the oral, peritoneal, and ocular routes, and that there are differences in the distribution of antigenpositive cells and in the location and severity of the cerebral lesions. Thus, EHV-9 may gain access to the CNS through a non-olfactory route as these animals inoculated via these nonnasal routes did not exhibit EHV-9 induced rhinitis, and the olfactory bulbs showed milder lesions and fewer viral antigen-positive cells than observed in the animals infected via the

One of the striking finding was that animals infected via the ocular route had mild and localized lesions in the rhinencephalon, which indicated that the virus had traveled to the CNS through the optic nerve. Similar to what is reported about fatal infections by Cercopithecine herpes virus 1 (B virus) in humans via ocular exposure from biological fluid

The differences in the incubation period and paths of travel to the CNS among the various routes in resulting in a variety of clinical signs and histopathological features, suggests a dependency on the replication of the virus at the site of entry and its opportunity to access regional nerves to travel to the brain. A similar hypothesis was proposed in the case of pigs infected orally by EHV-9 (Narita et al., 2000), where the virus was thought to travel centripetally in the nerve fibers from the oral mucosa to the trigeminal ganglion, eventually entering the olfactory lobes (Chowdhury et al., 1997; Kritas, et al., 1994; Narita et al., 1976). Previously, EHV-9 antigen was found in the neurons and neural fibers but not in the glial cells in the brain, indicating that neurons are the susceptible cells to EHV-9 in the CNS of hamsters (Fukushi et al., 2000). However, other neurotropic herpesviruses such as herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) are known to infect glial cells as well as neurons (Johnson, 1998). Astrocytes infection of EHV-9 was described in the cases of a giraffe and a polar bear (Donovan et al., 2009; Hoenerhoff et al., 2006). Immunohistochemistry demonstrated the presence of EHV-9 antigen in the neurons and neuronal fibers including the axons and dendrites in the brain of the goat and the naturally infected bear which indicate a trans-synaptic spread of EHV-9 from neuron to neuron via the neuronal fibers (Taniguchi et al., 2000b; Donovan et al., 2009). Similar transmission has been shown in pseudorabies viral infection (Card et al, 1990). In suckling hamster study, there was necrosis of some of trigeminal ganglion cells as well as detection of the viral antigen in the same ganglion and in the connection between trigeminal sensory nerve root and the brain stem at the level of the pons and medulla oblongata. Based on previous findings, the neurotropism might be the most characteristic property of EHV-9, differentiating it from other neurotropic herpesviruses. Similar transneural passage was

suggested after intranasal infection with IBR virus (Narita et al, 1979).

animals are inoculated with the same quantity of virus (El-Habashi et al, 2010a).

**4. Conclusion: EHV-9 infection as a model of cross-species viral** 

**transmissions** 

nasal route.

from macaque monkeys (CDC, 1999).


**7** 

*Japan* 

**Equine Herpesvirus 9 (EHV-9) Induced** 

Tokuma Yanai, Atsushi Kodama, Hiroki Sakai, Hideto Fukushi,

Equine Herpesvirus 9 (EHV-9) is a new member of the equine herpesviruses which was isolated from Thomson's gazelles (*Gazella thomsoni*) that died of fulminant encephalitis in a Japanese zoo (Fukushi et al., 1997; Yanai et al., 1998). Previously, experimental infections of EHV-9 were conducted in various species of animals other than primates to clarify the infectivity and virulence of this virus and to assess the emerging aspects of EHV-9 in zoo and domestic animal populations. EHV-9 caused fatal infections with fulminant encephalitis characterized by neuronal degeneration and necrosis as well as intra-nuclear inclusion bodies in rodents (Fukushi et al., 1997; Fukushi et al., 2000), goats (Taniguchi et al., 2000), pigs (Narita et al., 2000, 2001), dogs (Yanai et al., 2003a) and cats (Yanai et al., 2003b). Based on several experimental studies of EHV-9 involving various domestic animals such as dogs and cats often found in close proximity to humans, there were grave concerns that EHV-9 could be transmitted to humans through contact with affected animals or zebras through certain routes. In order to assess the risk of EHV-9 to humans, we tried to determine the infectivity of EHV-9 in non-human primates, including common marmosets (*Callithrix jacchus*) and cynomolgus macaques (*Macaca fasciocularis*), which have strong similarities to

One female and four male common marmosets, aged 2 to 4 years old and weighing 285- 368g, were used for this assessment (Kodama et al., 2007). Four of the marmosets were inoculated intranasally with 1ml of EHV-9 virus solution containing 106 plaque-forming units. The other was inoculated with 1 ml of MEM as a negative control. The inoculated animals were humanely euthanatized on Days 3, 4 and 5 following inoculation, respectively, at a point when they were in poor condition or dying from a neuronal disorder. The virus was recovered and identified by polymerase chain reaction (PCR) with the primers targeting the EHV-9 specific region of the EHV-9 gene. The PCR primers used for amplification were 5'-CTGGGTTATAGATTGTCGCCTC-3' and 5'-CCCAGAAAGTATTACACGCGAT-3'. The neutralization test was done using the 50% plaque reduction method with the MDBK cell

**1. Introduction** 

humans, using the nasal route.

**2. Marmosets**

monolayer.

**Encephalitis in Nonhuman Primates** 

Takeshi Kuraishi, Seisaku Hattori and Chieko Kai

*The University of Tokyo, Gifu University* 

