**3. Cynomolgus macaques**

One male and four female cynomolgus macaques were obtained from a commercial breeder (Kodama et al., 2011). All of the animals were free from pathogens such as salmonella and *Mycobacterium spp*. and passed the viral antibody tests for B virus and measles virus. The animals were divided into two groups consisting of Nos. 1 and 2 in one group and Nos. 3 and 4 in the other, and the two groups were inoculated intranasally with 1 ml of EHV-9 virus solution containing 103 and 106 plaque-forming units, respectively. The virus fluid was prepared by propagating the fifth passage of the original stock, which is in Madin-Darby bovine kidney (MDCK) cells, in fetal horse kidney cells. The infectivity of the inoculums was confirmed by virus plaque assay with MDCK cells. As a control, cynomolgus monkey No. 5 was inoculated with 1 ml of minimal essential medium. Animals Nos. 1 and 3 were euthanized in accordance with animal welfare regulations on 6 dpi. The other animals were euthanized on 10 dpi.

In contrast with the control animal, the inoculated animals began avoiding light starting from 4 dpi. Blood studies showed no hematological abnormalities in any of the animals.

At necropsy, no significant abnormalities were observed in any organs from any of the animals.

Histopathologically, no significant pathological changes were observed in any of the organs from any of the animals. Immunohistochemistry revealed no positive reactions in any of the organs or tissues, including the nasal cavities or brains of the animals. In the amplification of the gene for gB (ORF33) using specific primers for EHV-9 by PCR, no bands were detected in any of the samples from the right olfactory bulb, right cerebrum or right cerebellum, the blood samples, or the nasal swabs. It was demonstrated that the nasal swabs had no infectivity. The EHV-9 virus was not neutralized by the serum from any of the animals in the neutralization test.

The results in this study suggest that EHV-9 may be non-pathogenic for adult cynomolgus monkeys. The etiology for the clinical symptom was not apparent because there were no histopathological changes. However, the symptom may have been a response to the EHV-9 inoculation.

Although lethal encephalitis was induced via the nasal, oral, intraperitoneal and ocular routes of EHV-9 inoculation in hamsters (El-Habashi et al., 2010), the intranasal route may be the most probable one. In addition, EHV-9 replicated in the olfactory epithelium and olfactory glandular epithelium in common marmosets (Kodama et al., 2007). Based on those results, successful infection to the olfactory epithelium might be among the essential factors for following EHV-9 induced encephalitis. The proportion of surface area covered by the olfactory epithelium in macaques may be considerably smaller than that in rodents and dogs (Herkema, 1991). Thus, there may be constitutional barriers to the entrance of the EHV-9 into the olfactory epithelium. However, because it has been suggested that the relative amount of the olfactory epithelium in common marmosets is much closer to that of macaques than that of rats, the constitutional distinction may not be associated with EHV-9 infection induced via the intranasal route (Wako et al., 1999).
