**3.2 Inapparent encephalitis in several experimental hosts**

EHV-9 also cause inapparent encephalitis in horses, cattel and pigs. Although animals inoculated with EHV-9 showed high fever and slight depression, the animals did not show neurological symptoms. These inapparent encephalitis were characterized as meningoencephalitis, gliosis and cuffing with EHV-9 antigen bearing neural cells. Histopathological characteristics will be described and discussed.

#### **3.2.1 Horses**

Two young adult horses were inoculated intranasally with 10 ml of virus solution containing 107 PFU and euthanized two weeks after inoculation (Taniguchi et al., 2000b). The animals showed no clinical symptom except a moderate fever (higher than 39oC). The brains showed a moderate degree of nonsuppurative encephalitis characterized by

perivascular aggregates of lymphocytes (Fig. 11A) and gliosis (Fig. 11B). Neither neuronal necrosis nor intranuclear inclusions were observed in affected horses.

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

#### **3.2.2 Cattle**

140 Non-Flavivirus Encephalitis

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).

A B

**3.2 Inapparent encephalitis in several experimental hosts** 

Histopathological characteristics will be described and discussed.

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 intra-

EHV-9 also cause inapparent encephalitis in horses, cattel and pigs. Although animals inoculated with EHV-9 showed high fever and slight depression, the animals did not show neurological symptoms. These inapparent encephalitis were characterized as meningoencephalitis, gliosis and cuffing with EHV-9 antigen bearing neural cells.

Two young adult horses were inoculated intranasally with 10 ml of virus solution containing 107 PFU and euthanized two weeks after inoculation (Taniguchi et al., 2000b). The animals showed no clinical symptom except a moderate fever (higher than 39oC). The brains showed a moderate degree of nonsuppurative encephalitis characterized by

nasally. IHC.

**3.2.1 Horses** 

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.

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

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, after initial propagation at the site of viral entry.

Virology and Pathology of Encephalitis in Alien Hosts by Neurotropic Equine Herpesvirus 9 143

Fig. 13. A: Sagittal section of the entire body of a suckling hamster from the neck to the tail showing all internal organs. IHC. Bar, 10 nm. B: Expression of EHV-9 antigen in peritoneal macrophages at 6 h PI. Bar, 100 micrometer. C: Expression of EHV-9 antigen in the nucleus and cytoplasm of neuron in abdominal ganglion at 24 h PI. IHC. Bar, 100 micrometer.

Several routes of viral entry into the central nervous system (CNS) have been postulated in the neurotropic herpesvirus, which include the neural, olfactory, and hematogenous routes (Johnson, 1998). Other neurotropic herpes viruses, including herpes simplex virus-1 (HSV-1) and porcine herpesvirus-1, may enter the CNS via the intravenous, intramuscular, and intraperitoneal routes (Johnson, 1998). It has been previously hypothesized in hamsters and pigs that a possible route of EHV-9 infection is from the nasal mucosa along the olfactory pathway, vomeronasal organ, and/or trigeminal nerve (Fukushi et al., 2000; Narita et al., 2000) and then trans-synaptically via its connections to the hippocampus, amygdala, and cerebral cortex. Induction of encephalitis by intranasal inoculation in different animals including mice, rats (Fukushi et al., 1997), hamsters (Fukushi et al., 2000), goats (Taniguchi et al., 2000b), pigs (Narita et al., 2000), dogs and cats (Yanai et al., 2003a, b), common marmosets (Kodama et al., 2007) and cattle (El-Habashi et al., 2011) suggests that EHV-9 gains access to the brain via olfactory neurons of the olfactory mucosa specially there was inflammatory reaction in the olfactory mucosa of some of these animals and olfactory bulbs, cerebrum especially the frontal lobe, mid brain and medulla oblongata in most of these animals. Another possible route might be hematogenous dissemination from infected lungs. It is plausible to consider that EHV-9 may have entered the CNS from the nasal mucosa along the olfactory pathway to limbic structures, as was found in other virus, like

It was proved that EHV-9 migrates from nasal cavity to the brain through the olfactory nerve after initial propagation in the olfactory receptor neurons in suckling hamster (El-Habashi et al., 2010b). At 48 h PI, EHV-9 antigen was detected in most of the olfactory receptor neurons as well as in the central processes of the olfactory epithelial neurons, olfactory nerve and olfactory bulb. The olfactory epithelium offer direct free surface on the internal lining of the nasal cavity, after propagation in olfactory receptor neurons, the virus could travel directly through olfactory nerve to the brain while the terminal nerve endings of the maxillary branch of the trigeminal nerve lie in the submucosa and could be only infected with the virus if the epithelial surface is damaged and consequently the axons are directly exposed to the virus and this suggested from suckling hamster experiment as well as common marmoset which showed necrotizing rhinitis as well as late access of the virus to the trigeminal nerve, pons and medulla oblongata (El-Habashi et al, 2010b; Kodama et al., 2007). One study compared various routes of experimental EHV-9 inoculation in Syrian hamsters (Fukushi et al., 2000), including intranasal, intravenous, intraperitoneal, intramuscular, intraocular, and subcutaneous routes, but only intranasal inoculation

ABC

Bornavirus and rabies virus infection (Gosztonyi et al., 1993).

To access transmission of EHV-9 in the nasal cavity and brain, a sagittal model using suckling hamsters was developed, and proved useful in detecting viral transmission as well as extension of pathological lesions using the sagittal section of the head (El-Habashi et al., 2010b). Suckling hamsters were inoculated intranasally with EHV-9, and were sacrificed at 6, 12, 18, 24, 36, 48 and 60 h PI. Sagittal sections of the whole heads were made to access viral kinetics and identify the progress of the neuropathological lesions. At 12-24 h PI the virus attached and propagated in the olfactory epithelium and migrated from one cell to another. At 48 h PI, the olfactory epithelium shows irregularity, necrosis, and erosion in the mucosa (Fig. 12 A), and immunohistochemistry showed encephalitis extending into the olfactory bulb, as well as virus antigen in the olfactory nerve. The trigeminal ganglion showed neuronal necrosis and neurophagia of the trigeminal ganglion cells at 48 h PI (Fig. 12B). One of the most striking findings was the presence of the viral antigen in the connection of the trigeminal sensory nerve root to the brain stem, the pons and medulla oblongata, as well as weak positive reactions in the trigeminal nerve at 60 h PI (Fig. 12C). These results suggested that the sagittal model using suckling hamsters might be useful in accessing the kinetics of neuro-virulent viruses, including EHV-9.

Fig. 12. A: At 48 h PI, the olfactory epithelium shows irregularity, necrosis, and erosion in the mucosa. HE stain. B: At 48 h PI, the trigeminal ganglion shows neuronal necrosis, neurophagia of the trigeminal ganglion cells. HE stain. C: Results of immunolabeling with EHV-9 antibody at 60 h PI. Presence of the viral antigen in the connection of the trigeminal sensory nerve root to the brain stem. IHC.

The kinetics and neuropathogenicity of equine herpesvirus 9 (EHV-9) were studied in hamsters by means of intraperitoneal inoculation (El-Nahass et al., 2011) (Fig. 13). Five-weekold Syrian hamsters and 5-day-old Suckling hamsters were inoculated with 50 and 15 μl of 2 x 106 pfu/ml of EHV-9 virus solution, respectively. After inoculation, EHV-9 antigens were detected in the peritoneal macrophages, which were possibly the primary site of virus attachment and propagation at 6 h PI (Fig. 13B). At 12 h PI, the viral antigen was observed in both the abdominal ganglions (mainly the celiac ganglions) and the peripheral nerves derived from the spinal cord. The virus antigen was seen in the dorsal root (spinal) ganglions (Fig. 13C) and in different parts of the spinal cord especially the mid-lumbar and cervical spinal cord at 24 and 36 h PI respectively. At 96 h PI, the virus antigen was detected in the most caudal part of the brain as well as the intestinal myenteric plexuses. PCR conducted on the blood, spinal cord and brain samples revealed EHV-9 DNA in both the spinal cord, at 24 h PI, and in blood, at 36 h PI. Based on these results, EHV-9 possibly traveled from the myenteric plexus or abdominal ganglions via the peripheral nerves and spinal cord, and finally reached the brain after initial propagation in the abdominal macrophages.

To access transmission of EHV-9 in the nasal cavity and brain, a sagittal model using suckling hamsters was developed, and proved useful in detecting viral transmission as well as extension of pathological lesions using the sagittal section of the head (El-Habashi et al., 2010b). Suckling hamsters were inoculated intranasally with EHV-9, and were sacrificed at 6, 12, 18, 24, 36, 48 and 60 h PI. Sagittal sections of the whole heads were made to access viral kinetics and identify the progress of the neuropathological lesions. At 12-24 h PI the virus attached and propagated in the olfactory epithelium and migrated from one cell to another. At 48 h PI, the olfactory epithelium shows irregularity, necrosis, and erosion in the mucosa (Fig. 12 A), and immunohistochemistry showed encephalitis extending into the olfactory bulb, as well as virus antigen in the olfactory nerve. The trigeminal ganglion showed neuronal necrosis and neurophagia of the trigeminal ganglion cells at 48 h PI (Fig. 12B). One of the most striking findings was the presence of the viral antigen in the connection of the trigeminal sensory nerve root to the brain stem, the pons and medulla oblongata, as well as weak positive reactions in the trigeminal nerve at 60 h PI (Fig. 12C). These results suggested that the sagittal model using suckling hamsters might be useful in accessing the kinetics of

Fig. 12. A: At 48 h PI, the olfactory epithelium shows irregularity, necrosis, and erosion in the mucosa. HE stain. B: At 48 h PI, the trigeminal ganglion shows neuronal necrosis, neurophagia of the trigeminal ganglion cells. HE stain. C: Results of immunolabeling with EHV-9 antibody at 60 h PI. Presence of the viral antigen in the connection of the trigeminal

The kinetics and neuropathogenicity of equine herpesvirus 9 (EHV-9) were studied in hamsters by means of intraperitoneal inoculation (El-Nahass et al., 2011) (Fig. 13). Five-weekold Syrian hamsters and 5-day-old Suckling hamsters were inoculated with 50 and 15 μl of 2 x 106 pfu/ml of EHV-9 virus solution, respectively. After inoculation, EHV-9 antigens were detected in the peritoneal macrophages, which were possibly the primary site of virus attachment and propagation at 6 h PI (Fig. 13B). At 12 h PI, the viral antigen was observed in both the abdominal ganglions (mainly the celiac ganglions) and the peripheral nerves derived from the spinal cord. The virus antigen was seen in the dorsal root (spinal) ganglions (Fig. 13C) and in different parts of the spinal cord especially the mid-lumbar and cervical spinal cord at 24 and 36 h PI respectively. At 96 h PI, the virus antigen was detected in the most caudal part of the brain as well as the intestinal myenteric plexuses. PCR conducted on the blood, spinal cord and brain samples revealed EHV-9 DNA in both the spinal cord, at 24 h PI, and in blood, at 36 h PI. Based on these results, EHV-9 possibly traveled from the myenteric plexus or abdominal ganglions via the peripheral nerves and spinal cord, and finally reached the brain

neuro-virulent viruses, including EHV-9.

sensory nerve root to the brain stem. IHC.

after initial propagation in the abdominal macrophages.

ABC

Fig. 13. A: Sagittal section of the entire body of a suckling hamster from the neck to the tail showing all internal organs. IHC. Bar, 10 nm. B: Expression of EHV-9 antigen in peritoneal macrophages at 6 h PI. Bar, 100 micrometer. C: Expression of EHV-9 antigen in the nucleus and cytoplasm of neuron in abdominal ganglion at 24 h PI. IHC. Bar, 100 micrometer.

Several routes of viral entry into the central nervous system (CNS) have been postulated in the neurotropic herpesvirus, which include the neural, olfactory, and hematogenous routes (Johnson, 1998). Other neurotropic herpes viruses, including herpes simplex virus-1 (HSV-1) and porcine herpesvirus-1, may enter the CNS via the intravenous, intramuscular, and intraperitoneal routes (Johnson, 1998). It has been previously hypothesized in hamsters and pigs that a possible route of EHV-9 infection is from the nasal mucosa along the olfactory pathway, vomeronasal organ, and/or trigeminal nerve (Fukushi et al., 2000; Narita et al., 2000) and then trans-synaptically via its connections to the hippocampus, amygdala, and cerebral cortex. Induction of encephalitis by intranasal inoculation in different animals including mice, rats (Fukushi et al., 1997), hamsters (Fukushi et al., 2000), goats (Taniguchi et al., 2000b), pigs (Narita et al., 2000), dogs and cats (Yanai et al., 2003a, b), common marmosets (Kodama et al., 2007) and cattle (El-Habashi et al., 2011) suggests that EHV-9 gains access to the brain via olfactory neurons of the olfactory mucosa specially there was inflammatory reaction in the olfactory mucosa of some of these animals and olfactory bulbs, cerebrum especially the frontal lobe, mid brain and medulla oblongata in most of these animals. Another possible route might be hematogenous dissemination from infected lungs. It is plausible to consider that EHV-9 may have entered the CNS from the nasal mucosa along the olfactory pathway to limbic structures, as was found in other virus, like Bornavirus and rabies virus infection (Gosztonyi et al., 1993).

It was proved that EHV-9 migrates from nasal cavity to the brain through the olfactory nerve after initial propagation in the olfactory receptor neurons in suckling hamster (El-Habashi et al., 2010b). At 48 h PI, EHV-9 antigen was detected in most of the olfactory receptor neurons as well as in the central processes of the olfactory epithelial neurons, olfactory nerve and olfactory bulb. The olfactory epithelium offer direct free surface on the internal lining of the nasal cavity, after propagation in olfactory receptor neurons, the virus could travel directly through olfactory nerve to the brain while the terminal nerve endings of the maxillary branch of the trigeminal nerve lie in the submucosa and could be only infected with the virus if the epithelial surface is damaged and consequently the axons are directly exposed to the virus and this suggested from suckling hamster experiment as well as common marmoset which showed necrotizing rhinitis as well as late access of the virus to the trigeminal nerve, pons and medulla oblongata (El-Habashi et al, 2010b; Kodama et al., 2007). One study compared various routes of experimental EHV-9 inoculation in Syrian hamsters (Fukushi et al., 2000), including intranasal, intravenous, intraperitoneal, intramuscular, intraocular, and subcutaneous routes, but only intranasal inoculation

Virology and Pathology of Encephalitis in Alien Hosts by Neurotropic Equine Herpesvirus 9 145

EHV-9 infection can be regarded as one of cross-species viral transmission. In nature, natural barriers exist to prevent the cross-species transmission as well as natural clearance such as predation by carnivors can hide the lethal cross-species transmission. An artificial situation of zoos or farms can cause cross-species transmission among carrier animals and susceptible animals. However, some species do not show clinical symptoms even though they can be infected by viruses. In EHV-9 infection, all of the animals infected by EHV-9 caused various degree of meningo-encephalitis. It is not clear what kind of factors are

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**5. References** 

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