Effect of Mouse Strain on Equine Herpesvirus -9 Infection

Research Abstract

English abstract Based on previous experimental studies in various animals inoculated via the nasal route, it was confirmed that the olfactory pathway (i.e. through the olfactory nerves), as well the trigeminal pathway (through the trigeminal nerve), were the major route of transmission of EHV-9 into the CNS. However, our recent study, in which different routes of inoculation were compared, clearly indicated that the 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 antigen-positive 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 animals inoculated via these non-nasal routes did not exhibit EHV-9 induced rhini􀀧s, and the olfactory bulbs showed milder lesions and fewer viral antigen-positive cells than were observed in the animals infected via the nasal route . These findings spurred the author to perform the investigation on intraperitoneal inoculation of EHV-9 described in Chapter 1. In this part, I first used the adult Syrian hamster as the animal model for evaluating the kinetics of EHV-9 induced encephali􀀧s . The results of this study showed the essential roleof the spinal cord in the propagation and transmission of EHV-9. However, the study failed to determine the following : -١The primary sites for virus attachment and propagation -٢Time scheduled pathogenesis -٣Whether or not the hematogenous routes play a role in virus transmission To elucidate these points, suckling Syrian hamsterswere used in Chapter 1. In this part, using this animal model, it was possible to definitely determine the actual ١٢٣ pathogenesis of EHV-9 following intraperitoneal inocula􀀧on of EHV-9. This study showed that the virus gained access to the brain through the neuronal pathway rather than the haematogenous pathway, with this finding being confirmed by performing PCR on blood, brain and spinal cord samples. Fingerprints of EHV-9 DNA were found in the spinal cord samples at 36 h PI, in the brainsamples at 96 h, and in the PI blood samples at 48 h PI. The results clearly showed thatEHV-9 DNA was detected earlier in the spinal cord than in the blood. EHV-9 induced encephali􀀧s following intraperitoneal inoculation of EHV-9 may occur ini􀀧ally through primary a􀀶achment and propaga􀀧on of EHV-9 virus in peritoneal cells, mainly macrophages (which was confirmed by applying immunocytochemistry in an abdominal wash),following which two possible pathways might be proposed (Plate I :( -١Infection of the peripheral nerve axons and coeliac plexus within the abdominal cavity, followed by propagation of the virus within the dorsal root (spinal ( ganglia. That would be followed by transmission and propagation of EHV-9 in the spinal cord. The latter plays an essential role in ascending transmission of the virus to the brain . -٢Infection of the myenteric plexus with EHV-9, leading to spreading of the virus to the brainstem via the vagus nerve . Also, as described in Chapter 1, it was possible for the first time to identify the tendency of EHV-9 to infect the livers of suckling animals, and consequently to identify the role of the liver in virus replication, especially during the initial stages of infection . In Chapter 2, the detailed pathogenesis of EHV-9 following oral inocula􀀧on is illustrated, first in adult ICR and then in suckling Syrian hamsters. In the former, it ١٢٤ seemed that the primary sites for virus attachment and propagation were lingual macrophages, as the virus was detected immunohistochemically star􀀧ng from 12 h PI . These cells help in the propagation, transmission and spreading of the EHV-9 virus to target cells, or may serve as reservoirs for long-term infection that is followed by the development of encephalitis, as well as detection of the virus immunohistochemically at 72 h PI, mainly in the pons, in the hippocampus,midbrain and cerebellum at 96 h PI ، and finally in the olfactory bulb (mainly the granular layer) at 120 h PI . The distribution of EHV-9 in the granular layer andmitral layer of the olfactory bulb seems to indicate that the virus travels through non-olfactory pathways. One of the predominant features that is found in adult ICR is the effect of EHV-9 virus infection on the gastrointestinal tract in the formof hyperkeratosis, moderate to severe gastritis and multifocal ulceration in the forestomach. This is considered very important in terms of future studies on the effect of EHV-9 on other systems of animal bodies, particularly the gastrointestinal system . In Chapter 2, using a suckling animal model, I tried to cover points rela􀀧ng to the pathogenesis of EHV-9 infec􀀧on that could not be achieved using adult ICR mice . In this section, the role of oral and lingual submucosa was confirmed, as well as the role of macrophages in the propagation and transmission of EHV-9 to the mandibular and maxillary branches of the trigeminal nerve at 36 h PI and at 48h PI in the nuclei and cytoplasm of pseudounipolar neurons of the trigeminal ganglia, the meninges and the brainstem (the root of the trigeminal nerve entrance). That was followed by the occurrence of encephali􀀧s in the midbrain and ponsfrom 48 h PI un􀀧l the end of the experiment (plates II and III). At the same time, EHV-9 DNA was detected in the brains of EHV-9 inoculated hamsters at 36 h PI, in the spinal cord at 96 h PI and finally ١٢٥ in the blood samples at 48 h PI, thus confirming that the EHV-9 virus is transmi􀀶ed through neuronal pathways following oral inoculation. In Chapter 3, I discussed the manner of EHV-9 infec􀀧vity in two mouse strains : the congenitally athymic strain (BALB/c-nu/nu) and phenotypically normal mice )BALB/c). The infectivity of these two mouse strains was found to be quite different . BALB/c-nu-nu mice are more susceptible to EHV-9 infec􀀧on than BALB/c mice ، which were found to be relatively resistant. That was confirmed through weak EHV-9 propagation in the olfactory epithelia, followed bycomplete virus clearance within the olfactory epithelia at 96 h PI in BALB/c mice. Furthermore, the applica􀀧on of RT-PCR of EHV-9 in formalin fixed tissues on the olfactoryepithelia of BALB/c mice produced increases in the rela􀀧ve transcrip􀀧on ac􀀧vity ofORF30 in the olfactory epithelia un􀀧l ٤٨h PI, followed by a sharp decrease in transcrip􀀧on ac􀀧vity in this gene at 96 h PI )plate IV). In contrast, in immunohistochemical testing, BALB/c-nu-nu mice demonstrated high levels of EHV-9 an􀀧gen within the olfactory epithelia from 24 h PI until the end of the experiment. In addition, the virus was detected immunohistochemically not only in the olfactory nerves of all inoculated animals but also within the olfactory bulb in one animal. A proportional increase in mRNA expression levels was seen un􀀧l 48 h PI, followed by a gradual slowing until the expression level reached 20-fold at 96 h PI. Comparison of the rela􀀧ve quan􀀧ty of ORF30 gene expression using the cross point method (CP) each hour post inocula􀀧on between BALB/c and BALB/c-nu-nu mice strains showedno statistical differences in rela􀀧ve gene expression values of ORF30 in the brain 􀀧ssues. In addi􀀧on, significant gene expression was observed in olfactory epitheliain BALB/c-nu-nu mice compared to BALB/c mice at 24, 36, 48, 72 and 96 h PI .

Research Keywords

Herpesvirus -9 ,mice