تأثير سلالة الفئران على عدوى فيروس هربس الخيول 9
ملخص البحث
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 rhinis, 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 encephalis .
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 inoculaon 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 encephalis following intraperitoneal
inoculation of EHV-9 may occur inially through primary aachment and propagaon
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 inoculaon is
illustrated, first in adult ICR and then in suckling Syrian hamsters. In the former, it
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seemed that the primary sites for virus attachment and propagation were lingual
macrophages, as the virus was detected immunohistochemically starng 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 relang to
the pathogenesis of EHV-9 infecon 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 encephalis in the midbrain and ponsfrom 48 h PI unl 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 transmied
through neuronal pathways following oral inoculation.
In Chapter 3, I discussed the manner of EHV-9 infecvity 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 infecon 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 applicaon of RT-PCR
of EHV-9 in formalin fixed tissues on the olfactoryepithelia of BALB/c mice produced
increases in the relave transcripon acvity ofORF30 in the olfactory epithelia unl
٤٨h PI, followed by a sharp decrease in transcripon acvity 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 angen 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 unl 48 h PI, followed by a gradual slowing until the
expression level reached 20-fold at 96 h PI. Comparison of the relave quanty of
ORF30 gene expression using the cross point method (CP) each hour post inoculaon
between BALB/c and BALB/c-nu-nu mice strains showedno statistical differences in
relave gene expression values of ORF30 in the brain ssues. In addion, 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 .
الكلمات المفتاحيه
Herpesvirus -9 ,mice