Nicole Werner-Keišs
Investigations on the expression
of structural proteins of the Borna disease virus in intracerebrally infected Lewis rats
1. In the introduction, an overview of the current knowledge on natural and experimental Borna Disease Virus (BDV) infections is given, followed by a characterization of the BDV and its proteins. The immunopathogenesis of Borna Disease (BD) and its interactions with the host are discussed. Additionally, the structure and functions of subtilisin-like proprotein-convertases (SPC) are briefly described.
2. Adult Lewis rats were inoculated intracerebrally with a rat-adapted BDV-preparation 5/25/92 and clinical signs were observed within an investigation period of 90 days p.i. Brain tissue was investigated histologically for the presence of inflammatory and degenerative changes. Various BDV-specific antigens were detected immunohistologically by using a monoclonal antibody directed against BDV-N and polyclonal, monospecific antibodies against BDV-GP and BDV-M.
Their spatio-temporal distribution in the rat brain was semiquantitatively analysed.
The proprotein convertases SPC4 and SPC6 were detected by polyclonal antibodies. In situ hybridisation for the detection of different viral RNAs was performed using DIG-labelled RNA probes specific for BDV-N RNAs (genomic RNA, mRNA) and RNAs specific for the two subunits of BDV-GP, the C-terminal and N-terminal part. In addition to the semiquantitative evaluation, hippocampal cells positive for BDV-N and –GP and the corresponding RNAs were counted and statistically analysed. Infectious virus was determined in brain tissue by titration in cell cultures applying indirect immunofluorescence test (IIFT).
3. The infected Lewis rats displayed the typical biphasic course of the disease with the first phase starting at day 14 p. i. and the second phase beyond day 31 p.i.
Onset of clinical signs corresponded to the detection of inflammatory lesions in the infected rat brains.
SUMMARY 146
4. All BDV-infected animals developed a non-purulent meningoencephalitis. At day 7 p.i., small mononuclear infiltrates were detected in the leptomeninx. Mononuclear perivascular infiltrates were present firstly at day 14 p.i. and were most prominent between day 31 and 42 p.i., accompanied by severe parenchymal mononuclear infiltrates. In later stages of the disease (day 50 p.i.), inflammatory infiltrates decreased and a mild to moderate hydrocephalus developed. Inflammatory infiltrates were mainly found in the cortex cerebri, the hippocampal formation, amygdala and thalamus. A reactive astrogliosis was observed starting approximately at day 24 p.i.
5. All three BDV-specific antigens were detected firstly at day 7 p.i. in the rat brain by immunohistochemistry. BDV-N was expressed in cytoplasm and nuclei of neurons, astrocytes, oligodendrocytes and ependymal cells as well as in neuronal processes and neuropil. At later time points p.i., BDV-M was detectable mainly in astrocytes and small neurons. In contrast to the disseminated occurrence of BDV-N and BDV-M throughout the entire brain, BDV-GP was expressed exclusively in the cytoplasm of single large neurons in the cerebral cortex, hippocampal formation, amygdala and thalamus. This might indicate a restricted BDV-GP expression. During the whole investigation period, expression of BDV-GP was significantly lower than the expression of N (p < 0,0001). The peak of BDV-GP detection occurred between day 18 and 24 p.i. which was earlier than maxima of BDV-N and BDV-M expression (starting at day 31 p.i. each). At later time points p.i., demonstration of BDV-GP and BDV-M decreased, while BDV-N remained expressed at maximum levels until the end of the investigation.
6. The investigated proprotein-convertases SPC4 and SPC6 were detected in BDV-infected as well as “Mock” - BDV-infected animals by immunohistochemistry at all time points investigated. The SPC expression did not correlate with BDV-infection and there was no difference between BDV- and “Mock”-infected brains. SPC4 was detected throughout the entire rat brain and was expressed in all cell compartments of neurons and glial cells. In contrast, SPC6A was found almost exclusively in the white matter, neuronal processes and neuropil of the corpus callosum, striatum/N. caudatus, thalamus, amygdala and mesencephalon.
SUMMARY 147 7. All BDV-specific RNAs were detected firstly at day 7 p.i. by ISH. All RNAs were
found throughout the entire rat brain, but expression of BDV-GP RNAs occurred in fewer cells in comparison to BDV-N RNAs and was predominantly present in the cortex cerebri, amygdale, hippocampal formation and thalamus. In contrast to the expression pattern of BDV-N mRNA, which was found mainly in neuronal processes and cytoplasm, but also in nuclei of neurons and ependymal cells, BDV-GP mRNA expression was restricted to the nuclei of cells. This points out a restriction of nuclear export of BDV-GP mRNA. In comparison with BDV-GP-N mRNA, the expression of BDV-GP was significantly lower (p = 0.0005), what is indicative of a restricted BDV-GP translation. In general, genomic RNA occurred only in nuclei of cells, but occassionally fibres could display a positive signal as well. The maximum of expression of BDV-N and BDV-GP RNAs extended from day 24 to 60 p.i. At the end of the investigation period, a slight decrease was noted. In total, significantly more cells were positive for BDV-N mRNA than for BDV-GP-N mRNA (p = 0,0007).
8. Infectious virus was isolated from BDV-infected brain tissue at day 50 and 75 p.i.
with a titer of 2x107 ID50/ml. This titer is an evidence for virus replication during the infection.
9. The present study describes the spatio-temporal expression of BDV-N, BDV-M and BDV-GP as well as the RNAs of BDV-N and BDV-M during experimental BDV infection. The results of this in vivo study show, that different regulating and controlling mechanisms are operative for each viral mRNA and the corresponding proteins during experimental BDV infection of adult Lewis rats. In particular strategies as regional and cellular tropism of viral protein expression, regulation of transcription of the subgenomic RNAs and restriction of viral GP-translation seem to be involved.
These in vivo mechanisms are most likely essential for an efficient and successful viral dissemination and persistence in the CNS of infected hosts.
ABBILDUNGEN 148