In the present study, experimental BDV-infection of Lewis rats and TNF-transgenic mice was used for the characterization of in vivo strategies of BDV to spread and persist within the CNS and to analyze basic principles of viral neurotropism. Thus, mechanisms of viral replication and transcription as well as virus-host interactions were investigated and modulated by overexpression of the proinflammatory cytokine tumor necrosis factor α (TNF).
Viral structural proteins provide essential functions for viral spread, determination of cell tropism and induction of antiviral defense. Moreover, they are also capable to interfere with viral replication and transcription so that the less well-known expression strategies of the BDV-glycoprotein (BDV-GP) and BDV-matrixprotein (BDV-M) were analyzed in detail.
The typical clinical biphasic course of Borna disease (BD) in Lewis rats was confirmed as expected. The TNF-transgenic presented unexpected epileptic seizures with an increasing incidence during the course of the infection. In initial studies, elevated levels of prodynorphin mRNA were recorded in non infected, TNF-transgenic mice brains, possibly indicating a modulated dynorphin system. The amount of prodynorphin mRNA decreased significantly after BDV-infection only in the transgenic animals. Therefore, dysregulations of neuromodulatory neuropeptides in combination with the inflammatory and reactive alterations seem to be involved in the occurrence of epileptic seizures.
Histologically, the anticipated non purulent meningoencephalitis with long lasting astrogliosis and microglia activation was detected after BDV-infection of Lewis rats and TNF-transgenic mice. The non transgenic mice developed only mild inflammatory lesions. In the brains of Lewis rats, the typical decrease of inflammatory alterations was noted in the chronic phase of infection. The transgenic mice showed a significant progression of the meningoencephalitis with inflammatory infiltrates mainly in TNF-overexpressing brain areas which was accompanied by an unusual hypertrophy and degeneration of astrocytes.
After BDV-infection of Lewis rats and TNF-transgenic mice, an upregulation of neuroprotective factors such as BDNF, ADNP, HO-1 and the transcription factor NFкB was found in the brain. HO-1 was already induced very early after infection and its expression decreased simultaneously with the increase of the inflammatory reaction, whereas ADNP and BDNF were still upregulated at time points of maximal inflammation until the chronic phase. For the first time, both factors were also detected in inflammatory cells so that neuroimmunemodulatory effects can be assumed after BDV-infection. This also indicates that various protective and insult-limiting factors are acting during the different phases of BDV-infection. In the mouse model, NFкB activation was detected as nuclear translocation in immune cells, but also in endothelial cells, astrocytes and microglia, but not in neurons.
SUMMARY 178
The non purulent meningoencephalitis with astrocytic hypertrophy and microglial activation as well as the epileptic seizures are unequivocally due to the TNF-overexpression. The basic levels of TNFto and TNFtg mRNA were approximately 2-fold higher in tg+/+ than in tg+/- animals and significantly increased compared to ntg mice. Homozygous mice showed seizures more frequently and earlier after infection and developed more severe inflammatory and degenerative lesions in the brain. Interestingly, TNFtg induced an upregulation of host-derived TNF mRNA. After BDV-infection, no significant increase of TNFto mRNA values in transgenic mice was detected. However, TNFR1 and TNFR2 mRNA levels were significantly up-regulated in all infected mice groups. These findings indicate that TNF-effects were mediated by increased receptor availability and not by further upregulation of TNF itself.
Selective expression strategies were observed for the viral structural proteins which differed significantly from the expression pattern of the viral nucleoprotein BDV-N. The well-known disseminated expression profile of BDV-N was confirmed. After BDV-infection of Lewis rats and mice, BDV-N and BDV-N +ssRNA were present in the entire brain in cytoplasm and nucleus of various brain cells. Therefore, supply of adequate amounts of BDV-N is essential for an appropriate replication and transcription during the acute and chronic persistent phase in both models. BDV-M and BDV-GP expression in the brain was dominated by spatio-temporal, regional and cell specific effects. The expression profile of both structural proteins exhibited significant differences but also common features when compared to each other.
Consistently, BDV-M and BDV-GP were less expressed in the chronic phase of infection.
BDV-GP expression was significantly lower in both models and only detected in the cytoplasm of single neurons in defined brain areas. In the brain of Lewis rats and mice, BDV-M was found mainly in the cytoplasm of various brain cells in the entire brain. Early after infection of Lewis rats, BDV-M expression was enhanced by upregulation of transcription, nuclear export of BDV-intron I +ssRNA and BDV-M synthesis in neurons, whereas BDV-M was found more often in astrocytes and BDV-intron I +ssRNA was more frequently retained in the nucleus in the chronic phase. This correlated well with the decrease of BDV-M expression. In general, nuclear export of BDV-intron I +ssRNA differed between time points p.i., showed regional and cellular preferences and was constantly present only in hippocampal neurones. In BDV-infected mice, a comparable expression strategy of intron I-containing transcripts was noted which allowed efficient translation of BDV-M with occurrence of BDV-M in the entire brain. After BDV-infection of Lewis rats, the restricted BDV-GP expression was achieved by a regionally different efficiency of transcription, restriction of nuclear export, temporal regulation of neuronal transcription and limitation of translation predominantly in the chronic phase of infection. The controlled supply of BDV-GP coding transcripts is most likely due to low splicing efficiency of intron I and nuclear retention of Intron II-containing +ssRNAs. Presumably, the latter represents an essential mechanism
SUMMARY 179 to regulate viral transcription and translation capacity not only of BDV-GP but also of BDV-M.
In addition, all these viral strategies varied depending on the infected cell type and time point after infection. The limited, neuronal expression of BDV-GP points to a role of BDV-GP mainly for the transneuronal spread of BDV. In the BDV-infected mice brains, even less GP positive cells were detected despite more frequent cytoplasmic localization of BDV-intron II +ssRNA, stable amounts of BDV-BDV-intron I +ssRNA, decrease of BDV-BDV-intron II containing transcripts in the chronic phase or more frequent splicing of intron II, respectively.
The viral transcription efficiency in BDV-infected rat and mice brains of all groups confirmed the 3’-5’-transcription gradient of BDV. Copy numbers of BDV-N specific transcripts were significantly the highest at all time points investigated, whereas BDV-intron I and BDV-intron II +ssRNAs were expressed up to 10-fold less. In the rat brain, amounts of BDV-intron I and BDV-intron II +ssRNAs were almost comparable, most likely indicating a low splicing efficiency. Only at day 24 pi., significantly more BDV-intron I +ssRNA was measured in neurones compared to BDV-intron II +ssRNA, while this constellation was found constantly in all mice groups. In general, viral transcription was differently regulated in the acute and chronic phase of the disease in hippocampus and N.caudatus/putamen and in neurons and astrocytes, respectively. Astrocytes seem to play a particular role in the persistent stage of infection. The RNA expression profile after BDV-infection of all mice groups likewise points to a regulated transcription. The significant increase of RNA containing cells in the ntg animals in comparison to a nearly constant number of RNA positive cells in both tg groups could indicate a limiting effect of TNF. However, the TNF-overexpression did not eliminate BDV from the brain. In the mice brain, similar as in the rat, spliced and unspliced subgenomic RNAs were exported from the nucleus.
The demonstration of BDV-LE +ssRNA and genomic viral RNA in the rat and mice brains suggests a balanced and region-specific replication strategy of BDV. In general, low amounts of BDV-LE +ssRNA seem to be sufficient to guarantee adequate quantities of viral genome for a disseminated virus infection and to ensure viral persistence. Despite low levels of BDV-LE +ssRNA, high titers of infectious virus were determined even in the chronic phase in both models.
In the rat and mouse brain, the preference of BDV for specific brain regions and cell types was definitely shown for the hippocampus and pyramid cells on the basis of selective neuroprotective expression profiles and transcription efficiencies. This hippocampal condition seems to provide particularly suitable prerequisites for viral transcription and replication. In more detail, in the hippocampus of Lewis rats, transcription efficiencies of BDV were constantly high, while in the N.caudatus/putamen transcription was less proficient and similarly elevated expression levels were achieved only delayed. Moreover, efficiency of translation, at least for BDV-GP, was also obviously diminished in the N.caudatus/putamen.
SUMMARY 180
In summary, new regulative mechanism of viral transcription, translation and replication could be shown after experimental BDV-infection of Lewis rats and TNF-transgenic mice. Thus, the expression of viral proteins and subgenomic RNAs depends on spatio-temporal, regional and cell-specific effects with a key role for restriction of nuclear export and/or maturation and splicing of +ssRNAs as well as limitation of translation. This is completed by temporarily different, region- and cell type-specific quantities of viral transcripts and replicative intermediates. Both viral preparations were able to spread within the entire brain and to persist, also under modulated conditions such as overexpression. However, TNF-overexpression did not cause significant changes in viral protein expression but interfered with limited numbers of BDV RNA positive cells and enhanced amounts of BDV-LE +ssRNA.
Therefore, in the mice, BDV seem to utilize related, but modified viral strategies as in the rat brain. The expression of neuroprotective factors and the reaction pattern of resident brain cells also displayed spatio-temporal and cell-specific effects. In general, the present study provides more detailed insights into the complexity of the pathogenetic principles of BD and substantiates that BDV-infections represent excellent models for the investigation of molecular aspects of persistent viral infections of the CNS and their corresponding neuropathogenesis.
ABBILDUNGEN 181