Transcriptome analysis following neurotropic virus infection reveals faulty innate immunity and delayed antigen presentation in mice susceptible to virus-induced demyelination.

Viral infections of the central nervous system cause acute or delayed neuropathology and clinical consequences ranging from asymptomatic courses to chronic, debilitating diseases. The outcome of viral encephalitis is partially determined by genetically programed immune response patterns of the host. Experimental infection of mice with Theiler's murine encephalomyelitis virus (TMEV) causes diverse neurologic diseases, including TMEV-induced demyelinating disease (TMEV-IDD), depending on the used mouse strain. The aim of the present study was to compare initial transcriptomic changes occurring in the brain of TMEV-infected SJL (TMEV-IDD susceptible) and C57BL/6 (TMEV-IDD resistant) mice. Animals were infected with TMEV and sacrificed 4, 7, or 14 days post infection. RNA was isolated from brain tissue and analyzed by whole-transcriptome sequencing. Selected differences were confirmed on a protein level by immunohistochemistry. In mock-infected SJL and C57BL/6 mice, >200 differentially expressed genes (DEGs) were detected. Following TMEV-infection, the number of DEGs increased to >700. Infected C57BL/6 mice showed a higher expression of transcripts related to antigen presentation via major histocompatibility complex (MHC) I, innate antiviral immune responses and cytotoxicity, compared with infected SJL animals. Expression of many of those genes was weaker or delayed in SJL mice, associated with a failure of viral clearance in this mouse strain. SJL mice showed prolonged elevation of MHC II and chemotactic genes compared with C57BL/6 mice, which presumably facilitates the induction of chronic demyelinating disease. In addition, elevated expression of several genes associated with immunomodulatory or -suppressive functions was observed in SJL mice. The exploratory study confirms previous observations in the model and provides an extensive list of new immunologic parameters potentially contributing to different outcomes of viral encephalitis in two mouse strains.

Interleukin-10 expression during the acute phase is a putative prerequisite for delayed viral elimination in a murine model for multiple sclerosis.

Reduced protective immunity leads to viral persistence and demyelination in Theiler's murine encephalomyelitis. The aim of the present study was to compare the phenotype of brain-infiltrating leukocytes and cytokine expression in susceptible SJL and resistant C57BL/6 mice during Theilervirus-induced acute polioencephalitis. In contrast to C57/BL6 mice, SJL mice show an increased number of Foxp3(+) regulatory T cells and CD45R(+) B cells associated with delayed viral elimination and elevated IL-10 mRNA transcripts in the brain. Results substantiate the hypothesis that an imbalanced cytokine milieu during the early infection phase contributes to ineffective antiviral immunity in animals with a susceptible genetic background.

Periventricular demyelination and axonal pathology is associated with subependymal virus spread in a murine model for multiple sclerosis.

OBJECTIVES: Theiler's murine encephalomyelitis virus (TMEV) infection of mice is a widely used animal model for demyelinating disorders, such as multiple sclerosis (MS). The aim of the present study was to identify topographical differences of TMEV spread and demyelination in the brain of experimentally infected susceptible SJL/J mice and resistant C57BL/6 mice. METHODS: Demyelination was confirmed by Luxol fast blue and cresyl violet staining and axonal damage by neurofilament-specific and ß-amyloid precursor protein-specific immunohistochemistry. Viral dissemination within the central nervous system (CNS) was quantified by immunohistochemistry and in situ hybridization. Further, the phenotype of infected cells was determined by confocal laser scanning microscopy. RESULTS: An early transient infection of periventricular cells followed by demyelination and axonopathies around the fourth ventricle in SJL/J mice was noticed. Periventricular and brain stem demyelination was associated with a predominant infection of microglia/macrophages and oligodendrocytes. CONCLUSIONS: Summarized, the demonstration of ependymal infection and subjacent spread into the brain parenchyma as well as regional virus clearance despite ongoing demyelination and axonal damage in other CNS compartments allows new insights into TME pathogenesis. This novel aspect of TMEV CNS interaction will enhance the understanding of region-specific susceptibilities to injury and regenerative capacities of the brain in this MS model.

[Morphological studies in different avian species on artefacts induced by euthanasia with T 61 " or Pentobarbital (Narcoren)]. / Morphologische Untersuchungen an verschiedenen Vogelarten zu Artefakten durch Euthanasie mit T 61 oder Pentobarbital (Narcoren).

In mammals (e. g. macaques, dogs, cats, rats, sheep) as well as in men (suicides) euthanasia performed by intravenous injection of T 61 leads to serious lesions in lung, kidney or/and liver (endothelial damage, hyperemia, oedema, necrosis). This is caused by the solvent dimethylformamide (DMF). In this study, in contrast, in different species of birds (e. g. blackbird, carrion crow, kestrel, common buzzard, homer pigeon, common wood pigeon, mallard duck) and various modes of applications and dosages T 61, 1.0-3.0 ml/kg body mass, did not induce comparable artefacts in tissues of internal organs in the narcotized animals. Microscopically, only hyperemia and oedema of lung, kidney and/or liver were found. However, milder but similar lesions were detected also in groups of birds euthanized by pentobarbital (200 mg/kg body mass) as well as in control groups (overdosed ketamine intramusculary, 100 mg/kg body mass, and rapid exsanguination). In conclusion, euthanasia of narcotized birds performed by intravenous or intracardial injections ofT 61 seemed to be suitable. The observed lesions could therefore not be interpreted as T61 induced artefacts.

Axonopathy is associated with complex axonal transport defects in a model of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease characterized by myelin and axonal pathology. In a viral model of MS, we tested whether axonopathy initiation and development are based on an impaired transport of neurofilaments. Spinal cords of Theiler's murine encephalomyelitis virus (TMEV)-infected and mock-infected mice and TMEV infected neuroblastoma N1E-115 cells were analyzed by microarray analysis, light microscopy and electron and laser confocal microscopy. In vivo axonal accumulation of non-phosphorylated neurofilaments after TMEV infection revealed a temporal development caused by the impairments of the axonal traffic consisting of the downregulation of kinesin family member 5A, dynein cytoplasmic heavy chain 1, tau-1 and ß-tubulin III expression. In addition, alterations of the protein metabolism were also noticed. In vitro, the TMEV-infected N1E-115 cells developed tandem-repeated swellings similar to in vivo alterations. Furthermore, the hypothesis of an underlying axonal self-destruction program involving nicotinamide adenine dinucleotide depletion was supported by molecular findings. The obtained data indicate that neurofilament accumulation in TME is mainly the result of dysregulation of their axonal transport machinery and impairment of neurofilament phosphorylation and protein metabolism. The present findings allow a more precise understanding of the complex interactions responsible for initiation and development of axonopathies in inflammatory degenerative diseases.

Transient peripheral immune response and central nervous system leaky compartmentalization in a viral model for multiple sclerosis.

Theiler's virus-induced demyelination represents an important animal model to study the chronic-progressive form of multiple sclerosis (MS). The aim of the present study was to identify specific genes and pathways in the deep cervical lymph node (cLN) and spleen of experimentally infected SJL-mice, using DNA microarrays. Analyses identified 387 genes in the deep cLN and only 6 genes in the spleen of infected animals. The lymph node presented 27.4% of genes with fold changes +/-1.5 at 14 days post infection (dpi) and a reduced transcription at later time points. K-means clustering analyses resulted in five clusters. Accordingly, functional annotation revealed that the B-cell immune response pathway was the most up-regulated cluster at the early phase. Additionally, an increase of CD68- and lysozyme-positive cells in the deep cLN was observed by immunohistochemistry. Polioencephalitis was most intense at 14 dpi, and the spinal cord demyelinating leukomyelitis started at 42 dpi. In summary, early gene expression is indicative of virus-trigged immune responses in the central nervous system (CNS)-draining lymph node. The decreased gene transcription in the deep cLN during the chronic phase and the low number of spleen genes supports the hypothesis of a compartmentalized inflammation within the CNS, as described in progressive MS.

Theiler's murine encephalomyelitis virus preferentially infects immature stages of the murine oligodendrocyte precursor cell line BO-1 and blocks oligodendrocytic differentiation in vitro.

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelination is an important animal model for multiple sclerosis. The presence of oligodendrocyte precursor cells (OPCs) within demyelinated lesions together with the limited extent of remyelination has raised the question of how OPCs are affected by TMEV. It is well established that oligodendrocytes, astrocytes and microglia are targets during the chronic phase of the disease. However, whether TMEV infection interferes with the capacity of OPCs to generate oligodendrocytes has remained unclear. In the present study, a bipotential murine OPC cell line termed BO-1 was used to determine the antigenic phenotype susceptible to TMEV and the impact of TMEV infection upon cell differentiation. We show here that retinoic acid increased oligodendrocytic differentiation and decreased proliferation and TMEV infection rates. TMEV under serum-free conditions infected about 75% and 60% of early OPCs (NG2(+) and A2B5(+)) and immature oligodendrocytes (CNPase(+)), respectively, but only approximately 18% of mature oligodendrocytes (MBP(+)). Infection with TMEV prior to application of retinoic acid significantly reduced the percentage of MBP(+) BO-1 cells. These data demonstrate that TMEV preferentially infects early stages of the oligodendrocytic lineage and blocks oligodendrocyte maturation. The first demonstration of TMEV-mediated effects on OPC differentiation may shed new light on the pathogenesis of TMEV-induced demyelination and offers an explanation for the limited remyelination observed in vivo.

Generation and characterization of a polyclonal antibody for the detection of Theiler's murine encephalomyelitis virus by light and electron microscopy.

The BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) causes a demyelinating leukomyelitis in mice, which serves as an important animal model for multiple sclerosis in humans. The present report describes the generation and characterization of a TMEV-specific polyclonal antibody by immunization of rabbits with purified TMEV of the BeAn strain. The specificity of the antibody was confirmed by Western blotting and sequence analysis of the recognized antigen by high resolution mass spectrometry. The presence of TMEV-specific polyclonal antibodies in post-immunization sera was tested on TMEV-infected L-cells (murine lung tumor cell line) using an immunofluorescence assay. Additionally, the rabbit serum enabled virus detection in formalin-fixed and paraffin-embedded TMEV-infected BHK(21) cell pellets and brain tissue of TMEV-infected mice by immunohistochemistry. Immune electron microscopy revealed colloid gold-labeled picornavirus-typical paracrystalline arrays and non-aggregated viral particles of TMEV-infected BHK(21) cells. The present report demonstrates the applicability of the generated marker for investigating TMEV cell tropism and viral spread at a cellular and subcellular level in future studies.