Canine distemper virus does not infect oligodendrocytes in vitro

Dissociated canine brain cell cultures were infected with virulent canine distemper virus (CDV). Double immunofluorescent labelling was done to simultaneously demonstrate viral antigen and specific glial cell markers. Virus containing oligodendrocytes were not found at any stage of the infection. A certain proportion of the infected cells were shown to be astrocytes. It was concluded that CDV has no obvious tropism for oligodendrocytes which could explain the mechanism of demyelination in distemper in vivo.     

Spread and distribution of viral antigen in nervous canine distemper

Summary Canine distemper virus (CDV) antigen was demonstrated immunocytochemically in the central nervous system (CNS) of 19 dogs killed from 16 to 170 days after infection. In the earliest lesions, infection of glial cells preceded demyelination, and the degree of myelin destruction correlated with the amount of viral antigen in the tissue. It was concluded that initial demyelination in distemper is directly viral-induced, but the nature of the infected glial cells remains uncertain. Ependymal infection and spread of virus in the subependymal white matter was often seen, suggesting invasion of CDV into the CNS along the CSF pathways. Inflammation during the latter stages of the infection appeared to be associated with viral clearance from the CNS in most dogs. In two dogs with chronic progressive neurologic distemper, viral antigen was still present in the brain suggesting that viral persistence and associated immunologic reactions may contribute to further myelin damage. With the exception of one dog that survived for 6 months after infection, viral antigen was no longer detected in the dogs that had reeovered.Supported by the Swiss National Science Foundation (Grant no. 3.809.81) and the Swiss M. S. Society     

Glial proteins in canine distemper virus-induced demyelination

A temporal series of demyelinating lesions in experimental canine distemper virus (CDV) infection was examined with immunohistological techniques demonstrating myelin basic protein (MBP), myelin-associated glycoprotein (MAG), and glial fibrillary acidic protein (GFAP) on serial sections. The earliest lesions were characterized by decreased MBP and MAG and increased GFAP. During the further progression of the disease, MBP and MAG losses continued to match each other. There was no indication of MAG loss preceding the disappearance of MBP. In the more advanced lesions there was a marked decrease of GFAP positive cells. Since these findings differed considerably from similar immunohistochemical studies in progressive multifocal leukoencephalopathy (PML) where demyelination results from oligodendroglial infection, it was concluded that the oligodendroglial cell body is not the primary target of CDV. The marked astroglial changes were also considered to contribute to demyelination in CDV infection but the mechanism by which this happens remains unknown.     

Oligodendroglial degeneration in distemper: apoptosis or necrosis?

Canine distemper virus (CDV) causes a multifocal demyelinating disease in dogs. It was previously shown that the initial demyelinating lesions are directly virus induced since a correlation between the occurrence of demyelination and CDV replication in white matter cells was observed. During the course of infection oligodendrocytes undergo distinct morphological alterations, partly due to a restricted CDV infection of these cells, and eventually disappear from the lesions. This phenomenon has been described in vivo as well as in vitro. However, the reason for the morphological alterations and the following oligodendroglial depletion remained unclear. Since virus infection can induce cell death, it was investigated whether apoptosis or necrosis plays a role in the pathogenesis of demyelination in canine distemper. In brain tissue sections from dogs with acute distemper apoptotic cells were not detected within the demyelinating lesions using morphological and biochemical cell death criteria. In chronic distemper, apoptotic cells – presumably inflammatory cells – were seen within the perivascular cuffs. These in vivo findings were correlated to the in vitro situation using CDV-infected primary dog brain cell cultures as well as Vero cells. Infection with culture-adapted CDV lead to massive necrosis but not to apoptosis. After infection with virulent CDV neither apoptosis nor necrosis was a predominant feature in either culture system. These findings suggest that virus-induced demyelination in canine distemper is not the direct consequence of apoptosis or necrosis. It is speculated that another mechanism must be responsible for the observed morphological alterations of oligodendrocytes, ultimately leading to demyelination. Received: 29 April 1998 / Revised, accepted: 27 August 1998     

Oligodendroglial pathology in canine distemper virus infection in vitro

Summary Dog brain cell cultures were infected with different canine distemper virus (CDV) strains to study the oligodendrocytes, which were characterized with eight different antibodies to cover the whole oligodendroglial population in the culture. A few weeks after infection all oligodendroglial cell types started to degenerate and disappeared from the culture. However, since no CDV protein could be demonstrated in the degenerating oligodendrocytes with extensive double-labelling studies, this lesion can not be explained as being a result of cytolytic infection. This conclusion was further supported in experiments with plaque-forming CDV, in which viral replication is restricted to the cytolytic areas only; oligodendrocytes also degenerated in virus-free areas between the plaques. The hypothesis of toxic factors released by other infected cell types in the culture leading to secondary damage of the oligodendrocyte could not be confirmed by transferring supernatants from infected to normal cultures. Whereas the presence of toxic factors can not be completely excluded, the possibility of an abortive infection of the oligodendrocytes with no or very limited viral protein synthesis is discussed.Supported by the Swiss National Science Foundation (grant no. 3.949.84) and the Swiss Multiple Sclerosis Society     

Primary demyelination in experimental canine distemper virus induced encephalomyelitis in gnotobiotic dogs

Summary Nine dogs with canine distemper encephalitis (CDE) were examined with immunological techniques including demonstration of antibodies against canine distemper virus (CDV) in the serum and against myelin basic protein (MBP) in serum and in CSF. Mitogen stimulation tests of lymphocytes were also done. The brains were examined pathologically and immunoglobulin and C₃ were demonstrated in lesions by means of immunohistological techniques.Six dogs with acute CDE had none or low antibody levels against CDV or MBP, and there was no immunoglobulin in demyelinating lesions. Some of these dogs had depressed lymphocyte mitogen responses. Two dogs with chronic CDE showed recovery of lymphocyte mitogen responses. One of these had a significant antibody response against CDV and MBP in the serum. Both dogs with chronic CDE had very high antibody titers against MBP in the CSF and demyelinating lesions contained immunoglobulin. These results suggest that acute demyelination in CDE is probably due to some direct viral activity and that the progression of demyelination in chronic CDE is associated with a local immune response.Supported by the Schweizer Nationalfonds grands nos. 3.805.79 (to M. V.) and 3.957-0.80 (to A. J. S.) and the Schweizerische Multiple Sklerose Gesellschaft     

Demyelinating, non-demyelinating and attenuated canine distemper virus strains induce oligodendroglial cytolysis in vitro

A virulent canine distemper virus (CDV) strain that causes demyelination in vivo has been shown to induce oligodendroglial degeneration in vitro. In order to investigate if this effect on oligodendrocytes is specific for demyelinating strains only, primary brain cell cultures were infected with either virulent demyelinating strains (A75/17 and CH84-CDV), a virulent non-demyelinating strain (SH-CDV) or a non-virulent strain (OP-CDV). All virulent viruses caused a persistent type infection with moderate cytolysis whereas the non-virulent strain was highly cytolytic. All strains induced a similar pattern of oligodendroglial degeneration. It was concluded that the ability to induce oligodendroglial degeneration, which is thought to be the in vitro correlate of demyelination in vivo, is inherent to CDV irrespective of the strain. The discrepancy between biological behaviour of CDV strains in brain cell cultures and in vivo can be explained by the more complex virus-cell interactions in vivo than in vitro.     

Experimental canine distemper encephalomyelitis in neonatal gnotobiotic dogs

Summary The ultrastructural morphogenesis of neuronal degeneration and necrosis and patterns of associated myelin and axonal degeneration were studied in gnotobiotic dogs neonatally infected with neurovirulent R 252 strain of canine distemper virus (CDV-R 252). Distemper virus-infected neurons underwent a distinct sequence of ultrastructural changes culminating in direct viral-induced necrosis beginning after 21 days post inoculation (DPI). Viral-induced neuronal cytolysis occurs apparently independently of anti-viral immune mechanisms of immunologic destruction. Viral nucleocapsid aggregates in postsynaptic axosomatic and axodendritic complexes and in structurally intact axons provided morphologic evidence for viral-induced functional modulation of synaptic transmission and possible trans-synaptic interneuronal viral spread. There were secondary degenerative axonal and myelin changes, particularly in heavily myelinated tracts. There was no evidence of primary demyelination. Active phagocytosis of degenerating axons and myelin debris in foci of virus-associated necrosis was apparently restricted to CDV-containing macrophages. Demonstration of a productive CDV infection of choroid plexus epithelium 10 DPI and thereafter was identified as an intracranial source of free infectious virus.Supported by the State of Ohio Canine Research Fund and grant no. 2 ROI NS 14821, National Institutes of Health. A. E. Metzler was supported by the Schweizerische Stiftung für Medizinische Biologische Stipendien, Basel, and the Janggen-Pohen Stiftung, St. Gallen, Switzerland     

Immunocytochemical localization of MAG, MBP and P0 protein in acute and relapsing demyelinating lesions of Theiler''s virus infection

Acute demyelinating and relapsing demyelinating lesions from spinal cords of mice infected with the WW strain of Theiler's encephalomyelitis virus (TMEV) were studied immunocytochemically with antisera to various myelin constituents. Acute lesions were studied for differences in the distribution of myelin basic protein (MBP) and myelin associated glycoprotein (MAG). Relapsing lesions, characterized by demyelination of areas previously remyelinated by Schwann cells, were studied for differences in the distribution of P0 and MAG. In both instances the earliest lesions were characterized by preferential disappearance of MBP and P0 respectively when compared to MAG. In well-developed lesions, MAG, MBP and P0 were absent in essentially equal proportion. These observations are in agreement with previous findings suggesting a primary loss of myelin rather than a direct attack on oligodendrocytes as the main pathogenetic mechanism of demyelination in this viral model.     

Ultrastructural and biochemical findings in brain cell cultures infected with canine distemper virus

Summary To study the pathomechanism of demyelination in canine distemper (CD), dog brain cell cultures were infected with virulent A75/17-CD virus (CDV) and examined ultrastructurally. Special attention was paid to the oligodendrocytes, which were specifically immunolabelled. In addition, cerebroside sulfotransferase (CST), an enzyme specific for oligodendrocyte activity was assayed during the course of the infection. Infection and maturation as well as CDV-induced changes were found in astrocytes and brain macrophages. Infection of oligodendrocytes was rarely seen, although CST activity of the culture markedly decreased and vacuolar degeneration of these cells occurred, resulting in their complete disappearance. We concluded that the degeneration of oligodendrocytes and demyelination is not due to direct virus-oligodendrocyte interaction, but due to CDV-induced events in other glial cells.Supported by the Swiss National Science Foundation Grant nos. 3.956.87 (M. V.) and 3.156.88 (N. H.), the Swiss Multiple Sclerosis Society (M. V.) and the Swiss Foundation of Encouragement of Research in Mental Retardation (N. H.)     

Microglial cell activation in demyelinating canine distemper lesions

Microglia cells are the principal immune effector elements of the brain responding to any pathological event. To elucidate the possible role of microglia in initial non-inflammatory demyelination in canine distemper virus (CDV) infection, microglia from experimentally CDV infected dogs were isolated ex vivo by density gradient centrifugation and characterized immunophenotypically and functionally using flow cytometry. Results from dogs with demyelinating lesions were compared to results from recovered dogs and two healthy controls. CDV antigen could be detected in microglia of dogs with histopathologically confirmed demyelination. Microglia of these dogs showed marked upregulation of the surface molecules CD18, CD11b, CD11c, CD1c, MHC class I and MHC class II and a tendency for increased expression intensity of ICAM-1 (CD54), B7-1 (CD80), B7-2 (CD86), whereas no increased expression was found for CD44 and CD45. Functionally, microglia exhibited distinctly enhanced phagocytosis and generation of reactive oxygen species (ROS). It was concluded that in CDV infection, there is a clear association between microglial activation and demyelination. This strongly suggests that microglia contribute to acute myelin destruction in distemper.     

Demyelinating canine distemper encephalomyelitis: measurement of myelin basic protein in cerebrospinal fluid

Beagle dogs were experimentally infected with the Cornell A75-17 strain of canine distemper virus. At three time points post-infection (PI), immunoreactive myelin basic protein (MBP) was measured in cerebrospinal fluid (CSF). Levels were correlated with neuropathological findings, interferon in CSF and virus isolation from the brain. CSF from animals inoculated with Cornell A75-17 strain often showed detectable immunoreactive MBP late in the disease course. As anticipated from earlier morphological studies, CSF drawn around day 20 PI lacked MBP while subsequent samples were positive. Dogs with severe demyelination had elevated values of immunoreactive MBP while dogs with only mild inflammation had little or none. Release of MBP or MBP peptides into CSF of dogs with canine distemper may be a valuable laboratory test in studies of the natural history of this disease and in assessing the response to treatment. Whether an immune response to MBP plays an immunopathogenic role in the chronic, demyelinating phase of canine distemper encephalitis remains to be determined.     

Observations on demyelinating lesions induced by Theiler's virus in CBA mice

Summary We examined demyelinating lesions in the spinal cord of CBA mice infected with the BeAn strain of Theiler's virus to see if it was possible to document the sequence of changes which result in demyelination. It was found that the lesions which develop in the late stages of the disease were progressive. Therefore, by examining the different zones of a single lesion, it was possible to follow a sequence of changes which lead to demyelination. There was a clear progression from normal myelin, to vacuolated myelin, to myelin phagocytosis, to demyelinated axons, to remyelinated axons. Virus was detected in degenerating oligodendrocytes in the area showing myelin vacuolation by both electron microscopy and immunocytochemistry, a finding which indicated that virus infection precedes demyelination. The area of normal myelin which surrounded the zone of vacuolated myelin was infiltrated by lymphocytes, indicating that lymphocytic infiltration preceded viral replication and oligodendrocyte degeneration. Our observations indicate that cells of the immune system may play a role in the initiation of virus replication which appears to be a prerequisite for demyelination.Supported by grants from the Multiple Sclerosis Society     

Astrocytic infection in canine distemper virus-induced demyelination

Summary Acute canine distemper virus (CDV)-induced demyelinating lesions were examined with double-labelling immunocytochemistry simultaneously demonstrating CDV antigen and glial fibrillary acidid protein (GFAP) as marker for astrocytes. It was shown that 64% of all astrocytes within the demyelinating lesions were infected and that 95% of all infected cells counted in the lesions were astrocytes. These results suggest that the astrocyte ist the main target for CDV and that astroglial infection may play an important role in the mechanism of demyelination.Supported by the Swiss National Science Foundation (grant no. 3.956.87) and the Swiss Multiple Sclerosis Society     

Oligodendroglial reaction following spinal cord injury in rat: Transient upregulation of MBP mRNA

The reaction of oligodendrocytes in response to traumatic injury of the CNS are poorly understood. In the present report we studied changes in the expression of a major constituent of CNS myelin, myelin basic protein (MBP), by immunohistochemistry and in situ hybridization from 6 h up to 2 weeks following partial transection of the spinal cord in adult rats. MBP immunohistochemistry showed degeneration of myelin at the lesion center and signs of myelin breakdown in necrotic foci in the dorsal and ventral funiculi proximal and distal to the lesion. In situ hybridization revealed that mRNA for MBP was downregulated at the local lesion site within the first day following injury, probably reflecting oligodendrocytes to undergo cell death. From 2 days on, however, MBP mRNA was conspicuously upregulated at the border of the lesion area. This “reactive” response of surviving oligodendrocytes, as indicated by increased levels of MBP mRNA, peaked around 8 days. At this time, oligodendrocytes displaying strong MBP in situ signal formed stripe-like structures which were oriented radially toward the lesion center and arranged in parallel to neurofilament-positive axons. At around 2 weeks post-injury, MBP mRNA at the border of the lesion area was again downregulated to levels comparable to uninjured controls. These results show that traumatic injury of the spinal cord induces a “reactive” response of surviving oligodendrocytes adjacent to lesion sites. This response might represent an important component of local repair mechanisms. GLIA 23:278–284, 1998. © 1998 Wiley-Liss, Inc.