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     

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     

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.     

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.)     

Virulent and attenuated canine distemper virus infects multiple dog brain cell types in vitro.

Canine Distemper Virus (CDV) produces an encephalitis in dogs that varies with viral strain. We have studied the cell tropisms of two virulent strains (CDV-SH and CDV A75-17) and an attenuated strain, Rockborn (CDV-RO), in cultured canine brain cells. Infected cell types were identified by double immunofluorescent labeling of specific cell markers and viral antigens. All viral strains studied produced infection in astrocytes, fibroblasts, and macrophages. Neurons were not infected by CDV A75-17 but were rapidly infected by CDV-SH and CDV-RO. Multipolar oligodendrocytes were very rarely infected by any of the virus strains. In contrast, a morphologically distinct subset of bipolar oligodendrocytes were commonly infected by CDV-SH and CDV-RO. The kinetics of infection in the astrocytes, oligodendrocytes, neurons, and macrophages varied between strains. Both CDV-SH and CDV-RO rapidly infected bipolar oligodendrocytes, astrocytes, neurons, and macrophages by 14 days post infection while infection by CDV A75-17 was delayed until after 28-35 days post infection. The differences in the growth kinetics and cell tropisms for some brain cells, exhibited by the three viral strains examined in this in vitro study, may relate to the different CNS symptoms that these strains produce in vivo.     

Vimentin-positive astrocytes in canine distemper: a target for canine distemper virus especially in chronic demyelinating lesions?

In canine distemper demyelinating leukoencephalitis (DL), caused by canine distemper virus (CDV), astrocytes represent the main virus target. In these cells, glial fibrillary acidic protein (GFAP) is the main intermediate filament, whereas vimentin occurs early in the astrocytic lineage and is replaced gradually by GFAP. To further characterize the role of astrocytic infection in dogs with DL, an animal model for multiple sclerosis, formalin-fixed paraffin-embedded cerebella were investigated immunohistochemically and by immunofluorescence. The expression and morphological alterations of these intermediate filaments were also determined by immunofluorescence studies of CDV-infected canine mixed brain cell cultures. In acute distemper lesions, the astrocytic response was mainly composed of GFAP- and CDV-positive cells. In contrast, vimentin-positive astrocyte-like cells were present in advanced lesions, which represented the main cell type harboring the pathogen, indicating a change in cell tropism and/or susceptibility of glial cells during lesion progression in CDV encephalomyelitis. Canine cell cultures were composed of GFAP-positive astrocytes, vimentin-positive cells and other glial cells. Following infection with the CDV-R252 strain, GFAP-positive astrocytes, especially multinucleated syncytial giant cells, displayed a disrupted cytoskeleton, whereas vimentin-positive cells though more frequently infected did not show any alteration in the filament network. This indicates increased vulnerability of mature GFAP-positive astrocytes compared to immature, vimentin-positive astrocytes. The latter, however, exhibited increased susceptibility to CDV. To conclude, the present findings indicate a change in cell tropism of CDV and/or the occurrence of less differentiated astrocytes representing a permanent source for virus infection and spread in advanced lesions of DL.     

In vitro characterization and preferential infection by canine distemper virus of glial precursors with Schwann cell characteristics from adult canine brain

Aims: Canine distemper virus (CDV)‐induced demyelinating leukoencephalomyelitis is a naturally occurring model for multiple sclerosis. The aim of this study was to establish primary glial cell cultures from adult canine brain for the analysis of CDV spread and cell tropism. Methods: Cultures were inoculated with the CDV‐R252 and a CDV‐Onderstepoort strain expressing the green fluorescent protein (CDV‐OndeGFP). CDV antigen expression was studied using cell type‐specific antibodies at different days post infection. Glial cells expressing p75^NTR were purified using antibody‐based techniques and characterized with regard to antigen expression and proliferation. Results: Three weeks after seeding, cultures contained spindle‐shaped cells expressing p75^NTR, oligodendrocytic cells, astrocytes, microglia and fibroblasts. Both CDV strains induced a mild to moderate cytopathic effect that consisted of single necrotic and few syncytial giant cells, but displayed in part a differential cell tropism. Whereas CDV‐OndeGFP expression in microglia and astrocytes did not exceed 1% and 50%, respectively, CDV‐R252 infected 100% and 80% of both cell types, respectively. The cells most early infected by both CDV strains expressed p75^NTR and may correlate to cells previously identified as aldynoglia. Treatment of p75^NTR+ cells with Schwann cell mitogens and serum deprivation increased proliferation and A2B5 expression, respectively, indicating common properties compared with Schwann cells and oligodendrocyte precursors. Conclusions: Infection of adult canine astrocytes and microglia revealed CDV strain‐specific cell tropism. Moreover, this is the first identification of a glial cell type with Schwann cell‐like properties in adult canine brain and, more importantly, these cells displayed a high susceptibility to CDV infection.     

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     

Measles and canine distemper virus antibodies in patients with multiple sclerosis determined by radioimmunoassay

Antibodies against measles virus (MV) and canine distemper virus (CDV) were measured by solid-phase radioimmunoassay (RIA) of sera and cere-brospinal fluid (CSF) from 28 patients with multiple sclerosis (MS) and matched neurological controls. When the groups were compared for MV antibody titers and CDV antibody titers of sera and MV/CDV serum antibody titer ratios, no significant difference was found. The CDV antibody titers and the MV antibody titers were in good correlation. CDV antibodies showed RIA titration curves typical of low avidity antibodies. In tests for MV antibodies in CSF, 82% of the MS patients and 19% of the controls were positive, whereas 36% of the MS patients and 4% of the controls were positive in CDV RIA. The correlation between MV and CDV antibody levels, the low avidity of CDV antibodies and the fact that absorption of the specimens with MV antigen abolished all CDV antibody activity suggest that the CDV antibodies are MV antibodies cross-reacting with CDV. It is concluded that canine distemper virus is unlikely to be involved in the etiology of multiple sclerosis.     

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.     

Up-regulation of major histocompatibility complex class II antigen expression in the central nervous system of dogs with spontaneous canine distemper virus encephalitis

Major histocompatibility complex class II (MHC II) and canine distemper virus (CDV) antigen expression were compared by immunohistochemistry in the cerebellar white matter of ten dogs with naturally occurring canine distemper encephalitis. In addition, infiltrating mononuclear cells were characterized by employing poly- and monoclonal antibodies directed against human CD3, canine MHC II, CD5, B cell antigen and CDV-specific nucleoprotein. Positive antigen-antibody reaction was visualized by the avidin-biotin-peroxidase complex method on frozen sections. Histologically, neuropathological changes were categorized into acute, subacute, and chronic. In control brains, MHC II expression was weak and predominantly detected on resident microglia of the white matter and on endothelial, perivascular and intravascular cells. In CDV antigen-positive brains, MHC II was mainly found on microglia and to a lesser extent on endothelial, meningeal, choroid plexus epithelial, ependymal and intravascular cells. In addition, virtually all of the perivascular cells expressed MHC II antigen. CDV antigen was demonstrated most frequently in astrocytes. Of the perivascular lymphocytes, the majority were CD3-positive cells, followed by B cells. Only a small proportion of perivascular cells expressed the CD5 antigen. In addition, B cells and CD3 and CD5 antigen-positive cells were found occasionally in subacute and frequently in chronic demyelinating plaques. In acute encephalitis, CDV antigen exhibited a multifocal or diffuse distribution, and MHC II was moderately up-regulated throughout the white matter and accentuated in CDV antigen-positive plaques. In subacute encephalitis, moderate multifocal CDV antigen and moderate to strong diffuse MHC II-specific staining, especially prominent in CDV antigen-positive lesions, were observed. In chronic encephalitis, CDV antigen expression was restricted to single astrocytes at the edge of the lesions or was absent, while MHC II expression, especially prominent on microglia, was strongly up-regulated throughout the white matter, most pronounced in demyelinated plaques. In summary, in acute and subacute lesions without perivascular cuffs, MHC II expression correlated with the presence of CDV antigen. In contrast, in chronic lesions, MHC II expression on microglial cells was the most prominent despite a few CDV antigen-positive astrocytes, indicating that nonviral antigens may play an important role as triggering molecules for the process of demyelination. Received: 13 September 1995 / Revised: 26 February 1996 / Accepted: 1 April 1996     

Albumin leakage into cerebrospinal fluid of dogs lethally infected with R252 canine distemper virus

Cerebrospinal fluid (CSF) form nine lethally infected and three convalescent gnotobiotic dogs infected with the R252 strain of canine distemper virus (CDV) was evaluated prior to and following infection. Lethally infected dogs had a mean seven-fold increase in CSF albumin concentration compared to the preinoculation value, not present in dogs destined to survive. Immunochemical examination of tissue from these dogs revealed prominent perivascular localization of albumin. Examination of CSF cells demonstrated mild leukocytosis in both groups at the time when encephalopathic deaths occurred, with decreased lymphocyte percentages, particularly Thy-1-bearing lymphocytes, in lethally infected dogs. These dogs also had more extensive expression of viral antigens in CSF and peripheral blood leukocytes at the time of death than did surviving dogs, and failed to make antibody to viral antigens. The findings link terminal breakdown of the blood-brain barrier and extensive viral antigen expression in CSF leukocytes with experimental CDV infection resulting in death.     

Studies on the intrathecal humoral immune response in canine distemper encephalitis

Albumin and IgG were quantitated in paired cerebrospinal fluid (CSF) and serum samples from dogs with demyelinating canine distemper virus (CDV) infection by means of rocket immunoelectrophoresis. The IgG index as indicator for intrathecal immunoglobulin synthesis was normal in animals with non-inflammatory demyelinating lesions and elevated in dogs with inflammatory myelin lesions. Specific antibodies against CDV and myelin were quantitated in CSF and serum from 8 dogs with an elevated IgG index. Eight of these dogs had significant amounts of antimyelin antibody and 4 dogs had neutralizing anti-CDV antibody in the CSF. Whereas the pathogenetic significance of antimyelin antibodies remains uncertain, the intrathecal antiviral immune response provides a plausible explanation for immunopathologic destruction of myelin in distemper.     

Up-regulation of the hyaluronate receptor CD44 in canine distemper demyelinated plaques

CD44 antigen (CD44), the principle cell surface receptor for hyaluronate, is up-regulated in the human demyelinating disease multiple sclerosis on fibrous astrocytes. As astrocytes are the main target cell of canine distemper virus (CDV), the consequences of a CDV infection on the CD44 expression and distribution in brains with spontaneous demyelinating canine distemper encephalitis (CDE) were of interest. Thirteen acute, 35 subacute, and 11 chronic plaques of nine dogs with immunohistologically confirmed CDE and brains of control dogs were included in the study. For light microscopy, 5-μm-thick serial sections were stained with H & E and incubated with monoclonal antibodies (mAbs) against CD44 and canine distemper virus nucleoprotein and polyclonal antibodies (pAbs) against glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP). For immunoelectron microscopy, 90-nm-thick sections were double stained with anti-GFAP and anti-CD44 mAbs to specify CD44-expressing structures. In controls, CD44 was diffusely distributed in the white matter and single meningeal cells exhibited a marginal expression of the antigen. In acute and more prominently in subacute demyelinating encephalitis, there was a plaque-associated up-regulation of CD44 which paralleled GFAP. In chronic demyelinating lesions, a reduction of CD44 associated with a loss of GFAP-positive astrocytes was noted. Additionally, in chronic plaques, CD44 was expressed on the cell membrane of perivascular mononuclear cells. Immunoelectron microscopically, in controls, CD44 was rarely demonstrated on astrocytic cell processes. In contrast, in brains with CDE CD44 was found on the cell membrane of broadened astrocytic cell processes. In summary, CD44 is up-regulated on astrocytes in the early phase of CDE and seems to represent a marker for the activation of immune cells in the late phase of the infection. Received: 4 March 1999 / Revised: 9 June 1999 / Accepted: 28 June 1999     

Canine viruses and multiple sclerosis

Serum and cerebrospinal fluid from multiple sclerosis (MS) patients and control subjects were tested and compared for presence and titer of neutralizing antibody against the most common canine viruses. Canine viruses included canine distemper virus (CDV), canine adenovirus 1 (CAV-1), canine parainfluenza virus (CPIV), canine herpesvirus (CHV), canine coronavirus (CCV), and canine parvovirus (CPV). Neutralizing titers against measles virus (MV) and human adenovirus 8 (HA8) were also tested. Significantly elevated (p less than 0.05) antibody levels in sera from MS patients were found only against MV and CDV, but this depended upon the study population and the method of evaluation. The CDV-neutralizing component in serum could be absorbed on MV-infected cells. Results of this study failed to establish a link between canine viruses and MS.     

Canine distemper virus persistence in demyelinating encephalitis by swift intracellular cell-to-cell spread in astrocytes is controlled by the viral attachment protein

The mechanism of viral persistence, the driving force behind the chronic progression of inflammatory demyelination in canine distemper virus (CDV) infection, is associated with non-cytolytic viral cell-to-cell spread. Here, we studied the molecular mechanisms of viral spread of a recombinant fluorescent protein-expressing virulent CDV in primary canine astrocyte cultures. Time-lapse video microscopy documented that CDV spread was very efficient using cell processes contacting remote target cells. Strikingly, CDV transmission to remote cells could occur in less than 6 h, suggesting that a complete viral cycle with production of extracellular free particles was not essential in enabling CDV to spread in glial cells. Titration experiments and electron microscopy confirmed a very low CDV particle production despite higher titers of membrane-associated viruses. Interestingly, confocal laser microscopy and lentivirus transduction indicated expression and functionality of the viral fusion machinery, consisting of the viral fusion (F) and attachment (H) glycoproteins, at the cell surface. Importantly, using a single-cycle infectious recombinant H-knockout, H-complemented virus, we demonstrated that H, and thus potentially the viral fusion complex, was necessary to enable CDV spread. Furthermore, since we could not detect CD150/SLAM expression in brain cells, the presence of a yet non-identified glial receptor for CDV was suggested. Altogether, our findings indicate that persistence in CDV infection results from intracellular cell-to-cell transmission requiring the CDV-H protein. Viral transfer, happening selectively at the tip of astrocytic processes, may help the virus to cover long distances in the astroglial network, “outrunning” the host’s immune response in demyelinating plaques, thus continuously eliciting new lesions.