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.     

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.     

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     

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.)     

Persistent infection of oligodendrocytes in Theiler's virus-induced encephalomyelitis

Mice infected with Theiler's murine encephalomyelitis virus (TMEV) develop a chronic relapsing demyelinating myelitis. To determine the localization of viral antigen in infected cells of the spinal cord, we studied TMEV-infected SJL/J mice using immunoelectron microscopic peroxidase-antiperoxidase techniques. Viral antigens were expressed in the cytoplasm of neurons and astrocytes 4 and 11 days postinfection. At 28 days postinfection, macrophages, astrocytes, and oligodendrocytes showed viral antigen in their cytoplasm. At 45, 83, 270, and 360 days postinfection, most infected cells were oligodendrocytes as revealed ultrastructurally by immunoperoxidase staining of prominent glial loops that connect with myelin lamellae. Some of these sheaths also showed Schmidt-Lanterman incisures that stained for viral antigen. Virus could be recovered at low titers for the duration of the illness. The findings indicate that TMEV induces persistent infection of oligodendrocytes which could then become the target of immune-mediated injury resulting in demyelination.     

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.     

A normal human brain cell aggregate model for neurobiological studies

A new in vitro model of normal human brain has been developed in which fetal human brain cells form three-dimensional aggregates that can be maintained for up to 60 days in culture. Cells appear fully differentiated at the time of initiation in culture; the predominant cells identified were astrocytes, neurons, and oligodendrocytes with myelin, with occasional ependymal cells and macrophages. The specific arrangement and numbers of neural cells within aggregates differed among brain specimens. Cell kinetics studies detected DNA synthesis throughout the culture interval. Aggregates cocultured with a human malignant glioma cell line (U251-MG) were progressively invaded by tumor cells. In aggregates infected with human cytomegalovirus (CMV), intracellular viral replication and morphologic changes characteristic of human brain infection with this pathogen were seen. This model of brain aggregates should prove valuable for multidisciplinary studies in human neurobiology, particularly in the fields of developmental neurobiology, neuro-oncogenesis, tumor cell invasion, and species-specific viral infection of the central nervous system.     

Vulnerability of rat and mouse brain cells to murine hepatitis virus (JHM-strain): studies in vivo and in vitro

The pathogenicity and cell tropism of mouse hepatitis virus (MHV-JHM-strain) in the developing mouse (Balb/c) and rat (Wistar and Lewis) brain were analysed. Intracranial infection of Balb/c mice at postnatal day 5 induced a lethal encephalitis in all animals. Of Wistar rats infected at day 2 or 5 after birth, 30 to 70%, respectively, survived. The distribution of viral antigen was studied in frozen brain sections of animals that died after infection; astrocytes were found to be the major virus-infected cell type throughout the central nervous system. More than 75% of the surviving rat pups developed paralysis, but viral antigen was detected in only few brain cells and not in astrocytes. The cell tropism of MHV-JHM was examined further in virus-infected glial cell cultures derived from brains of rats or mice. In the glial cultures derived from Wistar rats, only oligodendrocytes were infected, whereas in cultures derived from mouse or Lewis rat brain viral antigen was detected in both astrocytes and oligodendrocytes. Infection of astrocytes led to the formation of syncytia and degradation of the cytoskeleton. Infected rat oligodendrocytes gradually disappeared from the cultures because of cell death. These phenomena indicate that, besides an indirect autoimmune response triggered by infected astrocytes, direct virus-induced injury to astrocytes or to oligodendrocytes can have a dominant role in the neuropathogenicity of mouse hepatitis virus. The present results underscore the importance of species and developmental stage of experimental animals in the neurotropism and pathogenicity of MHV-JHM.     

Functional expression of the seven-transmembrane HIV-1 co-receptor APJ in neural cells

APJ is a recently described seven-transmembrane (7TM) receptor that is abundantly expressed in the central nervous system (CNS). This suggests an important role for APJ in neural development and/or function, but neither its cellular distribution nor its function have been defined. APJ can also serve as a co-receptor with CD4 for fusion and infection by some strains of human immunodeficiency virus (HIV-1) in vitro, suggesting a role in HIV neuropathogenesis if it were expressed on CD4-positive CNS cells. To address this, we examined APJ expression in cultured neurons, astrocytes, oligodendrocytes, microglia and monocyte-derived macrophages utilizing both immunocytochemical staining with a polyclonal anti-APJ antibody and RT - PCR. We also analyzed the ability of a recently identified APJ peptide ligand, apelin, to induce calcium elevations in cultured neural cells. APJ was expressed at a high level in neurons and oligodendrocytes, and at lower levels in astrocytes. In contrast, APJ was not expressed in either primary microglia or monocyte-derived macrophages. Several forms of the APJ peptide ligand induced calcium elevations in neurons. Thus, APJ is selectively expressed in certain CNS cell types and mediates intracellular signals in neurons, suggesting that APJ may normally play a role in signaling in the CNS. However, the absence of APJ expression in microglia and macrophages, the prinicpal CD4-positive cell types in the brain, indicates that APJ is unlikely to mediate HIV-1 infection in the CNS.     

Virulent and avirulent strains of Semliki Forest virus show similar cell tropism for the murine central nervous system but differ in the severity and rate of induction of cytolytic damage

The pathogenicity of the avirulent, demyelinating A7 strain of Semliki Forest virus (SFV) and the virulent SFV4 strain (derived from an infectious clone) for the central nervous system of adult BALB/c mice following intranasal infection was compared. The techniques used included immunocytochemistry using anti-SFV antibody and antibodies to cell markers, in situ hybridization (ISH) using a biotinylated cDNA probe specific for SFV, and immunocytochemistry/ISH double labelling. Whereas SFV4 was lethal at 4 days post-infection, A7-infected mice appeared normal at all times. Neuronal necrosis in the pyriform cortex was present in both infections, but developed sooner and was more severe iollowing miection with SFV4 than with A7. Intact neurons and putative oligodendrocytes contained viral RNA and virus-specific antigen in SFV4 infected mice; viral RNA but not virus-specific antigen was detected in similar cells in A7-infected mice. These results confirm that SFV4 and A7 share similar cell tropisms for the murine central nervous system, but differ in the severity and rate of development of cytolytic damage. Intranasal infection is an efficient monitoring system for studies of the molecular basis of pathogenicity of SFV infection in mice.     

Multiple neural cell types are infected in vitro by border disease virus

Border disease (BD) of sheep results from a congenitally acquired nonarbotogavirus infection which causes a highly selective central nervous system (CNS) pathological lesion consisting of diffuse decreased myelination without inflammation or neuronal destruction. Thus, a selective disruption of oligodendroglial function appears to occur. In order to investigate the in vitro cell tropism of BD virus, primary cultures derived from fetal and adult ovine CNS and peripheral nervous system were inoculated with BD virus. Infected cell types were determined by dual immunofluorescent labeling for viral and cell type specific antigens. Infection of all the major cell types represented in these cultures, including oligodendrocytes, astrocytes, fibroblasts, dorsal root ganglion neurons and Schwann cells was found. Oligodendrocytes were only infected earlier and appeared to remain infected longer than astrocytes and fibroblasts. Infectious virus was produced by all cultures and continued to be produced even after the disappearance of nearly all immunocytochemically detectable viral antigen within cells. These studies suggest that the selective dysfunction of the oligodendrocyte in BD is not based on a selective viral tropism.     

FURTHER ULTRASTRUCTURAL OBSERVATIONS OF VIRUS MORPHOGENESIS AND MYELIN PATHOLOGY IN JHM VIRUS ENCEPHALOMYELITIS

Groups of 3, 17, and 28-day-old Swiss mice were inoculated intracerebrally with JHM virus, the neurotropic strain of mouse hepatitis virus (MHV), and studied serially by virologic and morphologic techniques. Beginning 2--5 days post-inoculation, all groups of infected mice developed CNS lesions which were destructive in the 3-day-old group and demyelinative in the 17 and 28-day-old animals. Infectious virus could be isolated from the brain, spinal cord, and liver. Electron microscopy demonstrated the virus to be pantropic in the CNS with virions occurring within ependymal cells, astrocytes, neurons, oligodendrocytes, endothelial cells, and cell of haematogenous origin. Giant cell formation was a constant feature. In regions of demyelination, oligodendrocytes exhibited a propensity to proliferative aberrant membrane. Myelin degradation was accompanied by membrane vesiculation and by the stripping action of macrophages. The lesions were not due to CNS elements in the inoculum since in animals inoculated with normal CNS suspensions from appropriate age groups failed to show lesions. The morphogenesis of JHM virions was followed ultrastructurally as was the formation of syncytia in the different cell types. In addition to delineating virus morphogenesis and myelin pathology, the results underscore the pantropic nature of JHM virus in the CNS, the synstemic nature of the infection, and that oligodendrocytes were the principal targets.     

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.     

Virus spread and initial pathological changes in the nervous system in genital herpes simplex virus type 2 infection in mice

Summary Mice were infected by the vaginal route with the MS strain of herpes simplex virus type 2 (HSV-2). Serial vaginal cultures were used to confirm infection and to select mice for this study. Two mice were killed by perfusion on days 2–6 post infection (p.i.) and lumbar and sacral cord with cauda were fixed and embedded for electron microscopy. Semithin Epon-sections were stained for viral antigen using a rabbit anti-HSV-2 antiserum and the Avidin-Biotin (ABC) method. Thin sections from antigen-positive blocks were examined by electron microscopy, and the number and types of infected cells detected by these two methods were compared. A good correlation was found between detection of infected cells by these methods. Infected cells included neurons of dorsal root ganglia and spinal cord, satellite cells of dorsal root ganglia, non-myelinating Schwann cells, astrocytes, oligodendrocytes and arachnoidal cells. Infected cells were first detected in the cauda on day 3 p.i. and in the spinal cord on day 5 p.i. The temporal and spatial distribution of infected cells was consistent with neural spread to and within the CNS. The pathological lesions showed a good correlation with the distribution and number of infected cells and are probably due to a direct virus effect. The similar sensitivity of the Epon-ABC method to electron microscopy in detecting infected cells indicates that this method may have useful applications in both experimental and diagnostic work.     

Pathogenicity of Semliki Forest virus for the rat central nervous system and primary rat neural cell cultures: possible implications for the pathogenesis of multiple sclerosis

The neurovirulent L10 strain of Semliki Forest virus (SFV) causes extensive neuronal damage in the central nervous system (CNS) of infected rats, and is probably the cause of death. The avirulent A7 and M9 strains do not cause extensive neuronal damage, but do induce immune-mediated CNS demyelination. In primary CNS cell cultures derived from rats, L10 multiplies more rapidly in neurons than avirulent strains, but infection with both virulent and avirulent strains causes depletion of oligodendrocytes from mixed glial cell cultures. It is proposed that the immune-mediated demyelination, which follows infection with avirulent strains, is induced by phagocytosis of myelin debris from infected oligodendrocytes, and the presentation of antigens derived from such debris to T-helper lymphocytes. Based on these and previous results, a scheme for the pathogenicity of defined strains of SFV is proposed. The applicability of this scheme to the understanding of human demyelinating disease such as multiple sclerosis is discussed.     

CD46 on glial cells can function as a receptor for viral glycoprotein-mediated cell-cell fusion

Membrane cofactor protein (CD46) is a regulator of complement activation that also serves as the entry receptor for human herpes virus 6 (HHV-6) and measles virus (MV) into human cells. While it is clear that oligodendrocytes and astrocytes are cell types commonly infected by these viruses, it is unclear whether oligodendrocytes express CD46, or which are the cellular mechanisms underlying the infection. We show that adult oligodendrocytes, as well as astrocytes and microglial cells, express CD46 on the cellular surface. Moreover, we employed a quantitative fusion assay to demonstrate that HHV-6A infection of T lymphocytes enables cell-cell fusion of these cells to astrocytes or to oligodendroglial cells. This fusion is mediated by the interaction between viral glycoproteins expressed on the membrane of the infected cells and CD46 on the glial targets, and is also observed using cells expressing recombinant MV glycoproteins. These data suggest a mechanism that involves cell-cell fusion by which certain viruses could spread the infection from the periphery to the cells in the nervous system.     

Differential induction of apoptosis in demyelinating and nondemyelinating infection by mouse hepatitis virus

The role of apoptosis in mouse hepatitis virus (MHV) infection is still controversial. To better assess the role of apoptosis in MHV infection, we used three different biologic phenotypes of MHV to examine their differential effect on the induction of apoptosis. MHV-A59 produces acute hepatitis, meningoencephalitis, and chronic demyelination. MHV-2 causes only acute hepatitis and meningitis, whereas Penn98-1 produces acute hepatitis and meningoencephalitis without demyelination. We detected TdT-mediated dUTP nick-end labeling (TUNEL) staining in the livers and meninges of MHV-A59-, MHV-2-, and Penn98-1-infected mice. TUNEL staining in brain parenchyma was only detected in MHV-A59- and Penn98-1-infected mice. We detected apoptosis by electronmicroscopy in olfactory neurons during acute infection with MHV-A59. The kinetics and distribution of TUNEL staining correlated with the pathologic damage and colocalized with viral antigen in some cells. At 1 month, TUNEL staining was found exclusively in areas of demyelination in the spinal cord of MHV-A59-infected mice; however, it was not found in nondemyelinated mice infected with MHV-2 or Penn98-1, or in mock-infected mice. TUNEL-positive cells were identified as macrophage/microglial cells, some astrocytes, and some oligodendrocytes, by colabeling with cell-specific markers. The presence of TUNEL staining in oligodendrocytes suggests that apoptosis may play an important role in MHV-induced demyelination.     

Localization of HIV-1 in human brain using polymerase chain reaction/in situ hybridization and immunocytochemistry

Human immunodeficiency virus type 1 (HIV-1) infects the brains of a majority of patients with the acquired immunodeficiency syndrome (AIDS), and has been linked to the development of a progressive dementia termed “HIV-associated dementia.” This disorder results in severe cognitive, behavioral, and motor deficits. Despite this neurological dysfunction, HIV-1 infection of brain cells does not occur significantly in neurons, astrocytes, or oligodendrocytes, but is restricted to brain macrophages and microglia. To identify possible low-level or latent infection of other brain cells, we combined the techniques of the polymerase chain reaction with in situ hybridization for the detection of HIV DNA, and used immunocytochemistry to identify the HIV-expressing cells. In the 21 adult brains studied (15 AIDS and 6 seronegative control brains), we found that polymerase chain reaction/in situ hybridization was both sensitive and specific for identifying HIV-infected cells. In all brains, the majority of infected cells were macrophages and microglia. In several brains, however, a substantial minority of cells harboring HIV DNA were identified as astrocytes. Neurons, oligodendrocytes, and endothelial cells were not infected with HIV, even in cases with HIV-associated dementia. These findings confirm previous data regarding the importance of macrophage/microglial infection, and essentially exclude neuronal infection in pathogenetic models of HIV-associated neurological disease. These data also demonstrate that latent or low-level infection of astrocytes occurs in AIDS, a finding that may be of importance in understanding HIV neuropathogenesis.