Infection of human oligodendroglioma cells by a recombinant measles virus expressing enhanced green fluorescent protein

One of the hallmarks of the human CNS disease subacute sclerosing panencephalitis (SSPE) is a high level of measles virus (MV) infection of oligodendrocytes. It is therefore surprising that there is only one previous report of MV infection of rat oligodendrocytes in culture and no reports of human oligodendrocyte infection in culture. In an attempt to develop a model system to study MV infection of oligodendrocytes, time-lapse confocal microscopy, immunocytochemistry, and electron microscopy (EM) were used to study infection of the human oligodendroglioma cell line, MO3.13. A rat oligodendrocyte cell line, OLN-93, was also studied as a control. MO3.13 cells were shown to be highly susceptible to MV infection and virus budding was observed from the surface of infected MO3.13 cells by EM. Analysis of the infection in real time and by immunocytochemistry revealed that virus spread occurred by cell-to-cell fusion and was also facilitated by virus transport in cell processes. MO3.13 cells were shown to express CD46, a MV receptor, but were negative for the recently discovered MV receptor, signaling leucocyte activation molecule (SLAM). Immunohistochemical studies on SSPE tissue sections demonstrated that CD46 was also expressed on populations of human oligodendrocytes. SLAM expression was not detected on oligodendrocytes. These studies, which are the first to show MV infection of human oligodendrocytes in culture, show that the cells are highly susceptible to MV infection and this model cell line has been used to further our understanding of MV spread in the CNS.     

Human herpesvirus type 6 indirectly enhances oligodendrocyte cell death

Accumulating evidence suggests that human herpesvirus type 6 (HHV-6) plays a pathogenic role in diseases of the central nervous system including multiple sclerosis (MS). Recent studies have indicated that HHV-6 DNA is detected with high frequency in MS lesions compared to normal-appearing white matter, implicating a role for HHV-6 in MS pathogenesis. It appears that T cells, which infiltrate into the brain in MS patients, and resident oligodendrocytes harbor HHV-6 virus in MS lesions. Because T cells infected with HHV-6 have elevated proinflammatory gene expression, we hypothesized that HHV-6 could be indirectly cytotoxic to glial cells, including oligodendrocytes. Supernatants from SupT1 cells infected with HHV-6 variant A (GS or U1102) or variant B (Z29) significantly reduced MO3.1 cell proliferation by 75% ± 10%, 78% ± 8% or 51% ±9%, respectively. HHV-6 viral supernatants (GS or U1102 or Z29) significantly increased MO3.1 or primary human oligodendrocyte precursor cells (OPCs) cell death, whereas primary human fetal astrocytes were not affected. Removal of HHV-6 virions or proteins by trypsin treatment from culture supernatants did not reverse the loss in oligodendrocyte proliferation or viability. Supernatants from HHV-6 GS or U1102 cultures were significantly more cytotoxic to MO3.1 cells or OPCs compared to supernatants from T cells infected with Z29. Dying oligodendrocytes did not have an apoptotic-like phenotype and toxicity was not inhibited by general inhibitor of apoptosis, ZVAD. Further, oligodendrocytes had minimal caspase-3 activation even in the presence of staurosporine, suggesting that cell death followed caspase-independent pathways. These results indicate that HHV-6 is indirectly cytotoxic to oligodendrocytes and that cell death is driven primarily by caspase-independent pathways.     

Absence of measles virus receptor (CD46) in lesions of subacute sclerosing panencephalitis brains

In this study we investigated pathological changes of the expression of the measles virus (MV) receptor, CD46, in subacute sclerosing panencephalitis (SSPE) brains. We analyzed CD46 expression in lesions of brain specimens from five SSPE patients in comparison to uninfected regions of the same brains and to normal human brains. The correlation between CD46 and MV infection, in individual cells in SSPE brains, was analyzed by double-staining procedures using monoclonal antibodies (mAbs) and in situ hybridization to detect MV-specific mRNAs. We found that CD46 was expressed at relatively low levels by neurons and astrocytes in normal brains in comparison to neuroblastoma and astrocytoma cell lines. Within heavily infected (MV-positive) brain lesions of all five SSPE cases, CD46 was either not detected or was expressed to a lesser degree by neural cells, irrespective of whether MV antigens were detectable or not. In contrast, normal levels of CD46 were found in SSPE brain tissue distant from the lesion. Using in situ hybridization, mRNAs of both MV nucleocapsid and MV hemagglutinin (MV-H) were detected in all SSPE lesions, while no or only small amounts of MV-H protein were detected. MV-infected neurons were never found to express CD46. Although a strict correlation between levels of the MV-H protein and the absence CD46 could not be seen, these findings suggest that the CD46 expression is reduced by the MV infection in lesions of SSPE brains. Received: 3 March 1997 / Revised, accepted: 30 April 1997     

Infiltration of immune T cells in the brain of mice with herpes simplex virus-induced encephalitis

Herpes simplex virus (HSV) infection of mice can induce viral encephalitis. Using two-fluorochrome immunofluorescence, our present study shows that though there is extensive myelin loss and necrosis in the brain stem of mice with HSV encephalitis, only some oligodendrocytes, astrocytes and microglial cells are infected. T cells that express CD4 or CD8 and a large number of CD4+, F4/80+ macrophages are present in perivascular infiltrates close to and in contact with HSV-infected cells in areas of massive myelin loss. These findings suggest that the resultant infiltration of immune cells into the brain during HSV-1 infection may cause as much damage as the virus itself.     

Experimental measles encephalitis in Lewis rats: dissemination of infected neuronal cell subtypes

Acute measles may lead in rare instances to the chronic progressive central nervous system disease process subacute sclerosing panencephalitis (SSPE). SSPE results from a persistent measles virus (MV) infection with incomplete virus replication involving the entire human brain. The experimental encephalitis model in Lewis rats was used to define affected cell populations after infection with the neurotropic MV strain CAM/RB. Distribution patterns of MV were analysed by appropriate cell markers in the brain sections of infected animals employing multiple immunofluorescence labelling and confocal laser scanning microscopy. MV was detected in neurones but not in astrocytes, oligodendrocytes, microglia, and endothelial cells. GABAergic and glutamatergic neurons displayed MV antigen whereas cholinergic and catecholaminergic neurons appeared devoid of MV immunoreactivity. Mapping of the rat brain has revealed MV-infected neurones predominantly in motor, somatosensory, auditory, and visual cortices as well as in the basal ganglia and thalamic nuclei of infected rats. The results indicate that MV apparently disseminates via GABAergic and glutaminergic neurones and their processes. The tightly restricted viral distribution pattern is consistent with both inefficient immune clearance from infected neurones and with the observed disease symptoms.     

Modulation of the cytokine network in human adult astrocytes by human herpesvirus-6A

Human herpesvirus-6A (HHV-6A) is a common pathogen whose role in CNS disorders including multiple sclerosis remains controversial. To understand how HHV-6A could influence inflammatory pathways in the CNS, we infected cultured human adult astrocytes and examined the expression of 268 cytokines, chemokines, growth factors and their receptors by gene profiling. HHV-6 infection alone had little effect on the astrocyte gene profile but strongly altered the astrocyte response to proinflammatory cytokines. Under those conditions astrocytes express higher levels of anti-inflammatory mediators including IL-10 and IL-11, chemotactic factors, growth factors and factors controlling type I interferon production. Our data suggest that HHV-6 itself does not evoke a pro-inflammatory response in astrocytes but rather triggers immune modulatory factors in the face of inflammation.     

Herpes simplex virus type I infection of the CNS induces major histocompatibility complex antigen expression on rat microglia

Rats were infected with herpes simplex virus type I (HSV-1) by corneal scarification. The spread of virus in the brain, the infiltration of leucocytes into infected areas, and the expression of major histocompatibility complex (MHC) glycoproteins by brain cells were assessed as a function of time by immunohistochemistry. Virus moved along neuronal pathways, achieving widespread distribution in the brain by days 8-10 when the illness appeared most severe. Granulocytes, T-lymphocytes, and monocytes infiltrated the tissue matrix at sites of infection. Microglial cells were induced to express MHC class I and class II glycoproteins. Reactive microglia near the sites of infection most vigorously expressed such glycoproteins. At the peak of the infection they were detectable on microglia throughout the brain, including areas apparently separated from active infection. Evidence of viral antigens, as well as microglial MHC expression, had largely disappeared by day 30. Neurons, astrocytes, and oligodendroglial cells failed to express MHC antigens.     

Approaches in the understanding of morbillivirus neurovirulence

Certain members of the morbillivirus genus, canine distemper virus, phocine distemper virus, and the cetacean viruses of dolphins and porpoises exhibit high levels of central nervous system (CNS) infection in their natural hosts. CNS complications are rare for measles virus (MV) and are not associated with rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) infection. However, both RPV and PPRV are neurovirulent in permissive murine strains. Human postmortem tissue, neural cell cultures, and animal models have been used to answer major questions concerning morbillivirus neurovirulence. Studies of the MV CNS complication subacute sclerosing panencephalitis (SSPE) indicate that virus could enter the CNS either by direct infection of endothelial cells or in infected leucocytes, followed by infection of predominately neurones and oligodendrocytes. It has been established that MV neurovirulence in mice is partially determined by the virus-receptor specificity. The two known MV receptors, CD46 and SLAM, have been examined in normal and SSPE brain tissue and the findings suggest that further receptors may be necessary to explain infection of the CNS with wild-type strains of MV. In both humans and mice (and in vitro), once infection of neurones has been established, virus spreads transneuronally. It is possible that all morbilliviruses transiently infect the CNS in their natural hosts, but development of disease is dependent on the efficiency of the immune response. Alternatively, for RPV and PPRV, virus entry may be restricted due either to absence of viral receptors or failure of virus to replicate or spread in the CNS.     

Elevated serum and cerebrospinal fluid levels of soluble human herpesvirus type 6 cellular receptor, membrane cofactor protein, in patients with multiple sclerosis

Membrane cofactor protein (CD46) is a member of a family of glycoproteins that are regulators of complement and prevent activation of complement on autologous cells. Recently, CD46 has been identified as the cellular receptor for human herpesvirus Type 6 (HHV-6). Elevated levels of soluble CD46 have been described in several autoimmune disorders, and may be implicated in the pathogenesis of these diseases. As several reports have supported an association of HHV-6 and multiple sclerosis, it was of interest to compare levels of soluble CD46 in the sera of multiple sclerosis patients to that of healthy controls, other neurological disease controls, and other inflammatory disease controls. Using an immunoaffinity column comprised of immobilized monoclonal antibodies to CD46, serum levels of soluble CD46 were found to be significantly elevated in multiple sclerosis patients compared with healthy and other neurological disease controls. Moreover, multiple sclerosis patients who tested positive for HHV-6 DNA in serum had significantly elevated levels of soluble CD46 in their serum compared with those who were negative for HHV-6 DNA. A significant increase in soluble CD46 was also found in the serum of other inflammatory disease controls tested compared to healthy controls. Additionally, a significant correlation was demonstrated between levels of soluble CD46 in the serum and cerebrospinal fluid of multiple sclerosis patients. Collectively, these data suggest that elevated levels of soluble CD46 may contribute to the pathogenesis of inflammatory diseases, including multiple sclerosis.     

IFN-gamma production and astrocyte recognition by autoreactive T cells induced by Theiler's virus infection: role of viral strains and capsid proteins

From mice infected with the DA strain of Theiler's murine encephalomyelitis virus (TMEV), CD8+ cytotoxic T lymphocytes (CTLs) could be detected after stimulation with TMEV infected antigen presenting cells (APCs). These CTLs killed not only TMEV infected but also uninfected syngeneic cells. Killing was associated with interferon (IFN)-gamma production in ELISPOT assays. The CTLs were efficiently induced by vaccinia virus encoding DA virus capsid proteins, but not by APCs infected with the GDVII strain of TMEV. The CTLs produced IFN-gamma in response to TMEV infected, but not uninfected, astrocytes. The CTLs could be maintained in the presence of interleukin (IL)-2. We hypothesized that, in DA virus infection, CD8+ CTLs specific for viral capsid protein could recognize self protein on oligodendrocytes by molecular mimicry, leading to demyelination.     

Measles infection of the central nervous system

Central nervous system (CNS) complications occurring early and late after acute measles are serious and often fatal. In spite of functional cell-mediated immunity and high antiviral antibody titers, an immunological control of the CNS infection is not achieved in patients suffering from subacute sclerosing panencephalitis (SSPE). The known cellular receptors for measle virus (MV) in humans, CD46 and CD150 (signaling lymphocyte activation molecule, SLAM), are important components of the viral tropism by mediating binding and entry to peripheral cells. Because neural cells do not express SLAM and only sporadically CD46, virus entry to neural cells, and spread within the CNS, remain mechanistically unclear. Mice, hamsters, and rats have been used as model systems to study MV-induced CNS infections, and revealed interesting aspects of virulence, persistence, the immune response, and prerequisites of protection. With the help of recombinant MV and mice expressing transgenic receptors, questions such as receptor-dependent viral spread, or viral determinants of virulence, have been investigated. However, many questions concerning the human MV-induced CNS diseases are still open.     

Characterization of the inflammatory response during acute measles encephalitis in NSE-CD46 transgenic mice.

Expression of the human measles virus receptor, CD46, in the murine central nervous system allows infection and replication by wild-type human measles virus (MV) strains (Rall, G.F., Manchester, M., Daniels L.R., Callahan, E., Belman, A., Oldstone, M.B.A., 1997. A transgenic mouse model for measles virus infection of the brain. Proc. Natl. Acad. Sci. U.S.A. 94, 2243-2248). MV replicates in neurons in focal lesions of the cortex, hippocampus and thalamus, leading to death of the animals. In MV-infected CD46 transgenic mice, infiltration of CD4+ and CD8+ T-lymphocytes, B-lymphocytes and macrophages was seen. Upregulation of MHC class I and class II molecules was observed, along with reactive astrocytosis and microgliosis. Increased chemokine mRNAs, especially RANTES and IP-10, and cytokine RNAs IL-6, TNF-alpha, and IL1-beta were observed. Apoptosis of neurons also was increased. No MV replication or inflammation was seen in similarly inoculated nontransgenic littermates. These results further characterize the MV-induced encephalitis in CD46 transgenic mice and highlight similarities to MV infection of the human CNS.     

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.     

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.     

Efficacy of antiviral compounds in human herpesvirus-6-infected glial cells

The beta-herpesvirus human herpesvirus-6 (HHV-6) is becoming increasingly recognized as an important pathogen in immunocompromised patients, particularly in post bone marrow transplant (BMT). Reactivation of latent HHV-6 resulting in encephalitis has been reported in BMT and stem cell transplant (SCT) patients. The development of HHV-6 encephalitis can be a fatal complication, the frequency of which is increasing likely due to improved diagnosis with quantitative polymerase chain reaction (PCR) of cerebrospinal fluid. There are currently no antiviral compounds approved for HHV-6, nor have any controlled clinical trials been conducted. The frequency and severity of HHV-6 encephalitis in both immunocompetent and immunocompromised patients necessitates studies on the usefulness of currently available anti-viral compounds. The authors compared the antiviral efficacy of four drugs currently used for cytomegalovirus (CMV) infection, a beta-herpesvirus sharing homology with HHV-6. In HHV-6A- and HHV-6B-infected T cells, acyclovir, ganciclovir, foscarnet, and cidofovir exhibited antiviral activity consistent with that published in other studies. In HHV-6-infected human astrocytes (U251), however, only foscarnet and cidofovir exhibited antiviral activity and this effect was restricted to infection with HHV-6 variant A. In pathological brain sections from patients with neurological disorders such as multiple sclerosis and epilepsy, HHV-6 has been localized to glial cells. Determination of antiviral activity in human glial fibrillary acidic protein (GFAP)-positive astrocytes of currently used antiviral compounds is essential for potential treatment of HHV-6 and neurological disorders. Our data highlight the necessity for further study of antiviral compound in HHV-6-infected glial cells as well as the development of more selective compounds for HHV-6.     

Increased susceptibility of human fetal astrocytes to human T-lymphotropic virus type I in culture.

Human T-lymphotropic virus type I (HTLV-I) has been considered as an agent responsible for tropical spastic paraparesis and HTLV-I associated myelopathy. However, the pathogenesis of the diseases remains unclear. In a previous study we demonstrated that HTLV-I could infect adult human astrocytes and oligodendrocytes in vitro, although the rates of infected cells were low, at a rate of 0.1% and 0.01-0.05% respectively. Since mother-to-child transmission has been proposed as one of the major pathways for the prevalence of HTLV-I endemic, in the present study we investigated the susceptibility of human fetal astrocytes to HTLV-I in culture. After two days of co-culturing fetal brain cells with irradiated MT-2 cells (an HTLV-I-producing T-cell line), immunofluorescence staining revealed many positive astrocytes for HTLV-I p19 antigen. Multinucleated giant cells doubly immunoreactive to glial fibrillary acidic protein and HTLV-I antigen were frequently observed and showed a characteristic feature of hairy or fluffy external appearance. The percentage of infected astrocytes became as high as 19.4% at Day 21 of co-culture and then decreased. Electron microscopic examination revealed type C virus-like particles in astrocytes. These results indicate that human fetal astrocytes are more susceptible to HTLV-I infection than adult human astrocytes in tissue culture.     

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.     

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.     

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.