Sindbis virus-induced neuronal death is both necrotic and apoptotic and is ameliorated by N-methyl-D-aspartate receptor antagonists.

Virus infection of neurons leads to different outcomes ranging from latent and noncytolytic infection to cell death. Viruses kill neurons directly by inducing either apoptosis or necrosis or indirectly as a result of the host immune response. Sindbis virus (SV) is an alphavirus that induces apoptotic cell death both in vitro and in vivo. However, apoptotic changes are not always evident in neurons induced to die by alphavirus infection. Time lapse imaging revealed that SV-infected primary cortical neurons exhibited both apoptotic and necrotic morphological features and that uninfected neurons in the cultures also died. Antagonists of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors protected neurons from SV-induced death without affecting virus replication or SV-induced apoptotic cell death. These results provide evidence that SV infection activates neurotoxic pathways that result in aberrant NMDA receptor stimulation and damage to infected and uninfected neurons.     

Canine distemper virus uses both the anterograde and the hematogenous pathway for neuroinvasion

Canine distemper virus (CDV), a member of the Morbillivirus genus that also includes measles virus, frequently causes neurologic complications, but the routes and timing of CDV invasion of the central nervous system (CNS) are poorly understood. To characterize these events, we cloned and sequenced the genome of a neurovirulent CDV (strain A75/17) and produced an infectious cDNA that expresses the green fluorescent protein. This virus fully retained its virulence in ferrets: the course and signs of disease were equivalent to those of the parental isolate. We observed CNS invasion through two distinct pathways: anterogradely via the olfactory nerve and hematogenously through the choroid plexus and cerebral blood vessels. CNS invasion only occurred after massive infection of the lymphatic system and spread to the epithelial cells throughout the body. While at early time points, mostly immune and endothelial cells were infected, the virus later spread to glial cells and neurons. Together, the results suggest similarities in the timing, target cells, and CNS invasion routes of CDV, members of the Morbillivirus genus, and even other neurovirulent paramyxoviruses like Nipah and mumps viruses.     

Antibodies to CD9, a tetraspan transmembrane protein, inhibit canine distemper virus-induced cell-cell fusion but not virus-cell fusion

Canine distemper virus (CDV) causes a life-threatening disease in several carnivores including domestic dogs. Recently, we identified a molecule, CD9, a member of the tetraspan transmembrane protein family, which facilitates, and antibodies to which inhibit, the infection of tissue culture cells with CDV (strain Onderstepoort). Here we describe that an anti-CD9 monoclonal antibody (MAb K41) did not interfere with binding of CDV to cells and uptake of virus. In addition, in single-step growth experiments, MAb K41 did not induce differences in the levels of viral mRNA and proteins. However, the virus release of syncytium-forming strains of CDV, the virus-induced cell-cell fusion in lytically infected cultures, and the cell-cell fusion of uninfected with persistently CDV-infected HeLa cells were strongly inhibited by MAb K41. These data indicate that anti-CD9 antibodies selectively block virus-induced cell-cell fusion, whereas virus-cell fusion is not affected.     

Canine distemper virus epithelial cell infection is required for clinical disease but not for immunosuppression

To characterize the importance of infection of epithelial cells for morbillivirus pathogenesis, we took advantage of the severe disease caused by canine distemper virus (CDV) in ferrets. To obtain a CDV that was unable to enter epithelial cells but retained the ability to enter immune cells, we transferred to its attachment (H) protein two mutations shown to interfere with the interaction of measles virus H with its epithelial receptor, human nectin-4. As expected for an epithelial receptor (EpR)-blind CDV, this virus infected dog and ferret epithelial cells inefficiently and did not cause cell fusion or syncytium formation. On the other hand, the EpR-blind CDV replicated in cells expressing canine signaling lymphocyte activation molecule (SLAM), the morbillivirus immune cell receptor, with similar kinetics to those of wild-type CDV. While ferrets infected with wild-type CDV died within 12 days after infection, after developing severe rash and fever, animals infected with the EpR-blind virus showed no clinical signs of disease. Nevertheless, both viruses spread rapidly and efficiently in immune cells, causing similar levels of leukopenia and inhibition of lymphocyte proliferation activity, two indicators of morbillivirus immunosuppression. Infection was documented for airway epithelia of ferrets infected with wild-type CDV but not for those of animals infected with the EpR-blind virus, and only animals infected with wild-type CDV shed virus. Thus, epithelial cell infection is necessary for clinical disease and efficient virus shedding but not for immunosuppression.     

Apoptosis in Alphavirus Encephalitis

Sindbis virus causes acute encephalitis in mice and serves as a useful model for encephalitic alphaviruses that infect humans. The outcome of infection is determined by whether infected neurons are resistant to virus-induced programmed cell death or activate their apoptotic pathway. The host immune response may also cause death of infected neurons. Determinants of neuronal apoptosis include the maturity of the neuron, the virulence of the infecting virus and the cellular immune response to infection. In many situations viral and cellular factors that decrease virus replication also decrease apoptosis. Antiviral antibody can downregulate virus replication in surviving neurons without affecting cell viability. Other innate and induced host immune responses can alter the outcome of infection without a change in virus production. Failure to induce apoptosis in infected neurons leads to long-term persistence of small amounts of viral RNA in the nervous system of infected mice despite the clearance of infectious virus. The molecular mechanisms that govern these pathogenesis factors are beginning to be elucidated.     

The incorporation of thymidine into deoxyribonucleic acid in mouse fibroblasts infected with polyoma virus

1. Mouse-fibroblast cultures in the stationary phase of growth show an increased rate of [(3)H]thymidine incorporation into DNA from 12 to 44hr. after infection with polyoma virus. 2. Intracellular virus progeny is first detected at about 24hr. after infection. 3. Calculations based on the [(3)H]thymidine-incorporation data and direct measurements of the DNA content of the cell cultures indicate that the amount of the excess of DNA synthesized by the infected cell cultures corresponds to about 10% of their total DNA. 4. The mitotic index of the cell cultures at 40hr. after infection was significantly higher than that of non-infected control cells. 5. Possible interpretations of the stimulation of DNA synthesis observed in polyoma-infected cell cultures are discussed.     

JC virus induces nonapoptotic cell death of human central nervous system progenitor cell-derived astrocytes

JC virus (JCV), a human neurotropic polyomavirus, demonstrates a selective glial cell tropism that causes cell death through lytic infection. Whether these cells die via apoptosis or necrosis following infection with JCV remains unclear. To investigate the mechanism of virus-induced cell death, we used a human central nervous system progenitor-derived astrocyte cell culture model developed in our laboratory. Using in situ DNA hybridization, immunocytochemistry, electron microscopy, and an RNase protection assay, we observed that astrocytes support a progressive JCV infection, which eventually leads to nonapoptotic cell death. Infected astrocyte cell cultures showed no difference from noninfected cells in mRNA expression of the caspase family genes or in any ultrastructural features associated with apoptosis. Infected cells demonstrated striking necrotic features such as cytoplasmic vacuolization, watery cytoplasm, and dissolution of organelles. Furthermore, staining for caspase-3 and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling were not detected in infected astrocyte cultures. Our findings suggest that JCV-induced cell death of these progenitor cell-derived astrocytes does not utilize an apoptosis pathway but exhibits a pattern of cell destruction consistent with necrotic cell death.     

Establishment of a persistent mutant of canine distemper virus

We produced a B95a lymphoid cell line persistently infected with canine distemper virus (CDV), in which virus-specific antigens were present in nearly 100% of cells without causing cytopathic effect. The virus recovered from this cell line was able to infect fresh B95a cells persistently, indicating that a persistent CDV was established.     

Neuropathogenesis of Japanese Encephalitis in a Primate Model

Background

Japanese encephalitis (JE) is a major cause of mortality and morbidity for which there is no treatment. In addition to direct viral cytopathology, the inflammatory response is postulated to contribute to the pathogenesis. Our goal was to determine the contribution of bystander effects and inflammatory mediators to neuronal cell death.

Methodology/Principal Findings

Material from a macaque model was used to characterize the inflammatory response and cytopathic effects of JE virus (JEV). Intranasal JEV infection induced a non-suppurative encephalitis, dominated by perivascular, infiltrates of mostly T cells, alongside endothelial cell activation, vascular damage and blood brain barrier (BBB) leakage; in the adjacent parenchyma there was macrophage infiltration, astrocyte and microglia activation. JEV antigen was mostly in neurons, but there was no correlation between intensity of viral infection and degree of inflammatory response. Apoptotic cell death occurred in both infected and non-infected neurons. Interferon-α, which is a microglial activator, was also expressed by both. Tumour Necrosis Factor-α, inducible nitric oxide synthase and nitrotyrosine were expressed by microglial cells, astrocytes and macrophages. The same cells expressed matrix metalloproteinase (MMP)-2 whilst MMP-9 was expressed by neurons.

Conclusions/Significance

The results are consistent with JEV inducing neuronal apoptotic death and release of cytokines that initiate microglial activation and release of pro-inflammatory and apoptotic mediators with subsequent apoptotic death of both infected and uninfected neurons. Activation of astrocytes, microglial and endothelial cells likely contributes to inflammatory cell recruitment and BBB breakdown. It appears that neuronal apoptotic death and activation of microglial cells and astrocytes play a crucial role in the pathogenesis of JE.     

Nectin4 is an epithelial cell receptor for canine distemper virus and involved in neurovirulence

Canine distemper virus (CDV) uses signaling lymphocyte activation molecule (SLAM), expressed on immune cells, as a receptor. However, epithelial and neural cells are also affected by CDV in vivo. Wild-type CDV strains showed efficient replication with syncytia in Vero cells expressing dog nectin4, and the infection was blocked by an anti-nectin4 antibody. In dogs with distemper, CDV antigen was preferentially detected in nectin4-positive neurons and epithelial cells, suggesting that nectin4 is an epithelial cell receptor for CDV and also involved in its neurovirulence.     

Canine Distemper Virus Infection Leads to an Inhibitory Phenotype of Monocyte-Derived Dendritic Cells In Vitro with Reduced Expression of Co-Stimulatory Molecules and Increased Interleukin-10 Transcription

Canine distemper virus (CDV) exhibits a profound lymphotropism that causes immunosuppression and increased susceptibility of affected dogs to opportunistic infections. Similar to human measles virus, CDV is supposed to inhibit terminal differentiation of dendritic cells (DCs), responsible for disturbed repopulation of lymphoid tissues and diminished antigen presenting function in dogs. In order to testify the hypothesis that CDV-infection leads to an impairment of professional antigen presenting cells, canine DCs have been generated from peripheral blood monocytes in vitro and infected with CDV. Virus infection was confirmed and quantified by transmission electron microscopy, CDV-specific immunofluorescence, and virus titration. Flow cytometric analyses revealed a significant down-regulation of the major histocompatibility complex class II and co-stimulatory molecules CD80 and CD86 in CDV-infected DCs, indicative of disturbed antigen presenting capacity. Molecular analyses revealed an increased expression of the immune inhibitory cytokine interleukin-10 in DCs following infection. Results of the present study demonstrate that CDV causes phenotypical changes and altered cytokine expression of DCs, which represent potential mechanisms to evade host immune responses and might contribute to immune dysfunction and virus persistence in canine distemper.     

CD9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus.

Canine distemper virus (CDV), a lymphotropic and neurotropic negative-stranded RNA virus of the Morbillivirus genus, causes a life-threatening disease in several carnivores, including domestic dogs. To identify the cellular receptor(s) involved in the uptake of CDV by susceptible cells, we isolated a monoclonal antibody (MAb K41) which binds to the cell surface and inhibits the CDV infection of several cell lines from various species. Pretreatment of cells with MAb K41 reduces the number of infectious centers and the size of the syncytia. Using affinity chromatography with MAb K41, we purified from HeLa and Vero cell extracts a 26-kDa protein which contained the amino acid sequence TKDEPQRETLK of human CD9, a member of the tetraspan transmembrane or transmembrane 4 superfamily of cell surface proteins. Transfection of NIH 3T3 or MDBK cells with a CD9 expression plasmid rendered these cells permissive for viral infection and raised virus production by a factor of 10 to 100. The mechanism involved is still unclear, since we were unable to detect direct binding of CDV to CD9 by using immunoprecipitation and a virus overlay protein binding assay. These findings indicate that human CD9 and its homologs in other species are necessary factors for the uptake of CDV by target cells, the formation of syncytia, and the production of progeny virus.     

Human Induced Pluripotent Stem Cell-Derived Models to Investigate Human Cytomegalovirus Infection in Neural Cells

Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs), neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate.     

Effect of enforced expression of human bcl-2 on Japanese encephalitis virus-induced apoptosis in cultured cells.

Infection by Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes acute encephalitis in humans and induces severe cytopathic effects in different types of cultured cells. This study attempted to determine whether apoptosis contributes to virus-induced cell death in a culture system by characterizing JEV lytic infection in baby hamster kidney BHK-21 cells, murine neuroblastoma N18 cells, and human neuronal progenitor NT2 cells. According to our results, the replication of JEV, and not the UV-inactivated virions per se, triggered apoptosis in these cell lines, as evidenced by nuclear condensation, DNA fragmentation ladder, and in situ end labeling of DNA strand breaks with terminal transferase (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay). Different strains of JEV, regardless of whether they are neurovirulent to mice, could induce apoptosis of the infected cells. In addition, enforced expression of the human protooncogene bcl-2 in BHK-21 cells, which did not influence virus production, appeared to delay the process of JEV-induced apoptosis, despite the fact that most infected cells were inevitably killed after prolonged cultures. However, Bcl-2 proteins expressed in N18 cells failed to block JEV-induced apoptosis, although they did prevent Sindbis virus-induced apoptosis from occurring in the same cells. This finding suggests that these two viruses may utilize similar but not identical mechanisms to kill their infected cells. The results presented here thus demonstrate that apoptosis can be a general mechanism for JEV-induced cell death and that enforced bcl-2 expression may be inadequate in protecting all cell types from JEV-induced apoptosis in cell cultures.     

Theiler's virus infection of primary cultures of bone marrow-derived monocytes/macrophages

Theiler's virus, a murine picornavirus, causes a persistent infection of macrophage/microglial cells in the central nervous systems of SJL/J mice. Viral replication is restricted in the majority of infected cells, whereas a minority of them contain large amounts of viral RNA and antigens. For the present work, we infected primary cultures of bone marrow monocytes/macrophages from SJL/J mice with Theiler's virus. During the first 10 h postinfection (p.i.), infected monocytes/macrophages were round and covered with filopodia and contained large amounts of viral antigens throughout their cytoplasm. Later on, they were large, flat, and devoid of filopodia and they contained only small amounts of viral antigens distributed in discrete inclusions. These two types of infected cells were very reminiscent of the two types of infected macrophages found in the spinal cords of SJL/J mice. At the peak of virus production, the viral yield per cell was approximately 200 times lower than that for BHK-21 cells. Cell death occurred in the culture during the first 24 h p.i. but not thereafter. No infected cells could be detected after 4 days p.i., and the infection never spread to 100% of the cells. This restriction was unchanged by treating the medium at pH 2 but was abolished by treating it with a neutralizing alpha/beta interferon antiserum, indicating a role for this cytokine in limiting virus expression in monocyte/macrophage cultures. The role of alpha/beta interferon was confirmed by the observation that monocytes/macrophages from IFNA/BR(-/-) mice were fully permissive.     

Virus infection induces neuronal apoptosis: A comparison with trophic factor withdrawal

Multicellular organisms can employ a number of defences to combat viral replication, the most dramatic being implementation of a cell autonomous apoptotic process. The overall cost to the viability of an organism of losing infected cells by apoptosis may be small if the dying cells can be substituted. In contrast, suicide of irreplaceable cells such as highly specialised neurons may have a more dramatic, even fatal consequence. Previous in vitro approaches to understanding whether neurotropic viruses cause neurons to apoptose have utilised transformed cell lines. These are not in the appropriate state of differentiation to provide an accurate indication of events in vivo. We have chosen to characterise the ability of a model CNS disease-causing virus, Semliki Forest virus (SFV), to infect and trigger apoptosis in primary cultures of nerve growth factor (NGF)-dependent sensory neurons. These cells are known to die when deprived of NGF and constitute a useful indicator of apoptosis. We observe that infection causes cell death which bears the morphological hallmarks of apoptosis, this occurs even in the present of survival promoting NGF and is concomitant with new virus production. Using the TUNEL (transferase dUTP nick end labelling) technique we show that SFV-induced apoptosis involves DNA fragmentation and requires caspase (CED-3/ICE cysteine protease) activation, as does apoptosis induced by NGF-deprivation. Extensive areas of apoptosis, as defined using a combination of ultrastructural analysis and TUNEL occur in infected neonatal mouse brains. The novel evidence that infection of primary neurons with SFV induces apoptosis with activation of one or more caspases defines a system for the further anlaysis of apoptosis regulation in physiologically relevant neurons.     

Glutamate Differently Modulates Metabotropic Glutamate Receptors in Neuronal and Glial Cells

Glutamate is an excitatory neurotransmitter implicated in learning and memory processes, but at high concentrations it acts as an excitotoxin causing degeneration and neuronal death. The aim of this work was to determine the excitotoxic effect of glutamate and the regulation of metabotropic glutamate receptors (mGluR) during excitotoxicity in neurons and C6 glioma cells. Results show that glutamate causes excitotoxic damage only in cortical neurons. Loss of cell viability in neurons was glutamate concentration- and time-dependent. Total mGluR levels were significantly reduced in these cells when exposed to glutamate. However, in C6 cells, which have been used as a model of glial cells, these receptors were regulated in a biphasic manner, decreased after 6 h, and increased after 24/48 h of treatment. Results show a cell dependent mGluR regulation by glutamate exposure which could mediate the vulnerability or not to glutamate mediated excitotoxicity.     

Early infection of the central nervous system by the GDVII and DA strains of Theiler's virus.

The DA strain of Theiler's virus, a murine picornavirus, causes a persistent infection of glial cells of the white matter of the spinal cord, associated with chronic inflammation and primary demyelination. The GDVII strain causes an acute fatal grey matter encephalomyelitis. We characterized the target cells of GDVII and DA viruses 4 days following intracerebral inoculation, and we compared the levels of viral RNA within these cells. GDVII virus infected approximately 10 times more cells than DA virus. Whereas GDVII virus infected neurons exclusively, DA virus infected also astrocytes and possible macrophage-microglial cells. The levels of viral RNA in neurons infected with GDVII and DA viruses were of the same order. These results show that DA virus infects glial cells already at the beginning of the disease and that the more efficient spread of GDVII virus is probably not due to a higher level of RNA replication per cell.