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.     

Microglial GLT-1 is upregulated in response to herpes simplex virus infection to provide an antiviral defence via glutathione

Herpes simplex virus (HSV) can enter the central nervous system and cause encephalitis (HSV-1) or meningitis (HSV-2). Microglia, the immunocompetent cells of the central nervous system, are potentially able to detect viral infections. Microglia have been shown to express the glutamate transporter GLT-1 during pathological events, leading to increased microglial glutamate uptake and glutathione synthesis. This study aims to address the role of GLT-1 and glutathione, a major antioxidant with antiviral properties, during HSV infections. Using neuron-enriched mixed primary cultures from rat, it was found that microglia have higher resistance to HSV infections than neurons or astrocytes after 24 h incubation with HSV. Purified microglia in culture were used to further address this. It was found that microglia were able to detect HSV and responded by releasing tumor necrosis factor-alpha (TNF-alpha) and upregulating GLT-1 after 24 h incubation with 1 PFU/cell HSV-1 or HSV-2. Furthermore, the microglial glutathione levels were not significantly diminished after 24 h. Inhibition of the microglial glutathione synthesis with 200 microM buthionine sulfoximide (BSO) led to significantly more infected cells after 24 h incubation with 1 PFU/cell HSV-1 or HSV-2. These data indicate that the higher resistance in microglia against HSV infections may be due to the expression of GLT-1, which can maintain the glutathione levels and provide a mechanism for microglial self-defense against HSV.     

Peripheral nervous system demyelination with herpes simplex virus

Inoculation of the cornea or footpad with herpes simplex virus Type I (HSV) has been shown to produce subsequent encephalitis or myelitis respectively. Although Schwann cells become infected, there is no destruction or demyelination in the peripheral nervous system (PNS). Demyelination only occurs in the central nervous system. Previous studies have shown that the Schwann cells infected with HSV do not produce enveloped viral particles. The studies presented here demonstrate that microinjection of HSV into the sciatic nerve of mice causes focal mononuclear cell infiltration and demyelination seven days after injection. The Schwann cells in this model produced enveloped virus. These studies demonstrate that when HSV is introduced into the extracellular space of the PNS, demyelination occurs.     

Herpes simplex virus types 1 and 2 in organotypic cultures of mouse central and peripheral nervous system. I. Light microscopic observations of myelin degeneration.

Mature mouse spinal cord-ganglion cultures, which contain both peripheral and central nervous system as one unit, were infected with herpes simplex virus type 1 (HSV 1) or type 2 (HSV 2) and observed by bright field microscopy for up to 72 hours. There was degeneration of both central and peripheral myelin in cultures infected with either virus, but the pattern of peripheral myelin degeneration associated with HSV 1-infected cultures was differrnt from that in HSV 2-infected cultures. Type 1 was charcterized by focal dilatations; type 2 by "sausage-shaped" swellings, and the cytopathic effect of HSV 2 both began (6 hours p.i.) and was completed (36 hours p.i.) earlier than in cultures infected with HSV 1 (12 hours and 48 hours p.i. respectively). In central nervous tissue, the apperance of degenerating myelin after infection with HSV 1 was indistinguishable from that in HSV 2-infected cultures, but the rate of myelin loss was greater in cultures infected with the type 2 virus. Evidence is presented which suggests that, at least in the peripheral nervous system,myelin degeneration did not appear to be dependent on neuronal or axonal dysfunction or death, but was a direct result of virus infection.     

Early loss of astrocytes in herpes simplex virus-induced central nervous system demyelination

Immunohistochemistry was used to study herpes simplex virus type 1-induced central nervous system demyelination in the trigeminal root entry zone of mice inoculated with herpes simplex virus type 1 by the corneal route. There was no change in peripheral nervous system myelin as shown by immunostaining for P0 glycoprotein. Double immunoperoxidase staining for herpes simplex virus type 1 antigens and glial fibrillary acidic protein showed that most of the infected cells were astrocytes. Glial fibrillary acidic protein immunostaining was completely lost in the inferior medial portion of the trigeminal root entry zone at 6 days after herpes simplex virus type 1 inoculation, a time when central nervous system myelin was preserved as indicated by immunostaining for myelin basic protein. The pattern of glial fibrillary acidic protein staining did not change and herpes simplex virus type 1 antigens were no longer detected after day 8. There was a progressive loss of myelin basic protein staining within the area unstained by glial fibrillary acidic protein antisera on days 8 to 14. This pattern of astrocyte loss before central nervous system demyelination is strikingly different from the reactive astrocytosis seen in other demyelinating lesions, such as acute experimental allergic encephalomyelitis, progressive multifocal leukoencephalopathy, or acute multiple sclerosis. Herpes simplex virus type 1 infection in mice provides an unusual model of acute central nervous system demyelination preceded by a loss of astrocytes.     

Multifocal CNS demyelination following peripheral inoculation with herpes simplex virus type 1

The peripheral inoculation of herpes simplex virus type 1 (HSV 1) in experimental animals induces central nervous system (CNS) demyelinating lesions, but the potential relevance of this model to multiple sclerosis is lessened by the unifocal nature of the lesion. In this study, inbred strains of mice were selected on the basis of varying resistance to mortality following lip inoculation with virus. A spectrum of CNS pathology was observed, ranging from focal collections of inflammatory cells at the trigeminal root entry zone in resistant strains (C57BL/6J), to unifocal demyelinating lesions in moderately resistant strains (BALB/cByJ), to multifocal demyelinating lesions throughout the brain in susceptible strains (A/J). Findings from viral titration studies of the CNS support a direct cytolytic effect of virus in the development of demyelinating lesions at the trigeminal root entry zone but cannot exclude an immune-mediated component. Furthermore, 50% tissue-culture-infective doses, immunofluorescence, and electron microscopic studies of primary cultures of oligodendrocytes, derived from the three strains of adult mice, identify differences in resistance to HSV 1 infection in vitro, suggesting that differences at this level may also contribute to the pathological appearance. Multifocal lesions in A/J mice were first observed when the infectious virus could no longer be isolated from the CNS and may be the result of an immune-mediated process "triggered" by the acute CNS infection in susceptible strains of mice.     

Disseminated hemorrhagic leukoencephalomyelitis with localization herpes simplex brain stem infection

Summary A case of widespread hemorrhagic and perivenous demyelinative leukoencephalomyelitis complicating a localized herpes simplex virus (HSV) brain stem infection is reported in a 28-year-old man. The presence of the virus is documented immunohistochemically and ultrastructurally. The spinal trigeminal tract at the level of the medulla oblongata contained viral antigen in the neurons, glia and in the vascular walls, including a few endothelial cells. The foci of demyelination showed deposits of gamma globulins and slight inflammatory infiltrations; the virus was absent from these lesions. It is postulated that HSV entered the central nervous system through the trigeminal nerve. Focal expression of the viral antigen on the endothelium in a sensitized host was the likely precipitating factor in the hyperacute autoimmune reaction, resulting in the widespread hemorrhagic and demyelinating lesions in the central nervous system.     

Expression of major histocompatibility complex antigens in the brains of patients with progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is caused by JC virus (JCV) infection of the central nervous system (CNS) in immunosuppressed patients. The immunopathogenesis of this chronic encephalitis is unknown. Because major histocompatibility (MHC) class I and class II antigens are important in modulating the immune response and viral clearance, we examined the tissue expression of MHC molecules in relation to CNS damage and presence of virus. By immunocytochemical staining, both MHC class I and class II antigens were expressed at high levels within PML lesions. Beta-2 microglobulin (beta-2m) was present on endothelial cells and JCV-infected oligodendroglia within the lesions. Also, many astrocytes with bizarre morphology expressed MHC class I antigens. In histologically normal regions of PML brains expression of beta-2m was noted only on endothelial cells. Expression of MHC class II also was focused within demyelinating lesions and was restricted to macrophages/microglia and occasional endothelial cells. When compared to other viral encephalitides (e.g. human immunodeficiency virus) these findings suggest that intra-CNS immune response to JCV is appropriate for antigenic presentation; however, the absence of responsive systemic T-cells may lead to chronic viral infection with progressive neuropathology.     

Observations on the distribution of canine distemper virus in the central nervous system of dogs with demyelinating encephalitis

Summary The distribution of canine distemper virus in the central nervous system was examined in 11 dogs with demyelinating encephalitis by the direct fluorescent antibody technique on paraffin sections of brain and spinal cord. In the grey matter there was a good correlation between the presence and severity of lesions and presence and amount of viral antigen. Large concentrations of virus were found in neurons and their processes. In most demyelinating lesions only small amounts of viral antigen were found, mostly located in astrocytes. The potential importance of the role of the astrocyte in demyelination in canine distemper virus infection is stressed.     

Robust expression of TNF-alpha, IL-1beta, RANTES, and IP-10 by human microglial cells during nonproductive infection with herpes simplex virus

Cytokine (TNF-alpha/beta, IL-1beta, IL-6, IL-18, IL-10, and IFN-alpha/beta/gamma) and chemokine (IL-8, IP-10, MCP-1, MIP-1alpha/beta, and RANTES) production during herpes simplex virus (HSV) 1 infection of human brain cells was examined. Primary astrocytes as well as neurons were found to support HSV replication, but neither of these fully permissive cell types produced cytokines or chemokines in response to HSV. In contrast, microglia did not support extensive viral replication; however, ICP4 was detected by immunochemical staining, demonstrating these cells were infected. Late viral protein (nucleocapsid antigen) was detected in <10% of infected microglial cells. Microglia responded to nonpermissive viral infection by producing considerable amounts of TNF-alpha, IL-1beta, IP-10, and RANTES, together with smaller amounts of IL-6, IL-8, and MIP-1alpha as detected by RPA and ELISA. Surprisingly, no interferons (alpha, beta, or gamma) were detected in response to viral infection. Pretreatment of fully permissive astrocytes with TNF-alpha prior to infection with HSV was found to dramatically inhibit replication, resulting in a 14-fold reduction of viral titer. In contrast, pretreatment of astrocytes with IL-1beta had little effect on viral replication. When added to neuronal cultures, exogenous TNF-alpha or IL-1beta did not suppress subsequent HSV replication. Exogenously added IP-10 inhibited HSV replication in neurons (with a 32-fold reduction in viral titer), however, similar IP-10 treatment did not affect viral replication in astrocytes. These results suggest that IP-10 possesses direct antiviral activity in neurons and support a role for microglia in both antiviral defense of the brain as well as amplification of immune responses during neuroinflammation.     

Herpes simplex virus latency in the nervous system – a new model

Permissive herpes simplex virus (HSV) infection in tissue culture results in host cell destruction. Latent HSV infection in vivo occurs in neurons of peripheral sensory ganglia (PSG) and it therefore can not take place in neurons in which the virus has completed a lytic replication cycle similar to that present in vitro. Our hypothesis, based on experimental data and observations in humans, suggests that establishment of latent infection and reactivation of HSV-1 does not involve neuronal cell loss. Latency is established in neurons in which the virus does not replicate and is determined, in part, by the tissue levels of a herpes transactivating protein (Vmw65) that is a component of the viral tegument. We also suggest that reactivation of latent infection does not involve destruction of neurons and is due to replication of virus at the peripheral mucocutaneous tissues to where virus or viral DNA have been transported from the nervous tissue. Alternatively, reactivation is initiated in the PSG using a replication cycle which does not involve irreversible damage to neurons. This model explains the lack of damage to neurons which continue to serve as permanent reservoirs of latent virus for the entire life of the host.     

Herpes simplex virus type I (HSV I)-induced multifocal central nervous system (CNS) demyelination in mice.

Multifocal central nervous system (CNS) demyelination develops in the brains of SJL/J, PL/J, and A/J mice following lip inoculation with a specific strain of herpes simplex virus I (HSV I). The lesions in all three inbred strains of mice share similar characteristics including demyelination, relative preservation of axons, and a mononuclear cell (MNC) infiltrate. The lesions, developing during the early phase of demyelination, also appear sequentially in the CNS (trigeminal root entry zone of the brainstem greater than cerebellum greater than cerebral hemispheres) of all three strains of mice but differ in the time of their initial appearance following infection as well as their morphology. In SJL/J mice, new areas of demyelination are observed for only 24 days following lip inoculation with virus. Late stage multifocal CNS demyelination persists throughout 28 weeks postinoculation (pi) in PL/J mice while in A/J mice the development of new areas of demyelination are restricted to 8 weeks pi. Although mononuclear inflammatory cells are present in the new areas of demyelination in either PL/J or A/J mice, viral antigens are not detected in the CNS beyond 12 days pi. In contrast, in situ hybridization studies using 35S-cDNA HSV probes and performed beyond day 12 pi identify probe-positive cells central to a number of the multifocal CNS demyelinating lesions in A/J mice. Results from studies with inbred and congenic strains of mice indicate that the major histocompatibility complex (H-2) does not determine the development of multifocal CNS demyelination following lip inoculation with HSV I but does influence the morphological appearance of the lesions that do develop.     

Toll-like receptor 7 is not necessary for retroviral neuropathogenesis but does contribute to virus-induced neuroinflammation

Toll-like receptor 7 (TLR7) recognizes guanidine-rich single-stranded (ss) viral RNA and is an important mediator of peripheral immune responses to several ssRNA viruses. However, the role that TLR7 plays in regulating the innate immune response to ssRNA virus infections in specific organs is not as clear. This is particularly true in the central nervous system (CNS) where microglia and astrocytes are often the first cells responding to virus infection instead of dendritic cells. In the current study, we examined the mechanism by which TLR7 contributes to ssRNA virus-induced neuroinflammation using a mouse model of polytropic retrovirus infection. The authors found that TLR7 was necessary for the early production of certain cytokines and chemokines, including CCL2 and tumor necrosis factor (TNF) and was also involved in the early activation of astrocytes. However, TLR7 was not necessary for cytokine production and astrocyte activation at later stages of infection and did not alter viral pathogenesis or viral replication in the brain. This suggests that other pathogen recognition receptors may be able to compensate for the lack of TLR7 during retrovirus infection in the CNS.     

Herpes simplex virus types 1 and 2 in organotypic cultures of mouse central and peripheral nervous system. 2. Electron microscopic observations of myelin degeneration.

Organotypic cultures of mouse spinal cord with attached dorsal root ganglia, which contain both central and peripheral myelin in the one unit of tissue, were infected with HSV 1 or HSV 2 and studied using electron microscopy. Intranuclear viral nucleocapsids and intracytoplasmic enveloped particles were found in the Schwann cells associated with peripheral myelin and in oligodendroglia associated with central myelin. Degeneration of peripheral myelin most commonly involved an asymmetrical swelling of the myelin lamellae, whereas degeneration of central myelin was characterized by a more generalized swelling resulting in separation of the myelin lamellae. Degeneration of both central and peripheral myelin was found in the presence of intact axons which were indistinguishable from those in controls.     

Expression of peripherin in the brain of macaques (Macaca mulatta and Macaca fascicularis) occurs in astrocytes rather than neurones and is associated with encephalitis

Peripherin is a member of the type III intermediate filament family, expressed in neurones of the peripheral nervous system of many species and in a discrete subpopulation of neurones of the central nervous system (CNS) during early development in rodents. Previous studies on rats have shown that peripherin immunoreactivity increased significantly in cell bodies of spinal motor neurones following axonal injury. Our study examined the expression of peripherin in the cerebrum of normal macaques (Macaca mulatta and Macaca fascicularis) and those with encephalitis of viral (simian immunodeficiency virus and simian virus 40) or autoimmune (experimental allergic encephalomyelitis) aetiology. Immunohistochemistry, immunoelectronmicroscopy, immunofluorescence and confocal microscopy were performed on tissue sections using antibodies against cell-specific markers and peripherin. Peripherin-positive cells were absent in the cerebrum of normal macaques of all ages examined, whereas animals with encephalitis had peripherin-positive cells associated with inflammatory infiltrates. Further evaluation revealed that these peripherin-positive cells were not neurones, but were predominantly astrocytes expressing glial fibrillary acidic protein. Our study suggests that peripherin is not neurone-specific in the CNS of macaques; peripherin is expressed in astrocytes of animals with encephalitis.     

Sex differences in susceptibility to viral infection of the central nervous system

We have characterized striking differences in recovery of male and female BALB/c and BALB/c-H-2^dm2 (dm2) mice from an experimental neurotropic viral infection of the central nervous system (CNS). Following intranasal inoculation of vesicular stomatitis virus (VSV), assays of tissue homogenates from female mice produced lower viral titers. There was also a significant reduction in the spread of virus from the rostral to caudal end of the brain in female mice. Enhanced recovery by female mice of both strains in response to this viral insult correlates with increased levels of Nitric Oxide Synthase (NOS) types I, II, and III expression, an increased prevalence of reactive astrocytes, earlier and enhanced levels of expression of Major Histocompatabilty Complex (MHC) class II molecules on astrocytes, endothelial and microglial cells, and increased T cell infiltration in the female BALB/c mouse. Taken together, these findings document sexual dimorphism in CNS immunity, and may provide an understanding of some of the mechanisms underlying many sex-biased diseases.     

The neurotropic herpes viruses: herpes simplex and varicella-zoster

Herpes simplex viruses types 1 and 2 (HSV1 and HSV2) and varicella-zoster virus (VZV) establish latent infection in dorsal root ganglia for the entire life of the host. From this reservoir they can reactivate to cause human morbidity and mortality. Although the viruses vary in the clinical disorders they cause and in their molecular structure, they share several features that affect the course of infection of the human nervous system. HSV1 is the causative agent of encephalitis, corneal blindness, and several disorders of the peripheral nervous system; HSV2 is responsible for meningoencephalitis in neonates and meningitis in adults. Reactivation of VZV, the pathogen of varicella (chickenpox), is associated with herpes zoster (shingles) and central nervous system complications such as myelitis and focal vasculopathies. We review the biological, medical, and neurological aspects of acute, latent, and reactivated infections with the neurotropic herpes viruses.     

Expression of insulin-like growth factors and corresponding binding proteins (IGFBP 1–6) in rat spinal cord and peripheral nerve after axonal injuries

Insulin-like growth factors (IGFs) exert trophic effects on several different cell types in the nervous system, including spinal motoneurons. After peripheral nerve injury, the increased expression of IGFs in the damaged nerve has been suggested to facilitate axonal regeneration. Here we have examined the expression pattern of mRNAs encoding IGF-1 and and -2, IGF binding proteins (IGFBPs) 1–6 in the rat spinal cord and peripheral nerve in three lesion models affecting lumbar motoneurons, i.e., sciatic nerve transection, ventral root avulsion, and a cut lesion in the ventral funiculus of the spinal cord. The expression was also studied in enriched Schwann cell and astrocyte cultures. The injured sciatic nerve expressed IGF-1 and IGF-2 as well as IGFBP-4 and IGFBP-5, whereas central nervous system (CNS) scar tissue expressed IGF-1, IGFBP-2, and IGFBP-5. IGFBP-6 mRNA was strongly upregulated in spinal motoneurons after all three types of lesions. IGFBP-6-like immunoreactivity was present in motoneuron cell bodies, dendrites in the ventral horn, and axons in the sciatic nerve. In line with the in vivo findings, cultured Schwann cells expressed IGF-1, IGF-2, IGFBP-4, and IGFBP-5 mRNAs, whereas cultured astrocytes expressed IGF-1, IGFBP-2, and IGFBP-5 mRNAs. These findings show that IGF-1 is available for lesioned motoneurons both after peripheral and central axonal lesions, whereas there are clear differences in the expression patterns for IGF-2 and some of the binding proteins in CNS and peripheral nervous system (PNS) scar tissue. The robust upregulation of IGFBP-6 mRNA in lesioned motoneurons suggests that this binding protein may be of special relevance for the severed cells. J. Comp. Neurol. 400:57–72, 1998. © 1998 Wiley-Liss, Inc.     

HveC (nectin-1) is expressed at high levels in sensory neurons, but not in motor neurons, of the rat peripheral nervous system

The entry of herpes simplex virus (HSV)-1 into cells is a complex process mediated in part by the binding of the HSV glycoprotein D (gD) to a specific cellular receptor identified as HveC, or nectin-1. We examined the distribution of HveC in sensory and motor neurons of the peripheral nervous system (PNS) by immunocytochemistry. HveC is expressed at high levels in sensory neurons of dorsal root ganglion and their peripheral axons, at lower levels in motor neurons of spinal cord, and without detectable expression in motor nerve terminals at the neuromuscular junction. These results have implications regarding the tropism of HSV to specific neuronal populations, and for the construction of HSV-based vectors for the peripheral nervous system.