Up-regulation of Fas ligand (FasL) in the central nervous system: A mechanism of immune evasion by rabies virus

Following its injection into the hindlimbs of mice, CVS, a highly pathogenic strain of rabies virus, invades the spinal cord and brain resulting in the death of the animal. In contrast, central nervous system (CNS) invasion by PV, a strain of attenuated pathogenicity, is restricted to the spinal cord and mice infected with this virus survive. Lymphocytes display transient migration into the infected CNS in fatal rabies and sustained migration in nonfatal rabies. The transient migration of T cells in fatal rabies is associated with an increase in T-cell apoptosis. We found that the early production of Fas ligand (FasL) mRNAs was up-regulated only in fatal rabies. FasL is produced by several neuronal cells and mainly in infected neurons. In mice lacking FasL (gld), infection with the neuroinvasive rabies virus strain was less severe, and the number of CD3 T cells undergoing apoptosis was smaller than that in normal mice. These data provide strong evidence that fatal rabies virus infection involves the early triggering of FasL production leading to the destruction of migratory T cells by the Fas/FasL apoptosis pathway. This mechanism could be in part responsible for the fact that T cells cannot control neuroinvasive rabies infection. Thus, rabies virus seems to use an immunosubversive strategy that takes advantage of the immune privilege status of the CNS.     

Abortive rabies virus central nervous infection is controlled by T lymphocyte local recruitment and induction of apoptosis

Nonfatal paralysis, induced by the attenuated Pasteur strain of rabies virus, is characterised by local and irreversible flaccid paralysis of the inoculated limbs. We characterised the spread and localisation of virus in the CNS of infected mice, determined the nature of cell injury and examined the role of the immune response. Data indicate that infection of BALB/c mice induced paralysis in 60% of infected mice, the others recovering without sequelae. In both groups of mice, virus was detected in restricted sub-populations of neurons from the brain and spinal cord, and intensity of the neuropathology correlated with levels of rabies RNA and apoptotic infected neurons. However, apoptosis of neurons and paralysis were not due to a direct deleterious effect of the virus, but induced by a T-dependent immune response, as evidenced by their absence in nude mice. Paralysed and asymptomatic mice developed a similar rabies virus-specific IgG2a antibody response, thus excluding the role of any modification of the humoral immune response. In contrast, three events were critically associated with the development of neurological symptoms: the amount of virus in the CNS, the level of apoptosis in both infected neurons and uninfected surrounding cells and the progressive parenchymal infiltration of CD4+ and CD8+ T cells at the site of infection. These data suggest that during nonfatal rabies infection, the levels of viral replication and primary degeneration of infected neurons by apoptosis could be responsible for the infiltration of T lymphocytes capable of inducing secondary degeneration of neural cells.     

Influence of factors secreted by wobbler astrocytes on neuronal and motoneuronal survival

During late postnatal development, mice with the autosomal recessive wobbler mutation (wr/wr) develop motoneuron degeneration associated with astrogliosis in the spinal cord. In vitro, primary wobbler astrocytes are also affected, exhibiting abnormal cell-cell contacts. To characterize further the wobbler disease, we investigated the in vitro effects of wobbler astrocytes on primary neuronal cultures from the spinal cords of 15-day-old wild-type mouse and rat embryos. Cocultures with the wobbler astrocytes, or direct addition of wobbler astrocyte-conditioned medium, led to a decrease in neuron number in primary mixed neuronal cultures, containing motoneurons and interneuron-like cells. In contrast, wobbler astrocyte-conditioned medium enhanced survival of highly purified motoneurons. These in vitro results suggest the possibility that wobbler astrocytes act not on motoneurons directly but, rather, through other spinal neurons to induce motoneuron degeneration in the wobbler disease.     

Endogenous and exogenous factors support neuronal survival and choline acetyltransferase activity in embryonic spinal cord cultures

Dissociated 4-day (stage 23) chick embryo lumbar cord cells were cultured at low or high cell densities for 1 or 5 days in the presence or absence of added spinal neuronotrophic factor (supplied as RN22 Schwannoma conditioned medium, RCM). In low density, 1-day cultures neuronal survival was dependent on added RCM whereas by 5 days no neurons survived, even in the presence of RCM. In high density 1-day cultures a substantial neuronal population could survive even without added RCM and a large proportion of this neuronal population would survive for 5 days. When conditioned media from high density lumbar cord cultures was supplied to low density unsupplemented cultures, a similar level of 5-day neuronal survival resulted. However, no neurons survived in RCM-supplemented 5-day high density cultures, indicating the presence in RCM of a material toxic for the neurons. Both the RCM and the high density lumbar culture-conditioned medium supported considerable choline acetyltransferase activity indicating the presence within these cultures of motoneurons.     

A human spinal cord cell promotes motoneuron survival and maturation in vitro

Primary cultures of motoneurons represent a good experimental model for studying mechanisms underlying certain spinal cord pathologies, such as amyotrophic lateral sclerosis and spinal bulbar muscular atrophy (Kennedy's disease). However, a major problem with such culture systems is the relatively short cell survival times, which limits the extent of motoneuronal maturation. In spite of supplementing culture media with various growth factors, it remains difficult to maintain motoneurons viable longer than 10 days in vitro. This study employs a new approach, in which rat motoneurons are plated on a layer of cultured cells derived from newborn human spinal cord. For all culture periods, more motoneurons remain viable in such cocultures compared with control monocultures. Moreover, although no motoneurons survive in control cultures after 22 days, viable motoneurons were observed in cocultures even after 7 weeks. Although no significant difference in neurite length was observed between 8-day mono- and cocultures, after 22 and 50 days in coculture motoneurons had a very mature morphology. They extended extremely robust, very long neurites, which formed impressive branched networks. Data obtained using a system in which the spinal cord cultures were separated from motoneurons by a porous polycarbonate filter suggest that soluble factors released from the supporting cells are in part responsible for the beneficial effects on motoneurons. Several approaches, including immunocytochemistry, immunoblotting, and electron microscopy, indicated that these supporting cells, capable of extending motoneuron survival and enhancing neurite growth, had an undifferentiated or poorly differentiated, possibly mesenchymal phenotype.     

Experimental rabies virus infection in Artibeus jamaicensis bats with CVS-24 variants

An experimental model of rabies was established in the fruit-eating bat species Artibeus jamaicensis. The infections caused by CVS-N2c and CVS-B2c, which are both stable variants of CVS-24, were compared after inoculation of adult bats in the right masseter muscle. CVS-N2c produced neurologic signs of rabies with paresis, ataxia, and inability to fly, while CVS-B2c did not produce neurologic signs. Bats were sacrificed and the distribution of rabies virus antigen was assessed in tissue sections with immunoperoxidase staining. Both viruses spread to the brain stem and bilaterally to the trigeminal ganglia by days 2 to 3. CVS-N2c had disseminated widely in the central nervous system (CNS) by day 4 and had involved the spinal cord, thalamus, cerebellum, and cerebral cortex. CVS-B2c had infected neurons in the spinal cord on day 5 and in the cerebellum, thalamus, and cerebral cortex on day 6. Infected pyramidal neurons of the hippocampus were observed on day 5 in CVS-N2c infection, but infected neurons were never noted in the hippocampus in CVS-B2c infection. CVS-N2c infected many more neurons and more prominently involved neuronal processes than CVS-B2c. CVS-N2c spread more efficiently in the CNS than CVS-B2c. Morphologic changes of apoptosis or biochemical evidence of DNA fragmentation were not observed in neurons with either virus after this route of inoculation. The different neurovirulent properties of these CVS variants in this model were not related to their in vivo ability to induce apoptosis.     

Identification of central nervous system neurons innervating the respiratory muscles of the mouse: a transneuronal tracing study

In recent years, the central control of breathing in mammals has been the subject of numerous studies. The aim of the present one was to characterize the neuronal network projecting to the main respiratory motoneurons, in adult mice. To this end, the morphology and location of the respiratory motoneurons and their sequential connections with other neurons were revealed using a transneuronal tracing technique by means of the rabies virus infection. The injections of the rabies virus in the respiratory muscles resulted in labeling the motoneurons and their serially connected interneurons at multiple levels of the mouse central nervous system: spinal cord, pons and medulla, cerebellum, mesencephalon, diencephalon, and telencephalon. Most of these labeled areas have been previously identified in the control of cardiorespiratory regulation, as well as in other autonomic functions. These anatomical data provide support for the integration of respiratory-related activities in complex behavioral responses. Furthermore, these data suggest similarities in the evolution of central respiratory networks in mammals.     

FGF-2 Is Sufficient to Isolate Progenitors Found in the Adult Mammalian Spinal Cord

The adult rat brain contains progenitor cells that can be induced to proliferatein vitroin response to FGF-2. In the present study we explored whether similar progenitor cells can be cultured from different levels (cervical, thoracic, lumbar, and sacral) of adult rat spinal cord and whether they give rise to neurons and glia as well as spinal cord-specific neurons (e.g., motoneurons). Cervical, thoracic, lumbar, and sacral areas of adult rat spinal cord (>3 months old) were microdissected and neural progenitors were isolated and cultured in serum-free medium containing FGF-2 (20 ng/ml) through multiple passages. Although all areas generated rapidly proliferating cells, the cultures were heterogeneous in nature and cell morphology varied within a given area as well as between areas. A percentage of cells from all areas of the spinal cord differentiate into cells displaying antigenic properties of neuronal, astroglial, and oligodendroglial lineages; however, the majority of cells from all regions expressed the immature proliferating progenitor marker vimentin. In established multipassage cultures, a few large, neuron-like cells expressed immunoreactivity for p75NGFr and did not express GFAP. These cells may be motoneurons. These results demonstrate that FGF-2 is mitogenic for progenitor cells from adult rat spinal cord that have the potential to give rise to glia and neurons including motoneurons.     

Effects of ciliary neuronotrophic factor on rat spinal cord neurons in vitro: survival and expression of choline acetyltransferase and low-affinity nerve growth factor receptors.

We have studied the effects of ciliary neuronotrophic factor (CNTF) and nerve growth factor (NGF) on cultures of E14 rat spinal cord cells maintained for 7 days. The trophic factors were supplied at the day of seeding and every other day thereafter. Treatments with CNTF (human recombinant or purified from rat sciatic nerve, 100 TU/ml) resulted after 7 days in an increase, relative to control cultures, of: (i) the total number of neurons (identified by neurofilament protein and neuron-specific enolase immunostaining) that were not stained with choline, acetyltransferase (ChAT) and low affinity nerve growth factor receptor (LNGFR) antibodies; (ii) the number of motoneurons (0.5% of the neuronal population) as identified by size (greater than 25 microns), morphology and immunostaining for ChAT and LNGFR; and (iii) a population of small- to medium-sized (less than 25 microns), ChAT- and LNGFR-positive neurons, representing 5-10% of the total neuronal population. NGF treatments (mouse submaxillary beta NGF; 10-3000 TU/ml) were without effect on all 3 neuronal populations. Experiments in which CNTF administration was delayed revealed that the population of ChAT- and LNGFR-negative neurons and the population of motoneurons, were both dependent on CNTF for their survival. The third population, small ChAT and LNGFR-positive neurons, was not dependent on CNTF for survival but was induced by CNTF to express its two markers. These observations indicate that CNTF is a neuronotrophic factor for motoneurons, but that the effect of CNTF is not restricted to that cell population. In addition to its survival promoting effect, CNTF has also a regulatory role on the expression of ChAT and LNGFR for some spinal cord neurons.     

In vitro cytotoxicity and demyelination induced by Theiler viruses in cultures of spinal cord slices

The cytopathic effects caused by Theiler viruses to myelinated organotypic spinal cord cultures was studied by light and electron microscopy. Heavily myelinated cultures, 2-3 weeks in vitro were infected with WW and GD VII viruses. Mock infection served as control. On light microscopy cytopathic effects and demyelination became evident about 16-17 hr after infection. Demyelination observed in WW virus-infected cultures was much more pronounced than in cultures infected with GD VII viruses. The myelin in mock-infected cultures remained undamaged. Electron microscopy revealed that in control cultures cells were intact, exhibiting numerous synapses and a network of axons enwrapped by multilayered myelin sheaths. Virus-infected spinal cord slices showed that a more severe cytotoxicity was caused by GD VII virus than by WW virus. The cytopathology included accumulation of cytoplasmatic vacuoles, margination of chromatin, synapse and cell disintegration, and various degrees of demyelination. Several GD VII virions were observed, arranged in crystalline arrays, mainly in electron-opaque cells, but not within axons. WW virions on the other hand were only occasionally encountered.     

Adenoviral gene transfer to the injured spinal cord of the adult rat

We have investigated gene transfer to the injured adult rat spinal cord by the use of a recombinant adenovirus. 105 or 5 x 106 plaque-forming units (pfu) of a replication-defective adenoviral vector carrying the green fluorescent protein (GFP) reporter gene were injected into a dorsal hemisection lesion at spinal level T8. Gene expression and inflammatory responses were studied 4, 8 and 21 days after surgery. Numerous cells within 3 mm on each side of the lesion were found to express high levels of GFP at 4 days after infection as shown by GFP fluorescence and immunohistochemistry. At 8 days, expression was still strong although weaker than at 4 days. After 21 days, transgene expression had almost ceased. Expression was neither higher nor more prolonged in animals that had received the higher vector dose. Delayed injection 1 week after spinal injury also did not increase transgene expression. Infected cell types were identified immunohistochemically. The most prominent transduced cells were spinal motoneurons. Additionally, we could identify other neurons, astrocytes, oligodendrocytes and peripheral cells infiltrating the lesion site. The glial and inflammatory reaction at and around the lesion was studied by cresyl violet histology, alpha-GFAP, OX42 and alpha-CD-8 immunohistochemistry. No significant differences from controls were found in the low virus group; in the high virus group a strong invasion of CD-8-positive lymphocytes was found. Open-field locomotion analysis showed virus-infected animals performing as well as control animals. Adenoviral gene transfer may be an efficient way to introduce factors to the injured spinal cord in paradigms of research or therapy.     

Specificity of rabies virus as a transneuronal tracer of motor networks: Transfer from hypoglossal motoneurons to connected second-order and higher order central nervous system cell groups

The specificity of transneuronal transfer of rabies virus [challenge virus standard (CVS) strain] was evaluated in a well-characterized neuronal network, i. e., retrograde infection of hypoglossal motoneurons and transneuronal transfer to connected (second-order) brainstem neurons. The distribution of the virus in the central nervous system was studied immunohistochemically at sequential intervals after unilateral inoculation into the hypoglossal nerve. The extent of transneuronal transfer of rabies virus was strictly time dependent and was distinguished in five stages. At 1 day postinoculation, labelling involved only hypoglossal motoneurons (stage 1). Retrograde transneuronal transfer occurred from 2.0–2.5 days postinoculation (stage 2). In stages 2–4, different groups of second-order neurons were labelled sequentially, depending on the strength of their input to the hypoglossal nucleus. In stages 4 and 5, labelling extended to several cortical and subcortical cell groups, which can be regarded as higher order because they are known to control tongue movements and/or to provide input to hypoglossal-projecting cell groups. The pattern of transneuronal transfer of rabies virus resembles that of alpha-herpesviruses with regard to the nonsynchronous labelling of different groups of second-order neurons and the transfer to higher order neurons. In striking contrast to alpha-herpesviruses, the transneuronal transfer of rabies is not accompanied by neuronal degeneration. Moreover, local spread of rabies from infected neurons and axons to adjoining glial cells, neurons, or fibers of passage does not occur. The results show that rabies virus is a very efficient transneuronal tracer. Results also provide a new insight into the organization of cortical and subcortical higher order neurons that mediate descending control of tongue movements indirectly via hypoglossal-projecting neurons. © 1995 Wiley-Liss, Inc.     

Effects of basic fibroblast growth factor on survival and choline acetyltransferase development of spinal cord neurons.

To investigate the biological role of basic fibroblast growth factor (bFGF) for the development of the spinal cord we studied the in vitro and in vivo effects of this protein on survival and choline acetyltransferase (ChAT)-activity of embryonic chick and rat spinal cord neurons. In vitro, bFGF (ED50 1-2.8 ng/ml) supported the survival of embryonic neurons from the ventral part of the rat spinal cord (ventral spinal cord, vsc), including motoneurons. Addition of bFGF (100 ng/ml) increased the ChAT-activity in embryonic chick vsc cultures to 150% as compared to untreated cultures (100%). The effect of bFGF was dose-dependent. In vivo-application of bFGF resulted in a similar increase of ChAT-activity in chick spinal cord. Since bFGF stimulates the ChAT-activity of spinal cord neurons in vivo and in vitro we therefore conclude that this protein may have a physiological function for the transmitter development of cholinergic spinal cord neurons.     

Pathogenesis of experimental rabies in mice: an immunohistochemical study

Summary The spread of rabies virus in the central nervous system of mice was examined after hindlimb footpad and intracerebral inoculation of the CVS strain of fixed rabies virus. All mice developed paralytic rabies. After intracerebral inoculation there was early simultaneous infection of neurons in the cerebral cortex and pyramidal neurons of the hippocampus, and later there was spread to the cerebellum. After high-dose intracerebral inoculation there was early infection ependymal cells lining the lateral ventricles and neurons adjacent to the central canal of the spinal cord, suggesting that rabies virus entry into the CNS occurs, at least in part, by a cerebrospinal fluid pathway. The sequence of involvement was different after hindlimb footpad inoculation. Infection became established in the cerebellum on day 5, in the cerebral cortex on day 6, and in the hippocampus on day 8. CA3 was initially affected, CA1 became infected 2 days later, and there was much less involvement of the dentate gyrus. Hippocampal infection occurred late relative to the rest of the brain after peripheral inoculation, but not after intracerebral inoculation. The hippocampus is not a good location for the detection of early brain infection after peripheral inoculation, although it may be involved when a natural rabies vector has the ability to transmit infection. These findings also raise questions about the mechanisms for the limbic dysfunction observed in clinical rabies.Supported by grant MA-10068 from the Medical Research Council of Canada     

Establishment of pure neuronal cultures from fetal rat spinal cord and proliferation of the neuronal precursor cells in the presence of fibroblast growth factor.

A primary culture system of nearly pure neuronal cells from 14-day-old fetal rat spinal cord has been developed by combining a preplating step, the use of a chemically defined serum-free medium, and borated polylysine-coated dishes that prevented the formation of cell aggregates. About 98% of the cells were found to be immunostained with neuron-specific enolase antibodies, confirming their neuronal nature. The cultures are composed essentially of a population of non-motoneurons and contain few motoneurons, characterized by their large size and multipolar aspect, the presence of acetylcholinesterase (AChE), and the intense immunoreaction for growth-associated protein GAP-43. Neuronal precursor cells are also present in these cultures and proliferate during the first 3 days. The addition of bovine brain basic fibroblast growth factor (bFGF) stimulates their proliferation over a period of 2 days, as determined by measurement of [125I]iododeoxyuridine incorporation and by immunocytochemical reaction after bromodeoxyuridine incorporation into nuclei. The proliferating cells were characterized as neurons by immunostaining against neuron-specific enolase. Recombinant human bFGF and bovine brain acidic FGF (aFGF) exerted similar effects. Other growth factors, including epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-beta 1), and thrombin, were without effect on the proliferative activity of these neuronal cells. bFGF has no effect on the survival of motoneurons and on the fiber outgrowth of the whole neuronal population. However, bFGF affects the development of bipolar AChE-positive neurons, probably belonging to the non-motoneuron population. The data indicate that bFGF and aFGF are mitogens for neuroblasts from rat spinal cord in culture and that bFGF influences the development of a subpopulation of spinal neurons that are AChE-positive.     

Infection of cultured rat myotubes and neurons from the spinal cord by rabies virus

Rabies virus multiplication was investigated in cultured primary rat myotubes and neurons. The susceptibility of these two cell types to fixed rabies challenge virus strain (CVS) was monitored by fluorescence and virus titration. Differentiated rat myotubes were susceptible to rabies virus infection, and showed an increasing accumulation of viral material from day one to day four. However, these cells did not release infective viral particles, nor did they accumulate infectious virions in the cytoplasm. In contrast, infected neurons released large amounts of infectious particles. Electron microscopy observation of infected myotubes showed minor alterations and the presence of typical viral inclusions in the cytoplasm without mature virions assembling viral membranes. Competition binding experiments show that alpha-bungarotoxin inhibits rabies virus infection from 10(-5) to 10(-7) M, whereas lower toxin concentrations failed to have any effect. These data do not confirm the hypothesis of a fixed rabies virus amplification step at the site of the viral entry. On the other hand, the high susceptibility of peripheral neurons to rabies virus infection is an argument for the direct uptake of virions by these cells. The restrictive viral multiplication in the myotubes is an alternative explanation for the local persistence of rabies virus at the site of inoculation.     

Neuronal expression of foreign genes with recombinant rabies virus variants

Rabies virus variants obtained by recombinant DNA techniques enabled us to use the high neurotropism of rabies virus to express foreign genes (e.g: Chloramphenicol Acetyl Transferase gene) in neuronal cell cultures as well as in rodent brain. The foreign gene was inserted in the viral pseudogene region; this insertion did not affect the neurotropism of rabies virus, as shown by infection of neuronal cell cultures without any major cytopathic effects for several days. Stereotaxic inoculation of these rabies virus variants into rat striatum indicated that insertion of the foreign gene did not alter the viral axonal transport and the subsequent widespread brain infection. These data allow to consider rabies virus as a vector for the selective expression of foreign genes in neurons.     

Adenoviral-mediated transfection of the lac-z geneinto rat dissociated embryonic central nervous systemcells before and after seeding

The adenovirus carrying a reporter gene—the Lac-Z gene—is well known toinfect central nervous system (CNS) cells in primary cell cultures. The percentage of infectedneurons with respect to the total number of neurons was studied in primary dissociated culturesas a function of the day of inoculation and the age of three rat CNS cultures : spinal cord,mesencephalon and cortex. Two methods of viral inoculation were compared : the firstinoculation was performed on the cultured cells at 2, 3 or 6 days in vitro (DIV) whereas the second inoculation was performed on the cell suspensions before seeding. All theinfected CNS cells had the same aspect as the control cultures. In the spinal cord and themesencephalic cultures, the glial cells were preferentially infected, especially when the cells wereinoculated at 6 DIV. In the cortical cultures, there were more infected neurons than infected glialcells. The number of CNS cells was lower when inoculation was performed at 6 DIV ascompared with 3 DIV. Very few infected GABA cells were found in the cultures. A highpercentage of infected neuronal cells relative to the total number of neuronal cells was foundwhen infection of the three types of cultures was performed on the dissociated embryonic cellsuspension before seeding.     

Expression of cholinergic phenotype by embryonic ventral horn neurons transplanted into the spinal cord in the rat

Solid grafts of E12 embryonic spinal ventral horn were transplanted into motoneuron-depleted adult lumbar spinal cord in the rat. A muscle was implanted parallel to the vertebral column with its nerve inserted into the lumbar cord at the site of transplantation so as to provide a target for innervation by the grafted neurons. Previous retrograde labelling studies have shown that modest numbers of grafted motoneuron-like cells participate in the muscle's reinnervation and these are often found outside the graft within the host spinal cord. However, Nissl stained sections show that larger numbers of neurons survive within tissue recognisable as being of graft origin. In this study we have examined the expression of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) by neurons within the graft. These enzymes are involved in cholinergic neurotransmission and are characteristic of motoneurons. Thirty-four to seventy days following transplantation the grafts contained numerous neurons with acetylcholinesterase (AChE) activity. Different patterns of AChE staining were observed which probably reflected the degree of differentiation and maturation within the graft. AChE positive neurons were found in isolation or in groups resembling developing motor pools. Most of the AChE-positive neurons appeared immature with scant cytoplasm. However, neurons could be found which appeared relatively mature with a regularly shaped nucleus, prominent nucleolus and Nissl bodies. The grafts contained few AChE-positive axons and no dense plexuses of varicose fibres around the neurons such as are found around motoneurons in the mature ventral horn. Comparisons between the size of AChE-positive neurons in the graft and the size of AChE-positive neurons in the developing ventral horn found that the size of grafted neurons to be intermediate between the sizes of spinal motoneurons at E19 and P0. Far fewer grafted neurons were found to be immunoreactive for choline acetyltransferase (ChAT) than histochemically reactive for AChE. This was consistent with our findings in the spinal cord during normal development where we found that fixation and staining procedures which labelled adult motoneurons failed to reliably demonstrate ChAT immunoreactivety in normal motoneurons prenatally, although AChE histochemical reactivity could be demonstrated as early as E16. We conclude that the grafts contain numbers of immature motoneurons which fail to proceed beyond a certain stage of development, perhaps because of a failure to form appropriate efferent and afferent connections.     

Different pathogenicity of encephalitic togaviruses in organotypic cultures of spinal cord slices.

The pathogenicity of two encephalitic Togaviruses, Sindbis virus (SV), an alphavirus, and West Nile virus (WNV), a flavivirus, was studied in organotypic cultures of fetal mouse spinal cord slices grown in roller tubes. After about 3 weeks in vitro, during which time the cultures became abundantly myelinated, they were infected either by 5 X 10(5) PFU SV or by 5 X 10(6) PFU WNV per culture. The viruses caused different patterns of cytopathogenicity: SV induced severe cytotoxicity in all glia cells and neurons with concomitant demyelination within 48 hr. In contrast, WNV, even 4 days after infection, caused only mild cytopathic effects mainly to neurons and astrocytes and a slight degree of damage to the myelin sheath. A most remarkable finding was the entrapment of WNV particles in the interperiod lines of the myelin sheaths. Treatment of cultures with mouse alpha and beta interferon prior to their infection with either virus protected the cultures from any viral damage. Long-term exposure of non-infected control organotypic cultures of fetal spinal cord slices to mouse interferons had no significant effect on neuronal and glial differentiation, and myelin formation.