Journal of Neuroscience Research 26:495-500 (1990)
Rapid Communication West Nile Virions Aligned Along Myelin Lamellae in Organotypic Spinal Cord Cultures A. Shahar, S. Lustig, Y. Akov, Y. David, P. Schneider, A. Friedmann, and R. Levin Department of Virology, Israel Institute for Biological Research, Ness-Ziona. Israel, (A.S.,S.L.,Y.A.,Y.D.,P.S.,R.I,.); Department of Genetics, Hebrew University, Jerusalem, lsrael (A.F.)
Organotypic spinal cord cultures infected with West Nile Virus (WNV) exhibited a remarkable arrangement of virions among lamellae of the myelin sheath. Virions were first observed in neurons and only at day 4 after infection appeared within the myelin lamellae. Virions were observed only in the central myelin, aligned along the interperiod lines and therefore attached to the outer side of the oligodendrocyte membrane. In spite of this peculiar location of the virions, their presence was not associated with severe damage to the axon or to the myelin sheath. The causes of the formation of this viral pattern, its morphological features, and the role which it might have in viral infection in vivo are considered. Key words: West Nile virus, interperiod lines, myelin sheath INTRODUCTION We have studied the replication of some encephalitic viruses in organotypic cultures of the central nervous system (CNS). Most of the viruses replicated in these cultures within the first 48 hr after infection. During this period they destroyed neuronal and glial elements and caused severe demyelination (Shahar ct a]., 1986). West Nile fever is caused by West Nile Virus (WNV), which belongs to the Flaviviridae family (Goldblum, 1959; Monath, 1986). It is widely disseminated by mosquitoes. In hyperendemic areas the infection occurs early in childhood, is relatively mild, and confers immunity. There are more chances for the development of meningo-encephalitis when the virus infection is contracted at an older age (Marburg et al., 1956). The disease in mice and rats is characterized by changes in behavior and orientation and by learning disabilities (Duffy et a]., 1958). Demyelination has not been described in experimental WNV infection and is not a typical feature of human flaviviral infections (Monath, 1986). WNV is known to have a potential for persistency in mammalian 0 1990 Wiley-Liss, Inc.
cell cultures, in rodents, and in monkeys (Pogodina et al., 1983). This virus cntity has a different pattern of pathogenicity: its highest stage of replication in organotypic spinal cord cultures occurs only 4 days after viral infection, and is associated with mild cytopathic effects and a slight degree of damage to the myelin sheath (Shahar ct al., 1990). At the time coinciding with highest viral replication in culture, WN virions were observed between adjacent myelin lamellae of the myelin sheath of several axons. Such a peculiar arrangement of viruses, aligned between my elin lamellae, has been reported only once, in a study on replication of Polyoma virus in oligodendroglia cells of a human brain in a case of Progressive Multifocal Leucoencephalopathy (Mazlo and Tariska, 1980). The purpose of this paper is to describe the arrangement of WN virions in the myelin sheath of cultured spinal cord axons and to consider the possible role that such arrangement in the myelin membrane may play in viral encephalitis.
MATERIALS AND METHODS Tissue Culture Spinal cords with attached dorsal root ganglia were dissected from 16-17 day-old mouse fetuses (SJLIOLA (F) Ncss-Ziona) and chopped into 400 p m slices with a McILwain tissue chopper. The slices were placed on 12 x 24 mm coverslips in a drop of plasma (Difco ly-
Received April 9, 1990; revised May 13, 1990; accepted May 14, 1990. Address reprint requests to Dr. A . Shahar, Section o f Electron Microscopy, Dept. o f Virology, Israel Institute for Biological Research, Ness-Ziona, Israel 70450.
496
Shahar et al.
ophilized), coagulated with thrombin (Hoffman LaRoche), and cultured in roller tubes as previously described. (Gahwiler, 1984; Shahar et al., 1990). Cultures were selected for viral infection after about 3 weeks. At that time they were abundantly myelinated, as judged by light microscopy with an oil Zeiss objective 40189 1601 15.
Viral Infection The source and history of WNV strains employed in this study were describe by Akov and Goldwasser (1966). WNV (5 X 10' PFU in 0.1 ml of Balanced Salt Solution) was inoculated into the culture medium of the spinal cord explants. After a 3 hr adsorption period (in a roller drum at 37" C) the medium was discarded and replaced with regular culture medium composed of Minimum Essential Medium (MEM), containing 2% of fetal calf serum.
Electron Microscopy Triplicate cultures were processed for electron microscopy every 24 hr during a week after viral infection. The cells were fixed in the tube with 2.5% glutaraldehyde for 3 hr. Prior to post fixation in 2% Osmium tetroxide for 1 hr, coverslips bearing the cultures were taken out from the tubes and put into plastic dishes for light microscopical analysis. After dehydration in graded alcohol, cultures were separated from coverglasser with the aid of a r u o r blade and embedded in epoxy resin (Epon 812). Observation was made with a Jeol 100s transmission electron microscope. Goniometry tilting of areas showing large numbers of virions was performed with the aid of a side-entry goniometer and a Jeol lOOCX electron microscope. Tilting of thin sections was made at ?60", 40", and 20". RESULTS Spinal cord explant cultures were infected with West Nile viruses added to the culture mcdium (Shahar et al., 1990). During the first 3 days after viral infection, viral particle? were found mainly in the cytoplasm of some neurons: there was slight cytopathic damage to the neurons but almost no detectable alterations to oligodendroglia cells or to the myelin sheath. Four days after inoculation, a time coinciding with the highest amount of viral replication in culture (Shahar ct al., 1990), WN virions were seen in the myclin sheaths of several axons. Virions were aligned along the interperiod lines between major dense lines of adjacent myelin lamellae. In other axons, which were possibly at the very early stage of myelination prior to viral inoculation, virions were arranged in lines among all lamellae
composing the myelin sheath (Fig. 1A,B). In other axons, which were probably already myelinated at the time of viral infection. virions were found only between the two outermost peripheral lamellae (Fig. 2A). In most cases virion5 were arranged in a long row and occupied most of the interperiod line space. Some times however, only single virions remained trapped between the peripheral lamellae (Fig. 2B). On the top of these lamellae containing the virions, one or two additional intact lamellae were usually formed (Fig. 2A). The goniometer tilting of virions, which appeared as if forming a monolayer, showed that these viruses werc actually arranged in long rows betwcen two adjacent lamellae. Ultrastructurally, the entrapment of viruses between myelin lamellae did not severely damage the axons or the myelin sheath, with axons preserving, in most cases, regular mitochondria and oriented cytoskeletal components (Figs. IB, 2A). In a small percentage of the myelin sheaths however, herniations and splitting of lamellae were observed (Fig. 1A. B). Finally, even after 1 week of viral infection, several undamaged myelinated axons could be seen. Furthermore, there was no correlation between the presence of viral particles in the axon and the virions present in the myelin sheath.
DISCUSSION The prescncc o l WNV between myelin lamellae raises a number of questions and hypotheses regarding their location and the possible role they might have in viral pathogenesis. One major question is whether the entrapped viruses are positioned inside or outside the oligodendrocyte cell. Our interpretation in this regard is in agreement with the one given by Mazlo and Tariska (1980), who described a similar arrangement of Polyoma virions in a human brain with Progressive Multifocal Leucoencephalopathy (PML). The fact that both WN virions in spinal cord cultures and Polyoma viruses in the PML brain were always seen aligned along the interperiod lines of the myelin sheath clearly indicatcd their extracellular location-that is, they were attached to the outer surface of the cell membrane. The interperiod lines are known to be formed by the adhesion of the two external surfaces of adjacent myelin lamellae. It is probably because of their exterior location that WN virions induce only minor damage to the myelin sheath, expressed as a slight separation of lamellae at the level of the interperiod lines. The same conclusion was arrived at in the ease of PML where the entrapped Polyoma viruses were considered to be un-involved in causing demyelination. Such a situation may be indicative of a persistent infection by WNV which is known to have potential for persistency. Another virus which infects the cells of the CNS is
Fig. I . Virions arranged in lines between all lamellae coniposing the myelin sheath of axons that were possibly at the very early stage of myelination prior to viral inoculation. Note
in A splitting of lamellae (arrow), and in B regular mitochondria (M), oriented cytoskeletal elements, and herniation of the myelin sheath.
498
Shahar et al.
Fig. 2. The outermost lamellae of axons, which were probably myelinated at the time of viral infection, showing a long row of entrapped virions in A and a few aligned viruses in B. Arrow in A indicates an additional intact lamellae, formed on the top of the lamellae containing the virions.
West Nile Virions Aligned Along Myelin Lamellae
499
Theiler’s virus. Since WNV is an enveloped virus and Theiler’s virus is a naked one, wc believe that the process of infection by these two viruses is not similar and follows a different pathogenic mechanism. According to Rodriguez et al. (1983), during the infection of the CNS by Theiler’s virus, viral antigens accumulate between the myelin lamellae, but no viral particles were observed in the stained area, as shown by imrnunoperoxidase staining. In distinction from WNV, Theiler’s virus attacks the myelin-forming cells and replicates in them, leading to the displacement of the myelin sheath and to a massive demyelination (Frankel et al., 1986, 1987; Shahar et al., 1986). The observation of viral particles in large number, attached to the myelin lamellae of only a small percentage of the myelinated axons, raises the following questions:
viral monolayer between them, might create a rather “conflicting” situation for the virions: on the one hand, they cannot be internalized by the cell, since the cell cytoplasm had been squeezed out when the inner surface of the lamellae adhered to form the major dense lines. On the other hand. since the virions are entrapped in the intcrperiod lines, they cannot leave the cell membrane to infect other cells. We speculate that the entrapped viruses might stay in this position, harmless and latent, for months or years. As a result of stress or damage to the myelin sheath, single virions could be released, enter other cells, and start another viral infection. Our finding could provide an explanation for a possible mechanism underlying slow viral and chronic relapsing diseases of the CNS.
I . Is it possible that there are specific “types” of oli-
REFERENCES
godendroglia (destined around selected axons) to which the virions are preferably attached? 2. Why do the virions concentrate in such large number on the myelin lamcllae of ccrtain axons, whilc their number in neurons (which are the first target cells) and in the culture medium is relatively small?
Akov Y, Goldwasser R ( I 966): Prevalence of antibody to Arbovinises in various animals in Israel. Bull WHO 34:901-909. Brinton h.IA ( I 986): Replication of Flaviviruses. In Schlesinger S, Schlesinger MJ (eds): “The Togiviridae and Flaviviridac.” N.Y. and London: Plenum Press pp 327-374. Duffy OD, Morphree OD, Morgan F“ (1958): Learning deficit in mature rats recovered froin early postnatal infection with West Nile virus. Proc Soc Exp Biol Med 98:242-244. Frankel G, Fnedmann A. Arnir. A, David Y, Shahar A (1986): Theilcr’s virus redication in isolated Schwann cell cultures. J Neurosci Res 15:127-136. Frankel G , Shahar A, David Y , Sicris C, Brenner T, Friedmann A (1987): Cytotoxicity induced by Theiler’s viruses in cclls of thc nervvus tissue. In: Shahar A , Goldberg A M (eds): “Model Systems in Neurotoxicology: Alternative Approaches to Animal Testing.” New York: Alan R . L i s , Inc. pp 363-372. Gahwiler BH (1984): Slicc cultures of cerebellar, hippocampal and e. Expericntia 40235-243. Nile fever i n the Middle East. Proc. 6th Int. Congress Tropical bled. hlalaria 8: I 12-1 25. Marburg K: Goldblum N , Sterk VV, Jasinka-Klingherg W, Klinghery MA (1956): The natural history of West Nile fever. 1. Clinical observations during an epidemic in Israel. Am J Hyg 64:2.59269. Mazlo M, Tariska 1 (1980): Morphological denionstration of the first phase of Polyoma virus replication in oligodendroglia cells of hurnan brain in progrcssive Multifocal Leucoencephalopathy (PML). Acta Neuropathol (Berlin) 49:133-143. Monath TP (1986): Pathology of Flaviviruses. In Schlesinper S, Schlesinger, MJ (eds): “The Togaviridae and Flaviviridae. ” N.Y. and London: Plenum Press pp 375-440. Pogodina VV, Frolova MP, Malenko GV, Fokina GI. Koreshkova LL, Kiseleva NG. Bochkova NG, Ralph NM (1983): Study of West Nile virus persistence in monkeys. Arch Virol 75:71-86. Rodriguez M. Leibowitz JL, Lampert PW (1983): Persistent infection of oligodendrocytes in Theiler’s vims-induced encephalomyelitis. Ann Neurol 13:426--433. Schlcsinger MJ, Schlesinger S (1986): Formation and assembly of Alphavirus glycoproteins. In Schlesinger S, Schlcsingcr MJ (eds): “The Togaviridae and Flaviviridae.” N.Y. and London: Plenum Press pp 121-148. Shahar A , Frankel G , David Y . Fricdmann A (1986): In vitro cyto-
The virions probably attach to specific receptors on the outer surface of the myelin lamcllae. The high density of attached virions, as also demonstrated by the goniometer tilting, might be due to a large number of receptors for WNV already present on the “specialized” oligos. Alternatively, the presence of the virus might induce the synthesis of more receptors by these cells. We found WNV attached only to thc myelin lamellae of the CNS, but not in myelin sheaths with basement membranes of Schwann cells from dorsal root ganglia, which were also present in the culture. The diffcrcntial localization of virions in outer or inner lamellae of myelinated axons might be due to the stagc of myelination at the time of infection: At an early stage, viral particles were trapped between all the lamellae composing the entire myelin sheath. In axons that were already myelinated before viral inoculation, virions appeared only between the outermost lamellae. In this case, there were one or two regular lamellae on the top of the lamellae containing the virions. A possibility exists that the interaction with viral particles triggered thc oligodendrocyte to form more membranes, i.e., to produce additional myelin lamellae. Induction of membrane formation in other cell types by some viruses was described by Schlesinger and Schlesinger (1986) and by Brinton (1986). Finally, this rare observation of virions attached to the outer surface of myelin lamellae, forming a kind of
500
Shahar et at.
toxicity and dernyelination induced by Theiler viruses in cultures of spinal cord slices. J Neurosci Res 16:671-681. Shahar A , Lustig S , Akov Y, David Y, Schneider P, Lcvin R (1990):
Different pathogenicity of encephalitic Togaviruses in organotypic cultures of spinal cord slices. J Neurosci Res 25:345352.
Rapid Communication West Nile Virions Aligned Along Myelin Lamellae in Organotypic Spinal Cord Cultures A. Shahar, S. Lustig, Y. Akov, Y. David, P. Schneider, A. Friedmann, and R. Levin Department of Virology, Israel Institute for Biological Research, Ness-Ziona. Israel, (A.S.,S.L.,Y.A.,Y.D.,P.S.,R.I,.); Department of Genetics, Hebrew University, Jerusalem, lsrael (A.F.)
Organotypic spinal cord cultures infected with West Nile Virus (WNV) exhibited a remarkable arrangement of virions among lamellae of the myelin sheath. Virions were first observed in neurons and only at day 4 after infection appeared within the myelin lamellae. Virions were observed only in the central myelin, aligned along the interperiod lines and therefore attached to the outer side of the oligodendrocyte membrane. In spite of this peculiar location of the virions, their presence was not associated with severe damage to the axon or to the myelin sheath. The causes of the formation of this viral pattern, its morphological features, and the role which it might have in viral infection in vivo are considered. Key words: West Nile virus, interperiod lines, myelin sheath INTRODUCTION We have studied the replication of some encephalitic viruses in organotypic cultures of the central nervous system (CNS). Most of the viruses replicated in these cultures within the first 48 hr after infection. During this period they destroyed neuronal and glial elements and caused severe demyelination (Shahar ct a]., 1986). West Nile fever is caused by West Nile Virus (WNV), which belongs to the Flaviviridae family (Goldblum, 1959; Monath, 1986). It is widely disseminated by mosquitoes. In hyperendemic areas the infection occurs early in childhood, is relatively mild, and confers immunity. There are more chances for the development of meningo-encephalitis when the virus infection is contracted at an older age (Marburg et al., 1956). The disease in mice and rats is characterized by changes in behavior and orientation and by learning disabilities (Duffy et a]., 1958). Demyelination has not been described in experimental WNV infection and is not a typical feature of human flaviviral infections (Monath, 1986). WNV is known to have a potential for persistency in mammalian 0 1990 Wiley-Liss, Inc.
cell cultures, in rodents, and in monkeys (Pogodina et al., 1983). This virus cntity has a different pattern of pathogenicity: its highest stage of replication in organotypic spinal cord cultures occurs only 4 days after viral infection, and is associated with mild cytopathic effects and a slight degree of damage to the myelin sheath (Shahar ct al., 1990). At the time coinciding with highest viral replication in culture, WN virions were observed between adjacent myelin lamellae of the myelin sheath of several axons. Such a peculiar arrangement of viruses, aligned between my elin lamellae, has been reported only once, in a study on replication of Polyoma virus in oligodendroglia cells of a human brain in a case of Progressive Multifocal Leucoencephalopathy (Mazlo and Tariska, 1980). The purpose of this paper is to describe the arrangement of WN virions in the myelin sheath of cultured spinal cord axons and to consider the possible role that such arrangement in the myelin membrane may play in viral encephalitis.
MATERIALS AND METHODS Tissue Culture Spinal cords with attached dorsal root ganglia were dissected from 16-17 day-old mouse fetuses (SJLIOLA (F) Ncss-Ziona) and chopped into 400 p m slices with a McILwain tissue chopper. The slices were placed on 12 x 24 mm coverslips in a drop of plasma (Difco ly-
Received April 9, 1990; revised May 13, 1990; accepted May 14, 1990. Address reprint requests to Dr. A . Shahar, Section o f Electron Microscopy, Dept. o f Virology, Israel Institute for Biological Research, Ness-Ziona, Israel 70450.
496
Shahar et al.
ophilized), coagulated with thrombin (Hoffman LaRoche), and cultured in roller tubes as previously described. (Gahwiler, 1984; Shahar et al., 1990). Cultures were selected for viral infection after about 3 weeks. At that time they were abundantly myelinated, as judged by light microscopy with an oil Zeiss objective 40189 1601 15.
Viral Infection The source and history of WNV strains employed in this study were describe by Akov and Goldwasser (1966). WNV (5 X 10' PFU in 0.1 ml of Balanced Salt Solution) was inoculated into the culture medium of the spinal cord explants. After a 3 hr adsorption period (in a roller drum at 37" C) the medium was discarded and replaced with regular culture medium composed of Minimum Essential Medium (MEM), containing 2% of fetal calf serum.
Electron Microscopy Triplicate cultures were processed for electron microscopy every 24 hr during a week after viral infection. The cells were fixed in the tube with 2.5% glutaraldehyde for 3 hr. Prior to post fixation in 2% Osmium tetroxide for 1 hr, coverslips bearing the cultures were taken out from the tubes and put into plastic dishes for light microscopical analysis. After dehydration in graded alcohol, cultures were separated from coverglasser with the aid of a r u o r blade and embedded in epoxy resin (Epon 812). Observation was made with a Jeol 100s transmission electron microscope. Goniometry tilting of areas showing large numbers of virions was performed with the aid of a side-entry goniometer and a Jeol lOOCX electron microscope. Tilting of thin sections was made at ?60", 40", and 20". RESULTS Spinal cord explant cultures were infected with West Nile viruses added to the culture mcdium (Shahar et al., 1990). During the first 3 days after viral infection, viral particle? were found mainly in the cytoplasm of some neurons: there was slight cytopathic damage to the neurons but almost no detectable alterations to oligodendroglia cells or to the myelin sheath. Four days after inoculation, a time coinciding with the highest amount of viral replication in culture (Shahar ct al., 1990), WN virions were seen in the myclin sheaths of several axons. Virions were aligned along the interperiod lines between major dense lines of adjacent myelin lamellae. In other axons, which were possibly at the very early stage of myelination prior to viral inoculation, virions were arranged in lines among all lamellae
composing the myelin sheath (Fig. 1A,B). In other axons, which were probably already myelinated at the time of viral infection. virions were found only between the two outermost peripheral lamellae (Fig. 2A). In most cases virion5 were arranged in a long row and occupied most of the interperiod line space. Some times however, only single virions remained trapped between the peripheral lamellae (Fig. 2B). On the top of these lamellae containing the virions, one or two additional intact lamellae were usually formed (Fig. 2A). The goniometer tilting of virions, which appeared as if forming a monolayer, showed that these viruses werc actually arranged in long rows betwcen two adjacent lamellae. Ultrastructurally, the entrapment of viruses between myelin lamellae did not severely damage the axons or the myelin sheath, with axons preserving, in most cases, regular mitochondria and oriented cytoskeletal components (Figs. IB, 2A). In a small percentage of the myelin sheaths however, herniations and splitting of lamellae were observed (Fig. 1A. B). Finally, even after 1 week of viral infection, several undamaged myelinated axons could be seen. Furthermore, there was no correlation between the presence of viral particles in the axon and the virions present in the myelin sheath.
DISCUSSION The prescncc o l WNV between myelin lamellae raises a number of questions and hypotheses regarding their location and the possible role they might have in viral pathogenesis. One major question is whether the entrapped viruses are positioned inside or outside the oligodendrocyte cell. Our interpretation in this regard is in agreement with the one given by Mazlo and Tariska (1980), who described a similar arrangement of Polyoma virions in a human brain with Progressive Multifocal Leucoencephalopathy (PML). The fact that both WN virions in spinal cord cultures and Polyoma viruses in the PML brain were always seen aligned along the interperiod lines of the myelin sheath clearly indicatcd their extracellular location-that is, they were attached to the outer surface of the cell membrane. The interperiod lines are known to be formed by the adhesion of the two external surfaces of adjacent myelin lamellae. It is probably because of their exterior location that WN virions induce only minor damage to the myelin sheath, expressed as a slight separation of lamellae at the level of the interperiod lines. The same conclusion was arrived at in the ease of PML where the entrapped Polyoma viruses were considered to be un-involved in causing demyelination. Such a situation may be indicative of a persistent infection by WNV which is known to have potential for persistency. Another virus which infects the cells of the CNS is
Fig. I . Virions arranged in lines between all lamellae coniposing the myelin sheath of axons that were possibly at the very early stage of myelination prior to viral inoculation. Note
in A splitting of lamellae (arrow), and in B regular mitochondria (M), oriented cytoskeletal elements, and herniation of the myelin sheath.
498
Shahar et al.
Fig. 2. The outermost lamellae of axons, which were probably myelinated at the time of viral infection, showing a long row of entrapped virions in A and a few aligned viruses in B. Arrow in A indicates an additional intact lamellae, formed on the top of the lamellae containing the virions.
West Nile Virions Aligned Along Myelin Lamellae
499
Theiler’s virus. Since WNV is an enveloped virus and Theiler’s virus is a naked one, wc believe that the process of infection by these two viruses is not similar and follows a different pathogenic mechanism. According to Rodriguez et al. (1983), during the infection of the CNS by Theiler’s virus, viral antigens accumulate between the myelin lamellae, but no viral particles were observed in the stained area, as shown by imrnunoperoxidase staining. In distinction from WNV, Theiler’s virus attacks the myelin-forming cells and replicates in them, leading to the displacement of the myelin sheath and to a massive demyelination (Frankel et al., 1986, 1987; Shahar et al., 1986). The observation of viral particles in large number, attached to the myelin lamellae of only a small percentage of the myelinated axons, raises the following questions:
viral monolayer between them, might create a rather “conflicting” situation for the virions: on the one hand, they cannot be internalized by the cell, since the cell cytoplasm had been squeezed out when the inner surface of the lamellae adhered to form the major dense lines. On the other hand. since the virions are entrapped in the intcrperiod lines, they cannot leave the cell membrane to infect other cells. We speculate that the entrapped viruses might stay in this position, harmless and latent, for months or years. As a result of stress or damage to the myelin sheath, single virions could be released, enter other cells, and start another viral infection. Our finding could provide an explanation for a possible mechanism underlying slow viral and chronic relapsing diseases of the CNS.
I . Is it possible that there are specific “types” of oli-
REFERENCES
godendroglia (destined around selected axons) to which the virions are preferably attached? 2. Why do the virions concentrate in such large number on the myelin lamcllae of ccrtain axons, whilc their number in neurons (which are the first target cells) and in the culture medium is relatively small?
Akov Y, Goldwasser R ( I 966): Prevalence of antibody to Arbovinises in various animals in Israel. Bull WHO 34:901-909. Brinton h.IA ( I 986): Replication of Flaviviruses. In Schlesinger S, Schlesinger MJ (eds): “The Togiviridae and Flaviviridac.” N.Y. and London: Plenum Press pp 327-374. Duffy OD, Morphree OD, Morgan F“ (1958): Learning deficit in mature rats recovered froin early postnatal infection with West Nile virus. Proc Soc Exp Biol Med 98:242-244. Frankel G, Fnedmann A. Arnir. A, David Y, Shahar A (1986): Theilcr’s virus redication in isolated Schwann cell cultures. J Neurosci Res 15:127-136. Frankel G , Shahar A, David Y , Sicris C, Brenner T, Friedmann A (1987): Cytotoxicity induced by Theiler’s viruses in cclls of thc nervvus tissue. In: Shahar A , Goldberg A M (eds): “Model Systems in Neurotoxicology: Alternative Approaches to Animal Testing.” New York: Alan R . L i s , Inc. pp 363-372. Gahwiler BH (1984): Slicc cultures of cerebellar, hippocampal and e. Expericntia 40235-243. Nile fever i n the Middle East. Proc. 6th Int. Congress Tropical bled. hlalaria 8: I 12-1 25. Marburg K: Goldblum N , Sterk VV, Jasinka-Klingherg W, Klinghery MA (1956): The natural history of West Nile fever. 1. Clinical observations during an epidemic in Israel. Am J Hyg 64:2.59269. Mazlo M, Tariska 1 (1980): Morphological denionstration of the first phase of Polyoma virus replication in oligodendroglia cells of hurnan brain in progrcssive Multifocal Leucoencephalopathy (PML). Acta Neuropathol (Berlin) 49:133-143. Monath TP (1986): Pathology of Flaviviruses. In Schlesinper S, Schlesinger, MJ (eds): “The Togaviridae and Flaviviridae. ” N.Y. and London: Plenum Press pp 375-440. Pogodina VV, Frolova MP, Malenko GV, Fokina GI. Koreshkova LL, Kiseleva NG. Bochkova NG, Ralph NM (1983): Study of West Nile virus persistence in monkeys. Arch Virol 75:71-86. Rodriguez M. Leibowitz JL, Lampert PW (1983): Persistent infection of oligodendrocytes in Theiler’s vims-induced encephalomyelitis. Ann Neurol 13:426--433. Schlcsinger MJ, Schlesinger S (1986): Formation and assembly of Alphavirus glycoproteins. In Schlesinger S, Schlcsingcr MJ (eds): “The Togaviridae and Flaviviridae.” N.Y. and London: Plenum Press pp 121-148. Shahar A , Frankel G , David Y . Fricdmann A (1986): In vitro cyto-
The virions probably attach to specific receptors on the outer surface of the myelin lamcllae. The high density of attached virions, as also demonstrated by the goniometer tilting, might be due to a large number of receptors for WNV already present on the “specialized” oligos. Alternatively, the presence of the virus might induce the synthesis of more receptors by these cells. We found WNV attached only to thc myelin lamellae of the CNS, but not in myelin sheaths with basement membranes of Schwann cells from dorsal root ganglia, which were also present in the culture. The diffcrcntial localization of virions in outer or inner lamellae of myelinated axons might be due to the stagc of myelination at the time of infection: At an early stage, viral particles were trapped between all the lamellae composing the entire myelin sheath. In axons that were already myelinated before viral inoculation, virions appeared only between the outermost lamellae. In this case, there were one or two regular lamellae on the top of the lamellae containing the virions. A possibility exists that the interaction with viral particles triggered thc oligodendrocyte to form more membranes, i.e., to produce additional myelin lamellae. Induction of membrane formation in other cell types by some viruses was described by Schlesinger and Schlesinger (1986) and by Brinton (1986). Finally, this rare observation of virions attached to the outer surface of myelin lamellae, forming a kind of
500
Shahar et at.
toxicity and dernyelination induced by Theiler viruses in cultures of spinal cord slices. J Neurosci Res 16:671-681. Shahar A , Lustig S , Akov Y, David Y, Schneider P, Lcvin R (1990):
Different pathogenicity of encephalitic Togaviruses in organotypic cultures of spinal cord slices. J Neurosci Res 25:345352.
