Acta neuropath. (Bet1.) 34, 175 -- 181 (1976)
Acta Neuropathologica 9 by Springer-Vertag1976
The Effects of Measles Virus and Various Strains of SSPE Virus on Organotypic Cultures of Nervous Tissue R. D. Sheppard 1, L. A. Feldman 2, L. H. Barbosa 3, C. S. Raine 4, and M. B. Bornsteina
Summary. The neurotropic effects, virologic behaviors and morphologic appearances of 4 strains of subacute sclerosing panencephalitis (SSPE) virus have been examined in organotypic cultures of hamster cerebellar tissue and have been compared with the Edmonston strain of measles virus in the same system. While measles virus caused extensive damage to nervous tissue, the SSPE strains, in general, exerted a less deleterious effect. All of the SSPE viruses replicated in this tissue. The SSPE strains showed morphologic variation ranging from normal measles-type virions to apparently nucleocapsid deficient forms. It is speculated that some of these differences between measles and SSPE virus m a y account for the differences in the in vivo conditions with which they are associated. Key words: Measles virus -- Subacute sclerosing panencephalitis -- Multiple sclerosis -- Neuroadapted -- Chronic neurological disorders.
Recently, immunologic, virologic, and morphologic data have implicated paramyxoviruses as being causally related to certain chronic neurological disorders. Notable examples are the isolations of cell-free measles-like viruses from brain and lymph node tissues of patients with subacute sclerosing panencephalitis (SSPE) (Chen et al., 1969; Horta-Barbosa et al., 1969; Payne et al., 1969; Katz et al., 1969; Parker et al., 1970); the rescue of parainfluenza type 1 from multiple sclerosis (MS) 1 Saul R. Korey Department of Neurology, Department of Pathology, (Neuropathology) and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, New York 10461. 2 The Department of Microbiology, College of Medicine and Dentistry of New JerseyNew Jersey Medical School, Newark, N. J. 07103. 3 The Infectious Diseases Branch, Collaborative and Field Research, National Institute of Neurological Diseases and Stroke, Bethesda, Maryland 20014. 4 Recipient of a Research Career Development Award from NIH-grant NS 70265. 5 Kennedy Scholar of the Rose F. Kennedy Center for Research in Mental Retardation and Human Development.
176
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brain cells following their fusion with indicator cells (ter Meulen et al., 1972) and the EM demonstration of paramyxovirus-like material in CNS tissues from MS patients (Prineas, 1972; Watanabe and Okazaki, 1973; Raine et al., 1974). As part of a series of studies on the interrelationships between paramyxoviruses and nervous tissue, we have previously investigated the effects of the Edmonston strain of measles virus on organotypic cultures of hamster cerebellar tissue (Rathe etal., 1972; Raine et al., 1973). In this model system, the Edmonston strain of measles virus was found to maintain its infectivity for at least 72 days post-inoculation (DPI), with maximal yields of infectious virus recoverable between 10 and 30 DPI, reaching titers of 105-106 plaque-forming units (PFU) per culture (Rathe et al., 1972). The virus was indiscriminating as to cellular affinity (i.e. tropism) an dboth neurons and glia were affected. By 21 DPI, each cerebellar explant was invariably reduced to a mass of fibrous astrocytes. Multinucleated giant cells and intranuclear and intracytoplasmic nucleocapsid material were characteristic features throughout the course of infection. The presence of intracytoplasmic nucleocapsid material preceded that of the intranuclear, and each intracytoplasmic filament was covered with a granular, fuzzy coating. Budding virus particles were continuously seen after 5 DPI and they characteristically demonstrated a discrete, regular alignment of viral nucleocapsid beneath the membrane. On the outer surface of the virus particle, an additional, diffuse electron-dense coating was present, which was thicker where it overlaid viral nucleocapsid (Fig. 1). However, in chronic, long-term cultures, somewhat atypical budding virions could be seen (20 ~ ) which were "empty", i.e. lacking the regular alignment of nucleocapsid, beneath the envelope which still possessed the fuzzy, outer coating. Similar studies have been performed with a hamster brain-adapted strain of SSPE virus (Mantooth) which had been passaged 13 times since isolation. This is referred to as Mantooth 13. In contrast to measles virus, the brain-adapted SSPE virus, Mantooth 13, exerted a less deleterious effect upon the CNS tissue. Chronically-infected cultures still contained neurons, synaptic contacts, and some myelin, in spite of an abundance of viral material (Rathe et al., 1973). Approximately 20--30 ~ of the cultures survived the initial 14 DPI. The tissue's response included
Fig. 1. An Edmonston measles virus budding from the surface of an infected cell in a hamster cerebellar culture 11 days P. I. The plasmalemma has acquired a dense undercoating to which are applied measles nucleocapsids, sectioned here transversely. On the outer surface of the bud are ridges of amorphous, fuzzy material situated opposite the underlying nucleocapsids. • 145000 Fig. 2. Intracytoplasmic nucleocapsid from a culture infected with Mantooth SSPE virus. Its structure is identical to that produced by Edmonston measles virus. The tubular nucleocapsids (seen in transverse section at arrow) possess an outer fuzzy coating. • t 50 000 Fig.3. Intranuctear nucleocapsid from a Mantooth SSPE virus infected culture also identical to that produced by Edmonston measles virus. The nucleocapsids are seen as tubular in transverse section and cross-striated in longitudinal section--indicative of a helically coiled filament. • 150000 Fig.4. Mantooth SSPE virions. Contrast with figure 1 and note absence of nucleocapsid associated with the envelope. • 70000
Measles and SSPE Viruses in Cultured CNS Tissue
Figs. 1 - - 4
177
178
R.D. Sheppard et al.
Table 1. Hamster cerebellar explants 7 D.I.V. exposed to between 2 • 104 and 4 • 105 PFU Virus
Isolated from
Grown in
Edmonston measles Human throat swab BSC-1 cells Mantooth SSPE
Mantooth SSPE Hall6 SSPE Fisher SSPE McClellan SSPE
Human brain
Passage level
Status
Many
Never exposed to nerve tissue Neuroadapted
HeLa cells 13 BSC-1 cells Suckling hamster brain Human brain HeLa cells 2 Human lymph node HeLa cells 3 Human lymph node HeLa cells 2 Human brain HeLa cells 4
Non-neuroadapted Non-neuroadapted Non-neuroadapted Non-neuroadapted
loss of myelin, neuronal (Purkinje cell) damage, formation of giant cells, widespread injury to glial and granule cells, acute cytopathic effect (CPE) and degeneration. Myelin loss preceded damage to neurons, and involved a "melting" or "fading" phenomenon. Giant cell formation occurred during the entire course of infection and was usually more frequent in cultures showing maximal CPE. As with measles, intracytoplasmic viral inclusions preceded the intranuclear forms. Intracytoplasmic nucleocapsids were always coated with a fuzzy, granular material identical to that seen during Edmonston measles infection (Figs. 2 and 3). Cultures surviving past 50 DPI retained neurons but rarely myelin. In the majority of instances, the virions produced by this strain of SSPE virus uniformly lacked the regular alignment of nucleocapsids beneath the membrane, but did possess the diffuse coating of material on the surface of the particle (Fig. 4). The purpose of our recent studies on cell-free SSPE virus strains was to examine; 1., whether these "empty" Mantooth virions were typical of SSPE in general; 2., whether they represented a change which had occurred during the many passages since its isolation from human brain and prior to its adaptation to hamster brain tissue, and 3., to observe other responses of the host-cell virus interrelationships. The passage history of the viruses used in these studies (Table 1) shows that the Edmonston measles virus had never been exposed to nervous tissue, that the Mantooth 13 SSPE virus had been adapted to nervous tissue, and that Mantooth 2 (the same isolate as Mantooth 13 but used at an earlier passage level) had never been exposed to or readapted to nervous tissue since its isolation from SSPE brain cells. For comparisons, 3 other non-neuroadapted SSPE virus strains were included, having been isolated from brain or lymph node tissue; Hall6 (Horta-Barbosa et al., 1971), McClellan, and Fisher (Horta-Barbosa et al., 1971). All the above viruses replicated in our CNS culture system. The titers of M 13, M2, and Edmonston measles began to increase at 4 DPI, but the SSPE strains began to level off at 10 DPI while measles continued to rise (Fig. 5). Higher yields of virus were demonstrable with the Edmonston measles virus than with either Mantooth strain. Both Mantooth passage levels produced comparable amounts
179
Measles and SSPE Viruses in Cultured CNS Tissue Fig.5. Growth curves for Edmonston measlesvirus (ED), Mantooth 2 SSPE virus (M 2) and Mantooth :13 SSPE virus (M 13) in hamster cerebeltar cultures
ED
~5 M2 M13
~4 o Q 3
|
10
i
20 DAYS P I
!
30
of infectious virus. Between 2 and 3 log. units of extracellular virus were produced per culture up to 30 DPI by both Mantooth 2 and Mantooth 13, while 3--4 log. units were produced extracellularly by Edmonston measles. Cell-associated (intracellular) virus yields were 90 ~o greater than extracellular in all cases. Mantooth 2 and Mantooth 13 produced 3--4 log. units of total virus (celt-associated plus extracellular) while measles produced 6 log. units of total virus per culture. (For details of methods to determine extra- and intracellular virus, see Feldman et al., 1968). Light and electron microscopy confirmed that Mantooth 2 exerted a much less deleterious effect on the host tissue than Mantooth 13 (Raine et al., 1974). The majority of Mantooth 2-infected cultures contained apparently normal-looking Purkinje cells, granule cells, and myelinated axons. When damage was observed following inoculation, it was manifested by degenerating Purkinje cells, occasional giant cells with mixed types of nuclei, i.e. glial and neuronal, and large intracytoplasmic viral inclusions. On the other hand, Mantooth 13 produced considerable damage as described earlier (Raine et al., 1973). The HallO, Fisher, and McClellan strains of SSPE all elicited responses similar to Mantooth 2 in the host tissue and produced infectious titers similar to those observed with the Mantooth 2 strain. Each displayed its own variations of virus particle morphology with respect to regular alignment of nucleocapsid beneath the membrane. These variations ranged from a total lack of nucleocapsid to a measles-like appearance of regularly aligned nucleocapsid. These variations are the subject of ongoing investigations. In conclusion, our studies have demonstrated that the cell-free SSPE strains, Mantooth 13, Mantooth 2, Hall6, Fisher, and McClellan, and the Edmonston strain of measles virus replicated in our culture system. Measles virus had the greatest effect on glia and neurons, Mantooth 13 SSPE virus was less aggressive toward glia, a n d Mantooth 2 SSPE virus had the least damaging effect upon glia. Furthermore, the neuroadapted Mantooth 13 SSPE virus had not changed its morphology during its various passages suggesting that this SSPE virus is a genetic variant or mutant of measles virus (Raine et al., 1974). Such variants may produce virus-host relationships which differ from those observed with wild Edmonston measles
180
R.D. Sheppard et al.
virus. This is of considerable interest since the 4 non-neuroadapted, early passagelevel SSPE viruses entered into a coexistence with the host tissue, producing tow levels of virus over a long period of time. Of further interest is the fact that amongst the different SSPE virus strains, there were variations in the morphology of the viral particles, while the responses of the host tissue were similar. The differences between measles virus and the SSPE virus strains as seen here may account for the variations in host responses in man, and the consequent production of a wide range of clinical disease patterns observed in SSPE in man.
Acknowledgments. We thank Dr. Robert D. Terry for his advice on this project, and Drs. Kenneth P. Johnson and Donald P. Byington (Cleveland) for generously supplying the hamster-adapted SSPE virus strain. Supported in part by grants NS 08952, NS 06735 and NS 03356 from the National Institutes of Health; the Nancy Louise Trynor Memorial Grant for Research in Multiple Sclerosis (433-D-11), the Alfred P. Sloan Foundation; the James S. & Louise M. Wordley Memorial Grant for Research in Multiple Sclerosis (555-C-3), and a grant from the Lions Clubs, Multiple District 16, New Jersey. Presented at the Symposium on Multiple Sclerosis held in Kyoto, Japan, August 1973. REFERENCES Chen, T. T., Watanabe, I., Zeman, W., Mealey, J. : Subacute sclerosing panencephalitis: Propagation of measles virus from brain biopsy in tissue culture. Science 163, 1193 (1969) Feldman, L.A., Sheppard, R.D., Bornstein, M. B. : Herpes simplex virus-host cell relationships in organized cultures of mammalian nerve tissues. J. Virol. 2, 621 (1968) Horta-Barbosa, L., Fuccillo, C. A., Sever, J. L., Zeman, W. : Subacute sclerosing panencephalitis: Isolation of measles virus from a brain biopsy. Nature (Lond.) 221, 974 (1969) Horta-Barbosa, L., Hamilton, R., Wittig, B., Fuccillo, D., Sever, J. : Subacute sclerosing panencephalitis: Isolation of suppressed measles virus from lymph node biopsies. Science 173, 840 (1971) Katz, M., Oyanagi, S., Loprowski, H. : Subacute sclerosing panencephalitis: Structures resembling myxovirus nucleocapsids in cells cultured from brains. Nature (Lond.) 222, 888 (1969) Parker, J. C., Jr., Klintworth, G. K., Graham, D. G., Griffith, J. F. : Uncommon morphologic features in subacute sclerosing panencephalitis (SSPE)--report of two cases with virus recovery from one autopsy brain specimen. Amer. J. Path. 61, 275 (1970) Payne, F. E., Baubtis, J. V., Itabashi, H. H. : Isolation of measles virus from cell cultures of brain from a patient with subacute sclerosing panencephalitis. New Engl. J. Med. 281, 585 (1969) Prineas, J. W.: Paramyxovirus-like particles associated with acute demyelination in chronic relapsing multiple sclerosis. Science 178, 760 (1972) Raine, C. S., Feldman, L.A., Sheppard, R.D., Bornstein, M. B. : Ultrastructure of measles virus in cultures of hamster cerebellum. J. Virol. 4, 169 (1969) Raine, C. S., Feldman, L. A., Sheppard, R. D., Bornstein, M. B. : Subacute sclerosing panencephalitis virus in cultures of organized central nervous tissue. Lab. Invest. 28, 627 (1973) Raine, C. S., Feldman, L. A., Sheppard, R. D., Barbosa, L. H., Bornstein, M. B. : Subacute sclerosing panencephalitis virus. Observations on a neuroadapted strain in organotypic central nervous system cultures. Lab. Invest. 31, 42 (1974) Raine, C.S., Powers, J.M., Suzuki, K.: Acute multiple sclerosis; confirmation of "paramyxovirus-like" intranuclear inclusions. Arch. Neurol. (Chic.) 30, 39 (1974)
Measles and SSPE Viruses in Cultured CNS Tissue
181
ter Meulen, V., Koprowski, H., Iwasaki, Y., Kackell, Y.M., Muller, D. : Fusion of cultured multiple sclerosis brain cells with indicator cells; Presence of nucleocapsids and virions and isolation of parainfluenzatype virus. Lancet 1972 II, 1 Watanabe, I., Okazaki, H.: Virus-like structure in multiple sclerosis. Lancet 1973 II, 569
Received September 4, 1975; Accepted October 31, 1975 Rachel Sheppard Dept. of Neurology Albert Einstein College of Medicine Rose F. Kennedy Center (Room 401) 1300 Morris Park Avenue Bronx, New York 10461 U.S.A.
Acta Neuropathologica 9 by Springer-Vertag1976
The Effects of Measles Virus and Various Strains of SSPE Virus on Organotypic Cultures of Nervous Tissue R. D. Sheppard 1, L. A. Feldman 2, L. H. Barbosa 3, C. S. Raine 4, and M. B. Bornsteina
Summary. The neurotropic effects, virologic behaviors and morphologic appearances of 4 strains of subacute sclerosing panencephalitis (SSPE) virus have been examined in organotypic cultures of hamster cerebellar tissue and have been compared with the Edmonston strain of measles virus in the same system. While measles virus caused extensive damage to nervous tissue, the SSPE strains, in general, exerted a less deleterious effect. All of the SSPE viruses replicated in this tissue. The SSPE strains showed morphologic variation ranging from normal measles-type virions to apparently nucleocapsid deficient forms. It is speculated that some of these differences between measles and SSPE virus m a y account for the differences in the in vivo conditions with which they are associated. Key words: Measles virus -- Subacute sclerosing panencephalitis -- Multiple sclerosis -- Neuroadapted -- Chronic neurological disorders.
Recently, immunologic, virologic, and morphologic data have implicated paramyxoviruses as being causally related to certain chronic neurological disorders. Notable examples are the isolations of cell-free measles-like viruses from brain and lymph node tissues of patients with subacute sclerosing panencephalitis (SSPE) (Chen et al., 1969; Horta-Barbosa et al., 1969; Payne et al., 1969; Katz et al., 1969; Parker et al., 1970); the rescue of parainfluenza type 1 from multiple sclerosis (MS) 1 Saul R. Korey Department of Neurology, Department of Pathology, (Neuropathology) and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, New York 10461. 2 The Department of Microbiology, College of Medicine and Dentistry of New JerseyNew Jersey Medical School, Newark, N. J. 07103. 3 The Infectious Diseases Branch, Collaborative and Field Research, National Institute of Neurological Diseases and Stroke, Bethesda, Maryland 20014. 4 Recipient of a Research Career Development Award from NIH-grant NS 70265. 5 Kennedy Scholar of the Rose F. Kennedy Center for Research in Mental Retardation and Human Development.
176
R.D. Sheppard et al.
brain cells following their fusion with indicator cells (ter Meulen et al., 1972) and the EM demonstration of paramyxovirus-like material in CNS tissues from MS patients (Prineas, 1972; Watanabe and Okazaki, 1973; Raine et al., 1974). As part of a series of studies on the interrelationships between paramyxoviruses and nervous tissue, we have previously investigated the effects of the Edmonston strain of measles virus on organotypic cultures of hamster cerebellar tissue (Rathe etal., 1972; Raine et al., 1973). In this model system, the Edmonston strain of measles virus was found to maintain its infectivity for at least 72 days post-inoculation (DPI), with maximal yields of infectious virus recoverable between 10 and 30 DPI, reaching titers of 105-106 plaque-forming units (PFU) per culture (Rathe et al., 1972). The virus was indiscriminating as to cellular affinity (i.e. tropism) an dboth neurons and glia were affected. By 21 DPI, each cerebellar explant was invariably reduced to a mass of fibrous astrocytes. Multinucleated giant cells and intranuclear and intracytoplasmic nucleocapsid material were characteristic features throughout the course of infection. The presence of intracytoplasmic nucleocapsid material preceded that of the intranuclear, and each intracytoplasmic filament was covered with a granular, fuzzy coating. Budding virus particles were continuously seen after 5 DPI and they characteristically demonstrated a discrete, regular alignment of viral nucleocapsid beneath the membrane. On the outer surface of the virus particle, an additional, diffuse electron-dense coating was present, which was thicker where it overlaid viral nucleocapsid (Fig. 1). However, in chronic, long-term cultures, somewhat atypical budding virions could be seen (20 ~ ) which were "empty", i.e. lacking the regular alignment of nucleocapsid, beneath the envelope which still possessed the fuzzy, outer coating. Similar studies have been performed with a hamster brain-adapted strain of SSPE virus (Mantooth) which had been passaged 13 times since isolation. This is referred to as Mantooth 13. In contrast to measles virus, the brain-adapted SSPE virus, Mantooth 13, exerted a less deleterious effect upon the CNS tissue. Chronically-infected cultures still contained neurons, synaptic contacts, and some myelin, in spite of an abundance of viral material (Rathe et al., 1973). Approximately 20--30 ~ of the cultures survived the initial 14 DPI. The tissue's response included
Fig. 1. An Edmonston measles virus budding from the surface of an infected cell in a hamster cerebellar culture 11 days P. I. The plasmalemma has acquired a dense undercoating to which are applied measles nucleocapsids, sectioned here transversely. On the outer surface of the bud are ridges of amorphous, fuzzy material situated opposite the underlying nucleocapsids. • 145000 Fig. 2. Intracytoplasmic nucleocapsid from a culture infected with Mantooth SSPE virus. Its structure is identical to that produced by Edmonston measles virus. The tubular nucleocapsids (seen in transverse section at arrow) possess an outer fuzzy coating. • t 50 000 Fig.3. Intranuctear nucleocapsid from a Mantooth SSPE virus infected culture also identical to that produced by Edmonston measles virus. The nucleocapsids are seen as tubular in transverse section and cross-striated in longitudinal section--indicative of a helically coiled filament. • 150000 Fig.4. Mantooth SSPE virions. Contrast with figure 1 and note absence of nucleocapsid associated with the envelope. • 70000
Measles and SSPE Viruses in Cultured CNS Tissue
Figs. 1 - - 4
177
178
R.D. Sheppard et al.
Table 1. Hamster cerebellar explants 7 D.I.V. exposed to between 2 • 104 and 4 • 105 PFU Virus
Isolated from
Grown in
Edmonston measles Human throat swab BSC-1 cells Mantooth SSPE
Mantooth SSPE Hall6 SSPE Fisher SSPE McClellan SSPE
Human brain
Passage level
Status
Many
Never exposed to nerve tissue Neuroadapted
HeLa cells 13 BSC-1 cells Suckling hamster brain Human brain HeLa cells 2 Human lymph node HeLa cells 3 Human lymph node HeLa cells 2 Human brain HeLa cells 4
Non-neuroadapted Non-neuroadapted Non-neuroadapted Non-neuroadapted
loss of myelin, neuronal (Purkinje cell) damage, formation of giant cells, widespread injury to glial and granule cells, acute cytopathic effect (CPE) and degeneration. Myelin loss preceded damage to neurons, and involved a "melting" or "fading" phenomenon. Giant cell formation occurred during the entire course of infection and was usually more frequent in cultures showing maximal CPE. As with measles, intracytoplasmic viral inclusions preceded the intranuclear forms. Intracytoplasmic nucleocapsids were always coated with a fuzzy, granular material identical to that seen during Edmonston measles infection (Figs. 2 and 3). Cultures surviving past 50 DPI retained neurons but rarely myelin. In the majority of instances, the virions produced by this strain of SSPE virus uniformly lacked the regular alignment of nucleocapsids beneath the membrane, but did possess the diffuse coating of material on the surface of the particle (Fig. 4). The purpose of our recent studies on cell-free SSPE virus strains was to examine; 1., whether these "empty" Mantooth virions were typical of SSPE in general; 2., whether they represented a change which had occurred during the many passages since its isolation from human brain and prior to its adaptation to hamster brain tissue, and 3., to observe other responses of the host-cell virus interrelationships. The passage history of the viruses used in these studies (Table 1) shows that the Edmonston measles virus had never been exposed to nervous tissue, that the Mantooth 13 SSPE virus had been adapted to nervous tissue, and that Mantooth 2 (the same isolate as Mantooth 13 but used at an earlier passage level) had never been exposed to or readapted to nervous tissue since its isolation from SSPE brain cells. For comparisons, 3 other non-neuroadapted SSPE virus strains were included, having been isolated from brain or lymph node tissue; Hall6 (Horta-Barbosa et al., 1971), McClellan, and Fisher (Horta-Barbosa et al., 1971). All the above viruses replicated in our CNS culture system. The titers of M 13, M2, and Edmonston measles began to increase at 4 DPI, but the SSPE strains began to level off at 10 DPI while measles continued to rise (Fig. 5). Higher yields of virus were demonstrable with the Edmonston measles virus than with either Mantooth strain. Both Mantooth passage levels produced comparable amounts
179
Measles and SSPE Viruses in Cultured CNS Tissue Fig.5. Growth curves for Edmonston measlesvirus (ED), Mantooth 2 SSPE virus (M 2) and Mantooth :13 SSPE virus (M 13) in hamster cerebeltar cultures
ED
~5 M2 M13
~4 o Q 3
|
10
i
20 DAYS P I
!
30
of infectious virus. Between 2 and 3 log. units of extracellular virus were produced per culture up to 30 DPI by both Mantooth 2 and Mantooth 13, while 3--4 log. units were produced extracellularly by Edmonston measles. Cell-associated (intracellular) virus yields were 90 ~o greater than extracellular in all cases. Mantooth 2 and Mantooth 13 produced 3--4 log. units of total virus (celt-associated plus extracellular) while measles produced 6 log. units of total virus per culture. (For details of methods to determine extra- and intracellular virus, see Feldman et al., 1968). Light and electron microscopy confirmed that Mantooth 2 exerted a much less deleterious effect on the host tissue than Mantooth 13 (Raine et al., 1974). The majority of Mantooth 2-infected cultures contained apparently normal-looking Purkinje cells, granule cells, and myelinated axons. When damage was observed following inoculation, it was manifested by degenerating Purkinje cells, occasional giant cells with mixed types of nuclei, i.e. glial and neuronal, and large intracytoplasmic viral inclusions. On the other hand, Mantooth 13 produced considerable damage as described earlier (Raine et al., 1973). The HallO, Fisher, and McClellan strains of SSPE all elicited responses similar to Mantooth 2 in the host tissue and produced infectious titers similar to those observed with the Mantooth 2 strain. Each displayed its own variations of virus particle morphology with respect to regular alignment of nucleocapsid beneath the membrane. These variations ranged from a total lack of nucleocapsid to a measles-like appearance of regularly aligned nucleocapsid. These variations are the subject of ongoing investigations. In conclusion, our studies have demonstrated that the cell-free SSPE strains, Mantooth 13, Mantooth 2, Hall6, Fisher, and McClellan, and the Edmonston strain of measles virus replicated in our culture system. Measles virus had the greatest effect on glia and neurons, Mantooth 13 SSPE virus was less aggressive toward glia, a n d Mantooth 2 SSPE virus had the least damaging effect upon glia. Furthermore, the neuroadapted Mantooth 13 SSPE virus had not changed its morphology during its various passages suggesting that this SSPE virus is a genetic variant or mutant of measles virus (Raine et al., 1974). Such variants may produce virus-host relationships which differ from those observed with wild Edmonston measles
180
R.D. Sheppard et al.
virus. This is of considerable interest since the 4 non-neuroadapted, early passagelevel SSPE viruses entered into a coexistence with the host tissue, producing tow levels of virus over a long period of time. Of further interest is the fact that amongst the different SSPE virus strains, there were variations in the morphology of the viral particles, while the responses of the host tissue were similar. The differences between measles virus and the SSPE virus strains as seen here may account for the variations in host responses in man, and the consequent production of a wide range of clinical disease patterns observed in SSPE in man.
Acknowledgments. We thank Dr. Robert D. Terry for his advice on this project, and Drs. Kenneth P. Johnson and Donald P. Byington (Cleveland) for generously supplying the hamster-adapted SSPE virus strain. Supported in part by grants NS 08952, NS 06735 and NS 03356 from the National Institutes of Health; the Nancy Louise Trynor Memorial Grant for Research in Multiple Sclerosis (433-D-11), the Alfred P. Sloan Foundation; the James S. & Louise M. Wordley Memorial Grant for Research in Multiple Sclerosis (555-C-3), and a grant from the Lions Clubs, Multiple District 16, New Jersey. Presented at the Symposium on Multiple Sclerosis held in Kyoto, Japan, August 1973. REFERENCES Chen, T. T., Watanabe, I., Zeman, W., Mealey, J. : Subacute sclerosing panencephalitis: Propagation of measles virus from brain biopsy in tissue culture. Science 163, 1193 (1969) Feldman, L.A., Sheppard, R.D., Bornstein, M. B. : Herpes simplex virus-host cell relationships in organized cultures of mammalian nerve tissues. J. Virol. 2, 621 (1968) Horta-Barbosa, L., Fuccillo, C. A., Sever, J. L., Zeman, W. : Subacute sclerosing panencephalitis: Isolation of measles virus from a brain biopsy. Nature (Lond.) 221, 974 (1969) Horta-Barbosa, L., Hamilton, R., Wittig, B., Fuccillo, D., Sever, J. : Subacute sclerosing panencephalitis: Isolation of suppressed measles virus from lymph node biopsies. Science 173, 840 (1971) Katz, M., Oyanagi, S., Loprowski, H. : Subacute sclerosing panencephalitis: Structures resembling myxovirus nucleocapsids in cells cultured from brains. Nature (Lond.) 222, 888 (1969) Parker, J. C., Jr., Klintworth, G. K., Graham, D. G., Griffith, J. F. : Uncommon morphologic features in subacute sclerosing panencephalitis (SSPE)--report of two cases with virus recovery from one autopsy brain specimen. Amer. J. Path. 61, 275 (1970) Payne, F. E., Baubtis, J. V., Itabashi, H. H. : Isolation of measles virus from cell cultures of brain from a patient with subacute sclerosing panencephalitis. New Engl. J. Med. 281, 585 (1969) Prineas, J. W.: Paramyxovirus-like particles associated with acute demyelination in chronic relapsing multiple sclerosis. Science 178, 760 (1972) Raine, C. S., Feldman, L.A., Sheppard, R.D., Bornstein, M. B. : Ultrastructure of measles virus in cultures of hamster cerebellum. J. Virol. 4, 169 (1969) Raine, C. S., Feldman, L. A., Sheppard, R. D., Bornstein, M. B. : Subacute sclerosing panencephalitis virus in cultures of organized central nervous tissue. Lab. Invest. 28, 627 (1973) Raine, C. S., Feldman, L. A., Sheppard, R. D., Barbosa, L. H., Bornstein, M. B. : Subacute sclerosing panencephalitis virus. Observations on a neuroadapted strain in organotypic central nervous system cultures. Lab. Invest. 31, 42 (1974) Raine, C.S., Powers, J.M., Suzuki, K.: Acute multiple sclerosis; confirmation of "paramyxovirus-like" intranuclear inclusions. Arch. Neurol. (Chic.) 30, 39 (1974)
Measles and SSPE Viruses in Cultured CNS Tissue
181
ter Meulen, V., Koprowski, H., Iwasaki, Y., Kackell, Y.M., Muller, D. : Fusion of cultured multiple sclerosis brain cells with indicator cells; Presence of nucleocapsids and virions and isolation of parainfluenzatype virus. Lancet 1972 II, 1 Watanabe, I., Okazaki, H.: Virus-like structure in multiple sclerosis. Lancet 1973 II, 569
Received September 4, 1975; Accepted October 31, 1975 Rachel Sheppard Dept. of Neurology Albert Einstein College of Medicine Rose F. Kennedy Center (Room 401) 1300 Morris Park Avenue Bronx, New York 10461 U.S.A.
