Infection of different cell lines of neural origin with subacute sclerosing panencephalitis (SSPE) virus

Measles virus is the causative agent of subacute sclerosing panencephalitis (SSPE). The viruses isolated from brain cells of patients with SSPE (called SSPE viruses) are defective in cell-free virus production in vitro. To investigate the cell tropism of three strains of SSPE virus (Osaka-1, Osaka-2, Osaka-3), SSPE virus-infected cell cultures were treated with cytochalasin D to prepare virus-like particles (CD-VLPs). All CD-VLPs formed syncytia after infection in CHO cells expressing CD150 but not in those expressing CD46. In addition, an antibody to CD46 did not block the infection of Vero cells by SSPE CDVLPs. The results were consistent with our previous suggestion that one or more unidentified receptors might be involved in the entry process. Infection with the CD-VLPs from three SSPE strains was further examined in different human cell lines, including those of neural origin, and was found to induce syncytia in epithelial cells (HeLa and 293T) as well as neuroblastoma cells (IMR-32 and SK-N-SH) with varying efficiency. SSPE CD-VLPs also infected glioblastoma cells (A172) and astrocytoma cells (U-251) but syncytial formation was rarely induced. These epithelial and neural cell lines were not permissive for the replication of wild-type MV. Together with our previous observations, these results suggest that the cell entry receptor is the major factor determining the cell tropism of SSPE viruses. Further studies are necessary to identify other viral and/or cellular factors that might be involved in the replication of SSPE virus in specific neural cells and in the brain.     

Complement-dependent cytotoxicity of sera obtained from subacute sclerosing panencephalitis patients

Human lymphoid cells (NC-37) persistently infected with either measles virus (Schwarz and TYCSA strains) or subacute sclerosing panencephalitis (SSPE) virus (Halle and Mantooth strains) were destroyed in the presence of complement by anti-measles sera as well as by sera from SSPE patients. The cytotoxic activity was demonstrated in both IgG and IgM fractions of measles convalescent sera, but only in IgG fraction of SSPE sera. Measles convalescent sera completely lost the cytotoxic activity to all the cell lines, when absorbed with any one of the cell lines, indicating that the viral surface antigens of these cell lines infected with measles or SSPE virus are identical. On the other hand, the cytotoxic activity of SSPE sera could not be readily absorbed with these cells. Thus, the affinity of SSPE sera for the viral surface antigens might be lower than that of measles convalescent sera.     

Subacute sclerosing panencephalitis virus dominantly interferes with replication of wild-type measles virus in a mixed infection: implication for viral persistence.

Interaction between the Edmonston or Nagahata strain of acute measles virus (MV) and the defective Biken strain of MV isolated from a patient with subacute sclerosing panencephalitis (SSPE) was examined by a cell fusion protocol. Biken-CV-1 cells nonproductively infected with Biken strain SSPE virus were fused with neomycin-resistant CV-1 cells. All the fused cells selected with the neomycin analog G418 expressed Biken viral proteins, as determined by an immunofluorescence assay. This procedure enabled the transfer of Biken viral genomes into cells previously infected with MV. In the fused cells coinfected by Biken strain SSPE virus and Edmonston or Nagahata strain MV, early MV gene expression was suppressed, as determined by immunoprecipitation with strain-specific antibodies. Maturation of Edmonston strain MV was also suppressed. When the coinfected fused cells were selected with G418, Biken viral proteins remained at a constant level for up to 7 weeks. Wild-type MV proteins gradually decreased to a barely detectable level after 4 weeks and became undetectable after 7 weeks. Immunofluorescence studies showed a steady decline in cells expressing wild-type MV proteins in the coinfected cultures. These results suggest that Biken strain SSPE virus dominantly interferes with the replication of wild-type MV. The possible mechanisms of dominant interference and the implication for evolution of a persistent MV infection are discussed.     

Growth of measles virus in various human lymphoid cell lines.

Neurovirulent TYCSA strain and attenuated Schwarz strain of measles virus and Halle strain of subacute sclerosing panencephalitis (SSPE) virus replicated in cultures of human lymphoid cell lines of the T-cell type, MOLT-3, MOLT-4 and CCRF-CEM. TYCSA and Halle strains grew rapidly, but Schwarz strain grew slowly in these cell lines. Furthermore, these three strains established persistent infection in CCRF-CEM cells but not in the other cell lines. In these persistently infected cultures an almost entire population of cells were shown to be infected and infectious virus was produced constantly for over 100 days. Cells persistently infected with Schwarz strain contained nucleocapsid structures in both the nucleus and cytoplasm and produced low titered infectious virus, whereas nucleocapsid structures were observed only in the cytoplasm of cells persistently infected with either TYCSA or Halle strain and the titers of infectious virus produced from these cells were high.     

Characterization of Measles Virus-Specific Proteins Synthesized In Vivo and In Vitro from Acutely and Persistently Infected Cells

Measles virus protein synthesis has been analyzed in acutely and persistently infected cells. To assess the role of measles in subacute sclerosing panencephalitis (SSPE), measles viral proteins synthesized in vivo or in vitro were tested for reactivity with serum from a guinea pig(s) immunized with measles virus and sera from patients with SSPE. Guinea pig antimeasles virus serum immunoprecipitates the viral polypeptides of 78,000 molecular weight (glycosylated [G]), 70,000 molecular weight (phosphorylated [P]), 60,000 molecular weight (nucleocapsid [N]), and 35,000 molecular weight (matrix [M]) from cells acutely infected with measles virus as well as from chronically infected cells, but in the latter case, immunoprecipitated M protein has a reduced electrophoretic migration. Sera of SSPE patients immunoprecipitated all but the G protein in acutely infected cells and only the P and N proteins from chronically infected cells. In immunoprecipitates of viral polypeptides synthesized in a reticulocyte cell-free translation system, in response to mRNA from acutely or persistently infected cells, the 78,000-molecular-weight form of the G protein was not detected among the cell-free products of either mRNA. Guinea pig antimeasles virus serum immunoprecipitated P, N, and M polypeptides from the products of either form of mRNA, whereas SSPE serum immunoprecipitated the P and N polypeptides but not the M polypeptide. The differences in immunoreactivity of the antimeasles virus antiserum and the SSPE serum are discussed in terms of possible modifications of measles virus proteins in SSPE.     

Immune response in subacute sclerosing panencephalitis: reduced antibody response to the matrix protein of measles virus.

Immune precipitation was used to study the humoral immune response of patients with subacute sclerosing panencephalitis (SSPE). Patients with SSPE have a progressive infection of the CNS by measles or a measles variant despite high serum antibody levels to measles virus as measured by standard serologic techniques. However, when the antibody response to individual measles virus proteins was measured, we found a striking reduction in the ability of sera from patients with SSPE to precipitate the matrix (M) protein as compared to the precipitation of the M protein by sera from normal adults who had natural measles infection in childhood, or by convalescent sera obtained 3 to 5 weeks after a naturally occurring measles infection. The decreased antibody response to the M protein in sera from patients with SSPE occurred despite a vigorous antibody response to the other viral proteins, suggesting a selective defect in the production of antibody to a single viral protein. The reduced anti-M antibody in sera from patients with SSPE was demonstrated whether immune precipitation was performed with wild-type measles virus or SSPE virus proteins. These results suggest that in SSPE only small amounts of the M protein are produced. This result may help explain how measles virus persists in the central nervous system of patients with SSPE.     

Characterization of canine distemper viruses adapted to human neural cells

The biochemical characteristics of canine distemper virus (CDV) adapted to three human neural cells (glioblastoma, oligodendroglioma, and neuroblastoma cells) were compared with those of the unadapted original virus. The specific gravity of the virions and nucleocapsids of the original and the three adapted viruses were not different. The molecular weights of genomic RNA and messenger RNAs encoding H, F, P, and NP proteins of the adapted viruses as estimated by Northern blot hybridization were similar to those of the original virus. By T1-resistant oligonucleotide analysis of the genomic RNA, the glioblastoma- and the neuroblastoma-adapted viruses gave two more spots than the original virus; the oligodendroglioma-adapted virus had a pattern identical to that of the original virus. By two-dimensional gel electrophoresis of virion proteins, we found a difference in the isoelectric point of the viral envelope proteins H and F between the original and the adapted viruses. These results suggest that viral genomic changes occurred during adaptation, resulting in the alteration of viral envelope proteins.     

Comparison of Subacute Sclerosing Panencephalitis and Measles Viruses: an Electron Microscope Study

The ultrastructure of CV-1 cells infected with subacute sclerosing panencephalitis (SSPE) viruses was compared with that of CV-1 cells infected with the wild or Edmonston strain of measles virus. Both SSPE viruses and the measles viruses produced two types of nucleocapsid structures: smooth filaments, 15 to 17 nm in diameter, and granular filaments, 22 to 25 nm. The smooth and granular filaments produced by SSPE and measles virus did not differ in appearance. In CV-1 cells infected with SSPE viruses, smooth filaments formed large intranuclear inclusions and granular filaments occupied a large area of the cytoplasm, but always spared the area under the cell membrane. Particles budding from the surface of these cells contained no nucleocapsids. In CV-1 cells infected with measles virus, only small aggregates of smooth filaments were seen in the nuclei. Granular filaments in the cytoplasm predominantly occupied the area under the cell membrane, and were aligned beneath the cell membrane in a parallel fashion and assembled into budding particles. These differences between SSPE and measles virus may be regarded as quantitative, but they do distinguish SSPE viruses from measles virus. Moreover, the formation of large nuclear inclusions filled with smooth filaments appears to be a characteristic process of SSPE, but not of measles, since this type of inclusion is invariably seen in SSPE brain tissues, brain cultures derived from them, and CV-1 cells infected with SSPE viruses.     

Expression of defective measles virus genes in brain tissues of patients with subacute sclerosing panencephalitis.

The persistence of measles virus in selected areas of the brains of four patients with subacute sclerosing panencephalitis (SSPE) was characterized by immunohistological and biochemical techniques. The five measles virus structural proteins were never simultaneously detectable in any of the brain sections. Nucleocapsid proteins and phosphoproteins were found in every diseased brain area, whereas hemagglutinin protein was detected in two cases, fusion protein was detected in three cases, and matrix protein was detected in only one case. Also, it could be shown that the amounts of measles virus RNA in the brains differed from patient to patient and in the different regions investigated. In all patients, plus-strand RNAs specific for these five viral genes could be detected. However, the amounts of fusion and hemagglutinin mRNAs were low compared with the amounts in lytically infected cells. The presence of particular measles virus RNAs in SSPE-infected brains did not always correlate with mRNA activity. In in vitro translations, the matrix protein was produced in only one case, and the hemagglutinin protein was produced in none. These results indicate that measles virus persistence in SSPE is correlated with different defects of several genes which probably prevent assembly of viral particles in SSPE-infected brain tissue.     

Selective inhibition of translation of the mRNA coding for measles virus membrane protein at elevated temperatures.

The elevation of culture temperatures of C6 cells that were persistently infected with the Lec strain of the subacute sclerosing panencephalitis (SSPE) virus (C6/SSPE) resulted in immediate selective inhibition of membrane (M) protein synthesis. This phenomenon was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cytoplasmic lysates and immunoprecipitation with monoclonal antibody against the M protein in short-time labeling experiments. The synthesis of various viral mRNAs in the presence of actinomycin D decreased gradually at similar rates after a shift to 39 degrees C. No specific disappearance of the mRNA coding for the M protein was observed when viral RNAs isolated from the infected cells were compared before and after a shift up by Northern blot analysis. Results of pulse-chase experiments did not show any significant difference in M protein stability between 35 and 39 degrees C. This rapid block of M protein synthesis was observed not only in Vero cells that were lytically infected with plaque-purified clones from the Lec strain, clones isolated from C6/SSPE cells and the standard Edmonston strain of measles virus but also in CV1, MA160, and HeLa cells that were lytically infected with the Edmonston strain. Poly(A)+ RNAs that were extracted from C6/SSPE cells before and after a shift to 39 degrees C produced detectable phospho, nucleocapsid, and M proteins in cell-free translation systems at 32 degrees C. Even higher incubation temperatures did not demonstrate the selective depression of M protein synthesis described above in vitro. All these data indicate that M protein synthesis of measles virus is selectively suppressed at elevated temperatures because of an inability of the translation apparatus to interact with the M protein-encoded mRNA.     

Neurovirulence in mice of neural cell-adapted canine distemper virus

Neurovirulence of the Onderstepoort strain of canine distemper virus (CDV) adapted to human neural cell lines was determined by the intracerebral inoculation of DDD mice at 3 and 5 weeks of age. Intensity of neurovirulence was estimated by histopathological changes in the central nervous system and clinical symptoms. The original virus propagated in Vero cells induced leptomeningoencephalitis, whereas neuroblastoma-adapted virus induced nerve cell degeneration and mild encephalitis with relatively low morbidity and fatality. In contrast, the viruses adapted to glioblastoma and oligodendroglioma caused high morbidity and fatality. The latter two viruses induced necrotizing encephalopathy including edema and hyperemia. In addition, the glioblastoma-adapted virus induced formation of giant cells. The oligodendroglioma-adapted virus caused demyelination and spongy state associated with degeneration of glial cells and axons. These observations are discussed in regard to a possible correlation between the neurovirulence of CDV in mice and its tropism for neural cells in vitro.     

Identification of a nonproductive, cell-associated form of measles virus by its resistance to inhibition by recombinant human interferon

Subacute sclerosing panencephalitis (SSPE) is a fatal disease in children and young adults that is caused by persistent infection of the central nervous system (CNS) by a nonproductive, cell-associated form of measles virus. Using an experimental model for SSPE (LEC viral strain in newborn hamsters), we have shown previously that establishment of such CNS infections involves selective elimination from the CNS of productively infected cells by host defensive mechanisms, coupled with the selective sparing of cells carrying nonproductive viral forms. That interferon (IFN) may play a role in this process was suggested by the disappearance of productively infected cells from the CNS tissues prior to the appearance of antiviral antibodies and by the demonstration of cell-associated, IFN-resistant viral variants in the virus stocks that were used. Results of this study support these conclusions by showing that similar IFN-resistant viral variants are present in the HBS strain of SSPE-derived measles virus and that these variants, in the presence of IFN, have properties that are similar to those of naturally occurring cell-associated strains of SSPE viruses, e.g., DR, IP3, and Biken. These IFN-resistant forms of HBS virus were isolated and were shown to maintain their resistance to inhibition by IFN after cloning. However, on removal of IFN, they reverted to productive forms similar to the parental HBS virus. The potential role of such viral forms in the pathogenesis of SSPE is discussed.     

Role of biased hypermutation in evolution of subacute sclerosing panencephalitis virus from progenitor acute measles virus.

We identified an acute measles virus (Nagahata strain) closely related to a defective virus (Biken strain) isolated from a patient with subacute sclerosing panencephalitis (SSPE). The proteins of Nagahata strain measles virus are antigenically and electrophoretically similar to the proteins of Edmonston strain measles virus. However, the nucleotide sequence of the Nagahata matrix (M) gene is significantly different from the M genes of all the acute measles virus strains studied to date. The Nagahata M gene is strikingly similar to the M gene of Biken strain SSPE virus isolated several years later in the same locale. Eighty percent of the nucleotide differences between the Nagahata and Biken M genes are uridine-to-cytosine transitions known as biased hypermutation, which has been postulated to be caused by a cellular RNA-modifying activity. These biased mutations account for all but one of the numerous missense genetic changes predicted to cause amino acid substitutions. As a result, the Biken virus M protein loses conformation-specific epitopes that are conserved in the M proteins of Nagahata and Edmonston strain acute measles viruses. These conformation-specific epitopes are also absent in the cryptic M proteins encoded by the hypermutated M genes of two other defective SSPE viruses (Niigata and Yamagata strains). Nagahata-like sequences are found in the M genes of at least five other SSPE viruses isolated from three continents. These data indicate that Biken strain SSPE virus is derived from a progenitor closely resembling Nagahata strain acute measles virus and that biased hypermutation is largely responsible for the structural defects in the Biken virus M protein.     

Immunoperoxidase Stain of Measles Antigen in Tissue Culture

A specific electron microscopy staining technique for measles antigen has been developed by using Vero cells infected with a subacute sclerosing panencephalitis (SSPE) measles virus strain and fixed in glutaraldehyde or formaldehyde. Peroxidase-labeled antibody was prepared according to the method of Avrameas (4). Sera from SSPE patients with high measles antibody titer as well as normal human sera with and without measles antibody were used. With both fixatives, specific labeling was obtained on the surface of infected cells, on the budding site, and on complete viral particles. The cell membrane staining sometimes had a patchy distribution in that the reaction was most intense on the surface projections in front of each nucleocapsid. This suggests modification of the cell membrane in association with the nucleocapsids. In contrast, no label was detected on the membranes of the cells during the latent period from penetration through maturation of the virus. In formaldehyde-fixed cultures, cytoplasmic inclusions were stained, and this label was located on the "fuzzy" material around the nucleocapsids. The smooth type of nucleocapsids, mainly seen in the nucleus, were never labeled. These findings suggest that the antigenic nature of the "fuzzy" nucleocapsids in the cytoplasm may be different from that of the "smooth" nucleocapsids. The immunoperoxidase method gives good resolution of viral antigenic sites at high magnifications under electron microscopy and may be of value in studies on the immunopathogenesis of SSPE and other chronic viral infections.     

Full-length sequence analysis of subacute sclerosing panencephalitis (SSPE) virus, a mutant of measles virus, isolated from brain tissues of a patient shortly after onset of SSPE

Subacute sclerosing panencephalitis (SSPE) virus, a measles virus (MeV) mutant, was isolated from brain tissues of a patient shortly after the clinical onset, and the entire viral genome was sequenced. The virus, named SSPE-Kobe-1, formed syncytia on B95a and Vero/SLAM cells without producing cell-free infectious virus particles, which is characteristic of SSPE virus. Phylogenetic analysis classified SSPE-Kobe-1 into genotype D3. When compared with an MeV field isolate of the same genotype (Ich-B strain), SSPE-Kobe-1 exhibited mutation rates of 0.8-1.6% at the nucleotide level in each of the proteincoding regions of the viral genome. It is noteworthy that the mutation rate of the M gene (1.2%) of SSPE-Kobe-1 was considerably lower than for other SSPE virus strains reported so far, but that the majority of the mutations (75%) were the uridine-to-cytidine biased hypermutation characteristic of the SSPE virus M gene. At the amino acid level, the viral proteins, such as N, P, C, V, M, F, H and L proteins, had point-mutations on 3, 7, 1, 4, 3, 9, 8 and 14 residues, respectively, compared with the Ich-B strain. In addition, the F and H proteins had mutated C-termini due to single-point mutations near or at the stop codons. Two of the three mutations in the M protein were Leu-to-Pro mutations, which are likely to affect the conformation and, therefore, the function of the protein. Because of the relatively small number of mutations, SSPE-Kobe-1 would be a useful tool to study genetic evolution of SSPE virus.     

Characterization of Canine Distemper Viruses Adapted to Neural Cells and Their Neurovirulence in Mice

Interaction of the Onderstepoort strain of canine distemper virus (CDV) with three established human neural cells, i.e. IMR-32 neuroblastoma, 118-MGC glioma and KG-1 oligodendroglioma, was examined, and adaptation of CDV to these cells was also attempted. The unadapted virus was found to grow at relatively low titers in the three neural cells inducing moderate to minimal cytopathic effects (CPE). The virus was successfully grown at high titers in these cells after 8 to 10 passages. Biological characteristics such as growth rate, morphology of CPE and plaque size changed after adaptation. Analysis by SDS-polyacrylamide gel electrophoresis, however, failed to show any difference in the molecular weight of component proteins among the unadapted and three adapted viruses. Inbred DDD strain of mice developed clinical signs after intracerebral inoculation with the unadapted virus but most of them survived with histological lesions of encephalitis. Neuroblastoma-adapted virus induced only transient clinical signs in some animals with mild encephalitic lesions in the gray matter. Increases in neurovirulence were found for viruses adapted to glioma and oligodendroglioma cells. Almost all mice inoculated with these two viruses at 3 weeks of age died within 8 days with histological lesions consisting of hyperemia, edema, severe degeneration of nerve cells and a few giant cells. Demyelinating lesions in the absence of inflammatory changes were observed in the cerebellum, pons and medulla oblongata of animals inoculated with oligodendroglioma-adapted virus.     

Localization of measles virus antigens in subacute sclerosing panencephalitis in ferrets

Young adult male ferrets were inoculated intracerebrally (i.c.) with a cell-associated encephalitogenic subacute sclerosing panencephalitis (SSPE) virus strain to study the pathogenesis of the disease at the ultrastructural level. Most became acutely ill in 8-13 days. Areas of the brain were examined with indirect immunoperoxidase labeling techniques to detect measles antigen. None of these animals showed the characteristic viral nucleocapsids or marked inflammatory response associated with SSPE. However, all had positive immunolabeling of unstructured virus antigen, especially in post-synaptic regions in all areas of the brain that were examined. One ferret, immunized with measles vaccine 40 days prior to challenge with SSPE, became ill 18 days post inoculation (p.i.). Perivascular cuffings of inflammatory cells and large cytoplasmic inclusions of fuzzy nucleocapsids were found in the brain and spinal cord. The study indicates that ferrets which become acutely ill after inoculation with cell-associated SSPE virus do so before there is a marked cellular immune response or formation of virus nucleocapsids.     

Measles Virus-Specified Polypeptide Synthesis in Two Persistently Infected HeLa Cell Lines

Measles virus-directed protein synthesis was examined in two HeLa cell lines (K11 and K11A) that are persistently infected with wild-type measles virus. Four viral proteins (H, hemagglutination protein; P, nucleocapsid-associated protein; NP, the major nucleocapsid protein; and M, the matrix protein) were readily detected in both cell lines by immune precipitation of [(35)S]methionine-labeled cell extracts followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three (H, NP, and M) of the four viral proteins in both K11 and K11A cells differed from the corresponding viral proteins synthesized in HeLa cells acutely infected with the parental wild-type virus. In addition, the M protein from K11A cells migrated significantly more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the M protein from K11 cells, and there appeared to be slight differences in the H and NP proteins between these two persistently infected cell lines. The altered viral proteins detected in K11 and K11A cells appeared to be the result of viral mutations rather than changes in the host cell, since virus recovered from these cells directed the synthesis of similar aberrant viral proteins in HeLa cells. Virus recovered from K11 cells and virus recovered from K11A cells were both temperature sensitive and grew more slowly than wild-type virus. HeLa cells infected with virus recovered from K11 cells readily became persistently infected, resembling the original persistently infected K11 cells. Thus, viral mutations are associated with persistent measles virus infections in cell cultures.     

Detection of antibody to M protein of measles virus in patients with subacute sclerosing panencephalitis: a comparative study on immunoprecipitation

Consistent results have not been obtained yet on the presence of antibody to the M protein of measles virus in the sera of patients with subacute sclerosing panencephalitis (SSPE). We performed a comparative study on various immunoprecipitation systems which appeared in the literature and found that the difference in the composition of the solubilizing buffer produced a large variety of results on the immunoprecipitation. [35S]Methionine-labeled Vero cells infected with the Edmonston strain of measles virus were solubilized by 10 different buffers and reacted with hyperimmune rabbit serum to whole virus, monospecific antisera to H, NP, and M proteins of the virus, normal adults' sera, and the sera from 16 SSPE patients. The immune complex was absorbed by protein A and both solubilization and precipitation rates were compared with each viral protein. Although viral proteins were solubilized by all buffers, the solubilization rate varied considerably. M protein was solubilized and was not coprecipitated nonspecifically with any of the other viral proteins. Purified protein A conjugated to Sepharose was preferable to Staphylococcus aureus for absorption of the immune complex since the latter absorbed both viral and host proteins nonspecifically. The precipitation rates of the viral proteins also varied according to the buffers. Better solubilization of the viral proteins seemed to reduce their rate of precipitation for which the presence of SDS may be responsible, and the presence of the protease inhibitors may also affect the results of immunoprecipitation. Detection of M protein in the immunoprecipitates was largely influenced by the kind of buffer used: some buffers could detect it clearly, but others could not defect it at all. Among the solubilizing buffers tested, Saleh's buffer (Virology 93: 369-376 (1979)),, which contains 0.5% DOC and 0.5% Triton X-100, was most reliable for detection of the anti-M antibody in the rabbit serum, because it showed a high solubilization and high precipitation rates of viral proteins without nonspecific absorption by protein A or coprecipitation of M proteins with any of the other proteins. Using this buffer, we could definitely detect M proteins in the immunoprecipitates from the sera of all six healthy adults and 15 out of 16 patients with SSPE. It was found, however, that the amount of M proteins in SSPE patients was lower than that in healthy adults and varied considerably.