Comparison of wild-type and subacute sclerosing panencephalitis strains of measles virus. Neurovirulence in ferrets and biological properties in cell cultures.

The neurovirulence of two wild type (wt) and seven Subacute Sclerosing Panencephalitis (SSPE) measles virus strains was tested in young adult ferrets by intracerebral (IC) inoculation of infected Vero cell suspensions. Wt strains Edmonston and Woodfolk and SSPE strains Mantooth, Halle, and LEC-S did not produce a detectable encephalitis in the ferrets, but caused a significant formation of serum antibodies against measles virus. SSPE strains LEC, IP-3, Biken, and D.R., on the other hand, were all neurovirulent in ferrets, particularly strain D.R. which caused an acute encephalitis in all inoculated animals. Strain Biken was of particular interest since it caused a subacute encephalitis in four of seven ferrets. The subacute encephalitis was characterized by a long incubation time, persistence of virus in the brain for at least 8 mo, widespread inflammatory lesions, and production of measles virus specific IgG in the brain. A study of the biological properties of the various measles virus strains showed that wt strains Edmonston and Woodfolk and SSPE strains Mantooth, Halle, and LEC-S produced free virus particles in significant titers both in Vero and ferret brain (FB) cultures. Cytopathic effect (CPE) with cell-fusion was marked in Vero cultures, whereas only minimal CPE and no cell-fusion were observed in the FB cultures. SSPE strains LEC, IP-3, Biken, and D.R., on the other hand, were mostly cell-associated in Vero and FB cultures, although atypical cell-free particles were produced by strains Biken and IP-3. All four strains showed cell-fusing activity in FB cultures, particularly strain D.R., which was the only strain that spread more actively by fusion in FB than in Vero cultures. The results are discussed in relation to the neurovirulence of the various measles virus strains in adult ferrets. Pronounced cell-fusing activity in FB cells and cell-association with minimal or no production of cell-free virus seem to be essential to establish a brain infection in the animals.

Measles virus nucleotide sequences: detection by hybridization in situ.

A tritium-labeled probe that detects measles virus nucleotide sequences was hybridized in situ to cells infected with measles virus and to sections of brain tissue from patients with subacute sclerosing panencephalitis and from patients with multiple sclerosis. The measles virus genome was detected in many cells in subacute sclerosing panencephalitis where this virus would have been missed by methods such as immunofluorescence. Measles virus sequences were also found in two foci in one of four cases of multiple sclerosis. This refined method of hybridization in situ, which can be useful in the search for covert virus infections of man, provides evidence that viruses may be involved in multiple sclerosis.

Subacute sclerosing panencephalitis: isolation of suppressed measles virus from lymph node biopsies.

Measles virus was isolated in mixed cultures of lymph node cells and HeLa cells. The agent was isolated by cocultivation from biopsy specimens of two of five patients with subacute sclerosing panencephalitis. The virus was identified by hemagglutination-inhibition, immunofluorescent, and neutralization tests. Biopsies from controls did not show evidence of measles virus.

Measles virus matrix protein synthesized in a subacute sclerosing panencephalitis cell line.

Measles virus generally produces acute illness. Rarely, however, persistent infection of brain cells occurs, resulting in a chronic and fatal neurological disease, subacute sclerosing panencephalitis (SSPE). Evidence indicates that expression of the measles virus matrix protein is selectively restricted in this persistent infection, but the mechanism underlying this restriction has not been identified. Defective translation of matrix messenger RNA has been described in one SSPE cell line. This report presents evidence that in a different SSPE tissue culture cell line IP-3-Ca, the matrix protein is synthesized but fails to accumulate. A general scheme is proposed to reconcile the different levels at which restriction of matrix protein has been observed.

Differences in early ultrastructural aspects of the replication of measles and subacute sclerosing panencephalitis viruses in a cell culture from a human astrocytoma.

Monolayer cultures from a human astrocytoma were infected with small amounts of Mantooth Subacute Sclerosing Panencephalitis (SSPE) and Edmonston measles viruses. The infected cells were studied with an electron microscope 48 hours and 96 hours post-inoculation (PI). By 48 hours PI, both viruses produced syncytia and cytoplasmic inclusions of granular nucleocapsids 20 to 25 nm in diameter which did not differ in appearance. With the Edmonston measles virus granular nucleocapsids assembled into budding particles were found just under the cell membrane while nucleocapsids of Mantooth SSPE virus spared the area under the cell membrane and were not incorporated into budding particles. Inclusions of smooth nucleocapsids, 15 nm in diameter, could be seen within the nuclei of Mantooth SSPE virus infected cells 96 hours PI; such nuclear inclusions were not found in the Edmonston measles virus infected cells. These results are compared with those obtained in other cell systems and are discussed with respect to recent findings in the field of SSPE.

Immunologic and virologic studies of measles inclusion body encephalitis in an immunosuppressed host: the relationship to subacute sclerosing panencephalitis.

An immunosuppressed child with acute lymphoblastic leukemia in clinical remission developed measles inclusion body encephalitis (MIBE). Although measles antigen and nonbudding measles virus nucleocapsids were detected in brain tissue, no virus was isolated. Immune precipitation of measles virus proteins with the patient's serum showed no detectable antibody to virus M protein, a finding that has been reported in subacute sclerosing panencephalitis (SSPE). The virologic and immune precipitation studies suggest a similar virus mutation in MIBE and SSPE. The pathogenesis of the two diseases may also be similar.

Quantitation of intrathecal measles virus IgG antibody synthesis rate: subacute sclerosing panencephalitis and multiple sclerosis.

A method for quantitating specific anti-viral antibodies in serum and cerebrospinal fluid (CSF) is established using enzyme-linked immunosorbent assay (ELISA). Quantitated antibody levels are used to determine intrathecal specific IgG synthesis rate for the particular antibody. Measles virus was used as a model for validating this quantitative technique: a mutated form of measles virus is a cause of subacute sclerosing panencephalitis (SSPE) and there is a possibility that measles virus is related to the cause of multiple sclerosis (MS). Matched serum and CSF samples were assayed. Concentration of anti-measles IgG was determined and intrathecal measles-specific IgG synthesis rate was calculated. For the SSPE samples, measles-specific IgG synthesis rate was elevated and comprised > 20% of the total intrathecal IgG synthesis rate; these results are consistent with the literature. The ELISA method can be performed routinely, providing a quick, simple, reproducible means of quantitating specific antibody concentrations, with sensitivity greater than 1 nanogram per milliliter. With this method, quantitation of IgG antibodies to any other viral antigen can be reliably and precisely determined.

Detection protocols for biotinylated probes: optimization using multistep techniques.

Recent studies using biotinylated in situ hybridization (ISH) have utilized a wide range of detection protocols for the biotinylated hybrids, leading to conflicting reports in the literature regarding sensitivity. In this study we compared 11 different detection protocols for biotinylated ISH using a measles virus-specific RNA probe on formalin-fixed, paraffin-embedded central nervous system tissue infected with measles virus. Maximum sensitivity was achieved with five-step detection protocols incorporating the use of a monoclonal antibody to biotin. Single-step detection protocols were found to be insensitive, as shown by their failure to detect viral nucleic acid in infected white-matter cells. Only by increasing the number of steps in the detection protocols were these infected cells demonstrable. Unless pre-hybridization, hybridization, and detection protocols are optimized, the results obtained in pathogenicity studies using ISH could be misinterpreted, leading to false conclusions about nucleic acid distribution. This also applies to the ever-increasing use of ISH for diagnostic purposes.

Measles virus gene expression in subacute sclerosing panencephalitis.

RNA was extracted from the diseased brain of a case of human subacute sclerosing panencephalitis (SSPE) and analysed for the expression of measles-specific RNA. Measles virus-specific mRNAs were present, but the amount of matrix (M) protein mRNA was greatly reduced in comparison to lytically infected cells and phospho- (P) protein mRNA was hardly detectable whereas the level of the corresponding intermediate-sized (is-) RNA was greatly increased. RNA obtained from the human brain was also translated in vitro and measles virus nucleocapsid and P protein was produced. However, in marked contrast to control reactions M protein was not detected in the products formed by translation in vitro. These results indicate an impaired measles virus M protein mRNA synthesis in infected brain tissue.

Measles virus nucleic acid sequences in human brain.

We constructed a measles virus genomic recombinant DNA library, and used clones coding for portions of the viral P, M and H proteins to probe for measles virus nucleic acid sequences in post-mortem multiple sclerosis, SSPE and control brains. By dot blot hybridization, the probes detected measles virus nucleic acid sequences in as little as 3 nanograms of total RNA extracted from measles virus-infected cells and also in highly diluted RNA extracted from SSPE brain, but did not detect measles virus sequences in RNA extracted from 11 multiple sclerosis or 8 control brains, even at a 1 000-fold higher concentration of RNA. By in situ hybridization, these probes detected measles virus nucleic acid sequences in virtually every cell and the surrounding neuropile of SSPE brain, but again did not detect such sequences in multiple sclerosis or control brains. Our findings using these highly specific probes confirm that measles virus is found in SSPE brains and indicate that measles virus genome is unlikely to be present in multiple sclerosis or normal brains.

Generation and properties of measles virus mutations typically associated with subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis (SSPE), a very rare but lethal disease caused by measles viruses (MV) persisting in the human central nervous system (CNS) is characterized by lack of viral budding, reduced expression of the viral envelope proteins and spread of MV genomes through the CNS despite massive immune responses. The five major MV genes from several SSPE cases were cloned and sequenced, the two transmembrane envelope glycoproteins hemagglutinin (H) and fusion protein (F) were expressed and their maturation, cellular localization and functionality analyzed. We conclude that 1) mutations in the MV genes arise not only individually, by errors of the MV polymerase, but also in clusters as hypermutations, presumably due to RNA unwinding/modifying activity altering accidentally formed double-stranded RNA regions, 2) MVs spread in SSPE brains after clonal selection, 3) the MV matrix (M) gene is most heavily mutated and dispensable, 4) the two genes encoding envelope transmembrane proteins give rise to functional but altered proteins (typically F is heavily altered in its cytoplasmic domain), 5) H protein is transported poorly to the cell surface, 6) F and H proteins maintain tightly interdepending fusion functions, presumably to allow local cell fusion and MV ribonucleoprotein (RNP) spread through the CNS.

Enrichment of measles virus-like RNA in the nucleocapsid fraction isolated from subacute sclerosing panencephalitis brains.

A procedure has been developed which facilitates the detection of measles virus RNA sequences in human brains. The procedure involves isolating subviral components (nucleocapsids) from brain tissues prior to RNA purification, followed by hybridization of these RNAs to cDNA synthesized from measles virus 50 S RNA template. Using these techniques we were able to obtain an RNA fraction which was manyfold enriched in measles virus-specific RNA, relative to unfractionated subacute sclerosing panencephalitis (SSPE) brain RNAs 70-100% of the measles virus-specific RNA present these SSPE brain samples were recovered in this enriched fraction.

Unusual features in a case diagnosed as subacute sclerosing panencephalitis (SSPE).

SSPE is characterised by progressive mental deterioration, myoclonic and similar motor disorders and final severe comatose states, increase of immunoglobuline G in the CSF, strongly elevated antibody titers to measles virus in serum and CSF and typical periodic K-complexes in the EEG. The disease appears commonly in childhood and has a fatal course. Cases with atypical signs have occasionally been reported. The case described in this paper shows a number of uncommon features: late onset, partial remission and stationary course, increased antibody titers to measles virus but relatively low in comparison to others, dissociation of cytoplasmic and nuclear fluorescent antibodies against SSPE brain tissue and an initial increase of antibodies against rubella virus. The patient was treated with isoprinosine. Improvement was observed before the start of this therapy and stabilized while treatment was being continued.

Antibodies to viral and non-viral antigens in subacute sclerosing panencephalitis and multiple sclerosis demonstrated by thin-layer polyacrylamide gel isoelectric focusing, antigen immunofixation and autoradiography.

Antibody activity in IgG zones separated by thin-layer polyacrylamide gel isoelectric focusing (PAG IEF) was determined in 3 patients with subacute sclerosing panencephalitis (SSPE), 4 patients with multiple sclerosis (MS) and 4 subjects with psychosomatic disorders, using antigen immunofixation and autoradiography. Viral (measles herpes simplex type 1, mumps) and non-viral (purified bovine myelin, bovine myelin basic protein, bovine oligodendrocytes, MS and normal human brain extract) were used as antigens. All oligoclonal and some of the polyclonal CSF IgG zones in the patients with SSPE contained measles virus antibodies, as did some of the oligoclonal and polyclonal CSF IgG zones in 3 of the patients with MS. No antibodies were detectable in CSF or serum IgG zones against any of the non-viral antigens tested.

Clinical significance of viral latency.

Evidence is accumulating to show that a number of viruses have the ability to adapt to man's defense mechanisms and survive in a latent state for what appears to be the life of the human host. Unfortunately, latent viral presence, which may appear clinically benign initally, may manifest itself later as severe and often fatal disease. Some members of the herpes virus family have latent potential and are discussed in detail. Clinical competence would suggest a thorough understanding of these late manifestations of occult viral presence.

Changing character of subacute sclerosing panencephalitis in the United States.

We analyzed National Registry data from 575 patients with subacute sclerosing panencephalitis (SSPE) in the United States to assess changes in patient characteristics and SSPE epidemiology. Racial proportions have changed in recent years with an increasing number of Hispanic patients reported in relation to a constant black:white ratio; however, the male:female ratio of approximately 2:1 has remained. The most striking feature of the data is the rapid decline in SSPE incidence. Corresponding to this decrease is an increase in the proportion of cases following measles vaccination. There also is a shorter incubation period for SSPE following vaccination than after measles infection.

Adult-onset subacute sclerosing panencephalitis: immunocytochemical and electron microscopic demonstration of the viral antigen.

We report a case of subacute sclerosing panencephalitis (SSPE) in a 52 year-old man, who developed rapidly progressive mental deterioration, myoclonic seizures, quadriplegia, and remained incapacitated until his death 4 years after the onset of symptoms. Immunocytochemical and electron microscopic studies are reported. Titers of measles virus antibodies were consistently high in both serum and cerebrospinal fluid, and periodic synchronous discharges were recorded on EEG. Suppressed cellular immunity was noted in skin test with phytohemagglutinin. The brain was extensively destroyed by inflammatory processes. There were either laminar or widespread areas of cortical necrosis associated with neuronophagia, neuronal loss, glial proliferation, and perivascular lymphocytic cuffing. Numerous intranuclear inclusions, in the neurons and glial cells, stained with immunoperoxidase using antiserum to SSPE virus; ultrastructurally, these inclusions were made of tubular nucleocapsids of paramyxovirus. Neurofibrillary changes were occasionally encountered in the pigmented neurons. The white matter showed extensive loss of myelinated fibers and increased numbers of astrocytes with bizarre nuclei. This well-documented case of SSPE in an adult might be related to a condition of impaired cellular immunity.

The relationship between measles virus infection and subacute sclerosing panencephalitis (SSPE).

On theoretical grounds we propose that the essential step in the development of subacute sclerosing panencephalitis (SSPE) after measles virus infection involves a reverse transcriptase-mediated change to a DNA form, probably brought about by co-infection with a leukovirus at a critical point in time. We further suggest that this new DNA then replicates either as the core of a new slow virus or as a membrane-attached viroid exhibiting a form of non-structural integration with host cell DNA resembling that found with the herpes viruses.

The role of the nucleus in the replication of measles virus.

The replication of measles and SSPE viruses in enucleate BSC-1 cells was measured by plaque assay, and the synthesis of virus-specific antigen was measured by indirect immunofluorescence. Titres of infectious virus produced in enucleate cells at 24 hours post inoculation (p.i.) were consistently 100-fold less, and the number of enucleate cells containing virus antigen at 30 hours p.i. was 10-fold less, than in nucleate control cultures. Enucleate cells at this time had 30% of the protein synthetic capacity of control nucleate cells, and supported the growth of Semliki Forest virus to titres equivalent to nucleate cell production.