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.

Defective measles virus in human subacute sclerosing panencephalitis brain.

Evidence is presented showing that the brain cells of patients with subacute sclerosing panencephalitis (SSPE) contain mutant measles (MV) genomes having the characteristics of 5' copy-back defective interfering (DI) RNAs. Using a polymerase chain reaction-based amplification specific for copy-back DIs, abundant, discrete cDNAs representing different-sized MV defective RNA species were generated from each SSPE brain. The defective genomes were cloned in two portions. The most common of these defective species were sequenced, confirming their MV genome origin and 5' copy-back nature. We deduced that the minimum DI stem length of these species was 95 nucleotides, further delimiting the prerequisite 5' regulatory region sequences specifying MV genomic replication/encapsidation functions. This calculation assumes a precise copy-back mechanism and complete complementarity of the panhandle structure. Since the SSPE-derived viral genome encodes dysfunctional viral envelope proteins, we hypothesize that SSPE brains may lack the high degree of selective pressure encountered in tissue culture MV infections. This allows for the coexistence of numerous replication-competent defective particles in each SSPE brain. A role for viral defective particles as modulators of this persistent measles virus infection of humans is proposed.

Restriction of fusion protein mRNA as a mechanism of measles virus persistence.

Vero cells persistently infected with the hamster neurotropic strain (HNT-PI) of measles virus are deficient in the release of extracellular virus and syncytia formation, suggesting that mutations occur within the viral envelope proteins. Nucleotide sequence comparisons indicated that the coding regions of the matrix (M) and fusion (F) genes of HNT-PI were relatively conserved compared with those of their lytic progenitor virus Philadelphia 26 (Ph26), whereas the hemagglutinin (H) gene differed by 4.2% at the amino acid level. Northern blot analyses demonstrated the predominance of bicistronic M/F transcripts in HNT-PI at a 5:1 ratio over F monocistronic mRNA. Accordingly, no F protein could be detected in the HNT-PI cell line, although both the M and H proteins were produced in amounts comparable to those of Ph26. When the Semliki Forest virus replicon was used, coexpression of the HNT-PI F and Ph26 H genes resulted in the formation of multinucleated syncytia in transfected Vero cell cultures. Since the HNT-PI F protein was fusogenic, the restriction of its monocistronic mRNA is postulated to be a contributing factor in the reduction of cell fusion and ultimately in the maintenance of the persistent infection.

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.

Effect of ribavirin on subacute sclerosing panencephalitis virus infections in hamsters.

The antiviral activity of ribavirin was studied in hamsters infected with subacute sclerosing panencephalitis (SSPE) virus. Ribavirn did not improve the survival of infected hamsters when administered intraperitoneally at the maximal nonlethal dose of 50 mg/kg/day for 10 days. However, when administered intracranially, ribavirin improved the survival of infected hamsters in a dose-dependent manner. The 50% effective dose was calculated to be 1.4 mg/kg/day, and the selectivity index, based on the ratio of the 50% lethally toxic dose (31 mg/kg/day) to the 50% effective dose, was 22. When begun 12 h, but not 36 h, postinfection, ribavirin at a dose of 10 mg/kg/day completely prevented mortality and inhibited the replication of SSPE virus in brains of infected hamsters. Intrathecal or intraventricular administration of ribavirin should be explored for potential use in the treatment of patients with SSPE.

Association of measles virus with neurofibrillary tangles in subacute sclerosing panencephalitis: a combined in situ hybridization and immunocytochemical investigation.

Neurofibrillary tangle formation, a cardinal characteristic of Alzheimer's disease, is also a feature of several other neurodegenerative disorders, including subacute sclerosing panencephalitis (SSPE). In the present study the association of measles virus genome with neurofibrillary tangle formation has been studied in five cases of SSPE, using in situ hybridization (measles genome) and immunocytochemistry (tau, ubiquitin and beta/A4 amyloid). In two cases with duration of disease less than one year, neurofibrillary tangle formation was not observed. However, in those cases in which the disease was of several years duration, numerous tau- and ubiquitin-positive neurofibrillary tangles were demonstrated. In the two cases of longest duration, double-labelling techniques demonstrated the frequent association of neurofibrillary tangle formation with neuronal measles virus genome positivity. Immunocytochemistry for beta/A4 amyloid failed to demonstrate amyloid in any of the five cases. These findings support the hypothesis that neurofibrillary tangle formation can occur independently of amyloid formation and that this mechanism may operate in both Alzheimer's disease and in virally-induced disease.

Clonal expansion of hypermutated measles virus in a SSPE brain.

Nucleotide sequence analysis was carried out to study genes encoding the matrix (M) protein of measles virus (MV) from several regions of the brain of a case of subacute sclerosing panencephalitis. This analysis revealed the presence of MV with "wild-type" sequences as well as variants which had undergone at least five biased hypermutation events (U to C and A to G in the positive strand sequences). Despite the presence of MV variants with genes encoding the intact matrix protein open reading frame, M protein could not be detected in any of the brain regions. The distribution of virus variants was studied by cDNA cloning and sequence analysis and by in situ hybridization. The hypermutated viruses appeared to expand clonally throughout the brain of patient B.

Functional analysis of matrix proteins expressed from cloned genes of measles virus variants that cause subacute sclerosing panencephalitis reveals a common defect in nucleocapsid binding.

We have developed an in vitro nucleocapsid-binding assay for studying the function of the matrix (M) protein of measles virus (MV) (A. Hirano, A. H. Wang, A. F. Gombart, and T. C. Wong, Proc. Natl. Acad. Sci. USA, 89:8745-8749, 1992). In this communication we show that the M proteins of three MV strains that cause acute infection (Nagahata, Edmonston, and YN) bind efficiently to the viral nucleocapsids whereas the M proteins of four MV strains isolated from patients with subacute sclerosing panencephalitis (SSPE) (Biken, IP-3, Niigata, and Yamagata) fail to bind to the viral nucleocapsids. MV Biken (an SSPE-related virus) produces variant M sequences which encode two antigenically distinct forms of M protein. A serine-versus-leucine difference is responsible for the antigenic variation. MV IP-3 (an SSPE-related virus) also produces variant M sequences, some of which have been postulated to encode a functional M protein responsible for the production of an infectious revertant virus. However, the variant M proteins of Biken and IP-3 strains show no nucleocapsid-binding activity. These results demonstrate that the nucleocapsid-binding function is conserved in the M proteins of MV strains that cause acute infection and that the M proteins of MV strains that cause SSPE exhibit a common defect in this function. Analysis of chimeric M proteins indicates that mutations in the amino-terminal, carboxy-proximal, or carboxy-terminal region of the M protein all abrogate nucleocapsid binding, suggesting that the M protein conformation is important for interaction with the viral nucleocapsid.

Measles virus infection of cells in perivascular infiltrates in the brain in subacute sclerosing panencephalitis: confirmation by non-radioactive in situ hybridization, immunocytochemistry and electron microscopy.

As part of continuing multidisciplinary studies on the neuropathogenesis of subacute sclerosing panencephalitis (SSPE), in situ hybridisation, immunocytochemistry and electron microscopy were used to detect measles virus nucleic acid, protein and nucleocapsids in brain perivascular infiltrates of three cases. Perivascular cuffing cells which contained measles virus nucleic acid and antigens were found in all cases. Infected cuffs occurred predominantly in areas of general parenchymal cell infection and in many of these a high proportion of the infiltrating cells were infected. Other cuffs in these areas were either uninfected or contained only a few infected cells. Occasional infected cells were also seen in cuffs in non-infected areas. In contrast, no specific immunocytochemical reactions or in situ hybridisation for measles virus was observed in brain tissue from a patient with herpes encephalitis. By electron microscopy viral nucleocapsid, consistent with measles virus, was found within the cytoplasm of plasma cells in the inflammatory cuffs in SSPE brain tissue. Possible explanations for our results are that infiltrates become infected on arrival in the CNS or alternatively, that the infected infiltrates reflect a generalised infection of the reticuloendothelial system. The frequent presence of uninfected cuffs favours the former explanation.

SSPE in Slovakia: immunocytochemical study.

In a retrospective study of two patients from Slovakia with clinical, virological and histopathological diagnosis of subacute sclerosing panencephalitis (SSPE), measles virus antigen was detected by immunocytochemical labelling studies. The formalin fixed, paraffin-embedded thin brain sections labelled with anti-measles antibodies and avidin-biotin complex peroxidase were counterstained with haematoxylin. Only a single area of brain was examined in each patient: cerebellum and parietal lobe. Viral antigen positive reaction was identified within Purkinje cells and extending along dendritic processes in cerebellum, and also in oligodendrocytes of subparietal white matter.

The matrix proteins of neurovirulent subacute sclerosing panencephalitis virus and its acute measles virus progenitor are functionally different.

Persistence of measles virus in the brains of patients with subacute sclerosing panencephalitis (SSPE) is accompanied by changes in the viral matrix (M) protein. To understand the significance of these changes, cell culture and cell-free assays were developed to compare the functions of the M proteins of an SSPE virus Biken strain and its acute measles virus progenitor Nagahata strain. The Nagahata viral M protein is associated with the intracellular viral nucleocapsids and the plasma membrane, whereas the Biken viral M protein is localized mainly in the cytosol. The lack of M protein in the Biken viral nucleocapsids is due to a failure of the Biken M protein to bind to the viral nucleocapsids. The Biken M protein also fails to bind to the Nagahata viral nucleocapsids. Conversely, the Nagahata M protein can bind to the Biken viral nucleocapsids, although this association is not as stable at physiological salt concentration. These results offer concrete evidence that the M protein of an SSPE virus is functionally different from that of its progenitor acute measles virus.

Subacute sclerosing panencephalitis is typically characterized by alterations in the fusion protein cytoplasmic domain of the persisting measles virus.

Our recent extensive analysis of three cases of subacute sclerosing panencephalitis (SSPE) revealed intriguing genetic defects in the persisting measles virus (MV): the fusion (F) genes encoded truncated cytoplasmic F protein domains (Cattaneo et al., Virology 173, 415-425, 1989). Now this MV genomic region has been investigated in eight additional SSPE cases by PCR amplification, replacement cloning into a vector containing the F gene of a lytic MV, in vitro expression, and sequencing. In all cases at least part of the clones showed mutations leading to F protein truncations, elongation, or nonconservative amino acid replacements. It is proposed that alteration of the F protein cytoplasmic domain may play a critical role in the development of SSPE.

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.

Virus-induced demyelination in man: models for multiple sclerosis.

Epidemiologists have long worked at the limit of their powers to invoke an infectious agent to explain some of the epidemiological features of multiple sclerosis (MS). No virus has yet been definitively assigned a causal role in the pathogenesis of MS. Many viruses, however, have been incriminated, but it has not been possible to reproduce every single observation. Nevertheless, important lessons have been learned from the quest to identify such agents. Other kinds of demyelinating disease within the central nervous system (CNS) have been clearly linked to direct or indirect effects of viral infection. An understanding of the pathogenesis of these models affords possible insights into the mechanisms by which viruses or other agents might be operative in MS and other chronic diseases of the nervous system.

Nucleotide sequence of the genes encoding the matrix protein of two wild-type measles virus strains.

The nucleotide sequences of the matrix protein (M) genes of two wild-type measles virus (MV) isolates (JM and CM) have been determined and shown to differ in 56 positions; 31 of these differences are located in the non-coding region and 25 in the coding region of the gene. Most (80%) of the mutations in the coding region are changes to the third base of a codon. A maximum parsimony analysis of the available M gene nucleotide sequences allowed the construction of a tree with at least three lineages or subtypes. One wild-type strain (JM) was very similar to a subacute sclerosing panencephalitis virus strain (case B); the second wild-type strain, CM, showed nucleotide sequence similarity with MV from a case of measles inclusion body encephalitis. Both wild-type virus sequences are distinct from those so far determined for vaccine strains.