Subacute Sclerosing Panencephalitis Strains Research Articles

Amino acid substitutions in the heptad repeat A and C regions of the F protein responsible for neurovirulence of measles virus Osaka-1 strain from a patient with subacute sclerosing panencephalitis

Measles virus (MV) is the causative agent of subacute sclerosing panencephalitis (SSPE). We previously reported that the F gene of the SSPE Osaka-2 strain is the major determinant of MV neurovirulence. Because the sites and extents of mutations differ among SSPE strains, it is necessary to determine the mutations responsible for the SSPE-specific phenotypes of individual viral strain. In this study, recombinant viruses containing the envelope-associated genes from the SSPE Osaka-1 strain were generated in the IC323 wild-type MV background. Hamsters inoculated with MV containing the H gene of the Osaka-1 strain displayed hyperactivity and seizures, but usually recovered and survived. Hamsters inoculated with MV containing the F gene of the Osaka-1 strain displayed severe neurologic signs and died. Amino acid substitutions in the heptad repeat A and C regions of the F protein, including a methionine-to-valine substitution at amino acid 94, play major roles in neurovirulence.

The first genetic characterization of a D4 measles virus strain derived from a patient with subacute sclerosing panencephalitis

Measles virus (MV) strains derived from patients with subacute sclerosing panencephalitis (SSPE), SSPE strains, possess numerous mutations when compared to viruses belonging to the same genotype and circulating in similar time period. Although many SSPE strains have been extensively characterized, none of them belongs to D4 genotype which currently predominates in Europe where it has caused a number of recent outbreaks/epidemics.We sequenced an MV derived from a patient with long-term SSPE; the virus was named MVs/Zagreb.CRO/30.06[D4] (SSPE). Initial genetic analysis showed that it belongs to D4 genotype. The sequences of genes encoding matrix and fusion proteins indicate premature protein terminations. Putative hemagglutin (H) protein is lengthened for 20 amino acids, which is the longest H protein elongation so far found in SSPE viruses.Nucleotides 1421 A, 1422 G, 1507 C and 1542 C in nucleoprotein gene open reading frame seem to be specific for this D4 strain, differentiating it from other D4 non-SSPE strains. Besides, a unique mutation at position 543 of H protein was found, histidine instead of tyrosine.As persistent MV infections are initially established by “normal” wild-type MV strains, the presented comparative analyses describe alterations that could be involved in the maintenance of persistent infection, disease development and progression.

Measles Viral Infection in PD-1 Gene Knockout Mice

Purpose: Subacute sclerosing panencephalitis (SSPE) is a neurodegerative disease due to persistent measles virus infection. We investigated the role of programmed death-1 (PD-1) molecule which is related with chronic viral infection in developing SSPE in mouse. Methods: We adopt the , , and wild type BALB/c 3 week old mice to make an animal model of SSPE by injecting measles virus (SSPE strain) intraventricularly. Three months after infusion of virus, the mice were sacrificed and examined if the typical pathologic lesions had been progressed. The sera were collected from each group of mice and the serum level of IL-21 was measured with ELISA kit. Results: The necrotic lesions on white matter and gliosis were found in focal areas in wild type BALB/c. The extent of lesion was smaller in heterotype BALB/c. Scanty lesions were found in mice. The sera level of IL-21 was not elevated in all three groups. Conclusion: Our data suggest that the PD-1 molecule may play a role in persistent viral infection.

Measles and SSPE: occurrence and pathogenesis

Measles is an acute febrile exanthematous condition that is usually a self-limiting disease, but it can be associated with several complications, one of which is subacute sclerosing panencephalitis (SSPE). It is a rare delayed complication of measles due topersistenceof the virus in the central nervous system. All of the genetic analyses of viral material derived from brain tissue of SSPE patients have revealed sequences of wild-type measles virus (MV). There is no evidence that measles vaccine can cause SSPE. Several mutations have been described in genes coding for proteins in SSPE strains of MV. Several host cell modifications, mechanisms of virus reactivation and immunopathology in pathogenesis of SSPE have been explained recently, broadening the understanding of this fatal disease.

Molecular characterization of measles virus strains causing subactute sclerosing panencephalitis in France in 1977 and 2007

Measles virus strains from two subacute sclerosing panencephalitis (SSPE) cases diagnosed in 1977 (Laine strain) and in 2007 (Hoedts strain) were studied. Phylogenetic analysis based on C-terminal part of the nucleoprotein and the entire H gene showed that Hoedts strain, circulating in France presumably in the 1980s, belonged to genotype C2. However, Laine strain, suspected to have circulated between 1940s and 1960s, could not be assigned to any known measles virus genotypes. Sequences analysis of the Laine strain suggested that it originated from a measles virus that may have circulating at the same period as the Edmonston strain. The analysis of the whole genome of both SSPE strains revealed biased hypermutations in M, F, and H gene. Some of these mutations like the L165P found in the M protein sequence of the Laine strain, the amino acid position 94, where a mutation M94V was found in the F protein sequence of the Hoedts strain are known to play an important role in the glycoprotein interaction and to impair the ability of measles virus strain to produce cell-free infectious viral particles.This is the first study on molecular characterization of the entire coding region of measles virus isolated from SSPE cases in France.

The F gene of the Osaka-2 strain of measles virus derived from a case of subacute sclerosing panencephalitis is a major determinant of neurovirulence.

Measles virus (MV) is the causative agent for acute measles and subacute sclerosing panencephalitis (SSPE). Although numerous mutations have been found in the MV genome of SSPE strains, the mutations responsible for the neurovirulence have not been determined. We previously reported that the SSPE Osaka-2 strain but not the wild-type strains of MV induced acute encephalopathy when they were inoculated intracerebrally into 3-week-old hamsters. The recombinant MV system was adapted for the current study to identify the gene(s) responsible for neurovirulence in our hamster model. Recombinant viruses that contained envelope-associated genes from the Osaka-2 strain were generated on the IC323 wild-type MV background. The recombinant virus containing the M gene alone did not induce neurological disease, whereas the H gene partially contributed to neurovirulence. In sharp contrast, the recombinant virus containing the F gene alone induced lethal encephalopathy. This phenotype was related to the ability of the F protein to induce syncytium formation in Vero cells. Further study indicated that a single T461I substitution in the F protein was sufficient to transform the nonneuropathogenic wild-type MV into a lethal virus for hamsters.

Effect of the alterations in the fusion protein of measles virus isolated from brains of patients with subacute sclerosing panencephalitis on syncytium formation

Measles virus (MV) is the causative agent of subacute sclerosing panencephalitis (SSPE) and viruses isolated from brains of the patients contain numerous mutations. We have previously demonstrated that the hemagglutinin (H) protein of MV SSPE strains can interact with the signaling lymphocyte activation molecule (SLAM) and an unidentified molecule on Vero cells, but not with CD46, as a receptor. The mechanism by which MV SSPE strains can induce cell–cell fusion in SLAM-negative Vero cells is not understood. We report here on the effect of mutations in the fusion (F) proteins of three MV SSPE strains on syncytium formation. The F proteins of the three SSPE strains were functional and co-expression with H protein from the MV wild-type or SSPE strains in this study induced formation of large syncytia in Vero cells as well as in cell lines expressing SLAM or CD46. Expression of chimeric F proteins of SSPE strains showed that amino acid substitutions in the F protein extracellular as well as cytoplasmic domain contributed to enhanced cell–cell fusion in Vero cells. These findings suggest a common molecular mechanism and a key role of the F protein for syncytium formation in cells expressing an unidentified third receptor for MV.

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.

Receptor use by vesicular stomatitis virus pseudotypes with glycoproteins of defective variants of measles virus isolated from brains of patients with subacute sclerosing panencephalitis.

The vaccine or Vero cell-adapted strains of measles virus (MV) have been reported to use CD46 as a cell entry receptor, while lymphotropic MVs preferentially use the signalling lymphocyte activation molecule (SLAM or CD150). In contrast to the virus obtained from patients with acute measles, little is known about the receptor that is used by defective variants of MV isolated from patients with subacute sclerosing panencephalitis (SSPE). The receptor-binding properties of SSPE strains of MV were analysed using vesicular stomatitis virus pseudotypes expressing the envelope glycoproteins of SSPE strains of MV. Such pseudotype viruses could use SLAM but not CD46 for entry. The pseudotype viruses with SSPE envelope glycoproteins could enter Vero cells, which do not express SLAM. In addition, their entry was not blocked by the monoclonal antibody to CD46, pointing to another entry receptor for SSPE strains on Vero cells. Furthermore, the unknown receptor(s), distinct from SLAM and CD46, may be present on cell lines derived from lymphoid and neural cells. Biochemical characterization of the receptor present on Vero cells and SK-N-SH neuroblastoma cells was consistent with a glycoprotein. Identification of additional entry receptors for MV will provide new insights into the mechanism of spread of MV in the central nervous system and possible reasons for differences between MVs isolated from patients with acute measles and SSPE.

Subacute measles encephalitis in an immunocompetent adult

Subacute sclerosing panencephalitis (SSPE) and subacute measles encephalitis (SME) are both rare complications of measles virus infection. SSPE typically affects immunocompetent children, has an insidious onset and follows a steadily progressive course. SME mainly occurs in immunosuppressed children and has a rapidly progressive course. We describe a 43 year old immunocompetent man who presented with a rapidly progressive fatal encephalopathy. Histological examination of the brain showed a meningoencephalitis with inclusion bodies. Complement fixing antibody to measles virus was present in his serum and CSF. Measles virus RNA was found in the brain, spinal cord and eye, but not in the CSF. Analysis of the nucleoprotein gene isolated from this patient did not show similarity to SSPE strains of the measles virus. This patient demonstrates that subacute encephalitis secondary to measles virus infection can develop in an immunocompetent adult host.

Alterations and diversity in the cytoplasmic tail of the fusion protein of subacute sclerosing panencephalitis virus strains isolated in Osaka, Japan

We determined the nucleotide sequence of the fusion (F) gene of three strains (Osaka-1, -2, and -3) of nonproductive variants of measles virus (MV). These viral strains were isolated in Osaka, Japan, from brain tissues of patients with subacute sclerosing panencephalitis (SSPE). Phylogenetic analysis revealed a close relationship among the three strains of SSPE virus. The cytoplasmic tail of the F protein, predicted from sequence analysis of the gene, is altered in all three SSPE strains when compared to the MV field strains. However, the extent and mode of alteration are different in each strain. The F protein of the Osaka-1 strain has six nonconservative amino acid substitutions and a 29-residue elongation of its cytoplasmic tail. The F protein of the Osaka-3 strain has two nonconservative substitutions and a 5-residue truncation of its C-terminus. Although the termination codon is not altered in the F protein of the Osaka-2 strain, five or six amino acids are changed in the cytoplasmic tail of the F protein of the two sibling viruses of this strain. The significance of the altered cytoplasmic domain of the SSPE viruses in the SSPE pathogenesis is discussed.

Hemadsorption expressed by cloned H genes from subacute sclerosing panencephalitis (SSPE) viruses and their possible progenitor measles viruses isolated in Osaka, Japan.

Most subacute sclerosing panencephalitis (SSPE) viruses, including our Osaka-1, -2, and -3 strains isolated in Osaka, have shown negative hemadsorption (HAD) by African green monkey red blood cells. This property has been thought to be characteristic of SSPE virus as compared to the positive reaction of the standard Edmonston strain of measles virus (MV). However, this assumption has become quite obscure because MV mutates frequently at the genetic level during its multiplication and also because recent field strains isolated by lymphoblastoid cell lines have shown negative HAD. To investigate the above issue, the nucleotide sequences of the hemagglutinin (H) genes from SSPE virus Osaka-1, -2, or -3 strains were compared to those of various MV field strains isolated in Osaka by Vero cells. The H gene sequences of three SSPE strains were relatively conserved without such biased hypermutation as had been observed in the matrix (M) gene of three SSPE strains. However, this analysis of the H gene sequence of the SSPE viruses enabled us to deduce possible progenitor MVs, which are in agreement with the deduction from the M gene analysis we reported previously. The HAD of Vero cells transfected with the cloned H cDNAs from the SSPE strains and their progenitors suggested that negative HAD of the SSPE viruses has been maintained as one of original properties of the progenitor MVs rather than having been acquired as an altered one during long-term persistent infection in the brains of patients with SSPE.

Demonstration of immunoglobulin in brains of ferrets inoculated with an SSPE strain of measles virus: use of protein A conjugated to horseradish peroxidase.

Measles virus-specific immunoglobulin G (IgG) has been found in the brains of patients with subacute sclerosing panencephalitis (SSPE), a slowly progressing central nervous system (CNS) disease affecting children. IgG/albumin ratios indicate that the antibodies are probably synthesized in the CNS. In a ferret model system, protein A conjugated to horseradish peroxidase (PrAPx) was used to localize Ig's in brains of animals inoculated with a cell associated strain of SSPE. Ig's were found in plasma cells in various stages of antibody production both in perivascular inflammatory lesions and scattered throughout the cerebral cortex. These findings offer corroborative evidence that the Ig found in SSPE ferret brain and CSF is actively synthesized within the CNS. Antibody was also demonstrated in glial and neuronal cell bodies and processes and in postsynaptic profiles. These are the same sites where measles virus antigens are most frequently found and suggests the possibility of immune complex formation.

Expression of five viral antigens in cells infected with wild-type and SSPE strains of measles virus: correlation with cytopathic effects and productivity of infections.

Cells infected with four strains (LEC, Biken, IP-3 and DR) of subacute sclerosing panencephalitis (SSPE) virus were compared with wild type measles virus (Edmonston) with respect to titers of extracellular virus, morphology of the cytopathic effect (CPE) and occurrence of different measles virus antigens within infected cells as determined by immune fluorescence. Murine monoclonal antibodies with specifities for the nucleocapsid (NP), polymerase (P), matrix (M), hemagglutinin (H), and fusion (F) proteins as well as specific hyperimmune sera prepared in rabbits against the NP, H and M proteins were used in immune fluorescence analyses of the various strains. All of the strains produced large amounts of NP and P. Only the NP antigen occurred in nuclei of cells. The Edmonston and LEC strains also showed bright fluorescence with the antibodies against the H, F, and M antigens. Immune fluorescent intensity was variably reduced in cells infected with the Biken, IP-3, and DR strains labelled with anti M, H, and F antibodies. The Biken strain produced moderate titers of extracellular virus and moderate amounts of M, H, and F antigens whereas the DR strain produced no extracellular virus and contained no detectable M or F and only trace amounts of H antigen. The IP-3 strain was intermediate both in antigen expression and in production of extracellular virus.

Absence of M protein in a cell-associated subacute sclerosing panencephalitis virus.

Measles virus has been suggested to cause subacute sclerosing panencephalitis (SSPE), a slow central nervous system disease of children. However, several questions remain about the pathogenesis of SSPE. For example, it is not known whether alteration of the measles virus genome has a role in the initiation and persistence of the disease. Several studies have compared the RNA and protein composition of wild-type (wt) and SSPE strains of measles virus in a search for markers characteristic of the latter. All the studies used SSPE strains that had reverted to the budding, virion-producing form, similar to wt. We have shown, however, that only cell-associated non-budding strains of SSPE virus cause an SSPE-like persistent infection in young ferrets. Strong cell association and cell-fusing activity were essential for the virulence of measles virus in the brains of experimental animals and possibly humans. We have, therefore, compared the protein composition of virulent SSPE strains to that of the budding, non-virulent SSPE and wt strains. We report here that the M protein was not detectable in non-budding SSPE strains D.R., Biken and IP-3, and strain D.R. contained very little H protein.

Specificity of measles and canine distemper virus antibodies

Rabbits, guinea-pigs and a goat were inoculated with purified measles virus strains, and rabbits with canine distemper virus (CDV). Antisera from rabbits did not readily distinguish between measles virus strains, although guinea-pig antiserum was more selective for the homologous virus. No cross-reaction (neutralization, inhibition of haemagglutination or haemolysin) was observed between measles and CDV with these sera. Human sera from healthy adults, subacute sclerosing panencephalitis (SSPE) and multiple sclerosis (MS) patients occasionally contained CDV-neutralizing antibodies although the titre was always lower than that against measles virus. Several human sera neutralized the Edmonston strain better than the Hallé SSPE strain. However, a plaque-purified LEC strain (SSPE) was also neutralized as efficiently by certain human sera. The strain specificity was unmodified by removal of the haemagglutinin (HA) antibody from the sera. Immunoprecipitation and polyacrylamide gel electrophoresis of 35S-measles virus-induced proteins showed that all the polypeptides with the exception of the HA (80K) cross-reacted with CDV dog serum.

Measles and subacute sclerosing panencephalitis virus proteins: lack of antibodies to the M protein in patients with subacute sclerosing panencephalitis.

Studies on the immunoprecipitation of the proteins of several different measles and subacute sclerosing panencephalitis (SSPE) virus strains from lysates of infected cells have revealed a relative lack of antibodies to the nonglycosylated viral membrane protein (M) in the sera of patients with SSPE, in contrast to a high level of antibodies to other viral proteins. No evidence was found for the existence of a large number of antigenically unique M proteins. In contrast to SSPE sera, other hyperimmune human or rabbit sera precipitated M protein as well as the other viral proteins. The results suggest that in patients with SSPE there is either diminished synthesis of M, or it is not recognized normally by the immune system, and that an abnormality in M protein is involved in the pathogenesis of SSPE. Although differences in electrophoretic mobility of the M proteins and several other viral proteins were observed among different SSPE and measles strains, there was no pattern characteristic of SSPE strains, nor could these strains be distinguished by peptide mapping.

Cyclic Expression of Antigen and Infectious Virus in a BHK Cell Line (0-853) Persistently Infected with an SSPE Strain of Measles Virus

Establishment and characteristics of a baby hamster kidney cell line (BHK 0-853) persistently infected with a subacute sclerosing panencephalitis (SSPE) strain of measles virus (Lec strain) is described. The persistent infection was easily and repeatedly established and no special conditions were required. There was a predictable fluctuation in expression of virus intracellular and membrane antigens which varied from greater than 90% to less than 1% of the cells demonstrating these antigens during the first 6 or 7 passages. Thereafter, fluctuation of antigen and infectious virus expression continued in an unpredictable fashion.

The effects of measles virus and various strains of SSPE virus on organotypic cultures of nervous tissue.

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 may account for the differences in the in vivo conditions with which they are associated.

A non-productive subacute sclerosing panencephalitis (SSPE) virus of human and ferret: an ultrastructural study.

A non-productive syncytiogenic measles virus isolated from the brain of an SSPE patient was grown on Vero cells. The ultrastructure of the infected syncytia was studied by electron microscopic and immunoperoxidase techniques. It was compared with the isolate of the virus after passage in ferrets, the Edmonston strain of wild measles virus and the Halle productive strain of SSPE, all on Vero cells. The immunoperoxidase labeling of the cell membranes of the Edmonston measles virus infected cells was very heavy and uniform. In contrast, the labeling of the non-productive SSPE infected cells was clearly discontinuous. In the latter, there was a preponderance of intranuclear over cytoplasmic nucleocapsid formation, whereas cytoplasmic nucleocapsids were prevalent in the virion-producing strains. Many similarities between Vero cells infected with the wild measles virus and the Halle strain of SSPE were observed, although differences between this SSPE strain and strains reported by others were noted.