Detection of measles virus RNA in lymphocytes from peripheral-blood and brain perivascular infiltrates of patients with subacute sclerosing panencephalitis

To clarify the relation between lymphocytes and measles virus in subacute sclerosing panencephalitis, we used in situ hybridization and a cloned measles virus DNA probe, specific for nucleocapsid protein, to detect measles virus RNA sequences in circulating lymphocytes and brain perivascular cuffs of patients with subacute sclerosing panencephalitis. Seventy to 90 per cent of peripheral mononuclear cells from three such patients were found to contain measles virus RNA sequences. In contrast, only a few infected cells were observed in four seropositive adults (0.1 to 5 per cent) and three age-matched children (10 to 15 per cent) used as controls. In one sample of brain tissue from a patient with subacute sclerosing panencephalitis, viral RNA sequences were also detected in nerve cells and in numerous cells from the perivascular infiltrates. In contrast, no hybridization was observed in brain tissue from a patient with herpetic encephalitis and from a patient with postlymphoma encephalitis. We conclude that measles virus has a strong tropism for lymphocytes and nerve cells in subacute sclerosing panencephalitis and that lymphocytes may be involved in the pathogenesis of the disease.     

Detection and characterization of measles virus strains in cases of subacute sclerosing panencephalitis in Croatia

Two cases of subacute sclerosing panencephalitis (SSPE), diagnosed in Croatia in 2002, were investigated. The coding regions of the matrix (M), hemagglutinin (H) and nucleoprotein (N) genes of measles virus were sequenced following direct RT-PCR amplification of viral RNA extracted from brain tissue. Phylogenetic analysis of the sequences of H and N genes, showed that both strains belonged to genotype D6. No vaccine strain was detected although both patients had been previously immunized. The comparison of analyzed sequences of two SSPE causative viruses with corresponding sequences of D6 genotype and with each other revealed a number of mutations in N and H gene sequences. In comparison to the Edmonston reference strain, the M gene of the SSPE viruses showed the characteristic biased hypermutation and a premature termination codon in one of the patients.     

Immunoglobulin G subclass antibodies to measles virus in patients with subacute sclerosing panencephalitis or multiple sclerosis

Mouse monoclonal antibodies specific for human immunoglobulin G (IgG) subclasses and a sensitive immunoassay were used to evaluate the IgG subclass antibody response to measles virus antigens in cerebrospinal fluid and serum samples from 20 patients with subacute sclerosing panencephalitis (SSPE), 12 patients with multiple sclerosis (MS), and 11 controls with high measles virus antibody titers in serum. In patients with SSPE, measles virus-specific antibodies were found mainly in the IgG1 subclass and the IgG subclass distribution remained unchanged, irrespective of the clinical stage or duration of the disease. In patients with MS and in controls, measles virus activity was also associated mainly with IgG1. However, the activity was significantly lower than that found in patients with SSPE. The results suggest that there is no primary abnormality in humoral immune response to measles virus in patients with MS. The disproportionately high levels of the measles virus-specific IgG1 subclass found in patients with SSPE may be due to persistent antigenic stimulation or reflect a defect in immunoregulatory mechanisms in response to viral infection.     

Cytotoxic antibody activity in measles and subacute sclerosing panencephalitis (SSPE) infection

Using the release of [⁵¹Cr] from measles virus carrier cells, cytotoxic antibodies were titrated in the sera and cerebrospinal fluid of subacute sclerosing panencephalitis (SSPE) patients and in the sera of individuals having recovered from natural measles virus infection. A similar range of titers was present in SSPE sera and in sera from individuals within three months of measles virus infection. Cerebrospinal fluid from SSPE patients and late measles sera contained lower cytotoxic antibody titers. An area of little cytotoxicity was present at low dilutions of some sera. Absorption of sera with the viral hemagglutinin produced by Tween-80 ether treatment of measles virus demonstrated the presence of cytotoxic antibodies directed against both the hemagglutinin and another antigen of measles virus.     

Isolation and characterisation of a defective measles virus from a subacute sclerosing panencephalitis patient.

A cytopathic measles virus was isolated from a brain biopsy of a subacute sclerosing panencephalitis (SSPE) patient. The agent could be transferred to Vero cells by cocultivation, but the infectivity always remained cell-associated -ie, a defective virus infection. The cell-associated nature of the virus was retained through 25 passages in Vero cells. Intracerebral inoculation of hamsters (2-6 days old) with the cocultured Vero cells gave rise to 100% mortality in 5-7 days. The virus retained its cell-associated nature after passage in hamsters. Electron microscopy of the brain and Vero cocultures showed the presence of virus-like ribonucleoparticles mainly in the nucleus. The presence of viral antigens in the nucleus, cytoplasm, and on the plasma membranes was confirmed by immunofluorescence. Using a combination of immunological and biochemical techniques, it was shown that all the viral proteins were synthesized with the exception of the haemagglutinin. Inclusion of the fusion inhibitor SV4814 (CBZ-D phenylalanine-L-phenylalanine-L-arginine-NO2) in the culture medium led to the elimination of the SSPE infection.     

Immunoglobulin class distribution of measles virus antibodies in serum and spinal fluids of patients with subacute sclerosing panencephalitis

Sera and cerebrospinal fluids (CSF) from patients with subacute sclerosing panencephalitis (SSPE) were tested by indirect immunofluorescence for the presence of measles virus specific antibodies of the various heavy chain classes. IgG and IgA antibodies were detected in the CSF while IgG, IgM, IgA, IgD and IgE measles virus antibodies were found in a significant number of the patient sera. Sera from SSPE patients had slightly elevated levels of IgG, IgM, IgD and IgE while IgA was decreased. The heterogeneous heavy chain class distribution of measles antibodies suggests the possibility that non-complement fixing antibodies serve as blocking antibodies which aid in the persistence of intracellular measles virus infection in patients with SSPE.     

Light-chain profiles in sera from patients with subacute sclerosing panencephalitis

Sera from 2 subacute sclerosing panencephalitis (SSPE) patients were absorbed with a concentrated preparation of measles virus. Measles-specific IgG was eluted from the precipitates containing measles antigen-antibody complex. These IgGs, when subjected to immunofixation after isoelectric focusing showed a number of oligoclonal bands with one type of light-(L) chain. In urea polyacrylamide gel electrophoresis, the reduced and alkylated measles-specific IgG showed 1-3 homogeneous L-chain bands, whereas IgG isolated from unabsorbed sera and IgG isolated from supernatants of SSPE sera after absorption with measles virus showed a diffuse L-chain band. It can be concluded that in SSPE, measles virus is responsible for the synthesis of L-chain with restricted heterogeneity.     

Immunological study of the agent responsible for subacute sclerosing panencephalitis and biochemical characterization of measles antibody in subacute sclerosing panencephalitis

Antibody of restricted heterogeneity produced in high titers in response to a given antigen is commonly observed in patients affected with subacute sclerosing panencephalitis and is comparable to what is observed in rabbits hyperimmunized with bacterial vaccines. Subacute sclerosing panencephalitis immunoglobulins were isolated and partially sequenced. Their immunological activity was measured with measles virus, subacute sclerosing panencephalitis virus (LEC strain) and distemper virus.     

Intranuclear polypeptides of measles and subacute sclerosing panencephalitis virus-infected cultures

CV-1 cells infected with either measles virus or five different isolates of SSPE virus each manifest a unique overall pattern of intranuclear polypeptides as well as differences in growth and yields of infectious particles. Of the viral structural proteins within the nucleus, both the P and M species most commonly exhibit migration differences on polyacrylamide gels. Each virus also generates a nonstructural protein of 20,000 daltons. Two of the SSPE viruses grow more slowly than the other viruses and produce significantly lower yields of infectious particles. Their nuclear isolates contain the most slowly migrating M protein, as well as two large species of 80,000 and 135,000 daltons. These findings suggest that there are differences in nuclear polypeptides between the viruses, not reflected in virion structural protein composition nor in the infected host cell cytoplasm, and that these changes occur particularly in less productive, more indolent infections.     

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.     

High risk of subacute sclerosing panencephalitis following measles outbreaks in Georgia

ObjectiveTo describe cases and estimate subacute sclerosing panencephalitis (SSPE) risk following large-sale measles outbreaks in Georgia. A rare, fatal late complication of measles, SSPE is often overlooked in assessments focused on the acute illness. Georgia had 8377 and 11,495 reported measles cases during the 2004–2005 and 2013–2015 outbreaks, respectively, but SSPE burden has not been assessed.MethodsSSPE cases diagnosed during 2008–2017 were identified from hospitalization registries in major neurological departments likely to admit SSPE patients. Information on reported measles cases and deaths was obtained from the national measles surveillance system and published reports. The risk of SSPE (number of measles cases per one SSPE case) was calculated for cases associated with the 2004–2005 outbreak. Crude estimates were adjusted to account for potential under-reporting of measles, using 50%, 25% and 10% estimates of completeness of reporting.ResultsSixteen SSPE cases diagnosed during 2008–2017 were identified. Eleven (92%) of 12 SSPE cases with a known history of measles had infection at ≤2 years and one (8%) at 3 years of age. Crude estimate of SSPE risk for the 2004–2005 outbreak was 1:1396. Adjusted estimates were 1:2792, 1:1:5584 and 1:13 960, assuming 50%, 25% and 10% completeness of reporting measles cases, respectively.ConclusionsThe review demonstrated substantial risk of SSPE in Georgia, supporting recent data suggesting that risk of SSPE following measles infection is higher than previously thought. To prevent SSPE in Georgia, very high timely immunization coverage for measles should be achieved among children, and immunity gap among adults should be closed.     

Circulating immune complexes in patients with subacute sclerosing panencephalitis

Levels of immune complexes (ICs) in serum and cerebrospinal fluid (CSF) specimens from subacute sclerosing panencephalitis (SSPE) patients were measured using a solid-phase C1q radioimmunoassay. Single or serial serum specimens were available from 19 patients, while serial CSF specimens were available from two patients. ICs isolated from one CSF specimen by C1q immobilized to Affi-Gel were analyzed for measles virus antigens by binding of measles virus-specific antisera and by polyacrylamide gel electrophoresis followed by Western blotting. Of 78 serum specimens analyzed, 36 (46%) were positive for ICs. When the 8 patients with 3 or more serum specimens were analyzed, 6 had fluctuating levels of ICs. Two of 5 CSF specimens obtained from a patient during acute disease onset were IC-positive, while a second patient exhibited a rapid increase and decrease of IC levels in 14 CSF specimens obtained during an acute disease exacerbation. Composition analysis of ICs isolated from one of the CSF specimens revealed the presence of antigens corresponding in size to measles virus polymerase, nucleoprotein, and possibly, hemagglutinin polypeptides. These results show that SSPE patients frequently have ICs, that IC levels fluctuate in both serum and CSF, and that the ICs are at least partially composed of measles virus antigens.     

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.     

Molecular characterization of measles virus strains causing subacute 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.     

Expression and properties of the V protein in acute measles virus and subacute sclerosing panencephalitis virus strains.

Measles virus (MV) inserts one guanosine (G) residue at a specific site in a subpopulation of the mRNA transcribed from the phosphoprotein (P) gene to produce V mRNA. Using an antiserum against the unique carboxyl-terminal region of the predicted V protein, we found that a phosphorylated V protein was expressed in two acute MV strains (Edmonston and Nagahata) and three SSPE virus strains (Biken, Yamagata, and Niigata). The V protein of Biken strain SSPE virus was electrophoretically and antigenically indistinguishable from the V protein of Nagahata strain acute MV, the likely progenitor of the Biken strain. The V protein of these two viruses was not present in the intracellular viral nucleocapsids, but was found only in the cytosolic free protein pool. Pulse-chase experiments failed to show transport of the V protein to the plasma membrane. The V protein was also absent in the extracellular virions. The P protein synthesized from the cloned gene associated with the MV nucleocapsids in vitro, but the V protein had no affinity to the MV nucleocapsids. These results suggest that expression and properties of the V protein are conserved in chronic MV infection.     

Western blot analyses of measles virus antibody in normal persons and in patients with multiple sclerosis, subacute sclerosing panencephalitis, or atypical measles.

A version of the Western blot was developed to detect serum antibodies against measles virus polypeptides. With this technique, a seroepidemiological survey of antibodies to the several measles virus proteins in diverse measles-related conditions was conducted. The sera were obtained from individuals with a recent or long-past history of natural measles, from persons with a history of immunization with live attenuated measles vaccine, and from patients with multiple sclerosis, subacute sclerosing panencephalitis, or atypical measles. The findings indicated that live attenuated measles vaccine elicits an antibody response qualitatively resembling that of a natural infection. In addition, multiple sclerosis patients made less antibody to the measles virus M protein than did individuals with a long-past history of natural measles. Thus, the immunological reaction of multiple sclerosis patients to measles virus is qualitatively, as well as quantitatively, different from that of normal persons. Finally, persons with subacute sclerosing panencephalitis and atypical measles mounted abnormally high antibody responses to measles virus polypeptides, in particular the P protein.