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.     

Detection of measles virus antigen(s) in peripheral lymphocytes from patients with subacute sclerosing panencephalitis

Summary Peripheral blood lymphocytes from 5 patients with subacute sclerosing panencephalitis (SSPE) were stimulated with phytohemagglutinin (PHA) and analyzed for the presence of the measles virus antigen(s) by immunofluorescence (IF). For detection of viral antigen fluorescein-conjugated globulins from SSPE patients or from measles convalescents both with high anti-measles titer were used. Peripheral blood lymphocytes from 5 children with measles were used as a positive control.Measles virus antigen(s) were localized in PHA-transformed lymphocytes in all SSPE patients.The present results indicate that in SSPE measles-like virus infection may be found not only in the central nervous system, but also in the circulating lymphoid cells.With 2 Figures     

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.     

Subacute sclerosing panencephalitis

Subacute sclerosing panencephalitis (SSPE) is a slowly progressive brain disorder caused by mutant measles virus. SSPE affects younger age groups. SSPE incidence is proportional to that of measles. High‐income countries have seen substantial decline in SSPE incidence following universal vaccination against measles. SSPE virus differs from wild measles virus. Measles virus genome recovered from the autopsied brain tissues demonstrates clustered mutations in virus genome particularly in the M gene. These mutations destroy the structure and functioning of the encoded proteins. Complete infectious virus particle has rarely been recovered from the brain. Human neurons lack required receptor for entry of measles virus inside the neurons. Recent in vitro studies suggest that mutations in F protein confer hyperfusogenic properties to measles virus facilitating transneuronal viral spread. The inflammatory response in the brain leads to extensive tissue damage. Clinically, SSPE is characterized by florid panencephalitis. Clinically, SSPE is characterized by cognitive decline, periodic myoclonus, gait abnormalities, vision loss, and ultimately to a vegetative state. Chorioretinitis is a common ocular abnormality. Electroencephalography (EEG) shows characteristic periodic discharges. Neuroimaging demonstrates periventricular white matter signal abnormalities. In advanced stages, there is marked cerebral atrophy. Definitive diagnosis requires demonstration of elevated measles antibody titers in cerebrospinal fluid (CSF). Many drugs have been used to stabilize the course of the disease but without evidence from randomized clinical trials. Six percent of patients may experience prolonged spontaneous remission. Fusion inhibitor peptide may, in the future, be exploited to treat SSPE. A universal vaccination against measles is the only proven way to tackle this menace currently.     

Events following the infections of enucleate cells with measles virus.

The development of measles virus (Edmonston) and SSPE measles virus (Horta-Barbosa) has been examined in enucleate BSC 1 cells. New antigen synthesis in measles virus infected enucleate cells has been demonstrated by fluorescent antibody, by the formation of extensive syncytia from enucleate cells alone and by analysis of polypeptide formation by polyacrylamide gel electrophoresis. All polypeptides formed in nucleate cells were also present in enucleate cells but the amount synthesized was reduced to around 20% of that in nucleate cells. There was also a significant reduction in the amount of antigen detected by fluorescent antibody in enucleate as compared to nucleate preparations. Examination of RNA synthesis in infected enucleate cells revealed only a marginal increase in acid-insoluble material. Titration of the output of infectious virus from enucleate cells infected at both 37 and 31 degrees C indicated a consistent reduction of almost two log units compared to nucleate cells. That the enucleate cells were capable of replicating input genome at these times was demonstrated by the successful growth of respiratory syncytial virus, both at 37 and 31 degrees C. SSPE measles virus grew to higher yield in nucleate BSC 1 than measles virus but there was again a reduction of more than two log units in enucleate cells. All polypeptides synthesized in SSPE infected nucleate cells were apparent in enucleate cells.     

Human natural cytotoxic cells: activation by a measles virus-induced lymphocyte factor which increases the expression of the HNC-1A3 antigen

When human lymphocytes were exposed to measles virus antigen or to SSPE virus-infected cells, their cytotoxic activity against uninfected MA-160 cells was markedly enhanced. In contrast, mumps virus or herpes simplex virus-infected cells did not show such an enhancing effect and were rather inhibitory. SSPE cells and measles virus antigen, but not mumps-virus-infected cells or mumps virus antigen, also induced a two-fold increase in the number of lymphocytes bearing the HNC-1A3 membrane antigen, which we recently reported to be associated with a natural cytotoxic cell population. Pretreatment of SSPE cells or measles virus antigen with anti-measles antibody abolished the enhancing effect. Heat-treating the cells or the antigen at 56 degrees C for 30 min similarly abolished the effect. Supernatants of lymphocyte-SSPE cell cocultures were also effective in enhancing natural cytotoxicity. The soluble factor was a low molecular weight dialyzable molecule which was unaffected by boiling or trypsinization. It was released within 4 hours of coculture even in the presence of cycloheximide or following irradiation. Neither interferon nor leukotriene B4 appeared to be responsible for this enhancement. Virus-lymphocyte interaction may thus enhance natural cytotoxic cell activity through the rapid production or release of a virus-induced small molecular weight lymphocyte factor.     

Immunological evidence for the synthesis of all canine distemper virus polypeptides in chronic neurological diseases in dogs. Chronic distemper and old dog encephalitis differ from SSPE in man.

Immunoprecipitation of the polypeptides of canine distemper virus (CDV) from lysates of infected cells has revealed that the serum and cerebrospinal fluid (CSF) of dogs with chronic distemper and old dog encephalitis contain high levels of antibody to all the viral structural polypeptides, indicating that all of these polypeptides are being synthesized in the dogs. These findings, and the fact that infectious virus has been isolated from explants of brain tissue without cocultivation techniques, differ from those with measles virus in subacute sclerosing panencephalitis (SSPE) in which there is a lack of antibody to the virus membrane (M) protein and cocultivation of brain explants with permissive cells is required for virus isolation. These results indicate that CDV undergoes complete replication in the brain in the persistent infection resulting in chronic neurological disease, in contrast to the situation with measles virus in SSPE in which replication appears to be abortive.     

Measles and central nervous system disease

This ia a summary and review of the Workshop, which points out that there is sufficient evidence to relate SSPE to a measles virus. This evidence is both direct-virus isolation- and indirect-immunological data. However the pathogenic process responsible for SSPE has not yet been elucidated. In the case of multiple sclerosis the evidence of the etiologic role of measles virus is only indirect and not as firm as in SSPE. Both diseases have stimulated investigations of the biology of the measles virus or provided ideas for studies in the future.     

Comparison of morbillivirus proteins by limited proteolysis

Proteins of a number of measles and SSPE virus strains have been compared by limited proteolysis and they appear to be largely conserved amongst the various strains. Viruses derived from SSPE cannot be distinguished from other measles viruses by this technique. Small differences in the digest patterns of the M proteins have been observed between the Edmonston and other measles virus strains. Furthermore, in some strains where the M proteins migrate slower in SDS-PAGE the limited proteolysis patterns are slightly different from those in other MV and SSPE virus strains. The limited proteolysis pattern of some canine distemper virus (CDV) proteins have been determined and nucleocapsid breakdown products have been identified in infected cells. Comparisons of proteins of four strains of CDV have shown that these, too, are largely conserved, although the digest of proteins of CDV appear to show more pronounced differences than those present in the MV and SSPE virus group. Limited proteolysis can be used readily to distinguish MV from CDV isolates.     

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.     

SSPE-BIKEN: a naturally arising hemagglutination-defective mutant of measles virus

Biochemical and genetic methods have been used to investigate a defective variant of measles virus previously isolated from a patient with subacute sclerosing panencephalitis (SSPE). Since its isolation, this syncytiogenic strain (SSPE-BIKEN) has remained cell-associated; infected cells do not hemadsorb and do not release infectious virus. Immune precipitation and polyacrylamide gel electrophoresis were used to study the synthesis of measles virion proteins in SSPE-BIKEN-infected cells. All of the virion proteins were detected in immune precipitated whole cell extracts. However, the hemagglutinin (HA) protein was not detected on the cell surface by lactoperoxidase iodination. These results suggest that the failure of the HA protein to insert into the cell membrane accounts for the block in the release of infections virus. Radioactively labeled, noninfectious, virus-like particles have been purified from the media of SSPE-BIKEN-infected cells. These particles contain virus nucleocapsid, nucleocapsid-associated, and membrane proteins, but very little HA and hemolysin proteins. Genetic complementation between SSPE-BIKEN and a temperature-sensitive mutant of measles virus was observed and suggests that the SSPE isolate defect is due to a mutation. Additional evidence of a mutation is provided by the detection of low frequency revertant progeny in SSPE-BIKEN stocks. Our results support the hypothesis that genetic variants of measles virus are involved in the etiology of SSPE.     

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.     

Long-term effect of elevated temperatures on SSPE virus expression in persistently infected rat glial cells

Summary Cultivation of measles virus (SSPE virus, Lec strain) persistently infected C 6 rat glioma cells at 39°C resulted in the loss of detectable expression of measles virus proteins. Temperature shift-back led to reactivation of measles virus even after maintenance of the cells at 39°C for 15 days. In Northern blot analysis viral mRNA disappeared at 3 days after shift-up whereas 50 S viral genome-sized RNA was detectable until 6 days. The 50 S RNA decreased in quantity in rough correlation with dilution by cell passage at 39°C. The 50 S viral RNA was found in the nucleocapsid fraction. On day 9 after shift-down of persistently infected cells, maintained at 39°C for 15 days, 50 S viral RNA reappeared although mRNAs were not yet detected. Infectious center assays showed that the number of cells in the population at 39°C, which contained an SSPE virus genome that could be reactivated, declined after temperature shift. Moreover, cell cloning experiments, in which single cells of cultures maintained for various lengths of time at 39°C were incubated at 35°C and examined by immunofluorescence, reconfirmed the above results. This indicates that the reactivation of SSPE virus described here was due to re-infection of virus-antigen negative cells with progeny virus produced by a few latently infected cells in the population. The biological significance of this phenomenon in the central nervous system virus infection is discussed.On sabbatical leave from the Queen's University of Belfast, Belfast, Northern Ireland.     

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.     

Subacute sclerosing panencephalitis (SSPE) agent in hamsters. 3. Induction of defective measles infection in hamster brain

A hamster-adapted SSPE agent was shown to cause a productive infection in weanling hamster brain which changed to a cell-associated or defective infection coincident with the appearance of measles antibodies in serum. Antibodies to measles hemagglutinin, hemolysin and nucleocapsid antigens developed in serum which also contained neutralizing activity for regular measles virus. The agent recovered from brains prior to the appearance of serum antibodies was infectious in cell free media, capable of rapidly destroying Vero cell cultures and able to progressively destroy primary hamster brain cultures. In contrast, the agent recovered from brain after serum antibodies were present was infectious only within cells, destroyed Vero cells ineffectively and spread slowly through primary brain tissue cultures releasing minute amounts of extracellular virus intermittently even though no measles antibodies were present in the culture media. Nevertheless, infected giant cells in the primary brain cultures contained both the HA and HL measles antigens in their cytoplasmic membranes. This in vivo conversion of a productive to a cell-associated cerebral infection appeared to be caused by the host antibody response and may mirror the initial events of human SSPE and possibly other slow or latent measles infections of the CNS.     

Absence of detectable measles virus genome sequence in inflammatory bowel disease tissues and peripheral blood lymphocytes

A highly sensitive measles-specific RT-PCR-nested PCR system was established, which consistently amplified measles virus genome sequence from control samples containing as little as 5.5 × 10⁻³ pfu per reaction. This method failed to detect the presence of measles virus in 93 colonoscopic biopsies and 31 peripheral blood lymphocyte preparations, examined and obtained from patients with inflammatory bowel disease (IBD) and noninflammatory controls. All patients had detectable levels of serum neutralization antibody against measles virus. Each biopsy was estimated to have about one million cells, based on the amplification of the beta actin gene. The assay was calibrated by use of a known number of lymphocytes. The method applied was able to amplify measles virus RNA from a nucleic acid mixture equivalent to 18 cells derived from subacute sclerosing panencephalitis (SSPE) brain material. The level of measles RNA present, if any, in the biopsies is therefore at least 50,000-fold less than in SSPE. J. Med. Virol. 55:243–249, 1998. © 1998 Wiley-Liss, Inc.     

In situ immune responses in Crohn's disease: A comparison with acute and persistent measles virus infection

The implied aetiological association of measles virus with Crohn's disease would be supported by detection of an immune response to infected cells in affected tissues. This study sought to detect and characterise in situ immune responses to measles virus in both acutely and persistently infected tissues, and in particular, Crohn's granulomata. Tissue sections from patients with Crohn's disease (n = 17), tuberculosis (n = 9), acute intestinal ischaemia (n = 5), acute measles pneumonitis (n = 2), acute measles appendicitis (n = 1), subacute sclerosing panencephalitis (SSPE; n = 1), and measles inclusion body encephalitis (MIBE; n = 1), were examined. Single and double immunohistochemical labelling was performed to identify both cytotoxic lymphocytes (CD8, TIA, perforin, Leu 7, CD45RO, CD45RA) and macrophages (KP1). The relationship of these cells to measles infected cells was examined by double immunolabelling with anti-measles virus nucleoprotein antibody. In both acute measles appendicitis and SSPE, CD8⁺/TIA⁺ cytotoxic lymphocytes · (CTL) targeted infected cells. In the cases of Crohn's disease (13/17), MIBE, fatal pneumonitis, and one tuberculous granuloma, that were positive for measles virus, infected cells appeared to be targeted by macrophages rather than CTL. CTL in both tuberculous and Crohn's granulomata were similar in their peripheral distribution, number, and phenotype. The data suggest that measles-specific CTL responses may be attenuated in Crohn's disease compared with acute measles appendicitis and SSPE, and secondly, that an abnormal macrophage response to persistent measles virus infection of the intestine may result in granulomatous inflammation. J Med Virol 51:90–100, 1997 . © 1997 Wiley-Liss, Inc.     

Genesis and maintenance of a persistent infection by canine distemper virus.

Vero cells were persistently infected with canine distemper virus by continuous undiluted passage of virus harvests. The cells were refractory to superinfection by both measles virus and canine distemper virus. These persistently infected cells produced and released into the medium a labile component which had a potent and selective inhibitory effect on the replication of canine distemper and measles virus. The inhibitory agent was not inactivated by u.v.-irradiation or sedimented by ultracentrifugation. Antisera against canine distemper virus or SSPE sera were able to block this inhibitory effect. We propose that these persistently infected cells produce an excess of a virus-induced regulatory protein.     

SSPE measles virus: Non-productive and productive infection in vero cells and suckling mice

Suckling mice have proved successful for the isolation of measles virus from the brain biopsy specimens of two clinical eases of subacute sclerosing panencephalitis (SSPE). A productive infection, with cell-free virus, has been obtained in mouse brain tissues with both isolates. Subsequent infection of Vero cell cultures was obtained with mouse brain homogenates but not with mouse brain derived cell-free virus, thereby mimicking the situation with SSPE in which cocultivation of brain and Vero cells is needed to infect the Vero cells. A non-productive infection, with cell-associated virus, has been obtained in Vero cells for a limited period with one isolate (LW) and for an indefinite period with the other isolate (MD). Infectivity experiments with MD isolate suggest that the cell-associated character of this SSPE measles virus is not an indication of genomic defectiveness.     

Antigenic differences in the hemagglutinin of measles and related viruses

To date only one serotype has been described for the measles virus group. We have used a monoclonal antibody which binds the hemagglutinin of the Edmonston strain of measles virus to show that the Lee (SSPE) strain lacks an antigenic determinant present on other measles strains. This indicates that antigenic variation may occur in measles virus.