Approaches in the understanding of morbillivirus neurovirulence

Certain members of the morbillivirus genus, canine distemper virus, phocine distemper virus, and the cetacean viruses of dolphins and porpoises exhibit high levels of central nervous system (CNS) infection in their natural hosts. CNS complications are rare for measles virus (MV) and are not associated with rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) infection. However, both RPV and PPRV are neurovirulent in permissive murine strains. Human postmortem tissue, neural cell cultures, and animal models have been used to answer major questions concerning morbillivirus neurovirulence. Studies of the MV CNS complication subacute sclerosing panencephalitis (SSPE) indicate that virus could enter the CNS either by direct infection of endothelial cells or in infected leucocytes, followed by infection of predominately neurones and oligodendrocytes. It has been established that MV neurovirulence in mice is partially determined by the virus-receptor specificity. The two known MV receptors, CD46 and SLAM, have been examined in normal and SSPE brain tissue and the findings suggest that further receptors may be necessary to explain infection of the CNS with wild-type strains of MV. In both humans and mice (and in vitro), once infection of neurones has been established, virus spreads transneuronally. It is possible that all morbilliviruses transiently infect the CNS in their natural hosts, but development of disease is dependent on the efficiency of the immune response. Alternatively, for RPV and PPRV, virus entry may be restricted due either to absence of viral receptors or failure of virus to replicate or spread in the CNS.     

Absence of measles virus receptor (CD46) in lesions of subacute sclerosing panencephalitis brains

In this study we investigated pathological changes of the expression of the measles virus (MV) receptor, CD46, in subacute sclerosing panencephalitis (SSPE) brains. We analyzed CD46 expression in lesions of brain specimens from five SSPE patients in comparison to uninfected regions of the same brains and to normal human brains. The correlation between CD46 and MV infection, in individual cells in SSPE brains, was analyzed by double-staining procedures using monoclonal antibodies (mAbs) and in situ hybridization to detect MV-specific mRNAs. We found that CD46 was expressed at relatively low levels by neurons and astrocytes in normal brains in comparison to neuroblastoma and astrocytoma cell lines. Within heavily infected (MV-positive) brain lesions of all five SSPE cases, CD46 was either not detected or was expressed to a lesser degree by neural cells, irrespective of whether MV antigens were detectable or not. In contrast, normal levels of CD46 were found in SSPE brain tissue distant from the lesion. Using in situ hybridization, mRNAs of both MV nucleocapsid and MV hemagglutinin (MV-H) were detected in all SSPE lesions, while no or only small amounts of MV-H protein were detected. MV-infected neurons were never found to express CD46. Although a strict correlation between levels of the MV-H protein and the absence CD46 could not be seen, these findings suggest that the CD46 expression is reduced by the MV infection in lesions of SSPE brains. Received: 3 March 1997 / Revised, accepted: 30 April 1997     

Infection of human oligodendroglioma cells by a recombinant measles virus expressing enhanced green fluorescent protein

One of the hallmarks of the human CNS disease subacute sclerosing panencephalitis (SSPE) is a high level of measles virus (MV) infection of oligodendrocytes. It is therefore surprising that there is only one previous report of MV infection of rat oligodendrocytes in culture and no reports of human oligodendrocyte infection in culture. In an attempt to develop a model system to study MV infection of oligodendrocytes, time-lapse confocal microscopy, immunocytochemistry, and electron microscopy (EM) were used to study infection of the human oligodendroglioma cell line, MO3.13. A rat oligodendrocyte cell line, OLN-93, was also studied as a control. MO3.13 cells were shown to be highly susceptible to MV infection and virus budding was observed from the surface of infected MO3.13 cells by EM. Analysis of the infection in real time and by immunocytochemistry revealed that virus spread occurred by cell-to-cell fusion and was also facilitated by virus transport in cell processes. MO3.13 cells were shown to express CD46, a MV receptor, but were negative for the recently discovered MV receptor, signaling leucocyte activation molecule (SLAM). Immunohistochemical studies on SSPE tissue sections demonstrated that CD46 was also expressed on populations of human oligodendrocytes. SLAM expression was not detected on oligodendrocytes. These studies, which are the first to show MV infection of human oligodendrocytes in culture, show that the cells are highly susceptible to MV infection and this model cell line has been used to further our understanding of MV spread in the CNS.     

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

Summary 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.     

Cloning the antibody response in humans with inflammatory CNS disease: isolation of measles virus-specific antibodies from phage display libraries of a subacute sclerosing panencephalitis brain.

We have developed a strategy to identify the disease-relevant antigens in a chronic inflammatory CNS disease exhibiting intrathecally expressed oligoclonal IgG. Using subacute sclerosing panencephalitis (SSPE), a chronic inflammatory measles virus infection of the brain as a model system, we constructed a phage display antibody Fab library from the amplified products of IgG expressed in the brain. Selection of the library against measles virus-infected cell lysates yielded four distinct Fabs which, by ELISA and by immunostaining, reacted specifically with measles virus-infected cells. Three Fabs immunoprecipitated a 72 kDa protein from infected cell cultures corresponding to the measles virus phosphoprotein. The fourth Fab immunoprecipitated and recognized by immunoblotting a 60 kDa protein corresponding to the measles virus nucleoprotein. The results demonstrate that functional antibodies from an inflammatory CNS disease can be expressed in bacteria and used to identify disease-relevant antigens. This approach could be applied to chronic inflammatory CNS diseases of unknown cause such as multiple sclerosis.     

A non-productive subacute sclerosing panencephalitis (SSPE) virus of human and ferret

Summary A non-productive syncytiogenic measles virus isolated from the brain of an SSPE patients 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.     

Subacute sclerosing panencephalitis in two brothers

We report the occurrence of subacute sclerosing panencephalitis (SSPE) in two brothers two years after measles infection. The diagnosis was confirmed by compatible data from medical history, occurrence of autochthonic measles virus (MV) IgG production in the central nervous system (CNS), and pathognomonic EEG changes. Pathogenetically, SSPE is caused by a genome mutation of intracellularly persisting MV, causing viral nucleocapsides to accumulate in the brain cells. A specific predisposing immune defect is not known. The occurrence of two cases in one family is suggestive of a genetic predisposing factor.     

LONGTERM INFECTION OF CULTURED HAMSTER DORSAL ROOT GANGLIA WITH HALLÉ SSPE VIRUS

Longterm infection of cultured hamster dorsal root ganglia with Hallé SSPE virus The morphogenesis of Hallé SSPE virus in cultures of hamster dorsal root ganglia (DRG) over a period of 60 days was followed by light and electron microscopy and by immunoperoxidase techniques. The Hallé virus was originally isolated from the lymph node of a patient with SSPE, and had been passaged only in non-neural cell lines. In organotypic cultures of hamster DRG, all cells except the neurons became infected. Large intracytoplasmic and intranuclear inclusions were commonly found in fibroblasts, but never in Schwann cells. The latter cells rounded up and were lost from the culture by 20 days post-infection (PI). Cell-free virus was detected by 7 days PI, reached a maximum at 30–40 days PI, and even at 60 days PI, substantial amounts of virus were still recorded. Budding virus particles were seen throughout the course of the infection. The formation of syncytia was rare. The present results are compared with previous data on the growth of measles virus in hamster DRG, and the growth of measles and SSPE viruses in cultures of hamster cerebellum. It was concluded that among the available strains of SSPE and measles virus, the Hallé strain is unique in its failure to infect nerve cells.     

Antibody in SSPE serum and brain IgG against measles virus smooth nucleocapsids detected by immunoelectron microscopy

Summary SSPE patients characteristically have high complement-fixing (CF) and neutralizing (N) antibody titers against measles virus in their sera, CSF, and brain. However, using SSPE patients' sera, the immunoperoxidase (IP) labeling of smooth nucleocapsids in SSPE or measles virus infected Vero cells or measles virions has not been achieved using conventional EM fixatives. Using the periodate-lysine-paraformaldehyde (PLP) fixative developed by McLean and Nakane (1974), and the indirect IP technique, antibodies against smooth nucleocapsids in SSPE and measles virus infected Vero cell cultures have been detected in serum from SSPE patients and normal individuals with high measles antibody titers.     

Genetic characterization of measles virus genotype D6 subacute sclerosing panencephalitis case,Alberta, Canada

Subacute sclerosing panencephalitis (SSPE) is a progressive and eventually fatal neurological disease arising from a persistent infection with measles virus (MV) acquired at a young age. SSPE measles virus strains are defective and unable to produce progeny virions, due to multiple and extensive mutations in a number of key genes. We sequenced the full MV genome from our recently reported SSPE case, which typed as genotype D6, and compared it with other genotype D6 wild type and SSPE sequences. The Alberta D6 strain was significantly different from other reported SSPE D6 sequences. Mutations were observed in all the genes of the Alberta strain, with the greatest sequence divergence noted in the M gene with 17.6% nucleotide and 31% amino acid variation. The L gene showed the least variation with 1.3% nucleotide and 0.7% amino acid differences respectively. The nucleotide variability for 15,672 bases of the complete genome compared to the wild type and other SSPE D6 strains was around 3%.     

Characterization of measles virus strains causing SSPE: a study of 11 cases

Eleven subacute sclerosing panencephalitis (SSPE) cases diagnosed in the UK between 1965 and 2000 were investigated. The entire or partial matrix (M), hemagglutinin (H), and nucleoprotein (N) genes of measles virus (MV) were sequenced following direct RT-PCR amplification from brain tissues. All the M genes showed the characteristic biased hypermutations and a premature termination codon was detected in 5/11 M sequences. Based on the more highly conserved H and N genes observed in persistent MV studies, phylogenetic analysis showed that two of three strains from patients likely to have acquired infection in the 1950s were related to clade C (WHO designation) and one appears to be a novel genotype. Three strains from patients infected in the 1960s and 1970s were clearly related to a MV strain isolated in 1974 belonging to genotype D1. Four strains from patients infected in the 1980s clustered with genotype D7 strains. One sequence from a patient infected in 1990s was identified as genotype D6. No vaccine strains were detected although five of these patients had been previously immunized. The sequence data obtained from these historic strains do not support the view that vaccine strains are associated with SSPE and provide valuable information for further studies of MV epidemiology, evolution, and pathogenesis in SSPE.     

Subacute sclerosing panencephalitis: an update

Subacute sclerosing panencephalitis (SSPE) is a chronic encephalitis occurring after infection with measles virus. The prevalence of the disease varies depending on uptake of measles vaccination, with the virus disproportionally affecting regions with low vaccination rates. The physiopathology of the disease is not fully understood; however, there is evidence that it involves factors that favour humoral over cellular immune response against the virus. As a result, the virus is able to infect the neurons and to survive in a latent form for years. The clinical manifestations occur, on average, 6 years after measles virus infection. The onset of SSPE is insidious, and psychiatric manifestations are prominent. Subsequently, myoclonic seizures usually lead to a final stage of akinetic mutism. The diagnosis is clinical, supported by periodic complexes on electroencephalography, brain imaging suggestive of demyelination, and immunological evidence of measles infection. Management of the disease includes seizure control and avoidance of secondary complications associated with the progressive disability. Trials of treatment with interferon, ribavirin, and isoprinosine using different methodologies have reported beneficial results. However, the disease shows relentless progression; only 5% of individuals with SSPE undergo spontaneous remission, with the remaining 95% dying within 5 years of diagnosis.     

Immunolabeling of SSPE and wild-type measles viruses in ferret brain cell culture

Summary Immunocytochemical studies using horseradish peroxidase labeled antibody were undertaken in an attempt to determine whether there are detectable antigenic differences which correlate with the biological properties of different strains of SSPE and wild-type measles virus grown in ferret brain cell cultures. The rabbit anti-measles hyperimmune serum used in this experiment contained antibodies to all the measles virus proteins when tested by immunoprecipitation. When cells infected with the wild-type measles or productive SSPE virus strains were treated with this serum, heavy deposits of reaction product were seen on the cell membrane and virion envelope. When SSPE serum which contained relatively little antibody to the M protein was applied, a clear unlabeled area was evident just beneath the surface label. Cells infected with the non-productive SSPE strains were labeled by both sera in a spotty or discontinuous pattern on the outer surface of the cell membrane. The differences in membrane labeling seem to reflect differences in the expression of viral membrane proteins by the various SSPE and measles virus strains.     

Comparative analysis of virus-specific antibodies and immunoglobulins in serum and cerebrospinal fluid of subacute measles virus-induced encephalomyelitis (SAME) in rats and subacute sclerosing panencephalitis (SSPE)

The intrathecal humoral immune response was analysed in patients with subacute sclerosing panencephalitis (SSPE) and Lewis rats with subacute measles virus (MV)-induced encephalomyelitis (SAME). SSPE patients as well as SAME rats revealed oligoclonal, intrathecal antibody synthesis with MV specificity. SAME rats synthesized MV-specific antibodies intracerebrally to a higher extent than SSPE patients. Although a restricted isoelectric pattern of MV-specific antibodies was detected in the cerebrospinal fluid (CSF) of SSPE patients as well as of SAME rats, the heterogeneity within clusters of immunoglobulin bands was higher in the rat specimens. Increase in the blood-brain barrier permeability for albumin was exclusively detected in SAME rats but not in SSPE patients. These data suggest that the rat model offers excellent opportunities to study the initial humoral events in MV-induced encephalitides.     

Recombinant antibodies generated from both clonal and less abundant plasma cell immunoglobulin G sequences in subacute sclerosing panencephalitis brain are directed against measles virus

Increased immunoglobulin G (IgG) and intrathecally produced oligoclonal bands (OGBs) are characteristic of a limited number of inflammatory central nervous system (CNS) diseases and are often directed against the cause of disease. In subacute sclerosing panencephalitis (SSPE), the cause of disease and the target of the oligoclonal response is measles virus (MV). The authors previously showed that clonally expanded populations of CD38+ plasma cells in SSPE brain, the likely source of OGBs, are directed against MV. In characterizing the breadth of the plasma cell reactivities, the authors found that a large proportion of the less abundant plasma cells are also directed against MV. The intrathecal response may be useful in determining the causes of other inflammatory CNS diseases, such as multiple sclerosis, Behcet's disease, and neurosarcoidosis.     

Distribution of measles virus in the central nervous system of HIV-seropositive children

In an autopsy study the distribution of measles virus (MV) in the central nervous system (CNS) of 18 measles-infected children (13 HIV seropositive, 5 HIV seronegative), in Abidjan, Ivory Coast was examined using immunocytochemistry and in situ hybridization. Of these children 17 died from measles giant cell pneumonia. In 3 of the 13 HIV-seropositive patients MV antigens and genomic RNA was detected in the CNS. One of these positive patients had an MV encephalitis with abundant virus throughout most of the CNS. MV was not detected in the CNS of any of the 5 HIV-seronegative patients. These findings, albeit in a small number of cases, would suggest there may be an increased susceptibility to infection of the CNS with MV in HIV-positive children. In this respect entry and growth of MV in the CNS in HIV-seropositive individuals may be similar to the occurrence of measles inclusion body encephalitis in immunocompromised individuals. Furthermore, comparison of the HIV-MV encephalitis patient with two patients with subacute sclerosing panencephalitis (SSPE) demonstrated a paucity of virus in neuronal processes in the HIV-MV encephalitis. Unlike in SSPE, MV maturation by budding through the plasma membrane may occur, thereby minimizing build up of and intracellular movement of incomplete virus. Received: 6 March 1998 / Revised, accepted: 2 June 1998     

Measles virus matrix protein gene expression in a subacute sclerosing panencephalitis patient brain and virus isolate demonstrated by cDNA hybridization and immunocytochemistry

Summary Subacute sclerosing panencephalitis (SSPE) is a rare, fatal disease of children caused by a persistent measles virus infection of the central nervous system. A defect in synthesis of measles virus matrix (M) protein may be a factor in virus persistence in the brain. This study details attempts to detect expression of M protein in the brain of an SSPE patient, in the cell-associated virus isolated from this brain, and in brains of ferrets inoculated with the isolate. In situ hybridization with a tritiated cloned cDNA probe was used to search for RNA encoding M protein. Immunostaining with monospecific antiserum and the avidin-biotin-peroxidase technique was done to locate the polypeptide. The data obtained indicate that although nucleotide sequences coding for M protein were detected in the patient and ferret brains, expression of M protein in these tissues could not be detected. In the cultured SSPE virus isolate, the results were the same until the infected cells were examined by electron microscopy and a very limited expression of M protein was revealed. This suggests either diminished synthesis and/or rapid degradation of M protein in this cell-associated virus strain.This paper is dedicated to the late Dr. George A. Jervis, first Director of the Institute for Basic Research, Physician and Scientist, for his humanity and many contributions to the mentally retarded and developmentally disabled.     

Impaired human leukocyte antigen-restricted measles virus-specific cytotoxic T-cell response in subacute sclerosing panencephalitis

The capacity of peripheral blood lymphocytes to proliferate in response to measles virus and to generate measles virus-specific cytotoxic T lymphocytes (CTL) was examined in 4 patients with subacute sclerosing panencephalitis (SSPE). The lymphoproliferative response to measles virus was obtainable in the 4 SSPE patients. In contrast, the CTL response to measles virus was reduced in 3 of the 4 SSPE patients. This defect appeared to be in the generation of the measles virus-specific CTLs, since measles virus-infected target cells from the patients could be lysed by human leukocyte antigen-matched peripheral blood lymphocytes from healthy individuals. The SSPE patients with reduced measles virus CTL response had a normal ability to generate mumps virus, influenza virus, or alloantigen-specific CTLs. The lysis of measles virus-infected targets that was observed with these SSPE patients could be reduced by depleting the effectors of natural killer cells or by performing cold target blocking with K562 cells, indicating that the lysis of the measles virus-infected targets was probably mediated by natural killer cells. These results demonstrate a reduction in the cell-mediated immune response to measles virus as measured by the generation of measles virus-specific CTLs in 3 of the 4 SSPE patients studied. This defect could relate to the persistence of measles virus in these patients.