Subacute Sclerosing Panencephalitis Measles Virus: Study of Biological Markers

Comparative studies between two measles virus strains isolated from patients with subacute sclerosing panencephalitis (SSPE) and a prototype low tissue culture passage Edmonston measles virus are described. Differences were noted in several properties. The findings described in this report suggest that strains of measles virus associated with SSPE have different biological properties and apparently cannot be distinguished from laboratory and field strains of the virus.     

Measles Immunoglobulins in Subacute Sclerosing Panencephalitis

Normal responses of measles specific immunoglobulins M and G (IgM and IgG) were defined in 10 children with measles. Abnormal responses of measles IgM and IgG were found in both sera and cerebrospinal fluids from three cases of subacute sclerosing panencephalitis. In two patients the serum titres of measles IgM and IgG were abnormally high. The measles IgM was present during prolonged illnesses in serum and cerebrospinal fluid, which suggested a correlation with the known persistence of measles virus antigen in the brain of the three patients. It was concluded that both measles IgM and IgG may be produced within the central nervous system in subacute sclerosing panencephalitis.     

Characterization of Virus-Specific RNAs from Subacute Sclerosing Panencephalitis Virus-Infected CV-1 Cells

CV-1 cells infected with subacute sclerosing panencephalitis (SSPE) virus incorporated uridine-(3)H into at least four virus-specific RNA components in the presence of actinomycin D. The component sedimenting fastest had a sedimentation coefficient of 50s corresponding to a molecular weight of 6 x 10(6). The other three RNA components have sedimentation constants of 35s, 22s, and 18s corresponding to molecular weights of 2.5 x 10(6), 1.0 x 10(6), and 0.75 x 10(6), respectively. The base composition of the 50s RNA is distinct from that of cellular RNA and comparable with base compositions of viral RNAs of other paramyxoviruses. The base composition of the 18s RNA shows approximate complementarity with the 50s RNA. RNA-RNA annealing experiments using unlabeled 50s SSPE RNA with labeled 18s RNA from cells infected with SSPE virus or measles virus show 100% annealing with 18s SSPE RNA but only 60% annealing with 18s measles RNA. These experiments suggest some differences between the 18s RNAs of SSPE virus-infected cells and measles virus-infected cells.     

Mode of Subacute Sclerosing Panencephalitis (SSPE) Virus Infection in Tissue Culture Cells

Cell-free infectious viruses were successfully recovered by the aid of freezing and thawing from cultures infected with the Kitaken-1 and Biken strains of subacute sclerosing panencephalitis (SSPE) virus. Our results including those in a previous report which dealt with the Niigata-1 strain of SSPE virus show that cell-free viruses can be detected from all of the SSPE virus-carrying cultures established in Japan. It was also found that cell-free infectious viruses can be recovered efficiently by dispersing the virus-carrying cultures with EDTA. The inclusion of trypsin in the EDTA solution, however, caused a poor recovery of the infectious viruses. Infection of cells with the cell-free viruses readily established the virus-carrying cultures that have characteristics comparable to those of their original cultures. The culture infected with the Kitaken-1 strain produced infectious viruses in about ten times the amount of the other two infected cultures. The buoyant densities of the cell-free infectious viruses were almost the same among the three strains, the values being 1.120 to 1.132, but significantly less than that of 1.164 of measles virus. The low density can be ascribed to one of the characteristics of these SSPE viruses.     

Immune Complexes and Visceral Deposits of Measles Antigens in Subacute Sclerosing Panencephalitis

Immunofluorescence has been used to study visceral organs from a case of subacute sclerosing panencephalitis. Immune complexes were shown as granular deposits of IgG, complement, and measles antigens in renal glomeruli. Measles antigens were detected in the spleen, liver, and lymph nodes from many parts of the body.Immune-complex formation may be important in the aetiology of this disease and perhaps in causing some of its tissue damage. The rarity of subacute sclerosing panencephalitis may be due to an unusual pattern of immunological reactivity required in a patient before a measles infection can produce a subacute encephalitis.     

Isolation and Biological Characterization of a Measles Virus-Like Agent from the Brain of an Autopsied Case of Subacute Sclerosing Panencephalitis (SSPE)

Isolation of a cytopathic agent causing formation of syncytial giant cells in co-cultivated Vero cells from the brain of an autopsied case of subacute sclerosing panencephalitis (SSPE) is reported. The syncytia usually autolyzed from the center after growing to 1 to 2 mm in diameter and then detached from the culture vessels, and finally made macroscopically recognizable round plaques on the monolayer under liquid overlay. The agent was identified serologically as an agent related to measles virus, by both immunofluorescent tests and plaque reduction tests using anti-measles sera. However, the infected cells did not produce either virions or hemagglutinin, and failed to show hemadsorption and hemolysis of African green monkey red cells even after the 55th passage through Vero cells. Newborn mice, adult mice and hamsters showed neurologic signs after intracerebral inoculations of the infected cells, and most of them died from acute encephalitis. Guinea pigs were unsusceptible. From the brain of the animals with neurologic signs, a similar agent to the inoculated one was recovered. The new isolate appears to be a strain closely related to measles virus on the basis of serology, and was designated as SSPE-“Kitaken-1” strain.     

Comparison of Subacute Sclerosing Panencephalitis and Measles Viruses: an Electron Microscope Study

The ultrastructure of CV-1 cells infected with subacute sclerosing panencephalitis (SSPE) viruses was compared with that of CV-1 cells infected with the wild or Edmonston strain of measles virus. Both SSPE viruses and the measles viruses produced two types of nucleocapsid structures: smooth filaments, 15 to 17 nm in diameter, and granular filaments, 22 to 25 nm. The smooth and granular filaments produced by SSPE and measles virus did not differ in appearance. In CV-1 cells infected with SSPE viruses, smooth filaments formed large intranuclear inclusions and granular filaments occupied a large area of the cytoplasm, but always spared the area under the cell membrane. Particles budding from the surface of these cells contained no nucleocapsids. In CV-1 cells infected with measles virus, only small aggregates of smooth filaments were seen in the nuclei. Granular filaments in the cytoplasm predominantly occupied the area under the cell membrane, and were aligned beneath the cell membrane in a parallel fashion and assembled into budding particles. These differences between SSPE and measles virus may be regarded as quantitative, but they do distinguish SSPE viruses from measles virus. Moreover, the formation of large nuclear inclusions filled with smooth filaments appears to be a characteristic process of SSPE, but not of measles, since this type of inclusion is invariably seen in SSPE brain tissues, brain cultures derived from them, and CV-1 cells infected with SSPE viruses.     

Subacute Sclerosing Panencephalitis: Studies of Two Cases Treated with 5-Bromo-2-deoxyuridine

Two cases of subacute sclerosing panencephalitis treated with 5-bromo-2-deoxyuridine (BUDR) are reported. Both cases had a classical presentation with motor and mental deterioration, myoclonic jerks, paretic colloidal gold curve in CSF and periodic bursts of the EEG. The diagnosis was confirmed by brain biopsy with light and electron microscopic findings. Immunological studies revealed markedly elevated serum and CSF measles antibodies on serial determinations by the virus neutralization, complement fixation and hemagglutination inhibition techniques. Both cases were treated with 100 mg./kg./day of BUDR, intravenously, for five days, and Case 2 received a second course of treatment. Only minimal side effects were experienced from the use of BUDR; clinical symptoms showed sustained improvement in Case 1 and no further deterioration in Case 2. Both patients survived for more than 16 months. More extensive controlled trials with antiviral agents for the treatment of subacute sclerosing panencephalitis appear to be justified.     

Electron Microscopic Observations in Subacute Sclerosing Panencephalitis Brain Cell Cultures: Their Correlation with Cytochemical and Immunocytological Findings

Three cell cultures established from brain tissue obtained by biopsy of patients with subacute sclerosing panencephalitis (SSPE) were studied with the electron microscope in an attempt to correlate ultrastructural changes with those found by cytochemistry and immunocytology. These cells contained a large number of nucleocapsids resembling those of a paramyxovirus concentrated in the nuclear inclusions, but also seen free in the nucleus and occasionally in the cytoplasmic inclusions. Nuclear bodies associated with the nucleocapsids and granular filaments occupied a vast area of the cytoplasm. The nuclear inclusions containing nucleocapsids corresponded to the eosinophilic and fluorescent nuclear inclusions. The areas occupied by granular filaments corresponded to the diffuse cytoplasmic fluorescence. The ultrastructural changes were similar to those seen in the original brain biopsies. In addition papova-like virions were noted in brain cell cultures derived from a biopsy but not in the brain tissue itself. Their relationship to SSPE remains undetermined.     

Epidemiology of Subacute Sclerosing Panencephalitis (SSPE) in Germany from 2003 to 2009: A Risk Estimation

Subacute sclerosing panencephalitis (SSPE) is a fatal long-term complication of measles infection. We performed an estimation of the total number of SSPE cases in Germany for the period 2003 to 2009 and calculated the risk of SSPE after an acute measles infection. SSPE cases were collected from the Surveillance Unit for Rare Paediatric Diseases in Germany and the Institute of Virology and Immunobiology at the University of Würzburg. The total number of SSPE cases was estimated by capture-recapture analysis. For the period 2003 to 2009, 31 children with SSPE who were treated at German hospitals were identified. The capture-recapture estimate was 39 cases (95% confidence interval: 29.2–48.0). The risk of developing SSPE for children contracting measles infection below 5 years of age was calculated as 1∶1700 to 1∶3300. This risk is in the same order of magnitude as the risk of a fatal acute measles infection.     

Ribonuclease Sensitivity of Semliki Forest Virus Nucleocapsids

Treatment of Semliki Forest virus nucleocapsids with pancreatic ribonuclease (1 mug/ml, 37 C) digests the ribonucleic acid to acid-soluble fragments; the nucleocapsid protein forms a rapidly sedimenting aggregate.     

Mode of Subacute Sclerosing Panencephalitis (SSPE) Virus Infection in Tissue Culture Cells: III. Neurovirulence of Cell-Free SSPE Viruses of Niigata-1, Kitaken-1, and Biken Strains

Cell-free viruses recovered from virus-carrying cultures of the Niigata-1, Kitaken-1, and Biken strains of SSPE virus were examined for neurovirulence. The cell-free viruses were prepared by freezing and thawing or by EDTA treatment of the virus-carrying cultures and inoculated into adult mice intracerebrally. A considerable number of the inoculated mice showed clinical signs about 1 to 5 weeks after the inoculation. The first symptom was hyperreactivity, which was followed by paresis and myoclonus. All of the affected mice fell in paralysis and finally died. The virus could be recovered from the moribund mice by cocultivation of the brain cells with Vero cells. Immunofluorescence staining of the brain tissue revealed that infected cells containing viral antigens were distributed sparsely. No inflammatory feature, however, was observed in the brain as far as examined and neutralizing antibody against SSPE virus was not detected in sera from the mice inoculated with the cell-free SSPE viruses.     

中国对虾非包涵体型杆状病毒核衣壳的分离纯化

An effective procedure for isolation and purification of nucleocapsids of Penaeus chinensis non-occluded baculovirus (PcNOBV) which has destroyed the Chinese shrimp industry since 1993 was described. Gill, stomach, gut and cuticle epidermis under exoskeleton were excised from cultured P. chinensis diseased with typical white spot syndrome and homogenized in liquid nitrogen with TNE buffer containing PMSF and β-ME. The homogenized mixture was filtered through a 0.45μm millipore filter membrane to remove cell debris and ultracentrifuged to pellet the remaining material.The pellet was suspended in PMTNE buffer and laid onto a handmade CsCl gradient. An obvious viral band was observed in the middle of the gradient. Large amounts of virus nucleocapsids were visualized under electron microscope consistently corresponding to the milk-colored viral band. The viral envelope was all lost after purification. The nucleocapsid was bacilliform averaging 80±13nm×380±24nm in size. The negatively stained PcNOBV nucleocapsids revealed 13-16 conspicuous stripes located periodically perpendicular to the longitudinal axis of the nucleocapsids. Six to seven capsomers of 9 nm in diameter were visualized on each side of the stripe.     

Isolation and Characterization of Sendai Virus Temperature-Sensitive Mutants

Ten temperature-sensitive mutants of Sendai virus, a paramyxovirus, were isolated and partially characterized. The mutants replicated in chicken embryo lung cells at 30 C, but not at 38 C; wild-type virus grew equally well at both temperatures. Complementation tests divided the mutants into seven groups. Six groups synthesized neither infectious virus nor RNA when incubated at 38 C from the beginning of infection. Temperature shift-up experiments demonstrated that three of these complementation groups were blocked in early steps required for RNA synthesis, but these gene functions were not needed throughout the replicative cycle. In contrast, the other three RNA-negative complementation groups were defective throughout the replicative cycle in functions required for virus-specific RNA synthesis. Only one mutant, which complemented all of the above, synthesized RNA but not infectious virus when placed at 38 C; the hemagglutinin of this mutant functioned only at the permissive temperature.     

Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids

Chronic hepatitis B virus (HBV) infection, a serious public health problem leading to cirrhosis and hepatocellular carcinoma, is currently treated with either pegylated alpha interferon (pegIFN-α) or one of the five nucleos(t)ide analogue viral DNA polymerase inhibitors. However, neither pegIFN-α nor nucleos(t)ide analogues are capable of reliably curing the viral infection. In order to develop novel antiviral drugs against HBV, we established a cell-based screening assay by using an immortalized mouse hepatocyte-derived stable cell line supporting a high level of HBV replication in a tetracycline-inducible manner. Screening of a library consisting of 26,900 small molecules led to the discovery of a series of sulfamoylbenzamide (SBA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA. Structure-activity relationship studies have thus far identified a group of fluorine-substituted SBAs with submicromolar antiviral activity against HBV in human hepatoma cells. Mechanistic analyses reveal that the compounds dose dependently inhibit the formation of pregenomic RNA (pgRNA)-containing nucleocapsids of HBV but not other animal hepadnaviruses, such as woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Moreover, heterologous genetic complementation studies of capsid protein, DNA polymerase, and pgRNA between HBV and WHV suggest that HBV capsid protein confers sensitivity to the SBAs. In summary, SBAs represent a novel chemical entity with superior activity and a unique antiviral mechanism and are thus warranted for further development as novel antiviral therapeutics for the treatment of chronic hepatitis B.