Mouse hepatitis virus (JHM strain) type 4 induces acute encephalitis followed by death in many strains of laboratory mice. Immunohistochemical study in vivo and analysis of mouse neuronal cells in vitro both indicate that the target cells in this infection is the neuron. Further, examination of several inbred mouse strains and neuronal cells from them shows that disease expression is controlled by a single autosomal gene action at the level of the neuronal cell. Susceptibility is dominant but not H-2 linked. However, cultured neuronal cells and macrophages from SJL/J mice, which are resistant to this infection, fail to make significant amounts of infectious virus after an appropriate viral inoculation. Apparently the defect is not at the level of the virus-cell receptor, because these cells, in part, express viral antigens.

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Mutagenesis of mouse hepatitis virus with 5-azacytidine or 5-fluorouracil yielded several temperature-sensitive mutants. Mutants have been isolated that dramatically enhance the production of demyelinating disease over that previously noted with the wild-type virus. This reproducible model should now make possible the precise elucidation of -the pathogenic mechanism and molecular basis of this virus-induced demyelination.A virus etiology has been proposed for several human demyelinating diseases, including multiple sclerosis (1). Studies of the pathogenic mechanism and molecular biology of virus-induced demyelination have been hampered by the unavailability of a feasible and reproducible animal model having a high incidence of demyelination. Mouse hepatitis virus (MHV) type 4 (JHM), a coronavirus, normally produces a rapidly fatal encephalomyelitis (2, 3). Under some conditions, such as low virus dose, MHV can, somewhat unpredictably, produce nonfatal demyelination due to a selective destruction of the myelinsynthesizing oligodendrocytes (4, 5). The early events in the pathogenesis of wild-type MHV-induced demyelination were difficult to evaluate because most of the infected animals either succumbed to the fatal encephalomyelitis or did not develop demyelination. Because selection for temperature-sensitive (ts) mutants has been shown by workers in our laboratory and different laboratories to often result in an alteration in the pathogenesis of other wild-type virus diseases (6-9), we sought, through the use of ts mutants, to attenuate MHV and thereby shift the disease spectrum from the fatal acute disease to the demyelinative disorder. We report the establishment of a reproducible model for the study of virus-induced demyelination.MATERIALS AND METHODS Virus Assay. Mouse hepatitis virus type 4 (JHM), kindly provided by L. Weiner (University of Southern California) was plaque purified in NCTC-1469 cells (10). The infectious titer, expressed as plaque-forming units per ml (PFU/ml), was determined by plaque assay on NCTC-1469 cell monolayers after 48 hr at 340.Virus Mutagenesis. The virus was grown overnight in NCTC-1469 cells at 340C in the presence of 5-azacytidine (12 ,gg/ml) or 5-fluorouracil (150 Ag/ml). These mutagen concentrations resulted in a 99% reduction of progeny virus. Mutants, selected for an inability to produce multinucleated syncytia at 39.5°, were plaque purified an additional time. Animal Inoculations. Four-week-old BALB/c St mice were inoculated intracerebrally with 0.05 ml of virus. In other experiments, mice were infected by intranasal instillation of virus.Histology and Electron Microscopy. After fixation with Bouin's solution, tissues were embedded in paraffin, sectioned, and stained with hematoxylin-eosin. Alternatively, mice were perfused via the heart with 2.5% phosphate-buffered glutaraldehyde. The spinal cords were embedded in araldite and stained with para-phenylenediamine. For electron microscopy, sections were stained with uranyl acetate and lead citrate and examined with a ...

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Autoantibodies directed against a wide range of normal tissue antigens have been found in the sera of patients with autoimmune diseases. It is generally thought that different and specific autoantibodies react with different tissues but the possibility exists that some autoantibodies may react with common antigens found in different tissues and organs. Recently, we showed that mice infected with reovirus developed a polyendocrine disease with autoantibodies to the pancreas, anterior pituitary, thymus and gastric mucosa. Using hybridoma technology, we obtained a number of monoclonal autoantibodies which reacted with antigens in single organs. We now report the production and pattern of reactivity of seven multiple organ-reactive monoclonal autoantibodies. By using antibody-affinity columns, autoantigens also have been isolated and their molecular weights determined. The results suggest that monoclonal multiple organ-reactive autoantibodies react either with the same molecule present in several organs or with common antigenic determinants on different molecules in multiple organs. In either case, the existence of multiple organ-reactive antibodies may be a partial explanation for multiple organ autoimmunity.

Mice infected with reovirus type 1 develop an autoimmune polyendocrine disease. Spleen cells from these mice were fused with myeloma cells and the culture fluids were screened by indirect immunofluorescence for autoantibodies reactive with normal mouse tissues. A large panel of cloned, stable antibody-producing hybridomas has been obtained. Fourteen of the hybridomas make autoantibodies that react with cells in the islets of Langerhans, 24 with cells in the anterior pituitary, 11 with cells in gastric mucosa, and 5 with nuclei. Except for the antibodies to nuclei, the monoclonal autoantibodies are organ-specific. Some, however, show broad cross-species reactivity, recognizing similar antigenic determinants in mouse, rat, pig, and human organs, whereas other recognize determinants only in rodent tissues. Several of the antigens recognized by these monoclonal autoantibodies have been identified as hormones (for example, glucagon, growth hormone, and insulin).

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