Mouse hepatitis virus type 4 (JHM strains). induced fatal central nervous system disease. I. genetic control and murine neuron as the susceptible site of disease

Mice. The inbred mouse strains listed in Table I were obtained from the Scripps Clinic and Research Foundation vivarium, La Jolla, Calif.; the L. C. Strong Research Foundation, La Jolla; or The Jackson Laboratory, Bar Harbor, Maine. 4-wk-old mice were inoculated intracerebrally with 0.05 ml of virus. To quantitate the amounts of infectious virus in various tissues, mice were exsanguinated and their tissues removed aseptically, then stored at -70°C until assayed. A 10% homogenate of various tissues, clarified by centrifugation at 700 g, was serially diluted 10-fold, and the amounts of infectious virus were quantitated by plaque assay on susceptible cells. Cultured Cells. To obtain primary cultures of neurons, spinal cords were removed from 11-12-d-old embryos and dissected free of meningeal coverings, dissociated through steel or nylon mesh with 60-100 openings/in and plated on 0.01% poly(n-lysine)-coated 35-mm dishes at a concentration of 1 × 106 cells/dish. Media consisted of 10% fetal bovine serum in Eagle's minimal essential media that contained 1% glutamine and 0.5% additional glucose. The purity of various neuronal and glial cells in the cultures was assayed by detecting plasma membrane receptors for tetanus toxin, surface galaetocerebroside, and cytoplasmic accumulation of glial fibrillary acid protein (12, 14, 16) . Neuronal cells have tetanus-toxin receptors but lack the glial markers of galactocerebroside and glial fibrillary acid protein (12) . Macrophages were obtained from peritoneal cavities of mice inoculated intraperitoneally with 2 ml of 3.8% thioglycollate broth. Peritoneal exudate cells harvested 5 d later were plated, and nonadherent cells removed i-2 h later after vigorous shaking. Details concerning stimulating, obtaining, and culturing these cells are published elsewhere (17, 18) . Adherent cells were macrophages, as judged by their ability to phagocytize zymosan particles, or sheep erythrocytes previously reacted with antibody a~d C5-defieient mouse sera, and by their morphology (19) . Homogeneity was usually >95% with a range of 85-98%.