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

Immunochemzcal Labeling afMHV Antigens. Cultured cells were fixed with 4% paraformaldehyde and 0.5% glutaraldehyde in cacodylate buffer, pH 7.3, incubated with phosphate-buffered saline containing 1% bovine serum albumin and subjected to one cycle of freeze-thawing in a cryo-protective medium composed of 25% sucrose and 10% glycerol as described earlier (20) , ~ or were used unfixed. For in vivo studies, tissues were obtained from mice perfused with 4% paraformaldehyde and 0.5% glutaraldehyde by the intracardiac route. Both uninfected and MHV-4 inoculated mice were analyzed. Perfused tissues were collected in petri dishes containing fixative (see above), and 8-14-/Lm sections were cut with an Oxford vibratome (Oxford Instruments Inc., Columbia, Md.). Thereafter, ceils or tissue sections were incubated with monospecific antibodies to purified MHV virions or MHV antigens initially prepared in rabbits (kindly supplied by Dr. K. Holmes, Uniformed Services University of the Health Sciences, Bethesda, Md.), or in mice kindly provided by the National Cancer Institute, Bethesda, Md. resource program, or generated by immunization in this laboratory. All reagents gave equivalent results, and specificity was determined by appropriate absorption and blocking studies (21, 22) . After 30 rain, cells or tissues were washed gently in buffer and stained with protein A conjugated with peroxidase or with antibody to mouse or rabbit IgG conjugated with fluorescein-isothio-cyanate as reported elsewhere (21, 22) . In some instances cultured macrophages or neurons were fixed with acid alcohol or with ether-alcohol as described (22) .