Coronavirus induction of class I major histocompatibility complex expression in murine astrocytes is virus strain specific

Detection oflnterferons. The possible presence oflFN in the supernatants of infected astrocytes was determined by bioassay in a vesicular stomatitis virus (VSV) neutralization assay using vital dye uptake as a spectrophotometrically measured endpoint in L929 cells (50, 51) . Supernatants were collected from infected and control uninfected astrocytes on day 3 and 7 p.i., UV irradiated, and added in triplicate to L929 cells plated at 10 s cells/well in 96-well plates. After a 24-h incubation at 37~ and 7% CO2, supernatants were aspirated and cells infected with VSV (Indiana strain) at approximately 10 tissue culture infectious doses (TCIDs0)/well. Control wells included uninfected L929 cells (cell control) and VSV-infected L929 cells cultured in the absence of interferon (virus control). At 24-36 h p.i., when CPE was maximum in the control wells, cells were stained for 2 h at 37~ with 0.004% neutral red. They were then lysed to release retained dye using 0.01 M HC1 in 30% ethanol, and ODs measured at 540 nm in an automated microELISA reader (Dynatech Laboratories, Inc., Chantilly, VA). IFN concentrations were deduced by comparison of sample ODs with those of standard concentrations of recombinant mouse IFN-3' (Genzyme Corp., Cambridge, MA) and murine IFN-c~//3 (Lee Biomolecular, San Diego, CA).