The frequency with which cells latently harbor the viral genome was determined as described previously [36] . Briefly, 4-fold serial dilutions of splenocytes were plated in 96-well plates with 16 wells per dilution. Nested PCR for the ORF50 gene was performed and the resulting PCR product was run on an agarose gel. Wells positive for a PCR product by ethidium bromide staining contain at least one copy of the viral genome. Single-copy sensitivity was confirmed using serial dilutions of MHV68 BAC DNA.