The frequency of lung cells from CD8-alpha knock-out mice containing cHV68 genome was determined using a previously described nested PCR assay (LD-PCR) with single-copy sensitivity to detect gene 50 of cHV68 [77] . Briefly, freshly isolated cells were counted, resupsended in isotonic solution, and then diluted in 10 4 uninfected NIH 3T12 cells prior to serial dilution plating. Plated cells were lysed overnight in proteinase K, followed by two rounds of nested PCR. Reactions were performed on 12 replicates per dilution per sample and products resolved on a 2% agarose gel and identified by ethidium bromide staining. PCR sensitivity was quantitated using 10, 1, or 0.5 copies of a gene 50 containing plasmid (pBamH I N) diluted in 10 4 uninfected NIH 3T12 cells.