The reduction in lytic reactivation frequency could either be a consequence of a decreased number of latently infected splenocytes or, alternatively, a failure of latently infected cells to undergo lytic reactivation. To distinguish these possibilities, we first monitored viral DNA load in the spleens of infected mice using a previously described qPCR assay for the glycoprotein B (gB) gene [35] and found that MHV68.DHS infected splenocytes harbor fewer viral genomes than splenocytes infected with MHV68.WT or MHV68.MR ( Figure 7C ). We also analyzed the frequency of splenocytes harboring the viral genome by limiting dilution PCR. Briefly, serial dilutions of splenocytes were plated in a 96 well plate and subjected to nested PCR for the viral ORF50 gene [36] . Spiked template controls confirmed that in this assay we were achieving near single copy sensitivity for the viral genome, with a very low rate of false positives (data not shown). While approximately one out of 200 splenocytes latently harbor the MHV68.WT or MHV68.MR genome, the frequency of MHV68.DHS genome harboring splenocytes was less than one out of 10,000 splenocytes ( Figure 7D ). Thus, while host shutoff appears dispensable for acute replication in the lung, it plays an important role in the downstream events leading to efficient latency establishment in the spleen.