Epstein-Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV) are human gammaherpesviruses. Gammaherpesviruses are known to be host-specific and cause lifelong infection. Macrophages are infected by gammaherpesviruses, but this process is poorly defined; it is unknown if they undergo lytic replication, latency, or some combination of these. Prior studies in the van Dyk lab utilizing MHV68, a murine model for gammaherpesviruses, shows efficient viral entry in both macrophage-like cells and permissive control fibroblasts. Permissive control fibroblasts are fully susceptible to infection done by MHV68. However, macrophage-like cells show a defect in subsequent viral replication and viral gene expression. Based on prior lab findings using bulk cell analyses, we wanted to know if viral gene expression is uniformly low/absent in infected macrophage-like cells or if there is a small population of cells with robust viral gene expression. We hypothesized that viral gene expression was contributed by a small percentage of macrophage-like cells when compared to permissive control fibroblast cells, in which most cells show abundant viral gene expression. To analyze viral gene expression, we used single-cell analysis of viral RNA expression through flow cytometric detection of RNA hybridization. We analyzed RNA expression in J774 macrophage-like cells versus 3T12 permissive fibroblasts through a RNA PrimeFlow. Through this RNA assay, we used RNA probes specific to cellular beta-actin (degraded in active virus replication), TMERs (viral ncRNAs that are abundantly expressed in lytic and latent infection), and ORF18 (viral late RNA only expressed in active lytic infection). We found that viral gene expression in J774 macrophages was low and that there was not a population of cells that showed abundant viral expression. The decrease in ORF18 signal was more pronounced in J774 macrophages than TMER signal, consistent with either a defect in viral replication/late gene expression or with latent infection in J774 macrophages. Caveats to our interpretation are the need for additional optimization of our infection and RNA PrimeFlow protocols since overall viral gene expression appeared to be low in both cell types. Nonetheless, this experiment demonstrated how single-cell analysis can shed greater light on bulk analysis, particularly in cases of heterogeneous responses within cell populations. Our data allows us to potentially develop vaccines that can prevent latent infection done by the gammaherpesvirus.