24Virus-host interactions are frequently studied in bulk cell populations, obscuring 25 cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at 26 the single-cell level, combining a sensitive and robust fluorescent in situ hybridization 27 platform with multiparameter flow cytometry, to study the expression of 28 gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection 29 and reactivation in vitro. This method allowed robust detection of viral ncRNAs of 30 murine gammaherpesvirus 68 (γHV68), Kaposi's sarcoma associated herpesvirus and 31 Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying 32 the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA 33 regulation during γHV68 infection, we find heterogeneous and asynchronous gene 34 expression during latency and reactivation, with reactivation from latency identified by a 35 distinct gene expression profile within rare cells. Further, during lytic replication with 36 γHV68, we find many cells have limited viral gene expression, with only a fraction of 37 cells showing robust gene expression, dynamic RNA localization, and progressive 38 infection. Lytic viral gene expression was enhanced in primary fibroblasts and by 39 conditions associated with enhanced viral replication, with multiple subpopulations of 40 cells present in even highly permissive infection conditions. These findings, powered by 41 single-cell analysis integrated with automated clustering algorithms, suggest inefficient 42 or abortive γHV infection in many cells, and identify substantial heterogeneity in viral 43 gene expression at the single-cell level. 44 45 3 AUTHOR SUMMARY 46 The gammaherpesviruses are a group of DNA tumor viruses that establish 47 The Herpesviridae are a family of large dsDNA viruses that include multiple 63 prominent human and animal pathogens [1]. Although these viruses infect different cell 64 types, and are associated with diverse pathologies, they share conserved genes and 65 two fundamental phases of infection: lytic replication and latent infection [1]. Lytic 66 replication is characterized by a cascade of viral gene expression, active viral DNA 67 replication and the production of infectious virions. Conversely, latency is characterized 68 4 by limited viral gene expression and the absence of de novo viral replication. While 69 latent infection is a relatively quiescent form of infection, the herpesviruses can 70 reactivate from latency, to reinitiate lytic replication. 71 Among the herpesviruses, the gammaherpesviruses (γHV) are lymphotropic 72 viruses that include the human pathogens Epstein-Barr virus (EBV) [2] and Kaposi's 73 sarcoma associated herpesvirus (KSHV) [3]. Murine gammaherpesvirus 68 (γHV68, or 74 MHV-68; ICTV nomenclature Murid herpesvirus 4, MuHV-4), is a well-described small 75 animal model for the γHVs [4]. While these viruses establish a lifelong infection that is 76 often clinically inapparent, immune-suppressed individuals are part...