PROJECT SUMMARY/ABSTRACT Viruses cause an array of disease manifestations ranging from acute respiratory disease, intestinal diarrhea, hemorrhagic fevers, hepatitis, cancer, chronic autoimmune disease and in some instances death. In 1995, human herpesvirus 8(HHV8)/Kaposi’s sarcoma-associated herpesvirus (KSHV) was identified and hypothesized to be associated with cancer in humans. Since then, an accumulation of scientific evidence substantiates KSHV as the etiological agent responsible for classical and acquired immunodeficiency disease syndrome (AIDS)- related Kaposi’s sarcoma (KS), as well as other lymphoproliferative disorders (LPDs) in immunocompetent and human immunodeficiency virus (HIV)-infected humans. Despite all the scientific evidence it is difficult to fully understand how the virus causes disease without the ability to follow a natural infection. As such, alternative in vivo models that are readily accessible and can recapitulate KSHV infection and associated disease are absolutely needed to identify viral determinants of pathogenesis and how these specific determinants, either viral open reading frames (ORFs) or other viral-encoded macromolecules, are involved in KSHV-mediated pathogenesis. Here, we propose to utilize a closely related and relevant virus that can manifest similar biological outcomes in its natural host. The genome of the virus, rhesus rhadinovirus (RRV), has been characterized and shown to be essentially colinear and encode most of the viral ORFs thought to be associated with pathogenesis. More importantly, in vivo studies show that RRV infection of its natural host recapitulates many, if not most, of the properties of KSHV, including persistence and LPDs. The long-term goals of the proposed research project are to elucidate how KSHV interacts with its host, utilizing RRV and experimental infection of its host. This will be accomplished by generating novel RRV recombinants followed by a series of in vitro analyses to characterize the recombinants, and finally experimental in vivo infections and characterization of the host immune response to recombinant virus infection. Importantly, in vitro samples and in vivo samples will be molecularly analyzed by single-cell RNA sequencing, total RNA sequencing and Nanostring Technologies to define molecular and cellular pathways affected by the unique viruses. Combining these types of in vitro and in vivo experiments to address viral pathogenesis will enable researchers to dissect how viruses cause infection and disease in susceptible populations and how scientists can shift the pendulum in favor of the host.