Establishment and Quantification of <em>De Novo</em> Lytic Infection by Cell-free Kaposi's Sarcoma-Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV), a gammaherpesvirus implicated in multiple human malignancies, can undergo lytic replication during primary infection, a process that contributes to viral dissemination, immune evasion, and disease pathogenesis. However, the lack of robust in vitro systems for de novo lytic infection has limited insights into early infection events. Here, we present a tractable protocol that employs human colorectal cancer HCT 116 cells as targets for infection with cell-free virions derived from KSHV bacterial artificial chromosome 16 (BAC16)-reactivated iSLK producer cells. This model recapitulates key steps in primary infection, including viral entry, genome delivery, lytic gene expression, and progeny production. Infection is synchronized via spinoculation and monitored temporally. Virus production is quantified using a dual readout system comprising green fluorescence protein (GFP)-based infectious unit (IU) assays and qPCR of encapsidated viral DNA. These approaches enable detailed analysis of viral replication kinetics. While the current protocol focuses on the detection of late-stage events, the system is adaptable for studying early phases of infection through modified sampling time points. By offering a reproducible, scalable platform for productive de novo infection, this system addresses a methodological gap in KSHV research and supports mechanistic studies of herpesvirus-host interaction and antiviral strategies.