Kaposi's sarcoma-associated herpesvirus (KSHV) undergoes replication independently via latent and lytic pathways. Latent replication is mediated by latent-associated nuclear antigen (LANA), the sole viral trans element for genome maintenance and replication. According to previous studies, LANA tethers the KSHV genome to the host chromosome during latency and interacts with host factors to ensure proper latent replication. Studies using Southern blot experiments have revealed consistently that vector constructs containing the viral terminal repeat (TR) region as a cis element in latent replication are replicated in the presence of LANA. However, Southern blotting is a time-intensive, complicated technique that requires multiple reagents. In addition, it has a limited ability to detect slight changes in replication efficiency under different conditions owing to its relatively low sensitivity. In the current study, a real-time polymerase chain reaction method was developed for detecting transient KSHV replication and was found to be capable of further identifying several factors that affect latent replication. This technique should provide a useful tool for the detection of KSHV latent replication under various conditions, including overexpression of viral or cellular factors and chemical stimulation. (C) 2013 Elsevier B.V. All rights reserved.