In Situ Detection of EpsteinBarr Virus and Phenotype Determination of EBV-Infected Cells

Epstein-Barr virus (EBV) establishes a lifelong infection of B cells. Consequently, EBV-carrying B cells are present in the peripheral blood as well as in lymphoid and nonlymphoid tissues of most individuals. As a result, the detection by polymerase chain reaction of EBV genomes in DNA extracts from tumor tissues does not permit conclusions as to the precise cellular source of the virus. For a meaningful analysis of EBV infection, it often is necessary to determine the cellular location of the virus using morphology-based techniques. In situ hybridization for the detection of the small EBVencoded RNAs (EBERs) has become the standard method for the detection of latent EBV infection. Owing to their abundance, the EBERs represent ideal targets for in situ hybridization using radiolabeled or nonradioactive probes. EBV has been detected in tumors of various lineages, and proliferation of nonneoplastic B cells may occur in the background of EBV-negative tumors. Thus, the assignment of EBV infection to a specific cell type may require double labeling techniques for the simultaneous detection of the virus and of cell lineage-specific gene products. Because of the heterogeneous composition of many EBV-associated tumors, gene expression analysis of EBV-infected cells in tissue sections also may require double labeling techniques. Here, methods are described for the In situ detection and phenotypic characterization of EBV-infected cells in the authors’ laboratories.