In order to study single viral gene functions in the context of genome and analysis interactions between one gene with another, the HEK293/p2089 stable cell line was established by transfecting the plasmid of DNA (p2089) into EBV-negative 293 cells and selecting for resistance against hygromycin. The plasmid p2089, which was kindly provided by Prof.Hammerschmidt, contained the whole EBV genome of wild-type B95-8. Two eukaryotic expression vectors (pcDNA3.1(+)/BZLF1 and pcDNA3.1(+)/BALF4) were constructed and then transiently cotransfected into the HEK293/p2089 stable cells, so as to induct EBV lytic replication and product recombinant EBV particles visualized through GFP-expressing. To estimate the EBV production, Raji cells were incubated with supernatants from the induced 293 cells carrying p2089 DNA, as revealed by indirect visualization of the Raji cells. GFP-positive cells were evaluated by inverted fluorescence microscope or FACS analysis. The different virus supernatants were quantified with the help of “green Raji units” per ml as an absolute number of infectious particles. This technique makes it possible for the reconstitution of viral progeny or mutants by transfection of BAC plasmid into eukaryotic cells, and any genetic modification in E. coli, thereby facilitating the analysis of viral gene functions in the context of genome. This new technique has provided a useful tool for the study of pathogenesis mechanism of EBV, especially for that of cancer-associated.