Recombinant EBV (EBV-GFP) was generated from EBV-positive Burkitt lymphoma cells (EBV+Akata) and a cell-free infection was conducted19 (link),20 (link). EBV with GFP integrated was produced from Akata cells based on a previous study19 (link) with slight modifications. Briefly, Akata-EBV cells were crosslinked with 0.8% (v/v) goat anti-human IgG (Bersee) for 6 h; then the cells were cultured for 72 h with standard medium. EBV particles were collected by centrifugation at 54,000 × g for 2 h and resuspended with serum-free DMEM. The virus suspension was aliquoted in 0.5 ml aliquots and stored at −80 °C or used immediately for infection studies. For cell-free infection, NP69, HK1 and HEK293 were exposed to EBV for 3 h before detecting the number of copies of the viral genome. For detection of the infection efficiency, cells were cultured for an additional 72 h, after which the medium was replaced. The infection efficiency was roughly judged under a fluorescence microscope (Leica) and precisely measured using a flow cytometer (Spectral Cell Analyzer, Sony) on the basis of GFP expression.
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