EBV and KSHV open reading frames (ORFs) were cloned and expressed as previously described [25] . Briefly, the open reading frames were polymerase chain reaction (PCR) amplified using primers based on the GenBank sequences V01555, AJ507799, and U756981. The bacterial artificial chromosome plasmids Akata BXI (EBV) and BAC36 (KSHV) [26] were used as templates (gifts from L. Hutt-Fletcher and S-J Gao). PCR products were cloned into the vector pDONR201. ORFs representing different virus strains were amplified directly from clinical samples or from cell lines. Proteins containing extensive repeats that limited expression in yeast (eg, EBNA1 and LANA) were amplified as N-terminal and C-terminal fragments. Escherichia coli bacteria were transformed with the reaction products and DNA prepared for sequence verification. Correct ORF-containing plasmid DNAs were then moved into a yeast destination vector, pEGH-A, a derivative of the yeast glutathione S-transferase (GST) vector, pEGH. Yeast cultures were induced with 2% galactose, and GST fusion proteins were isolated and purified on glutathione beads. One hundred and seventy-four appropriately sized EBV and KSHV proteins (Table S1 ; available online) were successfully purified based on immunoblot analysis using anti-GST antibody and were printed using a 48-pin contact printer (Bio-Rad) in duplicate on modified glass (Full Moon Biosystems) microscope slides along with controls. Each slide had 784 spots: 348 EBV and KSHV proteins, 112 control proteins (Table S1 ; available online), and 324 blank spots. The controls were used for orientation and to detect nonspecific interaction with proteins such as the GST fusion partner. Protein arrays were stored at 280°C.