FIG. 5.

LMP-1 sequence flanking Met129 codon from viral DNA from healthy seropositive donors. A region of the LMP-1 ORF was amplified by nested PCR from viral DNA isolated from tonsils using primer sets b and c (Fig. 1; Table 1). A portion of the sequence obtained from the PCR products is shown and corresponds to EBV coordinates 168,971 to 168,835 of B95-8 virus. Individual donors are designated by the numbers to the left of each sequence. All else is as described in the legend to Fig. 3. Total genomic DNA was prepared from hyperplastic tonsils of 37 EBV-seropositive children from Switzerland as described in the text, using a QIAamp blood kit following the manufacturer's instructions (Qiagen, Inc.) (4). PCRs using primer set b (Fig. 1; Table 1) were performed on the Perkin-Elmer Gene Amp system 9700 with the following conditions for the primary PCR: 1 cycle of 93°C for 10 min; 2 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 2 min; 3 cycles of 94°C for 30 s, 66°C for 30 s, 72°C for 2 min; 25 cycles of 94°C for 30 s, 64°C for 30 s, 72°C for 90 s; 1 cycle of 72°C for 10 min. The second round of PCR was performed using the following conditions: 1 cycle of 93°C for 10 min; 5 cycles of 94°C for 30 s, 63°C for 30 s, 72°C for 2 min; 25 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 90 s; 1 cycle of 72°C for 10 min. Cycle sequencing was performed using primer set c (Fig. 1; Table 1) with the Perkin-Elmer Gene Amp system 9700 and ABI Prism 310.