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. 2009 Sep 28;206(10):2091–2099. doi: 10.1084/jem.20081761

Figure 4.

Figure 4.

EBER1 exists in sera from patients with active EBV infections and induces the production of type I IFN and inflammatory cytokines. (A) Detection of EBER1 by RT-PCR in patient sera. RNA was extracted from 100 µl sera or plasma from patients with IM, CAEBV, and EBV-HLH, and from EBV-positive and –negative healthy donors. EBER1 was detected by 35 cycles of RT-PCR. Sample numbers in A–C correspond to each other; samples that are followed by a number in an open circle contain higher amounts of EBER1 than those with a number alone. Three or more independent experiments were performed. (B) Quantification of EBER1 in patient sera. RNA was extracted from the sera and subjected to real-time RT-PCR to detect EBER1. Error bars indicate the SD of duplicate wells. The data presented are representative of three independent experiments. (C) Induction of IFN-β production by RNA extracted from patient sera. LCLs (4 × 105) were treated with RNA that had been extracted from 100 µl sera in 1 ml culture medium, incubated for 14 h, and subjected to 30 cycles of RT-PCR to detect IFN-β (left) or ELISA of the culture supernatants for detection of IFN-β (right). The data of ELISA are shown as the means ± SD of duplicate determination and representative results of three independent experiments are shown. (D) Effect of an anti-TLR3 antibody on serum-induced IFN-β production. LCLs (4 × 105) were preincubated with the anti-TLR3 antibody for 30 min at 37°C, before being treated with RNA extracted from 100 µl of serum from a patient with IM or 1.0 µg in vitro–synthesized EBER1 as a positive control in 1 ml culture medium, and cultured for 14 h. Production of IFN-β was determined by ELISA of the culture supernatants. Error bars indicate the S.D. of duplicate wells. The data presented are representative of three independent experiments. (E) Effect of the RNA from patients sera on the induction of IFN-β and proinflammatory cytokines. Human PBMCs (1 × 106) were treated with RNA extracted from 100 µl patients sera or 0.5 µg in vitro–synthesized EBER1 in 1 ml culture medium and cultured for 14 h. The induction of IFN-β and proinflammatory cytokines was determined by 30 cycles of RT-PCR. Three independent experiments were performed.