Cells and clinical samples
The EBV-positive cell line Akata (in a latent cycle) was cultured in RPMI 1640 medium (Gibco BRL) supplemented with 10% v/v heat-inactivated fetal calf serum and 2 mM L-glutamine (Life Technologies). Reactivation in Akata cells to induce a replicative cycle was induced by 7.5 µg/mL polyclonal rabbit anti-human IgG (A0423, Dako), in 0.5% fetal calf serum supplemented medium. Intracellular DNA was collected after 24 h of treatment and directly quantified by a methyl-sensitive qPCR.
The clinical samples were as follows: EBV-positive saliva from healthy subjects (n = 10), and whole blood from patients with EBV primary infection (n = 9), from patients with confirmed PTLD post HSCT (n = 8) and from patients with high EBV VL (> 5000 UI/mL) post-HSCT but no proof of PTLD (n = 5).
Genomic DNA extraction
Total genomic DNA was extracted and purified using DNeasy Blood and Tissue Kit (QIAGEN) for EBV-positive cell lines and saliva, and Blood and cell culture DNA Mini Kit (QIAGEN) for whole blood and plasma, according to the manufacturer’s instructions.
EBV regions of interest
The relative methylated/unmethylated status of specific sites on viral DNA can be used to quantify the relative proportion of latent vs nonreplicated/virion lytic DNA. A set of four regions (designed as BZLF1, BALF5, LF2 and BDLF2) comprising CCGG sequences were identified from previous work by Fernandez and colleagues [10] that were methylated on latent genomes and unmethylated in lytically replicated DNA. MspI and HpaII are isoschizomers with differing sensitivities to CpG methylation. MspI cleaves both unmethylated and methylated CCGG sites, whereas HpaII cleaves only the unmethylated form of the restriction site. Specific primers were designed around each region of interest to amplify DNA following incubation with each enzyme (see Additional file 1: Figure S1).
Methyl-sensitive qPCR
Total DNA was subjected to digestion, at 37 °C for 16 h in 1X Cut Smart buffer (Biolabs) with either 100 units of HpaII (R0171M, Biolabs) or 100 units of MspI (R0106M, Biolabs) [11], therefore providing a positive control (Additional file 1: Figure S1A). Then, the four regions of interest were quantified by real-time PCR using TaqPath QPCR (ThermoFisher) with specific primers (Additional file 1: Figures S1B, S2). Cellular ß-globin was co-amplified as a positive control and to allow normalization of EBV DNA quantitation. Reactions were performed on a Biorad CFX96 Touch Real-Time PCR machine. The methylation index was defined according to the 2−∆∆Ct method proposed by Livak and Schmittgen [12]. The methylation index is equal to 2∆∆CT, where ∆∆CT is equal to ∆CTuntreated (cycle threshold [CT] of the EBV target gene minus CT of the ß-globin reference gene measured on the untreated sample) minus ∆CTtreated (CT of the EBV target gene minus CT of the ß-globin reference gene measured on HpaII treated sample). Therefore, the methylation index is equal to 2(∆CTuntreated−∆CTtreated). When the target is methylated, HpaII treatment has no effect and the methylation index is close to 1. When the target is demethylated, i.e., accessible to degradation by HpaII, 2∆∆CT tends to 0. For reasons related to experimental variations in CT measurement, the methylation index may occasionally be higher than 1 for highly methylated targets. Since MspI is degrading both methylated and unmethylated target, 2∆∆CT should always be close to 0 for MspI treated samples, providing evidence that the HpaII/MspI restriction site is present in the target EBV sequence (Additional file 1: Figure S1A).
