Epstein-Barr Virus Stimulates Torque Teno Virus Replication: A Possible Relationship to Multiple Sclerosis

Borkosky, Silvia S.; Whitley, Corinna; Kopp-Schneider, Annette; zur Hausen, Harald; deVilliers, Ethel-Michele

EBV activity/replication was measured by quantifying mRNA production of a 200 bp fragment of the EBV envelope glycoprotein gene gp350/220 [79] . Primers and a hydrolysis probe used are listed in Table 1 . The RNA samples (5 ng) from the transfected cells were amplified using a one step RT-qPCR protocol (Applied Biosystems). Briefly, the RT-qPCR mixture contained: 12.0035 mml of Taqman Fast Virus 1-Step Master Mix, 1.5 ml each of forward and reverse primers (10 mM), 1.5 ml of 59FAM-39TAMRA-labelled probe (10 mM), 28 ml water and 5 ml RNA. An initial reverse transcription step (5 min at 50uC), was followed by RT inactivation/initial denaturation (20 s at 95uC). Reaction mixtures were subsequently amplified for 40 cycles (15 s at 95uC and 1 min at 60uC). Negative controls included NTC and samples without reverse transcriptase. Fluorescent signals were detected using an ABI 7300 sequence detection system (Applied Biosystems). EBV gp350/220 gene expression was quantified using an external RNA calibration standard which was generated as follows: The EBV late envelope glycoprotein gp350/220 gene was transcribed from B95-8 cellular RNA with SuperScript II reverse transcriptase (Invitrogen) using specific primers containing restriction sites for BamHI and EcoRI ( Table 1 ). The resulting 200 bp cDNA amplicon was cloned into the pSPT18 vector (SP6/T7 Trancription Kit, Roche) as described previously [79] . Sequence analysis verified the sequence. The cDNA-containing plasmid was linearized with EcoRI prior to subsequent in vitro transcription using SP6 RNA polymerase (SP6/T7 Trancription Kit, Roche). The size and quality of this transcript were determined by denaturing RNA electrophoresis. Serial dilutions (5-fold) of this 200 bp gp350/220 transcript in 5 ng BJAB RNA (EBV negative) in DNase/RNase free water were used to calibrate the EBV gp350/220 gene expression. Standards were stored in aliquots at 270uC for no longer than 1 week and each aliquot was used once.