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

Two sets of primers and the respective hydrolysis probes were designed on the TTV-HD14b and TTV-HD14c genomic region spanning nucleotide 1 to 430 (identical between isolates). Primers and probes for both TTV-HD14b and TTV-HD14c were obtained from Biomers.net. The two sets (qP31F-qP133R-qP113Pr and qP326F-qP430R-qP396Pr) were separated by 192 nucleotides. Genomic DNA samples (100 ng in 5 ml) were amplified in triplicate, each in a total volume of 20 ml containing 12.5 ml Taqman Universal master mix (Applied Biosystems), 0.5 ml forward primer (10 mM), 0.75 ml reverse primer (20 mM), 0.63 ml 59FAM-39TAMRA-labelled probe (10 mM) and 0.62 ml water. Reaction mixtures were amplified for 40 cycles (15 s at 95uC and 1 min at 60uC) after an initial activation of the DNA polymerase for 10 min at 95uC. Fluorescent signals were detected in an ABI 7300 sequence detection system (Applied Biosystems). Results for each time point (day 3, 7, 10, 14, 17 and 21) were standardized against the amplification of TTV-HD14b and TTV-HD14c in the EBV-negative BJAB cell line and expressed as fold change values. DNA samples obtained from untransfected cells, as well as nontemplate controls (NTC) were included in each reaction plate.