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

Having measured varying levels of TTV replication in the respective EBV positive cell lines, we quantified the EBV DNA to determine whether EBV copy number exerted an influence. Realtime quantitative PCR was performed at all time points (day 3-21 post transfection) and in all cultures transfected with both TTV genomes using DNA from an EBV-harbouring (2 copies/cell) cell line Namalwa as standard curve [75] . A representative example of EBV copy number/cell at day 14 post-transfection of TTV-HD14c is presented in Figure 3 . The EBV copy number in the P3HR-1 cell line was 583 copies/cell, whereas B95-8 cells harboured 161 EBV copies/cell. Interestingly, BJAB/EBV cells habor only 10 EBV copies/cell despite the high levels of TTV amplification. The lowest EBV copy number was found in Ramos/EBV containing from 2 EBV copies/cell. From this analysis we can conclude that although the presence of EBV seems to enhance replication of TTV-HD DNA, the EBV viral copy number does not reflect the differences in replication level between cell lines. EBV production measured by gp350/220 expression is consistent with the EBV copy number and does not correlate with TTV replication Quantification of EBV DNA genome does not provide information regarding the intracellular activity of the EBV DNA. P3HR-1 and B95-8 are well-characterized cell lines in which EBV production has been reported [76, 77] , whereas EBV viral capsid antigen (VCA) and synthesis was demonstrated in the BJAB/EBV cell line [78] . The actual activity of the EBV DNA is less clear in the Ramos/EBV cell line and unknown for the ND1 cell line. We therefore quantified the EBV envelope glycoprotein gp350/220 mRNA as marker for EBV lytic activity in all EBV positive cell lines used in this study [79] . The results obtained showed a similar pattern as the EBV genome quantification. For comparison of the EBV DNA copy number, the RNA quantification presented in Figure 3 represents day 14 after transfection of TTV-HD14c. B95-8 and P3HR-1 showed the highest EBV-gp350/220 copy numbers of 48268 and 25018 copies/5 ng of total cellular RNA, respectively (Figure 3 ). ND1 cells harboured 8141 EBV-gp350/220 copies/5 ng RNA, followed by BJAB/EBV and Ramos/EBV with 965 and 240 copies/5 ng total RNA, respectively. Consistent results were obtained with both TTV isolates and with different biological replicates. Thus, EBV was replicating (as measured by gp350/220 gene transcription) in all EBV-positive cell lines used in this study, but this productivity did not necessarily relate to the EBV copy number/cell. The level of this EBV activity however does not seem to influence the level of TTV-HD replication.