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

The quantitative analyses performed with one set of primers and probe for TTV-HD14b and TTV-HD14c are shown in Figure 2 (A and B) . Overall, TTV-HD replication was increased up to day 21 in all cell lines tested when compared to viral replication in BJAB. Comparable results were obtained with the second primer pair and probe selected for this study (data not shown) and a similar pattern of amplification was obtained with different biological replicates. Untransfected control and nontemplate control samples remained negative in all experiments. The presence of EBV in cell lines BJAB/EBV, P3HR-1 and Ramos/EBV resulted in elevated levels of TTV-HD14b and TTV-HD14c DNA replication. Replication of both TTV-HD14 isolates was significantly higher (p,0.05) in all cell lines when compared to BJAB at each time point of evaluation ( Figure 2 ). From day 7 onwards, a higher level of TTV-HD14b replication ( Figure 2A ) was noted in the BJAB cell line converted to EBVpositivity, than in the other cell lines. Viral levels seemed to increase early after transfection, after which it levelled off. Replication of TTV-HD14b versus TTV-HD14c varied significantly (p,0.05) in each cell type, except for day 17 after transfection where replication differences between isolates in Ramos/EBV and B95-8 were not statistically significant, as well as day 3 where replication of the two TTV isolates did not differ significantly. TTV-HD14c replication in ND1 (EBV-immortalized B cells from an MS patient) ( Figure 2B ) seemed to be stable over time in comparison to a decrease for TTV-HD14b replication in these cells. An initial increase in B95-8 decreases more rapidly for TTV-HD14c than for TTV-HD14b. TTV replication behaviour in the EBV-positive Ramos/EBV cell line was surprising. The increased TTV-HD14b replication in Ramos/EBV versus Ramos cells was statistically significant (p,0.0001) up to day 14, where after TTV-HD14b replication in Ramos cells exceeded that in Ramos/EBV cells (days 17 and 21). TTV-HD14c replication in Ramos/EBV cells increased from day 3 to day 17, with levels reaching statistical significance for day 3 to 7 (p,0.0001), but not for days 14 and 17. Replication on day 21 was similar in both isolates with higher replication in Ramos than in Ramos/EBV cells. A previous report did not detect any difference in the level of permissiveness for an HSV-1 infection between Ramos and EBVconverted Ramos/EBV cells [74] .