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

Transfection of 2 mg TTV-DNA into 5610 6 cells was performed by using electroporation (Nucleofector II transfection device, Amaxa Biosystems) and the Nucleotransfection kit V (cat# vco-101 Amaxa Biosystems) and following protocols recommended by the manufacturer for each respective cell type. Transfection efficiency was controlled for by parallel transfections with the plasmid pmaxGFP (Amaxa Biosystems). Negative controls of each cell line with and without nucleofector solution were included. Cells were transfected for harvesting on days 3, 7 and 10, whereas transfected cells harvested at days 14, 17 and 21 were passaged from transfected cultures for day 7, 10 and 14, respectively. Cells were passaged at 7 day intervals and fresh medium added when cells became too dense during this interval. Transfection assays were performed in parallel by 2 of us (S.B. and C.W.). Cultures were harvested on days 3, 7, 10, 14, 17 and 21 after transfection at an average density of 1-1,5610 6 /ml and total DNA and RNA isolated. Total DNA was extracted using phenolchloroform-isoamylalcohol [65] and subjected to DpnI (Fermentas) digestion to remove input DNA used for transfection. Total RNA was isolated from the transfected cells using the RNeasy mini kit (Qiagen, Hilden, Germany). All RNA samples were treated with DNaseI (Promega) to remove any residual DNA. The quality of the RNA samples was checked by running the samples in a 1% agarose gel.