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

Viral infections have been implicated in the pathogenesis of multiple sclerosis. Epstein-Barr virus (EBV) has frequently been investigated as a possible candidate and torque teno virus (TTV) has also been discussed in this context. Nevertheless, mechanistic aspects remain unresolved. We report viral replication, as measured by genome amplification, as well as quantitative PCR of two TTV-HD14 isolates isolated from multiple sclerosis brain in a series of EBV-positive and -negative lymphoblastoid and Burkitt's lymphoma cell lines. Our results demonstrate the replication of both transfected TTV genomes up to day 21 post transfection in all the evaluated cell lines. Quantitative amplification indicates statistically significant enhanced TTV replication in the EBV-positive cell lines, including the EBV-converted BJAB line, in comparison to the EBV-negative Burkitt's lymphoma cell line BJAB. This suggests a helper effect of EBV infections in the replication of TTV. The present study provides information on a possible interaction of EBV and TTV in the etiology and progression of multiple sclerosis

In vitro replication of TTV-HD14b and TTV-HD14c as measured by real-time quantitative PCR.

<p>Replication of (A) TTV-HD14b and (B) TTV-HD14c. qPCR values are expressed in ΔΔCt relative to BJAB (used as calibrator). Shown are mean ±95% confidence interval values of triplicate tests. A significant higher replication level of both TTV isolates was detected in all cell lines when compared to BJAB (p<%0.05).</p

Primers and probes used.

a<p>- F, forward; R, reverse; Pr, probe.</p>b<p>- Underlined letters indicate restriction sites.</p

In vitro replication of TTV-HD14b and TTV-HD14c as measured by long-PCR amplification.

<p>(A) PCR amplification of full-length TTV-HD14b in P3HR-1, BJAB, BJAB/EBV and full-length TTV-HD14c in ND1 and Ramos/EBV cell lines. Days after transfection are indicated. M - DNA size marker, C1 - untransfected cells, C2 - untransfected cells with nucleofector solution, <b>+</b> - positive control for long PCR amplification consisted either of re-ligated TTV-HD14b or TTV-HD14c mixed with Ramos cell line DNA. (B) Electron micrograph of B95-8 cells 3 days after transfection with linearized TTV-HD14b DNA. TTV-like particles can be seen within the nucleus of a cell. EBV particles are indicated by short arrows. Bar = 250 nm.</p

Quantification of EBV-genomes and -replication.

<p>Real-time qPCR in the EBV positive cell lines transfected with TTV-HD14c (day 14 post-transfection) was applied to determine the EBV genome copies per cell, as well as EBV replication as measured by gp350/220 mRNA.</p