Supplementary Materials and Methods
Generation of radioactive probes
The plasmids pBSJJJ1 and pBSJJJ2 containing EBER1- and EBER2-specific fragments,
respectively, were a kind gift of Prof. Gerald Niedobitek, Berlin, Germany. Single stranded RNA
probes were synthesized by in vitro transcription of EBER1 and EBER2 sequences cloned in pBSK
vectors. By choosing the appropriate direction of transcription, antisense or sense, RNA strands
were synthesized. Plasmids used in transcription reactions were first linearized and then
resuspended in distilled water at a concentration of 1 µg/µl. Labelled probes were obtained as
following: 12.5 µl of αP33 UTP (PerkinElmer) were dried in Speed-Vac centrifuge and incubated at
room temperature overnight with: 1 µg of linearized DNA, 4 µl of 5X transcription buffer, 2µl of
DTT (0,1 M) (Promega), 3 µl rNTPs mix (rATP, rCTP, rGTP, 3,3 mM each), 1 µl of RNA-inhibitor
(40 U/µl) (Promega), 1µl of RNA polymerase (20 U/µl), and H2O up to a final volume of 20 µl. On
the following day probes were incubated with 1 µl of RNAse-free DNAse (40U/µl) (Promega) at
37°C for 30 minutes. The reaction was stopped by adding 1 µl of 0.5 M EDTA and the synthetized
RNA riboprobes purified on Mini Quick Spin RNA column (Roche Applied Sciences) according to
the manufacturer’s instructions. Counts per minute of 1 of 20 µl of the total reaction were measured
in the beta counter (Coulter). Probes were stored at – 80°C.
Radioactive in situ hybridization
Ten μm-tick brain and lymphoid tissue cryosections and deparaffinized sections of the infectious
mononucleosis tonsil were post-fixed for 15 min with 4% paraformaldehyde (Sigma-Aldrich) and
washed three times in PBS. Paraffin sections were treated twice for 15 min with toluene before the
ethanol series. After washing in saline sections were incubated in 0.5 µg/ml of Proteinase K (Roche
Applied Sciences) in 100 mM Tris-HCl (pH 8), 50 mM EDTA for 10 min at 30°C. Upon
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permeabilization, sections were treated with 4% paraformaldehyde for 15 min, washed three times
in PBS and then in H2O, incubated in Triethanolamine (Sigma-Aldrich) 0.1 M (pH 8) for 5 min.
Then, 400 μl of acetic anhydride (Sigma-Aldrich) was added two times for 5 min each. Finally,
sections were rinsed in H2O for 2 min and air-dried.
Hybridization was performed by incubating sections with αP33-labelled riboprobes overnight at
60°C at a concentration of 107 cpm/slide. On the following day, sections were rinsed in 5 X SSC for
5 min, washed in 50% formamide (Sigma-Aldrich), 2X SSC for 30 min at 60°C, and incubated in
20 µg/ml Ribonuclease-A (Roche Applied Sciences) in 0.5 M NaCl, 10 mM Tris-HCl (pH 8), 5 mM
EDTA for 30 min at 37°C. Sections were washed in 50% formamide, 2X SSC for 30 min at 60°C,
rinsed two times in 2X SSC and finally dried through the ethanol series. LM-1 emulsion (GE
Healthcare) was applied in dark room, according to the manufacturer’s instructions. After 5, 9 or 18
days, sections were developed in dark room using D19-developer and fixer solutions (Kodak) and
mounted with DPX (BDH). Sections were counterstained with hematoxylin or 4',6-diamidino-2phenylindole (DAPI; Sigma-Aldrich). Sections were analyzed using Olympus Bx-51 microscope.
Legend to Supplementary Figure
In situ hybridization with radiolabelled and PNA EBER probes in control tissues.
Independent ISH experiments were performed with pooled αP33-labelled EBER1 and EBER2
antisense (as) probes (A, C, G: darkfield images with DAPI counterstain; E: brightfield image with
hematoxylin counterstain) and PNA EBER1 and EBER2 antisense probes (B, D, H; brightfield
images with hematoxylin counterstain) in adjacent sections of control lymphoid and brain tissues. a
EBER+ cells are present at very high density in the tonsil of a patient with infection mononucleosis
(A, B). Scattered EBER+ cells are present in a control abdominal lymph node (C-E; the inset in E
shows one EBER+ cell in the lymph node at higher magnification), while no EBER+ cells are
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detected in a non pathological brain from a patient dying for cardiac failure (G, H). ISH performed
with αP33-labelled EBER1 and EBER2 sense (s) probes (inset in A, F) and PNA irrelevant probe
(inset in B) yields no signal. Bars: 200 μm in A, B and insets; 100 μm in C-H, 10 μm in the inset in
E.
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