Báo cáo khoa học: Construction and biological activity of a full-length molecular clone of human Torque teno virus (TTV) genotype 6 pptx

Construction and biological activity of a full-length
molecular clone of human Torque teno virus (TTV)
genotype 6
Laura Kakkola
1
, Johanna Tommiska
1
, Linda C. L. Boele
2
, Simo Miettinen
1
, Tea Blom
1
,
Tuija Kekarainen
2
, Jianming Qiu
3
, David Pintel
3
, Rob C. Hoeben
2
, Klaus Hedman
1
and Maria So
¨
derlund-Venermo
1
1 Department of Virology, Haartman Institute and Helsinki University Central Hospital, University of Helsinki, Finland
2 Department of Molecular Cell Biology, Leiden University Medical Center, the Netherlands
3 Department of Molecular Microbiology and Immunology, University of Missouri–Columbia, Life Sciences Center, Columbia, MO, USA

TT-virus (TTV) recently named Torque teno virus [1]
was found in 1997 in Japan from a patient with post-
transfusion hepatitis of unknown etiology [2]. The
virus is non-enveloped and contains a single-stranded
circular DNA genome of approximately 3.8 kb [3,4].
To date, five major phylogenetic groups have been
defined [1]. Due to its genome organization and struc-
ture, TTV resembles the chicken anemia virus (CAV)
of the Circoviridae family. This family of veterinary
viruses comprises the genus Gyrovirus, including CAV,
and the genus Circovirus, including the porcine circo-
virus (PCV) and the beak and feather disease virus of
birds. The human TT-virus is currently classified as a
member of a new, floating genus, Anellovirus [1].
The TTV genome consists of an approximately
2.6 kb coding and an approximately 1.2 kb noncoding
region. The latter contains a GC-rich area, a promoter
and transcriptional enhancer elements [3,5–8]. The
transcriptional capacity of the minute viral genome is
greatly expanded by splicing [9,10], resulting in six dis-
tinct yet partially overlapping viral proteins [11]. Little
is known of their functions. However, the longest gene,
Keywords
Anellovirus; replication; Torque teno virus;
transcription
Correspondence
L. Kakkola, Department of Virology,
Haartman Institute, Haartmaninkatu 3,
PO Box 21, University of Helsinki,
FIN-00014, Finland

Fax: +358 9 19126491
Tel: +358 9 19126676
E-mail: laura.kakkola@helsinki.fi
Website: sinki.fi/english
Note
Nucleotide sequence data are available in
the DDBJ ⁄ EMBL ⁄ GenBank databases under
the accession number AY666122
(Received 11 April 2007, revised 10 July
2007, accepted 11 July 2007)
doi:10.1111/j.1742-4658.2007.06020.x
Torque teno virus (TTV) is a non-enveloped human virus with a circular
negative-sense (approximately 3800 nucleotides) ssDNA genome. TTV
resembles in genome organization the chicken anemia virus, the animal
pathogen of the Circoviridae family, and is currently classified as a member
of a new, floating genus, Anellovirus. Molecular and cell biological research
on TTV has been restricted by the lack of permissive cell lines and func-
tional, replication-competent plasmid clones. In order to examine the key
biological activities (i.e. RNA transcription and DNA replication) of this
still poorly characterized ssDNA virus, we cloned the full-length genome of
TTV genotype 6 and transfected it into cells of several types. TTV mRNA
transcription was detected by RT-PCR in all the cell types: KU812Ep6,
Cos-1, 293, 293T, Chang liver, Huh7 and UT7 ⁄ Epo-S1. Replicating
TTV DNA was detected in the latter five cell types by a DpnI-based restric-
tion enzyme method coupled with Southern analysis, a novel approach to
assess TTV DNA replication. The replicating full-length clone, the cell lines
found to support TTV replication, and the methods presented here will
facilitate the elucidation of the molecular biology and the life cycle of this
recently identified human virus.
Abbreviations

CAV, chicken anemia virus; DIG, digoxigenin; PBMC, peripheral blood mononuclear cells; PCV, porcine circovirus; TTV, Torque teno virus.
FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS 4719
ORF1, is assumed to encode a capsid protein that may
also participate in DNA replication [3,12] as is the case
with CAV [13].
The infection mechanisms and pathogenicity of TTV
are unknown. Putative replicative forms of TTV DNA
have been found in peripheral blood mononuclear cells
(PBMC), bone marrow and liver [14–16], suggesting
replication at these sites. Low-level infectivity of TTV
has been shown in activated PBMC and in a few
human cell lines [17–19]. The TTV promoter has been
shown to be active in both human [5,8,11] and non-
human cells [8,9]. TTV mRNAs have been detected in
human PBMC [19], bone marrow [10] and several
other organs [20]. However, the main host cells and
the target organs of this virus are still undefined.
The study of the biological functions of TTV is par-
ticularly challenging. Replication does not appear to
be very efficient in primary cells [17–19], nor in the few
cell lines supporting virus growth [17]. On the other
hand, the veterinary circoviruses CAV and PCV have
been studied successfully with infectious plasmid clones
[21–24]. With a full-length TTV plasmid clone of geno-
type 1, RNA transcription and splicing was studied in
Cos-1 cells. However, neither DNA replication, nor
cell permissiveness was demonstrated [9].
The present study aimed to construct a full-length
TTV clone that can be used as a tool for exploring the
viral determinants important in virus–cell interactions,

and to find permissive cell lines for further molecular
and cell biological studies of this peculiar human virus.
For this purpose, we have cloned and sequenced the
full-length genome of TTV genotype 6, and used it to
detect the key biological functions (i.e. RNA transcrip-
tion and DNA replication) in a number of different
cell lines.
Results
Genotype 6 cloning and sequence analysis
A full length molecular clone, pTTV, in plasmid pST-
Blue-1 was constructed of TTV genotype 6 (Fig. 1A).
The cloned genome was sequenced (GenBank acces-
sion number AY666122; nucleotide numbering accord-
ingly), and found to be 3748 nucleotides in length. A
TATA-box (TATAA) was located at nucleotides 83–87
and a poly(A) sequence ATTAAA at nucleotides
2978–2983. A GC-rich area was 107 nucleotides in
length. In the present study, two forms of the full-
length clone were used for transfection experiments;
the excised linear genome (linTTV) and the intact plas-
mid pTTV (Fig. 1B,C). The full-length plasmid clone
contains at its left end the NG136 primer sequence [25]
in which two nucleotides differ from the in vivo geno-
type 6 sequence. However, the BspEI-excised linear
construct excludes these primer-derived nucleotides.
Production of TTV RNA
Prior to the studies, all the cells were tested and found
to be TTV DNA negative by generic UTR-PCR and
by genotype 6 specific PCR.
All seven cell lines were analyzed with RT-PCR and

were found to produce identical TTV RNA upon
transfection with either pTTV or linTTV. In RT-PCR
analysis of the TTV clone-transfected cells, two ampli-
cons were observed (Fig. 2): one from the spliced
TTV mRNA (454 bp) and the other from TTV DNA
(555 bp). RNase treatment prior to RT-PCR abolished
the 454 bp amplicon; and DNase treatment abolished
the 555 bp but not the 454 bp amplicon (Fig. 2D). In
addition, RT-PCR without the RT step, and RT-PCR
of the input DNA constructs, yielded only the 555 bp
amplicon (Fig. 2D). Furthermore, the sequence data of
both amplicons showed that in the 454 bp amplicon
the intron had, indeed, been spliced out. These experi-
ments substantiated that the 454 bp amplicon origi-
nated from the transcribed viral RNA and not from
DNA. The TTV RNA was shown by RT-PCR to per-
sist in subcultured cells for at least 11 days. Nontrans-
fected cells and cells transfected with the backbone
plasmid pSTBlue-1, remained negative for TTV RNA
(Fig. 2B,C) confirming the absence of endogenous,
transcriptionally active TTV. RT-PCR of retinoblas-
toma mRNA yielded the expected amplicons (data not
shown) demonstrating mRNA integrity. The results
were identical for all the cell lines.
Replication of TTV DNA
All seven cell lines were transfected with linTTV and
with the intact pTTV. For detection of TTV replication,
total DNA from the transfected cells was treated with
the restriction enzymes BamHI and DpnI, and subjected
to Southern analysis. Two different probes were used

(Fig. 1): the one labelled with
32
P differentiates by size
the replicating TTV DNA from the input; and the other
labelled with digoxigenin (DIG) additionally documents
the susceptibility of the input DNA to DpnI.
In cells transfected with linTTV, the input DNA (after
BamHI digestion) was seen with the DIG-probe as a
3004-bp fragment (Figs 1B and 3A,B, marked with filled
circles), which was further digested by DpnI into a
fragment of 2162 bp (Figs 1B and 3A,B, marked with
filled squares). On day 3 post transfection, a full-
length TTV DNA of 3748 bp (after BamHI digestion,
Biological activity of a full-length TTV DNA clone L. Kakkola et al.
4720 FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS
detected with either probe) emerged in 293T, Huh7 and
UT7 ⁄ Epo-S1 cells, and less pronounced also in 293 and
Chang liver cells (Fig. 3A,C, marked with an arrow).
However, no such bands appeared in KU812Ep6 and
Cos-1 cells. That BamHI digestion yielded a full-length
fragment indicates that circularization of the input lin-
ear construct had occurred. Furthermore, this 3748 bp
fragment was resistant to DpnI (Fig. 3A,C, marked with
an arrow), indicating TTV DNA replication. The linear-
ized backbone plasmid, not separated from the excised
linear TTV construct, did not replicate in these cells. As
an additional specificity control for the DpnI assay,
three (HindIII, EcoNI or ScaI) restriction enzymes,
other than BamHI, were used in Southern analysis,
resulting in identical DpnI-resistant bands on day 3. The

DpnI-resistant DNA progressively accumulated in the
transfected cells from day 0 to day 3, as shown for 293T
cells in Fig. 4. However, on days 3, 5 and 8–10 post
transfection upon cell passage, the amount of DpnI-
resistant (replicating) TTV DNA declined, and was
detectable in Southern analyses for up to day 5. Interest-
ingly, in those cells that permitted replication of the
excised linear construct, high molecular weight double
bands (sensitive to DpnI) were visible (Fig. 3). Single-
stranded DNA (ssDNA) could, however, not be visual-
ized by Southern analysis, suggesting that its production
remained below the detection limit. Of note, when com-
paring the 293 cells, with and without the SV40 large
T antigen, the amount of DpnI-resistant replicating
TTV DNA detected on day 3 post transfection (relative
to a standard amount of total cellular DNA) was invari-
ably much lower in 293 than in 293T cells (Fig. 3A, and
more pronounced in Fig. 3C), suggesting a possible
helper function for the SV40 large T antigen.
In cells transfected with the intact pTTV, the
input DNA (after BamHI digestion) was seen with
the DIG-probe as a 3165 bp fragment (Figs 1C and
3A,B, marked with filled circles), which was further
digested by DpnI into a fragment of 2162 bp
(Figs 1C and 3A,B, marked with filled squares). On
day 3 post transfection, the same 3165 bp fragment
(Fig. 3A, marked with an asterisk) was DpnI resis-
tant, indicating replication of the complete pTTV.
The results with the
32

P-probe verified this: the input
TTV genome
3748 bp
IEpsB
I
Ep
s
B
IHmaB
3004 bp
2162 bp
DIG
32
P
pSTBlue-1
3851 bp
I
H
m
aB
32
P
2162 bp
3165 bp
IH
ma
B
IHmaB
DIG
32

P
32
P
pTTV
total length
7775 bp
BC
3748/1
TTV
genotype6
•
NsiI
*
BspEI
pSTBlue-1
BspEI BspEI
tscr
tlat
•
TTVGCF NG136
A
Fig. 1. The full-length clone and the con-
structs for transfection. (A) The cloning
strategy of TTV into the pSTBlue-1 plasmid.
The GC-rich area is shown as a striped box
and the overlapping area in the clone as
spotted boxes. The three products used in
the construction of the TTV clone are indi-
cated with black lines. The key restriction
enzymes (see text for details), primers

(arrows: forward TTVGCF, reverse NG136),
the TATA-box (*), the poly(A) (d), the tran-
scription initiation (tscr) and the translation
initiation (tlat) sites are shown. Schematic
representations of (B) linear BspEI-excised
construct (linTTV), and (C) pTTV construct
used in transfection experiments. The viral
genome is represented by an empty bar and
the backbone plasmid by a thin black line.
The DIG- and the
32
P-labelled probes are
indicated. BamHI (vertical bars) and DpnI
(r) restriction enzymes were used for the
analysis of DNA replication. The predicted
TTV DNA products in replication analyses:
linTTV-derived 3004 bp fragment and pTTV-
derived 3165 bp fragment after BamHI
digestion are marked with d; linTTV and
pTTV derived 2162 bp fragments after
BamHI ⁄ DpnI-digestion are marked with j.
L. Kakkola et al. Biological activity of a full-length TTV DNA clone
FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS 4721
pTTV was seen (after BamHI digestion) as two
DpnI-sensitive restriction fragments of 3165 bp and
4610 bp (Fig. 3C) and, on day 3 post transfection,
these fragments had become DpnI resistant (Fig. 3C).
In all pTTV-transfected cells, as detected with either
probe, a full-length DpnI-resistant 3748 bp fragment
(which would indicate rescue and replication of the

TTV genome from the backbone plasmid) remained
absent.
As opposed to the five other cell lines, in KU812Ep6
and Cos-1 cells, no replicating DNA (or, in some
experiments with the latter cells, barely exceeding
detection threshold) were detected upon transfection
with either construct (Fig. 3A). No apparent cyto-
pathological changes were microscopically detected in
the cells supporting TTV DNA replication.
The results were the same regardless of the DNA iso-
lation method (total cellular; Hirt extraction) and of the
detection probes (DIG- and
32
P-labelled probe) (data
shown for 293 and 293T cells in Fig. 3A,C). The non-
transfected cells and the backbone plasmid-transfected
cells were always negative for TTV DNA. In Southern
analysis, the input DNA served as an internal control to
verify restriction enzyme activity: the input DNA was
sensitive to DpnI (Fig. 3A,B, marked with filled squares)
whereas the newly synthesized DNA was resistant
(Fig. 3A, marked with an arrow) but remained digest-
ible with other restriction enzymes (data not shown).
Circularization of the linear construct
In the linTTV-transfected cells on day 3, the emer-
gence of the 3748 bp TTV DNA after BamHI diges-
tion (Fig. 3A) indicated that the input linear
TTV DNA had circularized. To confirm this, two
other restriction enzymes (HindIII and ScaI, Fig. 5A)
that, like BamHI, cut the TTV genome only once, were

used with identical results (i.e. a single product of
approximately 3.7 kb was detected; data not shown).
The circularization was further confirmed by digestion
of the DNA samples with pairs of restriction enzymes
that cut on both sides of the linearization breakpoint
(Fig. 5). BamHI ⁄ SalIorXhoI ⁄ SalI double-digestions
of the input linear construct yielded three restriction
fragments of approximately 2100, 740 and 890 bp with
the first enzyme pair, and of 2200, 630 and 890 bp
with the latter. However, the same double-digestions
of replicating TTV DNA from 293T cells on day 3
post transfection yielded additional fragments of
approximately 1630 bp and 1520 bp, respectively
(Fig. 5B), indicating fusion of the linearization break-
point ends. Taken together, these results show that, in
the linTTV-transfected 293T cells, circular forms of the
TTV genome had been formed.
Effect of aphidicolin on TTV DNA replication
To reconfirm that TTV DNA replication had occurred
and to investigate whether it utilizes the cellular replica-
tion machinery, aphidicolin (an inhibitor of eukaryotic
C
B
D
A
nt 112
DNA
nt 112 nt 182 nt 284
RT1F
nt 129-147

RT1R
nt 683-660
RNA
nt 1 nt 3748
poly-A
DDDDD
non-transf.
cells
Mw
Mw
day1 day3 day5 day10
linTTV
500
400
300
bp
day3
Mw
pSTBlue-1pTTV
Mw
500
400
bp
DDDDD
day1 day3 day6 day10
600
day3
Mw -RT D
linTTV day3
400

500
600
bp
+RT
400
500
600
bp
Mw
-RT R D-RT RD
input
linTTV
input
pTTV
+RT+RT
R
Fig. 2. RT-PCR of TTV RNA. (A) A schematic drawing of TTV
RT-PCR. Transcription initiates at nucleotides 112, and splicing
removes nucleotides 182–284 [11]. RT-PCR primers are shown
with arrows. Amplicon sizes are 555 bp for DNA and 454 bp for
spliced mRNA. RT-PCR results of representative 293T cells trans-
fected with (B) the linear excised TTV (linTTV) and (C) intact pTTV.
Nontransfected cells and cells transfected with the backbone plas-
mid (pSTBlue-1) were included as controls. (D) RT-PCR controls
from 293T cells transfected with linTTV (day 3) and from the input
constructs. +RT, normal RT-PCR; –RT, without the RT-step; R,
RNase-treated; D, DNase-treated.
Biological activity of a full-length TTV DNA clone L. Kakkola et al.
4722 FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS
3639

C
A
B
B B/DU
bp
4899
3639
2799
Mw
input
linTTV
BB/DU
bp
3639
2799
Mw
input
pTTV
4899
4899
3639
2799
293T
BB/D
B
B/D B B/D B B/D B B/D
day1 day3 day1 day3 day3
linTTV pTTV
bp
pSTBlue-1

Mw
293
B B/D B B/D B B/D B B/D B B/D
day1 day3 day1 day3 day3
linTTV pTTV
bp
pSTBlue-1
4899
3639
2799
Mw
4899
2799
bp
B B/D B B/D B B/D B B/D B B/D
day1 day3 day1 day3 day3
Mw
Cos-1
linTTV pTTV
pSTBlue-1
KU812Ep6
B
B/D
B B/D B B/D B B/D B
day1 day3 day1 day3
linTTV
pTTV
cells
bp
B/D

4899
3639
2799
Mw
3639
UT7/Epo-S1
day1 day4 day1 day4
linTTV pTTV
cells
bp
B B/D B B/D B B/D
B
B/D B B/D
4899
2799
Mw
B
B/D B B/D B B/D B B/D B B/D
day1 day3 day1 day3 day3
bp
4899
3639
2799
Mw
Huh7
linTTV pTTV
pSTBlue-1
Chang liver
B
B/D B B/D B B/D B B/D B B/D

day1 day3 day1 day3 day3
linTTV pTTV
pSTBlue-1
bp
4899
3639
2799
Mw
linTTV
293T
293
input
pTTV
pTTV linTTV pTTV
bp
4625
3165
Fig. 3. Southern analysis of TTV DNA replication. (A) 293T, 293, KU812Ep6, UT ⁄ Epo-S1, Huh7, Cos-1 and Chang liver cells transfected with
the excised linear (linTTV) or the intact pTTV construct. The key products of the replication assay are marked in the 293T-cell figure: the input
linTTV yielding a 3004 bp fragment and the input pTTV yielding a 3165 bp fragment after BamHI digestion are marked with d; the input
linTTV and pTTV yielding 2162 bp fragments after BamHI ⁄ DpnI digestion are marked with j; the DpnI-resistant circularized full-length
TTV DNA is marked with an arrow; DpnI-resistant pTTV is marked with *. (B) Southern analysis of the input constructs. The products of the
restriction enzyme digestions are marked as those in the 293T-cell figure. (Note the absence of a 3748 bp product.) U, undigested. (C) South-
ern analysis of Hirt-extracted (BamHI ⁄ DpnI-digested) DNA from the 293T and 293 cells (with and without T antigen, respectively) transfected
with pTTV or linTTV. Input pTTV digested with BamHI as a control. Arrows indicate DpnI-resistant full-length TTV DNA. For detection of
TTV DNA, either a DIG- (A,B) or a
32
P- (C) labelled probe was used.
L. Kakkola et al. Biological activity of a full-length TTV DNA clone
FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS 4723

nuclear DNA replication) [26,27] was used. The 293T
cells were transfected either with linTTV or with
pTTV, and were grown in the presence (versus
absence) of aphidicolin. Upon aphidicolin treatment,
TTV DNA replication was blocked (Fig. 6), indicating
nuclear DNA replication.
Discussion
Most of the data published on TTV are from PCR
studies of clinical patient materials. However, to
apprehend the full impact of this human virus, more
information on the molecular biology and host–cell
interactions of this virus is needed. To this end, we
have cloned and sequenced the full-length genome of
TTV genotype 6, and studied the key biological activi-
ties of this virus. The cloned viral genome was
3748 nucleotides in length; 105 nucleotides shorter
than the TTV prototype TA278 (genotype 1, accession
number AB017610). In genomic organization, geno-
type 6 turned out to resemble the other known TTV
types: a TATA-box and a poly(A) sequence flanking
the coding region, and a GC-rich area of 107 bp
located in the noncoding region.
The biological activity of our full-length plasmid
clone was analyzed in seven cell lines. In a previous
study, the TTV expression profile (i.e. mRNAs tran-
scribed and proteins translated) in 293 cells was deter-
mined by using this full-length clone [11]. In the
present study, cells of all seven types, transfected with
either pTTV or linTTV, produced TTV RNA. The
RNA transcripts were unequivocally documented with

RT-PCR designed to flank a common splice site in the
TTV genome. In a previous study, TTV RNAs were
B
B/D B B/D B B/D B B/D
day0 day1 day2 day3
bp
4899
3639
2799
Mw
Fig. 4. Accumulation of DpnI-resistant full-length TTV DNA (marked
with an arrow) in 293T cells from day 0 to day 3 post transfection.
293T cells were transfected with linTTV, and DIG-labelled probe
was used to detect the TTV DNA.
A
B
lin day3 Mw
BamHI/SalI
lin day3Mw
XhoI/SalI
1953
1882
1515
1482
992
718
bp
2799
3639
pSTBlue-1

pSTBlue-1
H
maB
I
laS
I
1631 bp
l
aS
I
ohX
I
1521 bp
744 bp
TTV
genome
3748 bp
SalI
H
m
aB
I
ohX I
a
cS
I
d
niH
I
I

I
887 bp
634 bp
BspEI BspEI
2227 bp
2117 bp
Fig. 5. Circularization of the linear construct in 293T cells. (A) Sche-
matic representation of the restriction enzyme cutting sites and the
corresponding restriction-fragment sizes. (B) The input linear con-
struct (lin) and the total cellular DNA (from the linTTV-transfected
293T cells on day 3 post transfection) were digested with two
enzyme pairs (BamHI ⁄ SalIorXhoI ⁄ SalI), each pair cutting at both
sides of the linearization breakpoint. Southern detection was per-
formed with a probe prepared by nick translation (the band corre-
sponding to the cloning vector pSTBlue-1 is indicated). The
fragments from the circularized TTV DNA are marked with arrows.
pTTV linTTV pTTV linTTV
- APC + APC
A
B
BB/DBB/D
bp
4899
3639
2799
Mw
-APC + APC
Fig. 6. The effect of aphidicolin (APC) on TTV DNA replication.
293T cells were transfected with pTTV or the linear (linTTV) con-
struct in the absence (–APC) or presence (+APC) of aphidicolin. (A)

Southern analysis with the
32
P-probe of Hirt-extracted (Bam-
HI ⁄ DpnI-digested) DNA. (B) Southern analysis with the DIG-labelled
probe of BamHI- (B) and BamHI ⁄ DpnI-digested (B ⁄ D) DNA on
day 3 post transfection. Arrows are pointing to the sites of replicat-
ing, DpnI-resistant TTV DNA.
Biological activity of a full-length TTV DNA clone L. Kakkola et al.
4724 FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS
detected in Cos-1 cells transfected with a plasmid clone
of TTV genotype 1 [9]. The cloning strategy used by
this group was similar to ours: the plasmid clone con-
tained overlapping genomic regions and the cloning
breakpoint was in the noncoding region. Their geno-
type 1 clone and our genotype 6 clone differed in the
lengths of the overlaps and in the locations of the
cloning breakpoints. However, upon transfection, both
constructs produced RNA, indicating that cloning did
not impair the function of the promoter. The promoter
area of TTV genotype 1 has been shown to be active
in many cell types, including Huh7 and cells of ery-
throid origin [5,8]. Our results with genotype 6 were
similar, displaying TTV RNA expression (i.e. promoter
activity) in diverse cell types.
For the detection of replicating TTV DNA, we used
restriction enzyme analysis combined with Southern
hybridization. This straightforward approach was dem-
onstrated to be useful for the study of TTV DNA rep-
lication. TTV DNA replication was detected in the
majority of cell types. When the excised linear geno-

type 6 DNA was transfected into 293T, 293, UT7 ⁄
Epo-S1, Chang liver and Huh7 cells, circularized and
replicating (DpnI resistant) forms of TTV DNA accu-
mulated up to days 3–4. It is possible that the DpnI
resistance might arise from replication-independent
demethylation of the input DNA, or from DNA repli-
cation not related to TTV. However, both the input
linTTV and the cotransfected (together with linTTV)
backbone plasmid always remained sensitive to DpnI.
This indicates that the DpnI-resistance of TTV DNA
was not due to replication-independent demethylation
of the input DNA, or due to general DNA replication,
but instead was a result of TTV DNA-specific replica-
tion. That the production of the DpnI-resistant forms
could be abolished with a polymerase inhibitor further
verifies that replication had occurred within the cells.
The high-molecular weight double bands that appeared
restrictively in the cells that supported TTV DNA rep-
lication (after transfection with linTTV), could theoret-
ically originate from concatamers formed from the
transfected DNA. The bands were DpnI sensitive and
visible also in aphidicolin treated cells, suggesting that
they are formed without the cellular replication
machinery. Whether these intracellular DNA forms are
required for initiation of replication remains to be
studied. Of our seven cell lines, 293T showed the high-
est yield of replicating viral DNA, even exceeding that
of the Chang cells, which have been recently reported
to sustain TTV infection [17].
Upon transfection of five cell types with the intact

pTTV, DpnI-resistant (i.e. replicating) DNA was
detected. However, rescued forms of the TTV genome
were not seen, which could indicate inefficient excision
from the backbone plasmid. With other ssDNA-virus
clones, the extent of rescue and replication seems to
vary. For example, a rodent parvovirus, HaPV, repli-
cates and produces infectious virions when transfected
as an intact plasmid clone containing one copy of the
viral genome [28] whereas, among the circoviruses,
PCV gives a higher virus titer when cloned as tandem
repeats [29] and CAV requires either tandem-repeat
cloning or excision of the viral genome from the plas-
mid before transfection [30]. The rescue and replication
of the viral genomes could involve cellular and ⁄ or viral
proteins, with mechanisms possibly dependent on the
particular virus (e.g. nicking of DNA strands, recombi-
nation, and rolling circle replication) [31,32]. In com-
parison with other ssDNA virus molecular clones, ours
appear to resemble that of CAV; in order to produce
genome-size replicating DNA, the viral genome needs
to be excised from the backbone plasmid before trans-
fection.
With PCV, it has been shown that the mere origin
of replication, cloned into the plasmid, can lead to
replication of the entire plasmid, if transfected into
PCV-infected cells [33]. This raises the question
whether replication of our pTTV (and maybe also
linTTV) might have been assisted by coinfection with
a homologous virus. However, all our cell lines were
shown to be TTV DNA negative by generic PCR. This

suggests that the replicating TTV DNA in the present
study was not produced with the help of endogenous
TTV. Interestingly, the amount of replicating
TTV DNA was much lower in 293 than in 293T cells,
suggesting that the SV40 large T antigen might provide
some helper functions. CAV and PCV have been
shown to replicate efficiently in heterologously infected
cells: CAV in MDCC-MSB-1 cells transformed with
Marek’s virus [34] and PCV in pk-15 cells infected
with swine papova virus [35–37]. It is therefore
possible that TTV indeed might benefit from some
helper functions of other viruses for efficient produc-
tive infection.
By contrast to the other cell lines in the present
study, the TTV clone showed little or no replication in
KU812Ep6 and Cos-1 cells. The former are of ery-
throid origin, as are UT7 ⁄ Epo-S1, and could thus be
thought to support TTV replication. However, accord-
ing to our unpublished data, the viability of
KU812Ep6 cells declines during electroporation. TTV,
a minute virus apparently lacking DNA polymerase,
most likely depends on the cellular S-phase for replica-
tion. Indeed, our results with aphidicolin, which inhib-
its eukaryotic nuclear DNA replication and blocks the
cell cycle at early S-phase, strongly suggest that TTV
L. Kakkola et al. Biological activity of a full-length TTV DNA clone
FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS 4725
utilizes the cellular DNA polymerase and S-phase for
replication. The requirement for rapid cell division,
together with the diminished viability of the electropo-

rated KU812Ep6 cells, could explain the lack of repli-
cation of our full-length clone in those cells. The
deficiency of TTV replication in monkey kidney-
derived Cos-1 cells could represent species specificity.
However, because mRNAs were produced, the poten-
tial species barrier does not appear to affect transcrip-
tion. In previous studies, the sites of TTV replication
in humans have been suggested to be the hematologi-
cal compartment and the liver [15,16]. Our results,
demonstrating replication of cloned TTV genotype 6
DNA in erythroid UT7 ⁄ Epo-S1, and hepatic Huh7
and Chang liver cells, further support this concept. In
any case, the species and cell-type specificity of TTV
replication needs to be examined further. The genetic
variation among TT viruses is extremely high. To what
extent the various genotypes share biological functions
and possibly influence each others in coinfections
remains to be investigated. At least the RNA tran-
scription of genotypes 1 and 6, which belong to the
same genogroup, appears to be similar [9,11].
The production of infectious virions from the trans-
fected cells could not be verified unequivocally with
the methods in use, and thus remains to be investi-
gated. In our experiments, upon cell subculture, even
though the more sensitive RT-PCR continued to be
positive, the levels of replicating TTV DNA in South-
ern analysis declined below detection limit. These
results suggest that, if infectious virions were pro-
duced, the infection did not spread efficiently to the
neighbouring cells, and ⁄ or that the amounts of prog-

eny virions were relatively low. It is possible that the
cells used in the present study did not express the
unknown TTV receptor, or that TTV simply does not
grow well in ordinary tissue culture, thereby resem-
bling many other viruses such as human parvovi-
rus B19, hepatitis C virus and human papillomavirus.
A low infectivity of TTV is concordant with previous
studies showing that infection of Chang liver cells gave
rise to only low amounts of progeny virus [17] and
that, even in ex vivo PBMC cultures, the production of
TTV is scanty and requires cell activation [18,19].
Because TTV in vivo infects healthy individuals chroni-
cally, strict regulation of virus multiplicity and of cell
damage is mutually beneficial for the virus and its
host.
The research on TTV-like animal circoviruses has
been greatly assisted by the availability of full-length
plasmid clones producing infectious virions in vitro
and clinical disease in vivo [21–24,29]. Indeed, the
genetic map (defining viral mRNAs and proteins) of
TTV has been recently elucidated with this full-
length TTV clone [11]. In the present study, we
demonstrated TTV-promoter activity in all cell types
studied and, by a novel approach, identified five cell
lines that supported TTV replication. In the absence of
knowledge on TTV proteins, replication and cell bio-
logy, the full-length plasmid clone and replication
assays presented here will be valuable tools for the
examination of the mechanisms pertaining to the
molecular biology, the cell tropism and the clinical sig-

nificance of this recently discovered human virus.
Experimental procedures
Cell lines and transfection methods
Seven cell lines, Cos-1, Huh7, Chang liver, KU812Ep6,
UT7 ⁄ Epo-S1, 293T and 293, were used in this study. Cos-1
cells (African green monkey kidney; transformed with
SV40) were maintained in DMEM containing 10% fetal
bovine serum, 10 mm Hepes buffer (Gibco BRL ⁄ Invitro-
gen, Carlsbad, CA, USA) and antibiotics. The cells were
transfected 1 day after subculture, at approximately 95%
confluency, with 30 lL Lipofectamine2000 (Invitrogen Life
Technologies, Carlsbad, CA, USA) and 5 lg DNA per
60 mm
2
plate. Human hepatoma cells, Huh7, were main-
tained in MEM, with 10% fetal bovine serum and antibiot-
ics, and were transfected as the Cos-1 cells. Human liver
cells, Chang liver (ECACC 88021102), were maintained as
Huh7 cells, and transfected as Cos-1 cells (except that the
amount of Lipofectamine2000 was 16 lL). Human kidney-
derived 293T cells (expressing the SV40 T antigen, origi-
nally described as 293 ⁄ tsA1609neo [38]), and 293 cells [39]
were maintained in DMEM, 10% fetal bovine serum and
antibiotics. The 293T and 293 cells were transfected as the
Cos-1 (except that the amount of Lipofectamine2000 was
25 lL); or by the calcium phosphate technique [40]. The
human erythroid leukemia cell line KU812Ep6 [41], kindly
provided by Dr Miyagawa (Fujirebio Inc., Tokyo, Japan),
was maintained in suspension in RPMI1640, 10% fetal
bovine serum, 6 UÆmL

)1
erythropoietin (‘Eprex’; Janssen-
Cilag, Berchem, Belgium) and antibiotics. For transfection,
the cells were harvested on days 3–4. For one reaction,
2 · 10
6
cells were resuspended in 0.5 mL medium and 5 lg
DNA was added. The cells were electroporated immediately
at 300 or 350 V, and 960 lF (‘Gene Pulser’; Bio-Rad, Her-
cules, CA, USA). A human erythroblastoid cell line
UT7 ⁄ Epo-S1 [42,43], kindly provided by Dr Morita (To-
hoku University School of Medicine, Japan), was main-
tained in suspension in Iscove’s modified Dulbecco’s
medium containing 10% fetal bovine serum, 2 UÆmL
)1
erythropoietin and antibiotics. For transfection, the cells
were harvested on days 3–4 and transfected by the
AMAXA Nucleofector system, using kit R and program
Biological activity of a full-length TTV DNA clone L. Kakkola et al.
4726 FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS
T-24 (Amaxa Biosystems, Cologne, Germany). All the cells
were grown at 37 °Cin5%CO
2
; and were monitored with
PCR for the presence of endogenous TTV (generic UTR-
PCR, and genotype 6 specific PCR) [44].
Full-length cloning
The TTV genome was cloned in three overlapping fragments
(Fig. 1A). The previously cloned 3.3 kb region of TTV iso-
late HEL32 [44] was completed to contain the full-length gen-

ome of the isolate. The GC-rich part of the TTV genome was
amplified from the original serum [44] with seminested prim-
ers: forward TTVGCF (nucleotides 3206–3225) 5¢-CAGA
CTCCGAGATGCCATTG-3¢, first reverse TTVGCR1
(nucleotides 216–199) 5¢-CGAATTGCCCCTTGACTG-3¢
and second reverse TTVGCR3 (nucleotides 157–140) 5¢-GG
GATCACCCTTCGAGGT-3¢. Both PCR reactions (volume
25 lL) contained 200 lm of each dNTP (Roche, Basel, Swit-
zerland), 400 nm of each primer, 1.5 mm MgCl
2
, 10%
dimethylsulfoxide, 1 m betaine (Sigma, St. Louis, MO, USA)
and 17.5 U of enzyme mix (‘Expand High Fidelity PCR
System’; Roche). The PCR comprised annealing at 55 °C for
30 s and extension at 72 °C for 2 min for the first ten cycles,
with addition of 5 sÆcycle
)1
of extension time for the remain-
ing 20 cycles. The gel-purified PCR amplicon was cloned into
pSTBlue-1 AccepTor vector (Novagen, Madison, WI, USA)
in Escherichia coli DH5a cells.
To facilitate the cloning of the entire TTV genome into a
single plasmid, a third overlapping piece of 396 bp was
amplified by PCR from the original serum with primers
forward NG054 (nucleotides )2–18) [25] and reverse
TTVGCR6 (nucleotides 394–375) 5¢-CGTTCGAGTT
GGGTTCCATT-3¢.
Restriction enzyme digestions and ligations of the three
genomic parts resulted in a plasmid that contains the
entire TTV genome (pTTV), flanked by overlapping

175 bp areas, inserted between the EcoRI sites in the
pSTBlue-1 vector. In addition, a BspEI restriction enzyme
can be used to excise from the TTV clone the complete,
single-unit (without overlaps) TTV genome in linear
form.
Sequencing of the GC-rich region was carried out at the
DNA sequencing facility of the Institute of Biotechnology,
University of Helsinki, Finland. All other sequencing reac-
tions in this study were done using the ABI Prism 3100
Genetic Analyzer (Applied Biosystems, Foster City, CA,
USA) in the sequencing core facility of the Haartman Insti-
tute, University of Helsinki, Finland.
Plasmid constructs for transfection
For functional analysis of the TTV clone, the cells were
transfected with TTV plasmids of two different forms: an
uncut plasmid clone, pTTV, containing overlaps (Fig. 1C),
and a linear construct, linTTV [full-length genome (without
overlaps) excised with BspEI, but not purified from the
backbone plasmid; Fig. 1B]. The pSTBlue-1 backbone
vector was used as a negative control. The cells were
collected for RNA and DNA analyses on days 1–11 post
transfection.
The transfection efficiencies were optimized with pEGFP-
Luc vector (Clontech, Mountain View, CA, USA) encoding
green fluorescent protein. On day 1 post transfection, the
percentage of green fluorescent protein-positive cells was
estimated with a fluorescence microscope (‘Axioplan2’;
Zeiss, Oberkochen, Germany).
RNA isolation and RT-PCR
Total RNA of the cells was extracted with TRIzol Reagent

(Invitrogen Life Technologies). To remove residual DNA
when necessary, the RNA samples were treated with DNase
(1.5 U ⁄ 15 lL; ‘RQ1 RNase-Free DNase’; Promega, Madi-
son, WI, USA) for 70 min at 37 °C.
RT-PCR was performed with a RobusT II RT-PCR Kit
(Finnzymes, Espoo, Finland) in the presence of RNase
inhibitor (‘RNaseOut’; Invitrogen Life Technologies, or
‘RNase Inhibitor’; Roche). For TTV-specific amplification,
primers flanking the first common intron of 101 bp [11]
were designed: forward RT1F (nucleotides 129–147)
5¢-GCAGCGGCAGCACCTCGAA-3¢ and reverse RT1R
(nucleotides 683–660) 5¢-GTCTAGCAGGTCCTCGTCTG
CGAG-3¢. This separates the possible DNA-derived ampli-
cons from the RNA-derived ones by size (555 bp and
454 bp, respectively; Fig. 2A). The PCR program consisted
of reverse transcription for 30 min at 42 °C, followed by
PCR: 94 °C for 2 min, and 35 cycles at 94 °C for 30 s,
65 °C for 30 s, 68 °C for 45 s, with final extension at 68 °C
for 3 min. RT-PCR for the cellular retinoblastoma mRNA
was used as a control for RT-PCR and RNA isolation [45].
RT-PCRs were carried out on DNase-treated and non-
treated samples. The RNA experiments were performed at
least twice.
DNA replication analyses
Total cellular DNA was isolated by cell lysis (‘Protein-
ase K’, 2.4 U ⁄ 200 lL reaction; Fermentas, Burlington,
Ontario, Canada) and by shearing through an 18G needle,
as previously described [28]. The isolated DNA was
extracted with phenol and chloroform, precipitated with
ethanol and Na-acetate, and resuspended into water. Total

cellular DNA was also alternatively isolated with QIAamp
DNA Blood Mini Kit (Qiagen, Hilden Germany). Low
molecular weight DNA was alternatively extracted with the
Hirt protocol [46].
The DNA samples (2.5–5 lg of Hirt-extracted or 30–
40 lg of total DNA) were digested with BamHI. Subse-
quently, half the digest was treated with DpnI (which cuts
only prokaryotic DNA; New England BioLabs, Ipswich,
L. Kakkola et al. Biological activity of a full-length TTV DNA clone
FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS 4727
MA, USA). The BamHI- and BamHI ⁄ DpnI-digested DNA
samples were separated by gel electrophoresis. The DNA
was transferred to nylon membranes (‘Hybond-N+’, Amer-
sham Biosciences, Piscataway, NJ, USA), and was hybrid-
ized overnight. For hybridization, a DIG-labelled TTV-
specific probe, covering nucleotides 1803–2200 (Fig. 1), was
prepared as described [44] using the outer primer pair of
genotype 6 PCR. The DIG label was detected with
5-bromo-4-chloro-3-indolyl-phosphate (BCIP, Roche) and
4-nitro blue tetrazolium chloride (NBT, Roche) forming a
coloured precipitate. For the circularization experiments,
another DIG-labelled probe was prepared to cover the
entire pTTV construct by DIG-Nick Translation Mix
(Roche). In some experiments, a
32
P-labelled probe consist-
ing of a 560 bp fragment of the untranslated region (NsiI-
BspEI fragment from pTTV, Fig. 1) was used. The probe
was labelled to high specific activity with [a-
32

P]dCTP by
the random primer method [47], and the signal was detected
by film exposure.
For aphidicolin treatment, 2 lgÆmL
)1
of aphidicolin
(Sigma) was added to the medium, and 293T cells were
transfected by the calcium phosphate method. The cells
were incubated in aphidicolin for 16 h, washed, and grown
further with aphidicolin. On day 2 post transfection, the
DNA was isolated by the Hirt method, 10 lg of DNA was
digested (BamHI ⁄ DpnI), separated on an agarose gel, and
detected with the
32
P-probe. Alternatively, the 293T cells
were transfected with the Lipofectamine2000 method and
grown in the presence of aphidicolin. On day 3 post trans-
fection, the total DNA was isolated, 30 l g of DNA was
digested (BamHI ⁄ DpnI), and analyzed with Southern
hybridization with the DIG-probe.
Acknowledgements
The authors thank Drs Ilkka Julkunen (National Pub-
lic Health Institute, Helsinki, Finland), Eiji Morita
and Kazuo Sugamura (Tohoku University School of
Medicine, Sendai, Japan), and Eiji Miyagawa (Fujire-
bio Inc., Tokyo, Japan) for cell lines, and Pa
¨
ivi Norja
and Heidi Bonde
´

n for excellent technical assistance.
Dr Malcolm Richardson is acknowledged for revision
of the manuscript. This study was supported by the
Finnish Technology Advancement Fund, the Finnish
Academy (project 76132), the Helsinki Biomedical
Graduate School, the Alfred Kordelin Foundation, the
Paulo Foundation, the Sigrid Juse
´
lius Foundation, the
Research and Science Foundation of Farmos, the Ella
and Georg Ehrnrooth Foundation, the Finnish Kon-
kordia Fund, the Helsinki University Central Hospital
Research and Education Fund, The Maud Kuistila
Memorial Foundation, The Finnish Foundation for
Research on Viral Diseases, and the Medical Society
of Finland (Finska La
¨
karesa
¨
llskapet).
References
1 Biagini P, Todd D, Bendinelli M, Hino S, Mankertz A,
Mishiro S, Niel C, Okamoto H, Raidal S, Ritchie BW
et al. (2004) Anellovirus in virus taxonomy. Eight
Report of the International Committee on Taxonomy of
Viruses (Fauquet, CM, Mayo, MA, Maniloff, J, Dessel-
berger, U & Ball, LA, eds), pp. 335–341. Elsevier ⁄
Academic Press, London.
2 Nishizawa T, Okamoto H, Konishi K, Yoshizawa H,
Miyakawa Y & Mayumi M (1997) A novel DNA virus

(TTV) associated with elevated transaminase levels in
posttransfusion hepatitis of unknown etiology. Biochem
Biophys Res Comm 241, 92–97.
3 Mushahwar IK, Erker JC, Muerhoff AS, Leary TP,
Simons JN, Birkenmeyer LG, Chalmers ML, Pilot-
Matias TJ & Dexai SM (1999) Molecular and bio-
physical characterization of TT virus: evidence for a
new virus family infecting humans. Proc Natl Acad Sci
USA 96, 3177–3182.
4 Okamoto H, Nishizawa T, Kato N, Ukita M, Ikeda H,
Iizuka H, Miyakawa Y & Mayumi M (1998) Molecular
cloning and characterization of a novel DNA virus
(TTV) associated with posttransfusion hepatitis of
unknown etiology. Hepatol Res 10, 1–16.
5 Kamada K, Kamahora T, Kabat P & Hino S (2004)
Transcriptional regulation of TT virus: promoter and
enhancer regions in the 1.2-kb noncoding region,
Virology 321, 341–348.
6 Miyata H, Tsunoda H, Kazi A, Yamada A, Khan MA,
Murakami J, Kamahora T, Shiraki K & Hino S (1999)
Identification of a novel GC-rich 113-nucleotide region
to complete the circular, single-stranded DNA genome
of TT virus, the first human circovirus. J Virol 73,
3582–3586.
7 Okamoto H, Nishizawa T, Ukita M, Takahashi M,
Fukuda M, Iizuka H, Miyakawa Y & Mayumi M
(1999) The entire nucleotide sequence of a TT virus iso-
late from the United States (TUSO1): comparison with
reported isolates and phylogenetic analysis. Virology
259, 437–448.

8 Suzuki T, Suzuki R, Li J, Hijikata M, Matsuda M,
Li TC, Matsuura Y, Mishiro S & Miyamura T (2004)
Identification of basal promoter and enhancer elements
in an untranslated region of the TT virus genome.
J Virol 78, 10820–10824.
9 Kamahora T, Hino S & Miyata H (2000) Three spliced
mRNAs of TT virus transcribed from a plasmid con-
taining the entire genome in COS1 cells. J Virol 74,
9980–9986.
10 Okamoto H, Nishizawa T, Tawara A, Takahashi M,
Kishimoto J, Sai T & Sugai Y (2000) TT virus mRNAs
detected in the bone marrow cells from an infected
individual. Biochem Biophys Res Comm 279, 700–
707.
Biological activity of a full-length TTV DNA clone L. Kakkola et al.
4728 FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS
11 Qiu J, Kakkola L, Cheng FYeC, So
¨
derlund-Venermo M,
Hedman K & Pintel DJ (2005) Human circovirus TT
virus genotype 6 expresses six proteins following transfec-
tion of a full-length clone. J Virol 79, 6505–6510.
12 Erker JC, Leary TP, Desai SM, Chalmers ML &
Mushahwar IK (1999) Analyses of TT virus full-length
genomic sequences. J Gen Virol 80, 1743–1750.
13 Niagro FD, Forsthoefel AN, Lawther RP, Kamalana-
than L, Ritchie BW, Latimer KS & Lukert PD (1998)
Beak and feather disease virus and porcine circovirus
genomes: intermediates between the geminiviruses and
plant circoviruses. Arch Virol 143, 1723–1744.

14 Okamoto H, Takahashi M, Nishizawa T, Tawara A,
Sugai Y, Sai T, Tanaka T & Tsuda F (2000) Replicative
forms of TT virus DNA in bone marrow cells. Biochem
Biophys Res Comm 270, 657–662.
15 Okamoto H, Ukita M, Nishizawa T, Kishimoto J,
Hoshi Y, Mizuo H, Tanaka T, Miyakawa Y & Mayumi
M (2000) Circular double-stranded forms of TT virus
DNA in the liver. J Virol 74, 5161–5167.
16 Zhong S, Yeo W, Tang M, Liu C, Lin XR, Ho WM,
Hui P & Johnson PJ (2002) Frequent detection of the
replicative form of TT virus DNA in peripheral blood
mononuclear cells and bone marrow cells in cancer
patients. J Med Virol 66, 428–434.
17 Desai M, Pal R, Deshmukh R & Banker D (2005) Rep-
lication of TT virus in hepatocyte and leucocyte cell
lines. J Med Virol 77, 136–143.
18 Maggi F, Fornai C, Zaccaro L, Morrica A, Vatteroni
ML, Isola P, Marchi S, Ricchiuti A, Pistello M &
Bendinelli M (2001) TT virus (TTV) loads associated
with different peripheral blood cell types and evidence
for TTV replication in activated mononuclear cells.
J Med Virol 64, 190–194.
19 Mariscal LF, Lo
´
pez-Alcorocho JM, Rodrı
´
guez-In
˜
igo E,
Ortiz-Movilla N, de Lucas S, Bartolome

´
J & Carren
˜
oV
(2002) TT virus replicates in stimulated but not in non-
stimulated peripheral blood mononuclear cells. Virology
301, 121–129.
20 Pollicino T, Raffa G, Squadrito G, Costantino L, Cac-
ciola I, Brancatelli S, Alafaci C, Florio MG & Rai-
mondo G (2003) TT virus has a ubiquitous diffusion in
human body tissues: analyses of paired serum and tissue
samples. J Viral Hepat 10, 95–102.
21 Claessens JA, Schrier CC, Mockett AP, Jagt EH &
Sondermeijer PJ (1991) Molecular cloning and sequence
analysis of the genome of chicken anaemia agent. J Gen
Virol 72, 2003–2006.
22 Meehan BM, Creelan JL, McNulty MS & Todd D
(1997) Sequence of porcine circovirus DNA: affinities
with plant circoviruses. J Gen Virol 78, 221–227.
23 Meehan BM, McNeilly F, Todd D, Kennedy S, Jew-
hurst VA, Ellis JA, Hassard LE, Clark EG, Haines DM
& Allan GM (1998) Characterization of novel circovirus
DNAs associated with wasting syndromes in pigs. J Gen
Virol 79, 2171–2179.
24 Noteborn MH, de Boer GF, van Roozelaar DJ, Karr-
eman C, Kranenburg O, Vos JG, Jeurissen SH, Hoeben
RC, Zantema A, Koch G et al. (1991) Characterization
of cloned chicken anemia virus DNA that contains all
elements for the infectious replication cycle. J Virol 65,
3131–3139.

25 Okamoto H, Takahashi M, Nishizawa T, Ukita M,
Fukuda M, Tsuda F, Miyakawa Y & Mayumi M
(1999) Marked genomic heterogeneity and frequent
mixed infection of TT virus demonstrated by PCR with
primers from coding and noncoding regions. Virology
259, 428–436.
26 Ikegami S, Taguchi T, Ohashi M, Oguro M, Nagano H
& Mano Y (1978) Aphidicolin prevents mitotic cell
division by interfering with the activity of DNA
polymerase-alpha. Nature 275, 458–460.
27 Pedrali-Noy G & Spadari S (1997) Effect of aphidicolin
on viral and human DNA polymerases. Biochem
Biophys Res Commun 88, 1194–1202.
28 So
¨
derlund-Venermo M, Riley LK & Pintel DJ (2001)
Construction and initial characterization of the newly
identified rodent parvovirus HaPV. J Gen Virol 82,
919–927.
29 Fenaux MP, Halbur PG, Haqshenas G, Royer R,
Thomas P, Nawagitgul P, Gill M, Toth TE & Meng XJ
(2002) Cloned genomic DNA of type 2 porcine circo-
virus is infectious when injected directly into the liver
and lymph nodes of pigs: characterization of clinical
disease, virus distribution, and pathologic lesions.
J Virol 76, 541–551.
30 Meehan BM, Todd D, Creelan JL, Earle JA, Hoey EM
& McNulty MS (1992) Characterization of viral DNAs
from cells infected with chicken anaemia agent:
sequence analysis of the cloned replicative form and

transfection capabilities of cloned genome fragments.
Arch Virol 124, 301–319.
31 Berns KI (1990) Parvovirus replication. Microbiol Rev
54, 316–329.
32 Todd D, Creelan JL, Meehan BM & McNulty MS
(1996) Investigation of the transfection capability of
cloned tandemly-repeated chicken anaemia virus DNA
fragments. Arch Virol 141, 1523–1534.
33 Mankertz A, Persson F, Mankertz J, Blaess G & Buhk
H-J (1997) Mapping and characterization of the origin
of DNA replication of porcine circovirus. J Virol 71,
2562–2566.
34 Goryo M, Suwa T, Matsumoto S, Umemura T & Itak-
ura C (1987) Serial propagation and purification of
chicken anaemia agent in MDCC-MSB1 cell line. Avian
Pathol 16, 149–163.
35 Newman JT & Smith KO (1972) Characteristics of a
swine papovavirus. Infect Immun 5, 961–967.
L. Kakkola et al. Biological activity of a full-length TTV DNA clone
FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS 4729
36 Tischer I, Gelderblom H, Vettermann W & Koch MA
(1982) A very small porcine virus with circular single-
stranded DNA. Nature 295, 64–66.
37 Tumilowicz JJ, Hung CL & Kramarsky B (1979)
Concurrent replication of a papovavirus and a C-type
virus in the CCL 33 porcine cell line. In Vitro 15, 922–928.
38 DuBridge RB, Tang P, Hsia HC, Leong PM, Miller JH
& Calos MP (1987) Analysis of mutation in human cells
by using an Epstein–Barr virus shuttle system. Mol Cell
Biol 7, 379–387.

39 Graham FL, Smiley J, Russell WC & Nairn R (1977)
Characteristics of a human cell line transformed by DNA
from human adenovirus type. J Gen Virol 36, 59–74.
40 Graham FL & van der Eb AJ (1973) A new technique
for the assay of infectivity of human adenovirus 5
DNA. Virology 52, 456–467.
41 Miyagawa E, Yoshida T, Takahashi H, Yamaguchi K,
Nagano T, Kiriyama Y, Okochi K & Sato H (1999)
Infection of the erythroid cell line, KU812Ep6 with
human parvovirus B19 and its application to titration
of B19 infectivity. J Virol Meth 83, 45–54.
42 Morita E, Tada K, Chisaka H, Asao H, Sato H, Yaeg-
ashi N & Sugamura K (2001) Human parvovirus B19
induces cell cycle arrest at G2 phase with accumulation
of mitotic cyclins. J Virol 75, 7555–7563.
43 Shimomura S, Komatsu N, Frickhofen N, Anderson S,
Kajigaya S & Young NS (1992) First continous propa-
gation of B19 parvovirus in a cell line. Blood 79, 18–24.
44 Kakkola L, Hedman K, Vanrobaeys H, Hedman L &
So
¨
derlund-Venermo M (2002) Cloning and sequencing
of TT-virus genotype 6 and expression of antigenic open
reading frame 2 proteins. J Gen Virol 83, 979–990.
45 Brunstein J, So
¨
derlund-Venermo M & Hedman K
(2000) Identification of a novel RNA splicing pattern as
a basis of restricted cell tropism of erythrovirus B19.
Virology 274, 284–291.

46 Hirt B (1967) Selective extraction of polyoma DNA
from infected mouse cell cultures. J Mol Biol 26, 365–
369.
47 Feinberg AP & Vogelstein B (1983) A technique for
radiolabeling DNA restriction endonuclease fragments
to high specific activity. Anal Biochem 132, 6–13.
Biological activity of a full-length TTV DNA clone L. Kakkola et al.
4730 FEBS Journal 274 (2007) 4719–4730 ª 2007 The Authors Journal compilation ª 2007 FEBS