Download Analysis of the entire genomes of torque teno midi virus variants in

Journal of General Virology (2009), 90, 347–358
DOI 10.1099/vir.0.007385-0
Analysis of the entire genomes of torque teno midi
virus variants in chimpanzees: infrequent crossspecies infection between humans and
chimpanzees
Masashi Ninomiya,13 Masaharu Takahashi,1 Yu Hoshino,1 Koji Ichiyama,1
Peter Simmonds2 and Hiroaki Okamoto1
1
Correspondence
Division of Virology, Department of Infection and Immunity, Jichi Medical University School of
Medicine, Tochigi-Ken 329-0498, Japan
Hiroaki Okamoto
[email protected]
2
Virus Evolution Group, Centre for Infectious Diseases, University of Edinburgh, Summerhall,
Edinburgh EH9 1QH, UK
Received 16 September 2008
Accepted 22 October 2008
Humans are frequently infected with three anelloviruses which have circular DNA genomes of
3.6–3.9 kb [Torque teno virus (TTV)], 2.8–2.9 kb [Torque teno mini virus (TTMV)] and 3.2 kb [a
recently discovered anellovirus named Torque teno midi virus (TTMDV)]. Unexpectedly, human
TTMDV DNA was not detectable in any of 74 chimpanzees tested, although all but one tested
positive for both human TTV and TTMV DNA. Using universal primers for anelloviruses, novel
variants of TTMDV that are phylogenetically clearly separate from human TTMDV were identified
from chimpanzees, and over the entire genome, three chimpanzee TTMDV variants differed by
17.9–20.3 % from each other and by 40.4–43.6 % from all 18 reported human TTMDVs. A newly
developed PCR assay that uses chimpanzee TTMDV-specific primers revealed the high
prevalence of chimpanzee TTMDV in chimpanzees (63/74, 85 %) but low prevalence in humans
(1/100). While variants of TTV and TTMV from chimpanzees and humans were phylogenetically
interspersed, those of TTMDV were monophyletic for each species, with sequence diversity of
,33 and ,20 % within the 18 human and three chimpanzee TTMDV variants, respectively.
Maximum within-group divergence values for TTV and TTMV were 51 and 57 %, respectively; both
of these values were substantially greater than the maximum divergence among TTMDV variants
(44 %), consistent with a later evolutionary emergence of TTMDV. However, substantiation of this
hypothesis will require further analysis of genetic diversity using an expanded dataset of TTMDV
variants in humans and chimpanzees. Similarly, the underlying mechanism of observed infrequent
cross-species infection of TTMDV between humans and chimpanzees deserves further analysis.
INTRODUCTION
Torque teno virus (TTV) was first discovered in the serum
of a patient with cryptogenic hepatitis following transfusion in 1997 (Nishizawa et al., 1997; Okamoto et al.,
1998). In 2000, torque teno mini virus (TTMV) was
accidentally identified by PCR using TTV-specific primers
that partially matched homologous sequences but generated unexpectedly shorter amplicons (Takahashi et al.,
3Present address: Department of Gastroenterology, Tohoku University
Graduate School of Medicine, Sendai, Japan.
The nucleotide sequence data reported in this study have been assigned
DDBJ/EMBL/GenBank accession numbers AB449062–AB449064
(three complete chimpanzee TTMDV genomes) and AB449370–
AB449608 (239 partial TTV/TTMDV/TTMV sequences).
A supplementary table of primer sequences and two supplementary
figures are available with the online version of this paper.
007385 G 2009 SGM
2000). TTV and TTMV are both small, unenveloped
spherical viruses with circular single-stranded DNA
genomes of 3.6–3.9 and 2.8–2.9 kb, respectively (Miyata
et al., 1999; Mushahwar et al., 1999; Okamoto et al., 1998,
1999, 2000b; Takahashi et al., 2000; Peng et al., 2002). The
International Committee on Taxonomy of Viruses officially classified TTV and TTMV into the new floating
genus Anellovirus (Biagini et al., 2005).
Recently, a third group of TTV-like viruses, termed small
anellovirus (SAV) types 1 and 2 (SAV-1 and SAV-2) were
cloned from plasma samples of individuals at high risk for
human immunodeficiency virus (HIV) infection (Jones
et al., 2005). Further characterization revealed that these
viruses have a circular DNA genome of 3.2 kb, intermediate in size between TTV and TTMV, but with an
otherwise similar genomic organization (Ninomiya et al.,
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347
M. Ninomiya and others
2007a). We have provisionally designated this new virus as
torque teno midi virus (TTMDV), in preference to their
earlier designation as ‘small anellovirus’, since the original
sequences of SAV-1 and SAV-2 are likely to be incomplete.
Although TTV, TTMDV and TTMV have a presumed
common genomic organization with four open reading
frames (ORFs) (ORF1–ORF4) and a short region of high
GC content (approx. 90 %), they are substantially dissimilar in genomic length and genetic identity (Biagini et al.,
2001a, 2005; Hino & Miyata., 2007; Ninomiya et al., 2007a;
Okamoto et al., 1999, 2004; Simmonds, 2002; Takahashi et
al., 2000; Thom et al., 2003). TTV shows considerable
genetic diversity and is classified into at least 39 genotypes
with nucleotide divergence of .30 % from one another, or
into five major genetic groups (groups 1–5) with a
sequence divergence of .50 % from one another
(Hijikata et al., 1999; Okamoto et al., 2004; Peng et al.,
2002). In the case of TTMV, full-length genomic sequences
of 17 isolates have been determined thus far, and four
highly divergent genetic groups have been proposed
(Biagini et al., 2001b, 2007; Okamoto et al., 2000b;
Takahashi et al., 2000). Upon analysing more TTMDV
sequences, we noticed that they formed a large swarm of
isolates differing in length (3175–3230 nt) and in sequence
(33 % divergence over the entire genomic sequence)
(Ninomiya et al., 2007b). Our previous study using a
newly developed PCR method revealed high frequencies of
viraemia with TTV (99.2 %), TTMDV (82.4 %) and TTMV
(89.7 %) among apparently healthy subjects of 1–81 years
of age. Additionally, dual or triple infection of these three
anelloviruses was seen frequently, even among infants
(Ninomiya et al., 2008).
Infection with TTV is not restricted to humans; various
animal species including non-human primates, tupaias,
livestock and some companion animals carry a wide range
of highly divergent TTV-like viruses (Inami et al., 2000;
Okamoto et al., 2000a, b, 2001, 2002; Thom et al., 2003).
Notably, and in contrast with other non-human primates
and other animal species, chimpanzees are also infected
with TTMVs (Okamoto et al., 2000b). Chimpanzees have
been experimentally infected with human TTVs and a
simian TTV was detected in a human host (Iwaki et al.,
2003; Luo et al., 2000; Mushahwar et al., 1999; Okamoto
et al., 2000a, b; Tawara et al., 2000), suggesting the
occurrence of cross-species transmission of TTVs between
humans and chimpanzees. Phylogenetic analysis revealed
that both chimpanzee TTVs and TTMVs were interspersed
with human TTVs and TTMVs, respectively (Ninomiya
et al., 2007a; Thom et al., 2003).
These observations suggest that chimpanzees might also be
infected with TTMDV, contributing to dual or triple
infection of TTV, TTMDV and TTMV as in humans, but
this hypothesis currently remains undetermined. In the
present study, we aimed to investigate the presence of
TTMDV in chimpanzees and to clarify whether dual or
triple infection of the three described anelloviruses is
348
prevalent in chimpanzees, similar to humans. Contrary to
our initial expectations, human TTMDV was not detectable in any of 74 chimpanzees that had been raised in the
Primates Park in Japan. However, novel variants of
TTMDV that were phylogenetically clearly separate from
human TTMDV were detected in chimpanzees and the
entire genomic sequence was determined for three
chimpanzee TTMDV isolates. To further elucidate the
possibility of cross-species infection of TTMDV, the
prevalence rates of chimpanzee TTMDV and human
TTMDV were examined among chimpanzee and human
populations by using PCR with species-specific primers for
TTMDV. These new investigations into the host range and
specificity of TTV-like viruses contribute to a better
understanding of the evolution and pathogenesis of
anelloviruses.
METHODS
Serum samples. Serum samples obtained between 1991 and 1997
from 74 chimpanzees that had been kept at Kumamoto Primates Park
(Sanwa Kagaku Kenkyusho Co. Ltd, Kumamoto, Japan) (Okamoto et
al., 2000a) were used in the present study. Of these, 34 were wildcaught and the remaining 40 were bred at the Primates Park. The
animals studied were maintained and monitored under conditions
that met all relevant requirements for the humane care and ethical use
of primates in an approved facility. Eight of 74 chimpanzees were
positive for antibodies to hepatitis C virus (HCV) (anti-HCV)
(Abbott Japan) and none of them tested positive for hepatitis B
surface antigen (HBsAg) after testing with the MyCell kit (Institute of
Immunology Co.). Thirty-six chimpanzees had undergone transmission experiments. In addition, serum samples that had been collected
between 2001 and 2003 from a total of 100 apparently healthy
Japanese adults (mean age 54.9±11.9 years; 57 males and 43 females)
were used in the present study. These samples were also tested for
HBsAg and anti-HCV, and additionally for antibodies to HIV type 1
tested using the SERODIA-HIV kit (Fujirebio); all samples were
negative in all three tests. All serum samples were stored at 280 uC
until testing.
Detection of TTV, TTMDV and TTMV DNA in serum. The presence
of TTV, TTMDV and TTMV DNA was determined by using a
previously described PCR assay for differential detection of three
human anelloviruses (Ninomiya et al., 2008). The expected sizes of
the amplification products were 112–117 bp (TTV DNA), 88 bp
(TTMDV DNA) and 70–72 bp (TTMV DNA).
Molecular cloning of partial TTV, TTMDV and TTMV sequences.
The presence of chimpanzee-specific TTMDV DNA that could not be
amplified by the previously described second round PCR using
primers NG795 and NG796 (Ninomiya et al., 2008) was assessed by
preparing recombinant DNA clones containing a DNA fragment of
TTV, TTMDV or TTMV. Briefly, of the chimpanzees that tested
positive for TTV and TTMV DNAs, but negative for TTMDV DNA,
using the method described previously (Ninomiya et al., 2008), three
chimpanzees were randomly selected and their serum samples were
amplified with the mixed primers NG779/NG780 and NG781/NG782
(Fig. 2b; Supplementary Table S1, available in JGV Online). These
first-round amplicons (approx. 130 bp) were ligated into pT7 Blue TVector (Novagen). Independent clones obtained using this method
were directly sequenced by using the BigDye Terminator v3.1 Cycle
Sequencing kit (Applied Biosystems).
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Journal of General Virology 90
Analysis of torque teno midi virus in chimpanzees
Detection of chimpanzee TTMDV DNA in serum. Based on a
partial TTMDV DNA sequence obtained in the present study from
one chimpanzee (no. 210), we designed inverted-nested primers for
specific PCR amplification of the nearly full-length circular
chimpanzee TTMDV genomes. Briefly, long-distance PCR was carried
out using TaKaRa LA Taq polymerase with GC buffer I (TaKaRa
Bio). The first PCR round consisted of 35 cycles of (94 uC for 45 s,
with an additional 3 min in the first cycle, 60 uC for 45 s and 72 uC
for 3 min, with an additional 7 min in the last cycle) and used
primers NG797 and NG798. The second round consisted of 25 cycles
of (94 uC for 45 s, with an additional 3 min in the first cycle, 60 uC
for 45 s and 72 uC for 2 min, with an additional 7 min in the last
cycle) using primers NG799 and NG800 (Supplementary Table S1).
The approximately 3.2 kb amplification products were used for
determination of the full-length genomic sequences of TTMDV as
described below.
Determination of the entire genomic sequence of chimpanzee
TTMDV DNA. To determine the full-length nucleotide sequence of
three TTMDV isolates, the 1.0 kb genomic region including the GCrich area that overlapped with the previously amplified region at
both ends was amplified by PCR using nested primers that were
specific for each isolate (Supplementary Table S1) and TaKaRa LA
Taq polymerase with GC buffer I (TaKaRa Bio). In brief, the first
PCR round consisted of 35 cycles of 94 uC for 30 s, with an
additional 3 min in the first cycle, 60 uC for 30 s and 72 uC for 60 s,
with an additional 7 min in the last cycle. This was followed by a
second round consisting of 25 cycles using the same conditions as in
the first round. The 1.0 kb amplicon obtained from this PCR or the
3.2 kb amplicon (above) were cloned into pT7BlueT-Vector
(Novagen). Both strands of TTMDV DNA were sequenced by using
recombinant double-stranded DNA as templates and the BigDye
Terminator v3.1 Cycle Sequencing kit on an ABI 3100 Genetic
Analyzer (Applied Biosystems). Because of the difficulty in
sequencing GC-rich regions with stem and loop structures, when
necessary, the dGTP BigDye Terminator v3.0 Cycle Sequencing
Ready Reaction kit (Applied Biosystems) and DYEnamic ET
Terminator Cycle Sequencing kit (GE Healthcare) were also used.
Prevalence rates of Pan troglodytes (Pt)-TTMDV DNA in
chimpanzees and humans. In order to examine further the
prevalence of Pt-TTMDV infection in chimpanzees or in humans,
nested primers that were derived from a highly conserved area
among Pt-TTMDVs but not among TTVs, TTMDVs and TTMVs
were designed to specifically amplify a 121 bp product. Platinum
Taq DNA polymerase (Invitrogen) was used to amplify DNA in the
first round for 35 cycles (94 uC for 30 s, with an additional 2 min
in the first cycle, 59 uC for 30 s and 72 uC for 30 s, with an
additional 7 min in the last cycle) with primers NG824 and NG825,
followed by 25 cycles of the second round PCR (conditions the
same as in the first round) with primers NG826 and NG825
(Supplementary Table S1).
Analysis of nucleotide and amino acid sequences. Sequence
analysis was performed using Genetyx ver. 8 (Genetyx Corp.) and
ODEN (version 1.1.1) from the DNA Data Bank of Japan (National
Institute of Genetics, Mishima, Japan) (Ina, 1994). Sequence
alignments were generated by CLUSTAL W (version 1.8) (Thompson
et al., 1994). Phylogenetic trees were constructed by the neighbourjoining method (Saitou & Nei, 1987). Phylogenetic analysis of
sequences (Fig. 1; Supplementary Figs S1a, b and S2, available in JGV
Online) used uncorrected p-distances between nucleotide sequences.
The reliability of the phylogenetic results was assessed using 1000
bootstrap replicates (Felsenstein, 1985). The final tree was obtained
with TreeView program (version 1.6.6) (Page, 1996).
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RESULTS
Detection of TTV, TTMDV and TTMV DNAs in
serum samples of chimpanzees
The PCR assays for differential detection of three human
anelloviruses (Ninomiya et al., 2008) detected TTV and
TTMV DNA in serum samples from 73 of 74 chimpanzees
(98.6 %). This is similar to detection in humans, in which
the prevalence rates of TTV and TTMV DNA were 100 and
82 %, respectively. There was no discernible difference in
the prevalence of TTV and TTMV between wild-caught
and bred chimpanzees. However, TTMDV DNA was not
detectable in any of the 74 chimpanzees tested, despite a
high prevalence rate (75 %) of TTMDV DNA in humans
(Table 1).
Analysis of molecular clones of partial TTV,
TTMDV and TTMV sequences in chimpanzees
Anellovirus DNA from three randomly selected chimpanzees (nos. 108, 206 and 210) was amplified by using the
universal primers NG779/NG780 and NG781/NG782 in
the first round (Ninomiya et al., 2008); clones from the PtTTMDV isolates were identified and characterized phylogenetically. The phylogenetic tree constructed based on the
78–92 nt sequence (primer sequences at both ends
excluded) of the 72 clones obtained from chimpanzee
108 revealed the distribution of only TTV (n566) and
TTMV (n56) (Supplementary Fig. S1a). Similarly, 76 DNA
clones obtained from chimpanzee 206 were classified as
TTV (n562) and TTMV (n514) (Supplementary Fig.
S1b). However, one TTMDV-like clone that is remotely
related to all reported human SAV/TTMDV isolates was
identified from chimpanzee 210 (Fig. 1) and provisionally
designated Pt-Anello210-35.
Determination of the full-length nucleotide
sequence of TTMDV-like isolates from
chimpanzees
Inverted-nested primers that have nucleotide sequences
unique to the Pt-Anello210-35 clone (Fig. 2a), but are
markedly different from known human and chimpanzee
TTV isolates, human TTMDV isolates and human and
chimpanzee TTMV isolates within the corresponding
sequences, were designed for specific amplification of the
full-length circular Pt-Anello210-35 genome (Supplementary Table S1). When the long-distance inverted PCR
was performed in serum samples from seven chimpanzees
including chimpanzee 210, 3.2 kb amplicons were detected
in two chimpanzees (nos. 210 and 225). Five DNA clones,
each of 3.2 kb, were identified from the two PCR-positive
chimpanzees and were subjected to analysis by partial
nucleotide sequencing. Chimpanzee 225 had two distinct
groups of clones (Pt-TTMDV225-1 and Pt-TTMDV225-2)
that, in the partial 0.5 kb sequence, differed by 25 %. The
entire nucleotide sequence for a representative clone(s)
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349
M. Ninomiya and others
Fig. 1. Phylogenetic tree constructed by the
neighbour-joining method based on the partial
nucleotide sequences (78–92 nt) of TTV,
TTMDV and TTMV isolates obtained from
chimpanzee 210, using a macaque TTV-like
variant (Mf-TTV3) as an outgroup. Isolates are
all Pt-Anello210 strains, which are indicated
as, for example, ‘35’ for Pt-Anello210-35 for
simplicity. The representative TTV and TTMV
isolates, as well as all 22 reported SAV/
TTMDV isolates whose entire or near-entire
sequence is known, are indicated in bold type;
*, isolates of chimpanzee origin. Branch
lengths are proportional to uncorrected pdistances between sequences (see scale bar).
Bootstrap values are indicated for the major
nodes as a percentage obtained from 1 000
resamplings of the data. GenBank accession
numbers are given in parentheses.
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Analysis of torque teno midi virus in chimpanzees
Table 1. Detection of human anelloviruses (TTV, TTMDV and TTMV) and chimpanzee-specific TTMDV in serum samples obtained
from humans and chimpanzees
Sample
Chimpanzees
Experiment (2)
Wild-caught
Bred
Subtotal
Experiment (+)
Wild-caught
Bred
Subtotal
Total
Humans
Total no.
6
32
38
28
8
36
74
100
No. (%) of positive samples detected by:
Human-TTV PCR
Human-TTMDV PCR
Human-TTMV PCR
Pt-TTMDV-specific PCR
6 (100)
32 (100)
38 (100)
0
0
0
6 (100)
32 (100)
38 (100)
6 (100)
25 (78)
31 (82)
27
8
35
73
82
25
7
32
63
1
27
8
35
73
100
(96)
(100)
(97)
(99)
(100)*
0
0
0
0
75 (75)*
(96)
(100)
(97)
(99)
(82)*
(89)
(88)
(89)
(85)
(1)
*Data from Ninomiya et al., (2008).
from each of the two samples, and their 59 and 39 terminal
sequences corresponding to the primers that were used for
the 3.2 kb long-distance PCR, was confirmed by sequencing the 0.8–1.2 kb product amplified by the inverted PCR
with isolate-specific nested primers (Supplementary Table
S1). Consequently, the complete genomic sequence was
determined for the three TTMDV-like isolates in the
present study, named Pt-TTMDV210, Pt-TTMDV225-1
and Pt-TTMDV225-2, respectively. These isolates had
circular genomes of 3257, 3256 and 3269 nt, respectively.
These are similar in size to human TTMDV, longer than
human and chimpanzee TTMVs and shorter than human
and chimpanzee TTVs. Each chimpanzee TTMDV isolate
possessed four major ORFs, regions with high GC content,
a coding region defined as the sequence between the
beginning of ORF2 and the end of ORF4 that has a high
degree of genetic divergence and a non-coding region
between the end of ORF4 and the beginning of ORF2, with
a relatively conserved area (Fig. 3a).
that is rich in arginine at its N terminus and ORF2 encoded
the conserved motif W-X7-H-X3-C-X1-C-X5-H (Hijikata et
al., 1999; Okamoto et al., 2000b; Takahashi et al., 2000;
Ninomiya et al., 2007a).
Although the TTMDV-specific mRNAs have not yet been
analysed, the consensus motifs of donor and acceptor sites
(Breathnach et al., 1978; Mount, 1982) for three splicings
(Fig. 3b) were found in three chimpanzee TTMDV isolates,
similar to those in TTVs which are known to have three
distinct species of TTV mRNAs (2.9–3, 1.2 and 1 kb) with
three different splicings (Kamahora et al., 2000; Okamoto et
al., 2000c). The short splicing of 110–111 nt (Splice 1 in Fig.
3b) was assumed to be present in the three mRNAs of
chimpanzee TTMDV. The two longer splicings of 1386–
1404 nt (Splice 2 in Fig. 3) and 1558–1576 nt (Splice 3 in Fig.
3b) were hypothesized to exist in the two short mRNAs.
To elucidate the genetic relatedness of Pt-TTMDV with
other anelloviruses of human and non-human origin, a
phylogenetic tree was constructed based on inferred amino
acid sequences from ORF1 (Fig. 4b). To avoid measuring
distances between mis-aligned sequences, we excluded the
lysine/arginine-rich region at the start of the protein
(codons 1–68 in the prototype TA278 sequence), as it was
impossible to identify homologous amino acids in
sequences largely restricted to these two residues. We
similarly excluded the C-terminal region sequence beyond
amino acid 288 as sequences of non-human primates could
not be defensibly aligned with each other or with primatederived TTV sequences. The tree showed that chimpanzee
TTMDV is a virus species that is phylogenetically
distinguishable from TTVs and TTMVs of human and
chimpanzee origin as well as TTVs of macaque, douroucouli, tamarin, tupaia, dog, cat and pig, whose entire
In the deduced amino acid sequences encoded by ORF1
and ORF2, several motifs known to be characteristic of
human and chimpanzee TTVs and TTMVs as well as
human TTMDVs were also preserved in the three PtTTMDV isolates. ORF1 encoded a sequence of 658–663 aa
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Comparison of the three Pt-TTMDV isolates with
each other and the reported anelloviruses
The three Pt-TTMDV isolates obtained in the present study
were 79.7–82.9 % similar to each other over the entire
genome. They showed only 56.4–59.6 % identity with 18
human TTMDV isolates whose entire genomic sequence
has been determined (Ninomiya et al., 2007a, b). A
phylogenetic tree was constructed based on the entire
nucleotide sequence of the three Pt-TTMDV and 18
human TTMDV isolates, which revealed that Pt-TTMDV
isolates segregate into the same cluster with a bootstrap
value of 100 % and are clearly separate from human
TTMDV (Fig. 4a).
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M. Ninomiya and others
Fig. 2. Strategy for amplifying chimpanzee TTMDV genomes. (a) Positions and sequences of oligonucleotide primers (NG797,
NG798, NG799 and NG800) are indicated. The primers were used for inverted long-distance PCR and have sequences
specific to the Pt-Anello210-35 clone. Nucleotide positions (54–150) were determined according to the sequence of the
3257 nt-long Pt-TTMDV210 isolate obtained in this study. (b) Positions and sequences of oligonucleotide primers (NG824,
NG825 and NG826) used in the chimpanzee TTMDV-specific PCR are shown. The universal primers (NG779/NG780 and
NG781/NG782), which are located just downstream of the TATA box, that were used for amplification of three anellovirus
DNAs in the first round are also shown. For comparison, partial nucleotide sequences of representative isolates of human/
chimpanzee TTV, human/chimpanzee TTMV and human TTMDV (GenBank accession numbers are given in Fig. 1) are indicated
in (a) and (b). *, Indicates that the origin of the TTV and TTMV isolates is reported to be chimpanzee.
genomic sequence has thus far been determined (Okamoto
et al., 2000a, b, 2001, 2002). Of note, chimpanzee TTMDVs
were clearly separate from human TTMDVs, while chimpanzee TTVs (Pt-TTV6, s-TTV.CH65-1, s-TTV.CH65-2
and s-TTV.CH71) and chimpanzee TTMV (Pt-TTMV8-II)
were interspersed with human TTVs and human TTMVs,
respectively.
352
Detection of Pt-TTMDV DNA in serum samples
from chimpanzees and humans
In an attempt to examine more accurately the prevalence of
Pt-TTMDV in chimpanzees, nested primers that are
specific to Pt-TTMDV located in the highly conserved
genomic region just downstream of the TATA box were
designed for specific amplification of Pt-TTMDV DNA by
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Journal of General Virology 90
Analysis of torque teno midi virus in chimpanzees
Fig. 3. Genomic organization and putative
transcription profile of chimpanzee TTMDV
isolates. (a) The predicted genomic organization of a representative chimpanzee TTMDV
isolate (Pt-TTMDV210) compared with the
prototype TTMDV of human origin. The closed
arrows represent four major ORFs (ORF1–
ORF4). The open boxes (depicted with dotted
lines) located between an upstream closed
box and downstream closed arrow in ORF3
and ORF4, which encode joint proteins,
represent areas corresponding to introns in
the mRNAs (Kamahora et al., 2000; Okamoto
et al., 2000c). The shaded boxes indicate the
GC-rich stretches; the small closed circle
represents the position of the TATA box. (b)
Sequences of the donor and acceptor sites of
splicing in TTMDVs. The consensus sequence
of donor and acceptor sites in splicings
(Breathnach et al., 1978; Mount, 1982) is
shown above the splice sequences. M denotes
A or C, R denotes A or G, Y denotes T or C
and X denotes A, G, C or T. The sequences of
the donor and acceptor sites for TTMDVs are
indicated (i) in the first splicing (Splice 1),
which are shared by the 2.9–3, 1.2 and 1 kb
mRNAs of human TTVs VT416 (Kamahora et
al., 2000) and TYM9 (Okamoto et al., 2000c),
(ii) in the second splicing (Splice 2), which is
present in the 1.2 kb mRNA, and (iii) in the
third splicing (Splice 3), which is present in the
1.0 kb mRNA. Nucleotides matching the consensus sequence are shown in upper-case.
The regions of introns and the number of
nucleotides spliced out (in parentheses) are
shown on the right.
a PCR assay (Fig. 2b). By using this newly developed PCR
assay (Pt-TTMDV-specific PCR), Pt-TTMDV DNA was
detected in serum samples from 63 (85 %) of 74
chimpanzees (Table 1). Sequence analysis of the 20
randomly selected amplicons demonstrated the specificity
of the PCR assay (Supplementary Fig. S2): all 20
chimpanzee TTMDV isolates were grouped into the cluster
comprising Pt-TTMDV210, Pt-TTMDV225-1 and PtTTMDV225-2 isolates. No significant difference was noted
in the prevalence of chimpanzee TTMDV between wildcaught and bred chimpanzees or between chimpanzees that
had or had not taken part in a transmission experiment in
the past. Of interest, when the Pt-TTMDV-specific PCR
was applied to 100 human sera, one subject who tested
positive for human TTV and TTMDV DNA was found to
be co-infected with chimpanzee TTMDV. Reproducibility
of the PCR assay was verified by repeated assays and the
specificity of the assay was confirmed by sequence analysis
of the amplicon (Supplementary Fig. S2). The TTMDV
isolate (Hu-TTMDV1075) obtained from this subject was
http://vir.sgmjournals.org
segregated into the cluster consisting of three chimpanzee
TTMDV isolates whose entire nucleotide sequence was
determined and 20 other isolates with the prefix of PtTTMDV whose partial nucleotide sequence was determined.
DISCUSSION
Detection and characterization of TTMDV
In the present study, when the differential PCR assays for
three human anelloviruses were applied to serum samples
from chimpanzees, both TTV and TTMV DNA was
detected in all but one of the 74 chimpanzees tested,
indicating that chimpanzees are also frequently infected
with human TTVs and TTMVs. Phylogenetic analysis
indicated that chimpanzees are also infected with their own
TTVs and TTMVs that are closer to those of chimpanzee
origin (Fig. 1). Contrary to our expectations, DNA of
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M. Ninomiya and others
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Journal of General Virology 90
Analysis of torque teno midi virus in chimpanzees
Fig. 4. Phylogenetic trees constructed based on the entire genomic sequence of the 21 TTMDV isolates including the three
chimpanzee isolates obtained in the present study and the 18 human isolates obtained in our previous study (Ninomiya et al.,
2007a, b) (a) and amino acid sequences from the conserved region of ORF1 (b). The trees were constructed from pairwise
uncorrected amino acid p-distances by the neighbour-joining method (Saitou & Nei, 1987). The tree in (b) was constructed to
enable the more divergent non-primate TTV-like sequences to be defensibly aligned; it includes sequences from the 21 human
and chimpanzee TTMDV isolates as well as 71 representative TTV and 19 representative TTMV isolates from humans, four TTV
isolates and one TTMV isolate from chimpanzees and other anelloviruses from macaques (Mf-TTV3 and Mf-TTV9), a
douroucouli (At-TTV3), a tamarin (So-TTV2), a tupaia (Tbc-TTV14), a pig (Sd-TTV31), a dog (Cf-TTV10) and a cat (Fc-TTV4).
Selected bootstrap values for branches defining the main groups are shown near the nodes (percentage generated from 1000
samplings of the data). The scale represents the number of nucleotide or amino acid substitutions per position.
human TTMDV was not detectable in any of the
chimpanzees tested, regardless of the past history of
transmission experiments with human viruses. However,
taking into account that anelloviruses are markedly
heterogeneous, which may cause false-negative detection
of the viral genome due to mismatches of primer
sequences, it seems likely that chimpanzees have their
own TTMDVs. To search for TTMDV-like viruses in
chimpanzees, a total of 70, 72 or 76 molecular clones of
amplicons, obtained from DNA from sera of each of three
randomly selected chimpanzees, were subjected to
sequence analysis. One clone (Pt-Anello210-35) that was
remotely related to all known human TTMDV isolates was
found in one of the three chimpanzees. This finding led to
the identification of chimpanzee-specific TTMDV, which
was provisionally designated Pt-TTMDV, and resulted in
the determination of the entire nucleotide sequence of
three such isolates (Pt-TTMDV210, Pt-TTMDV225-1 and
Pt-TTMDV225-2) each with a 3.2 kb genome, identical to
that of human TTMDV isolates.
Although chimpanzee TTMDV differs from human
TTMDV by 40.4–43.6 % (mean542 %) over the entire
genome and by 60.6–68.9 % (mean564.3 %) in the entire
ORF1 amino acid sequence, it seems reasonable to classify
chimpanzee TTMDV into the third group of the genus
Anellovirus together with human TTMDV, based on the
following reasons. Phylogenetic analysis revealed that
chimpanzee TTMDV isolates segregate into an independent cluster supported by a high bootstrap value of 100 %,
but they are closest to the cluster of human TTMDV
isolates among all known anelloviruses in humans, nonhuman primates and non-primate animals whose ORF1
amino acid sequence has been determined (Fig. 4b).
Human and chimpanzee TTMDV isolates have in common
a circular 3.2 kb DNA genome, a genomic organization
comprising four major ORFs with high diversity but of
similar overall size, a well-conserved area within the noncoding region with GC-rich regions and several motifs of
deduced amino acid sequences encoded by ORFs that are
similar to TTVs and TTMVs, as well as a unique
transcriptional profile that has been demonstrated for
TTVs (Kamahora et al., 2000; Okamoto et al., 2000c).
A complication of primate studies is the cross-species
transmission of TTVs and TTMVs. Human and chimpanzee TTVs do not cluster into species-specific monophyla.
http://vir.sgmjournals.org
Rather, some genetic groups of human and chimpanzee
TTVs cluster to make human/chimpanzee clades, as has
been reported for T-cell leukemia virus type 1 and HIV
type 1 (Gao et al., 1999; Koralnik et al., 1994). Similarly,
TTMVs of chimpanzee origin have genotypes distinct
from, but interspersed between, human TTMV genotypes.
Interestingly, it has been reported that simian TTV
infections also occur in humans, indicating that TTV
may be of zoonotic origin (Iwaki et al., 2003). However, it
is also possible that some TTVs found in apes that are
classified as simian TTVs are actually human TTVs,
transmitted during capture, transportation or handling of
the animals, as has been speculated for human TTV
sequences that were detected in gibbons (Noppornpanth et
al., 2001). Cross-species transmission may occur through
the administration of human plasma-derived blood
products containing infectious TTV or related viruses,
which frequently occurs during animal experimentation.
Both transient and persistent infections of human TTV in
chimpanzees were observed in experimental transmission
studies (Mushahwar et al., 1999: Tawara et al., 2000). The
present study corroborates the previous studies because
human TTV sequences have been identified in samples
obtained from captive chimpanzees (Cong et al., 2000;
Okamoto et al., 2000a; Romeo et al., 2000), supporting the
transmissibility of human TTVs to chimpanzees. Therefore,
to investigate further their species specificity, samples
should ideally be collected from animals in the wild.
Although serum samples obtained from wild-caught and
bred chimpanzees that had been raised in a Primates Park
for more than 3 years were used in the present study, human
TTMDV was not detected in any of the chimpanzees tested
and chimpanzee TTMDV was detected in only one of 100
human subjects (Table 1). Phylogenetic analysis revealed
that chimpanzee TTMDV isolates segregated into a cluster
and were clearly separate from the human TTMDV isolates,
suggesting that cross-species infection of TTMDV occurs
rarely, if at all, between humans and chimpanzees. At
present, serological methods to detect past infection and
immunological responses to human TTMDV in chimpanzees are not available. The development of assays capable of
detecting type-specific antibody to different anellovirus
variants would be of use as a much more sensitive indicator
of the frequency of exposure and infection of chimpanzees
with human TTMDV.
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M. Ninomiya and others
Co-evolution of TTV-like viruses in primates
Observations of consistently congruent phylogenetic relationships between TTV-like viruses and their primate and
non-primate hosts in which they are detected has widely
suggested the possibility that these viruses have remained
host-specific, and that their evolution has followed that of
the animals they infect (Okamoto et al., 2000b, 2001, 2002;
Thom et al., 2003; Verschoor et al., 1999). Although there
are other potential explanations for these similarities in
host and virus phylogenies, such as the effects of
environmental overlap and of large-scale virus selection
associated with adaptation for replication and persistence
in a range of dissimilar mammalian species, the coevolution hypothesis has several well-established precedents in other persistent mammalian DNA viruses, such as
herpesviruses (McGeoch et al., 2006) and papillomaviruses
(Garcı́a-Vallvé et al., 2005).
TTV-like viruses infecting cats and dogs (two suborders in
the order Carnivora) and pigs (order Artiodactyla) are
highly divergent from each other and from those found in
primates (Okamoto et al., 2002). The latter form a
monophyletic group with variants recovered from the
tupaia branching first followed by New World (owl
monkey) and then Old World monkey species (macaque)
viruses and finally a large group of viruses obtained from
chimpanzees and humans. However, rather than being
monophyletic, the three groups of ape-derived TTV-like
viruses (TTV, TTMDV and TTMV) contain viruses from
both humans and chimpanzees. The co-evolution hypothesis would, if confirmed, place their divergence from
each other before the speciation of humans and chimpanzees, currently thought to have occurred approximately 5–7
million years ago (The Chimpanzee Sequencing and
Analysis Consortium, 2005).
Although the co-evolution hypothesis suggests that the last
common ancestor of human and chimpanzee must have
been infected with progenitor strains of all three viruses,
the current study identifies possible differences in their
evolutionary timescales. As groups, TTV and TTMV are
more diverse than TTMDV; the percentage identity over
the entire genome among TTVs was recorded to be
approximately 49 % (Peng et al., 2002) and approximately
43 % among TTMVs (Biagini et al., 2001b, 2007), while the
lowest similarity among 21 human and chimpanzee
TTMDV isolates was 56 % (this study; Ninomiya et al.,
2007a, b). Furthermore, while variants of TTV and TTMV
from chimpanzees and humans are interspersed, those of
TTMDV are monophyletic for each species, with much
more restricted sequence diversity within each; the lowest
similarities were 67 and 80 % within the 18 human and
three chimpanzee TTMDV variants characterized to date.
The available data therefore suggest that the TTMDV
group originated at a later time than the time of
chimpanzee/human speciation, rather than before it, as
seems likely for TTV and TTMV. However, we fully
acknowledge that at this early stage, the dataset of TTMDV
356
sequences that is available may not encompass the full
diversity of viruses within this group. Confirmation of the
restricted sequence diversity of TTMDV and thus the later
evolutionary origin for TTMDV that this would indicate
will require further analysis of the genetic diversity of this
virus group in humans, chimpanzees and, ideally, in other
higher apes.
In conclusion, the present study revealed that wild-caught
and bred chimpanzees that had been raised in a Primates
Park for more than 3 years were frequently infected with
human/chimpanzee TTV (viraemia rate 98.6 %) and
human/chimpanzee TTMV (98.6 %), but not with human
TTMDV. Instead, chimpanzees were found to be frequently
infected with a distinct set of TTMDV variants (85.1 %),
with a 3.2 kb-long genome, which is similar to the length
of human TTMDV and between that of TTV and TTMV.
Chimpanzee (Pan troglodytes)-specific TTMDV was tentatively abbreviated Pt-TTMDV and designated as another
member of the third group in the genus Anellovirus. As
expected, frequent dual or triple infection of these three
anelloviruses was observed in chimpanzees, similar to the
infections observed in humans. Chimpanzees harbour a
complex flora of TTV, TTMDV and TTMV that is similarly
diverse to that found in humans (Simmonds et al., 1999).
Unexpectedly, infrequent cross-species infection of
TTMDV between humans and chimpanzees was noted in
the present study (this is dissimilar to TTV and TTMV),
the underlying mechanism of which warrants further
analysis. Investigating the evolutionary mechanisms by
which TTVs, TTMDVs and TTMVs can establish persistent
infections in humans and chimpanzees would be a
productive area for fundamental virology in the future.
ACKNOWLEDGEMENTS
This work was supported in part by a grant from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.
REFERENCES
Biagini, P., Gallian, P., Attoui, H., Cantaloube, J.-F., Touinssi, M., de
Micco, P. & de Lamballerie, X. (2001a). Comparison of systems
performance for TT virus detection using PCR primer sets located in
non-coding and coding regions of the viral genome. J Clin Virol 22,
91–99.
Biagini, P., Gallian, P., Attoui, H., Touinssi, M., Cantaloube, J.-F., de
Micco, P. & de Lamballerie, X. (2001b). Genetic analysis of full-length
genomes and subgenomic sequences of TT virus-like mini virus
human isolates. J Gen Virol 82, 379–383.
Biagini, P., Todd, D., Bendinelli, M., Hino, S., Mankertz, A., Mishiro, S.,
Niel, C., Okamoto, H., Radial, S. & other authors (2005). Anellovirus.
In Virus Taxonomy, Eighth Report of the International Committee
on Taxonomy of Viruses, pp. 335–341. Edited by C. M. Fauquet, M. A.
Mayo, J. Maniloff, U. Desselberger & L. A. Ball. San Diego, CA:
Elsevier.
Biagini, P., Uch, R., Belhouchet, M., Attoui, H., Cantaloube, J.-F.,
Brisbarre, N. & de Micco, P. (2007). Circular genomes related to
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:50:37
Journal of General Virology 90
Analysis of torque teno midi virus in chimpanzees
anelloviruses identified in human and animal samples by using a
combined rolling-circle amplification/sequence-independent single
primer amplification approach. J Gen Virol 88, 2696–2701.
Breathnach, R., Benoist, C., O’Hare, K., Gannon, F. & Chambon, P.
(1978). Ovalbumin gene: evidence for a leader sequence in mRNA
and DNA sequences at the exon-intron boundaries. Proc Natl Acad Sci
U S A 75, 4853–4857.
Cong, M. E., Nichols, B., Dou, X. G., Spelbring, J. E., Krawczynski, K.,
Fields, H. A. & Khudyakov, Y. E. (2000). Related TT viruses in
chimpanzees. Virology 274, 343–355.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach
evidence for a new virus family infecting humans. Proc Natl Acad Sci
U S A 96, 3177–3182.
Ninomiya, M., Nishizawa, T., Takahashi, M., Lorenzo, F. R.,
Shimosegawa, T. & Okamoto, H. (2007a). Identification and
genomic characterization of a novel human torque teno virus of 3.2
kb. J Gen Virol 88, 1939–1944.
Ninomiya, M., Takahashi, M., Shimosegawa, T. & Okamoto, H.
(2007b). Analysis of the entire genomes of fifteen torque teno midi
virus variants classifiable into a third group of genus Anellovirus. Arch
Virol 152, 1961–1975.
using the bootstrap. Evolution 39, 783–791.
Ninomiya, M., Takahashi, M., Nishizawa, T., Shimosegawa, T. &
Okamoto, H. (2008). Development of PCR assays with nested primers
Gao, F., Bailes, E., Robertson, D. L., Chen, Y., Rodenburg, C. M.,
Michael, S. F., Cummins, L. B., Arthur, L. O., Peeters, M. & other
authors (1999). Origin of HIV-1 in the chimpanzee Pan troglodytes
specific for differential detection of three human anelloviruses and
early acquisition of dual or triple infection during infancy. J Clin
Microbiol 46, 507–514.
troglodytes. Nature 397, 436–441.
Nishizawa, T., Okamoto, H., Konishi, K., Yoshizawa, H., Miyakawa, Y.
& Mayumi, M. (1997). A novel DNA virus (TTV) associated with
Garcı́a-Vallvé, S., Alonso, A. & Bravo, I. G. (2005). Papillomaviruses:
different genes have different histories. Trends Microbiol 13, 514–521.
Hijikata, M., Takahashi, K. & Mishiro, S. (1999). Complete circular
DNA genome of a TT virus variant (isolate name SANBAN) and 44
partial ORF2 sequences implicating a great degree of diversity beyond
genotypes. Virology 260, 17–22.
Hino, S. & Miyata, H. (2007). Torque teno virus (TTV): current status.
Rev Med Virol 17, 45–57.
Ina, Y. (1994). ODEN: a program package for molecular evolutionary
analysis and database search of DNA and amino acid sequences.
Comput Appl Biosci 10, 11–12.
Inami, T., Obara, T., Moriyama, M., Arakawa, Y. & Abe, K. (2000).
Full-length nucleotide sequence of a simian TT virus isolate obtained
from a chimpanzee: evidence for a new TT virus-like species. Virology
277, 330–335.
Iwaki, Y., Aiba, N., Tran, H. T., Ding, X., Hayashi, S., Arakawa, Y.,
Sata, T. & Abe, K. (2003). Simian TT virus (s-TTV) infection in
patients with liver diseases. Hepatol Res 25, 135–142.
Jones, M. S., Kapoor, A., Lukashov, V. V., Simmonds, P., Hecht, F. &
Delwart, E. (2005). New DNA viruses identified in patients with acute
viral infection syndrome. J Virol 79, 8230–8236.
Kamahora, T., Hino, S. & Miyata, H. (2000). Three spliced mRNAs of
TT virus transcribed from a plasmid containing the entire genome in
COS1 cells. J Virol 74, 9980–9986.
Koralnik, I. J., Boeri, E., Saxinger, W. C., Monico, A. L., Fullen, J.,
Gessain, A., Guo, H. G., Gallo, R. C., Markham, P. & other authors
(1994). Phylogenetic associations of human and simian T-cell
leukemia/lymphotropic virus type I strains: evidence for interspecies
transmission. J Virol 68, 2693–2707.
Luo, K., Liang, W., He, H., Yang, S., Wang, Y., Xiao, H., Liu, D. &
Zhang, L. (2000). Experimental infection of nonenveloped DNA virus
(TTV) in rhesus monkey. J Med Virol 61, 159–164.
McGeoch, D. J., Rixon, F. J. & Davison, A. J. (2006). Topics in
herpesvirus genomics and evolution. Virus Res 117, 90–104.
elevated transaminase levels in posttransfusion hepatitis of unknown
etiology. Biochem Biophys Res Commun 241, 92–97.
Noppornpanth, S., Chinchai, T., Ratanakorn, P. & Poovorawan, Y.
(2001). TT virus infection in gibbons. J Vet Med Sci 63, 663–666.
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.
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.
Okamoto, H., Fukuda, M., Tawara, A., Nishizawa, T., Itoh, Y.,
Hayasaka, I., Tsuda, F., Tanaka, T., Miyakawa, Y. & Mayumi, M.
(2000a). Species-specific TT viruses and cross-species infection in
nonhuman primates. J Virol 74, 1132–1139.
Okamoto, H., Nishizawa, T., Tawara, A., Peng, Y., Takahashi, M.,
Kishimoto, J., Tanaka, T., Miyakawa, Y. & Mayumi, M. (2000b).
Species-specific TT viruses in humans and nonhuman primates and
their phylogenetic relatedness. Virology 277, 368–378.
Okamoto, H., Nishizawa, T., Tawara, A., Takahashi, M., Kishimoto, J.,
Sai, T. & Sugai, Y. (2000c). TT virus mRNAs detected in the bone
marrow cells from an infected individual. Biochem Biophys Res
Commun 279, 700–707.
Okamoto, H., Nishizawa, T., Takahashi, M., Tawara, A., Peng, Y.,
Kishimoto, J. & Wang, Y. (2001). Genomic and evolutionary
characterization of TT virus (TTV) in tupaias and comparison with
species-specific TTVs in humans and non-human primates. J Gen
Virol 82, 2041–2050.
Okamoto, H., Takahashi, M., Nishizawa, T., Tawara, A., Fukai, K.,
Muramatsu, U., Naito, Y. & Yoshikawa, A. (2002). Genomic
characterization of TT viruses (TTVs) in pigs, cats and dogs and
their relatedness with species-specific TTVs in primates and tupaias.
J Gen Virol 83, 1291–1297.
Miyata, H., Tsunoda, H., Kazi, A., Yamada, A., Khan, M. A., Murakami, J.,
Kamahora, T., Shiraki, K. & Hino, S. (1999). Identification of a novel
Okamoto, H., Takahashi, M. & Nishizawa, T. (2004). Torque teno
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.
virus (TTV): molecular virology and clinical implications. In Viral
Hepatitis: Molecular Biology, Diagnosis, Epidemiology and Control, pp.
241–254. Edited by I. K. Mushahwar. San Diego, CA: Elsevier.
Mount, S. M. (1982). A catalogue of splice junction sequences. Nucleic
Page, R. D. M. (1996). TreeView: an application to display
phylogenetic trees on personal computers. Comput Appl Biosci 12,
357–358.
Acids Res 10, 459–472.
Mushahwar, I. K., Erker, J. C., Muerhoff, A. S., Leary, T. P., Simons,
J. N., Birkenmeyer, L. G., Chalmers, M. L., Pilot-Matias, T. J. & Dexai,
S. M. (1999). Molecular and biophysical characterization of TT virus:
http://vir.sgmjournals.org
Peng, Y. H., Nishizawa, T., Takahashi, M., Ishikawa, T., Yoshikawa, A.
& Okamoto, H. (2002). Analysis of the entire genomes of thirteen TT
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:50:37
357
M. Ninomiya and others
virus variants classifiable into the fourth and fifth genetic groups,
isolated from viremic infants. Arch Virol 147, 21–41.
TLMV) intermediately related to TT virus and chicken anemia virus.
Arch Virol 145, 979–993.
Romeo, R., Hegerich, P., Emerson, S. U., Colombo, M., Purcell, R. H.
& Bukh, J. (2000). High prevalence of TT virus (TTV) in naive
Tawara, A., Akahane, Y., Takahashi, M., Nishizawa, T., Ishikawa, T. &
Okamoto, H. (2000). Transmission of human TT virus of genotype 1a
chimpanzees and in hepatitis C virus-infected humans: frequent
mixed infections and identification of new TTV genotypes in
chimpanzees. J Gen Virol 81, 1001–1007.
to chimpanzees with fecal supernatant or serum from patients with
acute TTV infection. Biochem Biophys Res Commun 278, 470–476.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
Initial sequence of the chimpanzee genome and comparison with the
human genome. Nature 437, 69–87.
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–
425.
Simmonds, P. (2002). TT virus and related human circoviruses. In
Clinical Virology, 2nd edn, pp. 613–621. Edited by D. D. Richman,
R. J. Whitley & F. G. Hayden. Washington, DC: American Society for
Microbiology.
The Chimpanzee Sequencing and Analysis Consortium (2005).
Thom, K., Morrison, C., Lewis, J. C. & Simmonds, P. (2003).
Distribution of TT virus (TTV), TTV-like minivirus, and related
viruses in humans and nonhuman primates. Virology 306, 324–333.
Simmonds, P., Prescott, L. E., Logue, C., Davidson, F., Thomas, A. E.
& Ludlam, C. A. (1999). TT virus – part of the normal human flora?
CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties and
weight matrix choice. Nucleic Acids Res 22, 4673–4680.
J Infect Dis 180, 1748–1750.
Verschoor, E. J., Langenhuijzen, S. & Heeney, J. L. (1999). TT viruses
Takahashi, K., Iwasa, Y., Hijikata, M. & Mishiro, S. (2000).
(TTV) of non-human primates and their relationship to the human
TTV genotypes. J Gen Virol 80, 2491–2499.
Identification of a new human DNA virus (TTV-like mini virus,
358
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:50:37
Journal of General Virology 90