Genomic and evolutionary characterization of TT virus (TTV) in tupaias and comparison with species-specific TTVs in humans and non-human primates

Hiroaki Okamoto1, Tsutomu Nishizawa1, Masaharu Takahashi1, Akio Tawara2, Yihong Peng1, Junichi Kishimoto3 and Yu Wang4

Immunology Division and Division of Molecular Virology, Jichi Medical School, Tochigi-Ken 329-0498, Japan1
First Department of Internal Medicine, Yamanashi Medical University, Yamanashi-Ken 409-3898, Japan2
Institute of Immunology, Tokyo 112-0004, Japan3
Hepatology Institute, People’s Hospital, Peking University, Beijing 100044, People’s Republic of China4

Author for correspondence: Hiroaki Okamoto. Fax +81 285 44 1557. e-mail hokamoto{at}jichi.ac.jp


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
TT virus (TTV) was recovered from the sera of tupaias (Tupaia belangeri chinensis) by PCR using primers derived from the noncoding region of the human TTV genome, and its entire genomic sequence was determined. One tupaia TTV isolate (Tbc-TTV14) consisted of only 2199 nucleotides (nt) and had three open reading frames (ORFs), spanning 1506 nt (ORF1), 177 nt (ORF2) and 642 nt (ORF3), which were in the same orientation as the ORFs of the human prototype TTV (TA278). ORF3 was presumed to arise from a splicing of TTV mRNA, similar to reported human TTVs whose spliced mRNAs have been identified, and encoded a joint protein of 214 amino acids with a Ser-, Lys- and Arg-rich sequence at the C terminus. Tbc-TTV14 was less than 50% similar to previously reported TTVs of 3·4–3·9 kb and TTV-like mini viruses (TLMVs) of 2·8–3·0 kb isolated from humans and non-human primates, and known animal circoviruses. Although Tbc-TTV14 has a genomic length similar to animal circoviruses (1·8–2·3 kb), Tbc-TTV14 resembled TTVs and TLMVs with regard to putative genomic organization and transcription profile. Conserved motifs were commonly observed in the coding and noncoding regions of the Tbc-TTV14 genome and in all TTV and TLMV genomes. Phylogenetic analysis revealed that Tbc-TTV14 is the closest to TLMVs, and is closer to TTVs isolated from tamarin and douroucouli than to TTVs isolated from humans and chimpanzees. These results indicate that tupaias are naturally infected with a new TTV species that has not been identified among primates.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
TT virus (TTV) was isolated from the serum of a patient with post-transfusion hepatitis of unknown aetiology in 1997 (Nishizawa et al., 1997 ). It has a negative-sense, single-stranded circular DNA genome of approximately 3·8 kb (Okamoto et al., 1998 , 1999a ; Miyata et al., 1999 ; Mushahwar et al., 1999 ). Because of the lack of significant sequence similarity between TTV and members of the Circoviridae family, including porcine circovirus (PCV) and beak and feather disease virus (BFDV) of parrots in the Circovirus genus, and chicken anaemia virus (CAV) in the Gyrovirus genus (Pringle, 1999 ; Todd et al., 2000 ), it has been proposed that TTV belongs to a new virus family that has been tentatively designated Circinoviridae (Mushahwar et al., 1999 ), Paracircoviridae (Takahashi et al., 2000b ) or the TTV family (Tanaka et al., 2001 ) by different research groups. TTV isolates have an extremely wide range of sequence divergence (Khudyakov et al., 2000 ; Mushahwar, 2001 ; Okamoto et al., 1999b ) and they are tentatively classified into 23 genotypes with sequence divergence of >30% from one another (Muljono et al., 2001 ) or into four major phylogenetic groups (Muljono et al., 2001 ; Tanaka et al., 2001 ). In addition, a smaller member of this virus family, TTV-like mini virus (TLMV), whose genomic length is approximately 2·8–3·0 kb, has recently been identified in humans and chimpanzees (Okamoto et al., 2000b ; Takahashi et al., 2000a , b ; Biagini et al., 2001 ).

TTV recovered from the sera and faeces of infected humans has been visualized by electron microscopy and found to be an unenveloped, small, spherical particle with a diameter of 30–32 nm (Itoh et al., 2000 ). Circular double-stranded TTV DNA in replicative intermediate forms has been detected in liver tissues and bone marrow cells, suggesting that TTV can replicate in these tissues (Okamoto et al., 2000d , e ). It has recently been demonstrated in vitro and in vivo that three distinct mRNAs of 2·9–3·0, 1·2 and 1·0 kb with common 5' and 3' termini are transcribed from the minus-stranded TTV DNA (Kamahora et al., 2000 ; Okamoto et al., 2000c ). These three mRNAs have in common a short splicing of approximately 100 nucleotides (nt). The 1·2 and 1·0 kb mRNAs possess an additional splicing of approximately 1700 and 1900 nt, respectively, leading to the creation of two novel open reading frames (ORF3 and ORF4).

There is increasing evidence that non-human primates and farm animals are infected with TTV (Abe et al., 2000 ; Leary et al., 1999 ; Okamoto et al., 2000a ; Romeo et al., 2000 ; Verschoor et al., 1999 ). The entire nucleotide sequences of species-specific TTVs that infect non-human primates such as chimpanzee (Pan troglodytes), Japanese macaque (Macaca fuscata), cotton-top tamarin (Saguinus oedipus) and douroucouli (Aotes trivirgatus) have been determined (Okamoto et al., 2000b ; Inami et al., 2000 ). In the present study, TTV was isolated from tupaias (tree shrews), which share characteristics with both primates and insectivores, and which have recently been classified in a single order called Scandentia, rather than in the order Primates or the order Insectivora (Martin, 1990 ). The complete DNA sequence of TTV of tupaia origin was determined and compared with species-specific TTVs and TLMVs from humans and non-human primates. The present study indicated that the TTV that infects tupaias has the shortest and simplest viral genome (2199 nt) among the TTVs and TLMVs thus far identified, and that although the tupaia TTV genome differs from all other TTV and TLMV genomes by more than 50% at both the nucleotide and amino acid levels, its putative genomic organization and transcription profile are similar to those of TTVs and TLMVs. The results obtained will be useful for gaining a better understanding of the genomic characteristics, evolutionary relationships and taxonomic classification of TTVs and TLMVs in humans, non-human primates and Scandentia.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Tupaias.
Nineteen tupaias (Tupaia belangeri chinensis) were caught in the wild in Yunnan Province, China, in 1989 and their sera were collected. The serum samples were obtained from the animals before they were used for medical experiments, and were kept at -20 °C until testing. They were negative for hepatitis B surface antigen (HBsAg) by passive haemagglutination (MyCell RPHA, Institute of Immunology Co.) and negative for antibody against hepatitis C virus (HCV) by passive haemagglutination (Abbott HCV PHA 2nd Generation, Dainabot).

{blacksquare} Extraction of nucleic acids and amplification by PCR.
Nucleic acids were extracted from 100 µl of serum using a High Pure Viral Nucleic Acid Kit (Roche) and dissolved in 50 µl of nuclease-free distilled water. An amount equivalent to 20 µl of serum was subjected to the following three PCR methods. Conventional PCR was carried out using the primer pair NG343 (sense: 5' GCA CTT CCG AAT GGC TGA GTT T 3') and NG344 (antisense: 5' TCC CGA GCC CGA ATT GCC CCT 3'), and Perkin-Elmer AmpliTaq Gold (Roche) for 40 cycles (95 °C for 30 s, with an additional 9 min in the first cycle; 58 °C for 30 s; and 72 °C for 40 s, with an additional 7 min in the last cycle). These primers were derived from the sequence of the untranslated region (UTR) of the human prototype TTV (TA278); the UTR is highly conserved among the TTVs that have been isolated from humans and non-human primates (Okamoto et al., 2000a , b ). The amplification product was subjected to electrophoresis on a 2·5% NuSieve 3:1 agarose gel (FMC BioProducts) to detect a band of approximately 120 bp.

The full-length TTV genomes of tupaia were amplified by PCR with inverted primers NG474 (sense: 5' CGA GCT GGG CGG GTG CCG GAG GCT G 3') and NG475 [antisense: 5' TCA GAG CGT GGT CTG ATS GCT CTG 3' (S=G or C)] in the presence of TaKaRa LA Taq with GC buffer I (TaKaRa Shuzo). DNAs extracted from the sera of tupaias were used as templates for the inverted PCR for 35 cycles (94 °C for 45 s, with an additional 3 min in the first cycle; 63 °C for 45 s, and 72 °C for 3 min, with an additional 7 min in the last cycle). The amplification product was electrophoresed on a 1% SeaKem GTG agarose gel (FMC BioProducts) to detect the full genomic TTV DNA band.

TTV DNA was detected by nested PCR with primers derived from the UTR sequence of the tupaia TTV genomes (Tbc-TTV5u, Tbc-TTV6u, Tbc-TTV8u, Tbc-TTV14u and Tbc-TTV15u) and Perkin-Elmer AmpliTaq Gold. Briefly, the first-round PCR (95 °C for 30 s, with an additional 9 min in the first cycle; 60 °C for 30 s; 72 °C for 40 s, with an additional 7 min in the last cycle) was performed for 35 cycles with primers NG478 (sense: 5' ATG CCG CCA GCG GTC AGA GC 3') and NG479 [antisense: 5' CCC TTG ACT YCG GCA GTG CG 3' (Y=T or C)], and the second-round PCR was performed for 25 cycles under the same conditions with primers NG480 [sense: 5' AGC GGT CAG AGC SAT CAG AC 3' (S=G or C)] and NG481 (antisense: 5' AGT GCG YGG CAC AGC CTC CG 3'). The amplification product of the first-round PCR was 88 bp (nt 213–300), and that of the second-round PCR was 66 bp (nt 221–286): the nucleotide positions are numbered with reference to the Tbc-TTV14 isolate of 2199 nt determined in the present study (see Results).

{blacksquare} Determination and analysis of TTV sequences.
The PCR amplification products obtained with primers NG343 and NG344, or primers NG474 and NG475, were separated on agarose gel electrophoresis, purified using Centri-Sep Spin Columns (Princeton Separations), and ligated into pT7BlueT-Vector (Novagen). Using the recombinant DNA obtained as a template, both strands were sequenced with the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Sequence analysis was performed with Genetyx-Mac version 10.1.4 (Software Development Co.) and ODEN version 1.1.1 (Ina, 1994 ) from the DNA Data Bank of Japan (National Institute of Genetics, Mishima, Japan). The nucleotide sequences were aligned to obtain the maximal homology using the MAlign program (Software Development). The phylogenetic relatedness among TTV and TLMV sequences was estimated by the neighbour-joining method (Saitou & Nei, 1987 ). The reliability of the phylogenetic results was assessed using 1000 bootstrap replicates (Felsenstein, 1985 ).

{blacksquare} Strandedness of TTV genome from tupaias.
Extracted nucleic acids were treated with S1 nuclease or mung bean nuclease (TaKaRa Shuzo) as described previously (Okamoto et al., 1998 , 2000e ). The genome was subjected to PCR with several pairs of primer sets specific for tupaia TTV to determine its strandedness.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Detection of TTV DNA in sera from tupaias
When PCR was performed using primers NG343 and NG344, which had been derived from a human TTV genome (TA278), TTV DNA was detected in the sera of 5 (26%) of the 19 tupaias. The PCR-amplified products from the 5 tupaias were subjected to sequence analysis. Based on a common 84 bp sequence in the 5 sequences (Tbc-TTV5u, Tbc-TTV6u, Tbc-TTV8u, Tbc-TTV14u and Tbc-TTV15u: accession nos AB057359–AB057363), nested primers (NG478–NG479 for the first round and NG480–NG481 for the second round) were newly designed to sensitively detect TTV DNA in the sera from the tupaias. On performing PCR with the nested primers 15 (79%) of the tupaias, including the above-mentioned 5, were found to be positive for TTV DNA, indicating that TTV infection is prevalent in tupaias.

Full-length nucleotide sequence of Tbc-TTV14 isolate
Using the DNA extracted from the sera of 5 tupaias as a template, the entire genomic sequence of TTV was amplified by PCR with inverted primers NG474 and NG475, which had been derived from the 84 bp sequence of 5 isolates amplified with primers NG343 and NG344. The TTV genome was found to be approximately 2·2 kb in size in all 5 samples. Then, the PCR-amplified product from the serum with the strongest PCR signal was molecularly cloned, and a TTV clone named Tbc-TTV14 was sequenced over the entire genome. The Tbc-TTV14 isolate had a circular genomic structure with a genomic length of 2199 nt, and possessed two major ORFs similar to the prototype human TTV (TA278) (Okamoto et al., 1998 ). ORF1 and ORF2 in the Tbc-TTV14 isolate had a coding capacity of 502 amino acids (aa) (nt 432–1937) and 59 aa (nt 343–519), respectively. The Tbc-TTV14 genome was deduced to be single-stranded from its behaviour on digestion with S1 nuclease or mung bean nuclease, similar to the genomic DNAs of TTVs and TLMVs from humans and non-human primates (Okamoto et al., 1998 , 2000e ; Takahashi et al., 2000b ).

The Tbc-TTV14 genome is shorter than the TTV genomes isolated previously from humans and non-human primates (3·4–3·9 kb) as well as the TLMV genomes (2·8–3·0 kb) thus far identified. Comparison of the Tbc-TTV14 genome with reported TTV and TLMV genomes from humans and non-human primates whose entire or partial nucleotide sequence is known (Biagini et al., 2000 , 2001 ; Erker et al., 1999 ; Hallett et al., 2000 ; Hijikata et al., 1999 ; Inami et al., 2000 ; Mushahwar et al., 1999 ; Okamoto et al., 1999a , 2000b ; Takahashi et al., 2000a , b ; Tanaka et al., 2001 ; Ukita et al., 2000 ) revealed that it is less than 50% similar to the previously reported TTV and TLMV genomes. These results indicate that the Tbc-TTV14 isolate differs considerably in genomic length and sequence from the known TTVs and TLMVs in humans and non-human primates.

Putative splicing sites and proposed genomic organization of the Tbc-TTV14 isolate
Three distinct species of TTV mRNAs (2·9–3·0, 1·2 and 1·0 kb) with three different splicings are observed in human TTVs of 3·8 kb (Kamahora et al., 2000 ; Okamoto et al., 2000c ). Although the TTV mRNAs of the TTVs from non-human primates and TLMVs have not yet been analysed, the consensus motifs of donor and acceptor sites (Breathnach et al., 1978 ; Mount, 1982 ) for three splicings (Splice 1, Splice 2 and Splice 3) were analysed in the present study, and were found in all of the TTVs from non-human primates as well as in TLMVs (Fig. 1). However, the consensus motifs for the short splicing (Splice 1) that is shared by all three mRNAs and is located at their 5' termini were not recognized in the Tbc-TTV14 sequence. In addition, consensus motifs for the third splicing (Splice 3), which is present in the shortest mRNA, were not identified in the Tbc-TTV14 sequence. Only the consensus motifs for the second splicing (Splice 2) were recognizable in the Tbc-TTV14 genome. Fig. 2 compares the proposed genomic organization of the Tbc-TTV14 isolate and those of TTVs from humans and non-human primates (chimpanzee, Japanese macaque, tamarin and douroucouli) as well as TLMVs from human (CBD231) and chimpanzee (Pt-TTV8-II) (Okamoto et al., 2000b ; Takahashi et al., 2000b ). The TTVs isolated from humans and non-human primates as well as the TLMVs possessed in common four ORFs (ORF1–ORF4) in exactly the same orientation: ORF1 was in frame 1; ORF2 and ORF3 were in frame 2; the 5' terminus of ORF4 was located in frame 2 and the 3' terminus of ORF4 was located in-frame 3. In contrast, Tbc-TTV14 lacked ORF4 and had only three ORFs (ORF1–ORF3). Interestingly, the donor site shared by the two longer splicings (Splice 2 and Splice 3) was located 1 nt upstream of the last codon position in ORF2, not only in Tbc-TTV14 but also in the TTVs and TLMVs from humans and non-human primates.



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Fig. 1. Sequences of the donor and acceptor sites of splicings in TTVs and TLMVs isolated from humans and non-human primates as well as in Tbc-TTV14. The consensus sequence of donor and acceptor sites in splicings (Breathnach et al., 1978 ; Mount, 1982 ) is shown at the top. M denotes A or C, R denotes A or G, and Y denotes T or C. The sequences of the donor and acceptor sites in the first splicing (Splice 1), which are shared by the 2·9–3·0, 1·2 and 1·0 kb mRNAs of human TTVs [VT416 (Kamahora et al., 2000 ) and TYM9 (Okamoto et al., 2000c )], those in the second splicing (Splice 2), which is present in the 1·2 kb mRNA, and those in the third splicing (Splice 3), which is present in the 1·0 kb mRNA, are indicated for TTVs [TA278 (accession no. AB017610) and TYM9 (AB050448)] and TLMVs [CBD231 (AB026930) and TGP96 (AB041962)] from humans; TTV [Pt-TTV6 (AB041957)] and TLMV [Pt-TTV8-II (AB041963)] from chimpanzees; TTVs [Mf-TTV3 (AB041958) and Mf-TTV9 (AB041959)] from Japanese macaques; TTV [So-TTV2 (AB041960)] from tamarin; TTV [At-TTV3 (AB041961)] from douroucouli; and Tbc-TTV14 obtained from a tupaia in the present study. Nucleotides matching the consensus sequence are written in upper-case letters. The regions of introns and the number of nucleotides spliced out are shown in parentheses on the right.

 


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Fig. 2. Predicted genomic organization of Tbc-TTV14 (a) as well as eight TTV and TLMV isolates from humans and non-human primates (bi). The Tbc-TTV14 isolate obtained in the present study is boxed. The circumference of each circle represents the relative size of the genome. The closed arrows represent ORFs (ORF1–ORF4). The open boxes located between an upstream closed box and the downstream closed arrow in ORF3 and ORF4 which encode joint proteins, represent areas corresponding to introns in the mRNAs (Splice 2 and Splice 3, respectively, in Fig. 1). The closed box indicates the GC-rich stretch and the small closed circle represents the position of the TATA-box. Accession numbers are given in the legend to Fig. 1.

 
UTR sequence of Tbc-TTV14
The UTR in Tbc-TTV14 was defined as the sequence between the end of ORF1 and the beginning of ORF2, and spanned 604 nt, occupying 27% of the entire genome (Fig. 2). On the other hand, the UTR in TTVs and TLMVs isolated from humans and non-human primates is defined as the sequence between the end of ORF4 and the beginning of ORF2, and spans 811–1131 nt among the TTVs and 453–525 nt among the TLMVs, occupying 24–30% and 16–18%, respectively, of the entire genome sequence. The GC-rich stretch and TATA-box were commonly observed in the central portion of the UTR of all TTV and TLMV isolates including Tbc-TTV14.

Fig. 3 compares the nucleotide sequence that extends from the TATA-box in the middle of the UTR to the initiation codon of ORF2 in Tbc-TTV14 and in the TTVs and TLMVs of humans and non-human primates. The nucleotide sequence in this particular region is the most conserved among the entire genomes of all TTV and TLMV isolates. Remarkably, all 13 TTV and TLMV isolates including Tbc-TTV14 shared two highly conserved sequences of 15 nt each (CGAATGGCTGAGTTT and AGGGGCAATTCGGGC), which were located in the 3'-terminal parts of the NG343 (sense) and NG344 (antisense) primers used in the initial PCR amplification of Tbc-TTV14 in the present study. Although one or two point mutations were found in these two 15 nt sequences in the KC009 isolate and Tbc-TTV14 isolates, these sequences were also maintained in all reported human TTV and TLMV isolates. In the sequence depicted in Fig. 3, a spliced sequence of 83–111 nt was recognized for TTVs and TLMVs in humans and non-human primates. However, as to the sequence from the TATA-box to the initiation codon of ORF2, Tbc-TTV14 had the shortest sequence (167 nt) compared with those of the other isolates in Fig. 3 (189–299 nt), and lacked motifs of the putative donor and acceptor sites for the short splicing that is shared by all three mRNAs of the TTVs and TLMVs that have been isolated from humans and non-human primates.



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Fig. 3. Alignment of the nucleotide sequences of TTVs and TLMVs obtained from humans and non-human primates as well as Tbc-TTV14 at a region that is highly conserved among TTVs and TLMVs. The nucleotide sequence between the TATA-box and the putative initiation codon of ORF2 in the UTR is compared among TTVs [TA278, PMV (accession no. AF261761), TUS01 (AB017613), KC009 (AB038621)] isolated from humans, TLMVs (CBD231, TGP96 and Pt-TTV8-II) isolated from humans and a chimpanzee, TTVs (Pt-TTV6, Mf-TTV3, Mf-TTV9, So-TTV2 and At-TTV3) isolated from non-human primates, and Tbc-TTV14. A dash indicates an identical nucleotide in comparison with the top sequence, and a slash indicates a deletion. A putative cap site with the sequence GAG (Okamoto et al., 2000c ) is marked with dots. The sequences corresponding to primers NG343 (sense) and NG344 (antisense) are boxed. Sequences that are spliced out in the mRNAs (Splice 1 in Fig. 1) are shaded. The Sp1 sequence (GGGCGG) is overlined.

 
Fig. 4 illustrates the phylogenetic tree that was constructed using the conserved sequences in the TTV and TLMV isolates indicated in Fig. 3. Reflecting the similarity in genomic length, Tbc-TTV14 was closest to TLMVs isolated from humans and a chimpanzee (CBD231, TGP96 and Pt-TTV8-II). Furthermore, Tbc-TTV14 was clearly closer to TTVs isolated from tamarin (So-TTV2) and douroucouli (At-TTV3) than to the TTVs isolated from humans (TA278, PMV, TUS01 and KC009), the chimpanzee (Pt-TTV6) and Japanese macaques (Mf-TTV3 and Mf-TTV9). These results were confirmed by another phylogenetic tree that had been constructed based on an analysis of a gap-stripped alignment (161 nt within the sequence of Fig. 3) that is only from sites present in all the taxa.



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Fig. 4. Phylogenetic tree constructed from the well-conserved UTR sequence by the neighbour-joining method (Saitou & Nei, 1987 ). The bootstrap values were 100% for all nodes for 1000 resamplings of the data. The accession number of each isolate is given in the legends to Fig. 1 and Fig. 3.

 
In the middle of the UTR of the Tbc-TTV14 genome, there is a GC-rich stretch of 78 nt, which is comparable in length to the GC-rich stretch (35–133 nt) in TTVs and TLMVs isolated from humans and non-human primates. Characteristic stem and loop structures constructed from the GC-rich stretch and neighbouring sequences have been proposed for species-specific TTVs and TLMVs from human and non-human primates (Okamoto et al., 2000b ; Takahashi et al., 2000a , b ), but a unique secondary structure was observed for Tbc-TTV14. The genome of the Tbc-TTV14 isolate contained two A/C-rich direct repeats (DR1 and DR2) of 28 nt each, upstream of the GC-rich stretch, and four stem–loop structures in the GC-rich sequence (Fig. 5). As in the TTV genome from douroucouli (At-TTV3) (Okamoto et al., 2000b ), the fourth large stem–loop structure was unique, in that it contained a loop with the sequence [TACCACAAA (the same nt underlined)], which is similar to a nonamer motif (TANTAYYMS) that is conserved in animal and plant circoviruses (with the exception of CAV) and which is required for replication (Bassami et al., 1998 ; Mankertz et al., 1998 ).



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Fig. 5. Predicted secondary structure formed by a central portion of the UTR including the GC-rich stretch in Tbc-TTV14. Watson–Crick base pairings are marked by dots in between pairs of bases. Arrows represent direct repeats (DR1 and DR2). The conserved sequence resembling the nonamer motif [TANTAYTMS (Bassami et al., 1998 ; Mankertz et al., 1998 )] in a loop of the fourth stem–loop structure, is boxed.

 
Coding region sequence of Tbc-TTV14
The Tbc-TTV14 isolate possessed only three ORFs (ORF1, ORF2 and ORF3). ORF1 encoded 502 aa and was rich in Arg at its N terminus. Three of the four conserved motifs (motifs 1, 2 and 3) present in the putative replication-associated proteins (Rep proteins), which are involved in rolling-circle replication, were also conserved in Tbc-TTV14: FTL at aa 204–206 (motif 1), YKSHFQW at aa 315–321 (motif 2) and YTVR at 384–387 (motif 3), similar to many plant and animal circoviruses (Bassami et al., 1998 ; Niagro et al., 1998 ), as well as to TTVs and TLMVs isolated from humans and non-human primates (Okamoto et al., 2000b ). Two His residues are generally conserved in motif 2, but one His was replaced by Lys in Tbc-TTV14, as in a reported TTV isolated from a non-human primate (At-TTV3) (Okamoto et al., 2000b ). ORF2 of Tbc-TTV14 encoded 59 aa. Although significant sequence similarity was not observed in the amino acid sequence between Tbc-TTV14 and TTVs or TLMVs, the conserved motif (W-X7-H-X3-C-X1-C-X5-H) in the N terminus of the ORF2 protein of reported TTVs and TLMVs (Hijikata et al., 1999 ; Takahashi et al., 2000b ) was also shared by Tbc-TTV14. In addition, the amino acid composition of the C terminus of the ORF2 protein of the Tbc-TTV14 isolate was similar to those of reported TTV and TLMV isolates (Fig. 6a), being rich in Gly, Glu and Pro.



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Fig. 6. Comparison of the predicted amino acid sequences among the TTVs and TLMVs isolated from humans and non-human primates as well as Tbc-TTV14. The accession number of each isolate is given in the legends to Fig. 1 and Fig. 3. The total number of amino acids encoded by the putative ORFs (ORF2, ORF3 and ORF4) is indicated in parentheses after each isolate name in (a), (b) and (c). (a) Comparison of the amino acid sequences encoded by ORF2. A dash indicates an identical amino acid to that in the sequence in the top row. Five conserved residues (Trp, His, Cys, Cys and His) are boxed. The number of amino acid residues remaining in the C-terminal part of the presumed translation product of ORF2 is indicated in parentheses at the right, along with the numbers of abundant amino acids (Gly, Glu and Pro). (b) Comparison of the 100 aa C-terminal sequences encoded by the putative ORF3. For visual clarity, Ser (S) residues are shown in bold upper-case letters, and Arg (R) and Lys (K) residues are shown in upper-case letters. The numbers of the indicated amino acids (Ser, Arg and Lys) which are abundant in the indicated region are shown on the right. (c) Comparison of the amino acid sequences encoded by the 3'-terminal end of the putative ORF4. A dash indicates an amino acid identical to that in the sequence in the top row, and a slash indicates a deletion. The eight conserved residues (Glu, Arg, Arg, Pro, Pro, Val, Phe and Leu) are boxed. Data are not shown for PMV due to premature termination, and Tbc-TTV14 due to lack of this ORF.

 
ORF3 of Tbc-TTV14 encoded a putative joint protein of 214 aa, of which 58 aa were the same as ORF2. In Fig. 6(b), the 100 C-terminal aa of Tbc-TTV14 ORF3 are aligned with those of TTVs and TLMVs isolated from humans and non-human primates. In Tbc-TTV14, as well as in all TTVs and TLMVs from humans and non-human primates, the C-terminal portion of ORF3 is rich in Ser (12–29 residues), and also in Arg and Lys (18–28 residues). ORF4, which encodes another joint protein, was not recognized in Tbc-TTV14, but was commonly observed in the TTVs and TLMVs isolated from humans and non-human primates. The C terminus of ORF4 contains a conserved motif, E-X8-R-X2-R-X4–6-P-X5–11-P-X1–8-V-X1-F-X1-L (Fig. 6c), whose function and virological significance need to be demonstrated in future studies.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
The TTVs isolated from humans and non-human primates (chimpanzees, Japanese macaques, tamarins and douroucoulis) and TLMVs isolated from humans and chimpanzees have a genomic length of 3·4–3·9 kb and 2·8–3·0 kb, respectively (Biagini et al., 2001 ; Inami et al., 2001; Miyata et al., 1999 ; Mushahwar et al., 1999 ; Okamoto et al., 1999a , 2000b , c ; Takahashi et al., 2000a , b ; Ukita et al., 2000 ). In the present study, a new TTV species with the smallest known genome (2·2 kb) was isolated from the sera of tupaias by PCR with primers derived from the highly conserved UTR sequence in human prototype TTV (TA278). Tupaias are small, squirrel-like mammals, are closely related to primates, and live in Southeast Asia. They had been classified in the order Primates or the order Insectivora, but have recently been classified in the order Scandentia. Tupaias show susceptibility to infection by herpes simplex virus types 1 and 2 (Darai et al., 1978 ) and human hepatitis viruses A, B and C (Walter et al., 1996 ; Zan et al., 1981 ; Xie et al., 1998 ). Although primers derived from the human TTV genome were used for the detection of TTV DNA in tupaias, human TTV was not isolated in the present study. Fifteen of 19 tupaias were infected with TTV as detected by PCR with primers derived from the TTV sequence in tupaias, indicating that tupaia-specific TTV is prevalent among tupaias.

The genomic DNA of tupaia TTV (Tbc-TTV14) was presumed to be circular and single-stranded, similar to that of the human prototype TTV (Miyata et al., 1999 ; Mushahwar et al., 1999 ; Okamoto et al., 1999a , 2000e ). The genomic length of the Tbc-TTV14 isolate was 2199 nt, comparable with animal circoviruses (PCV, BFDV and CAV), which have a circular, single-stranded DNA of 1·8–2·3 kb (Bassami et al., 1998 ; Niagro et al., 1998 ; Noteborn et al., 1991 ; Todd et al., 2000 ). However, Tbc-TTV14 differed significantly from animal circoviruses at the sequence and genomic organization levels. The genomic organization and putative transcriptional profile of Tbc-TTV14 resembled those of TTVs and TLMVs isolated from humans and non-human primates, although the sequence similarity of the Tbc-TTV14 isolate against TTV and TLMV isolates was less than 50%.

It has recently been demonstrated in vitro and in vivo that three distinct mRNAs of 2·9–3·0, 1·2 and 1·0 kb in size, which have common 5' and 3' termini, are transcribed from the 3·8 kb genomic DNA of human TTV (Kamahora et al., 2000 ; Okamoto et al., 2000c ). All of these mRNAs arise from splicing, and the shorter mRNAs of 1·2 and 1·0 kb possess additional splicing sites to link distant ORFs to create two new ORFs (ORF3 and ORF4), capable of encoding 260–286 aa and 249–289 aa, respectively, in the human TTVs. Based on the presence of consensus motifs of donor and acceptor sites of splicings in the genome (Fig. 1), such a transcription profile was presumed in the present study for TTV genomes from non-human primates and TLMV genomes, although the mRNAs of their genomes have not yet been observed. This suggests that this unique transcription profile, which is not observed among known members of the Circoviridae family (Niagro et al., 1998 ; Noteborn et al., 1995 ; Todd et al., 2000 ), is common to the TTVs and TLMVs that infect humans and non-human primates. However, the Tbc-TTV14 genome obtained in the present study lacked two of the three splicings: it possessed only one splicing (Splice 2), which is involved in the creation of ORF3. The complete preservation of coding capacity for ORF1 and ORF2 proteins as well as two joint proteins (ORF3 and ORF4), as illustrated in Fig. 2, would indicate that the proposed genomic organization and transcription profile are characteristic of all TTVs and TLMVs isolated from humans and non-human primates. In contrast, the genomic organization of Tbc-TTV14 was distinct from those of TTVs and TLMVs infecting humans and non-human primates, due to the lack of ORF4. Strict conservation of the putative ORF3 in tupaia TTV suggests that the ORF3 protein is indispensable for all TTVs and TLMVs in mammals. In fact, ORF3 has a Ser-rich tract accompanied by a cluster of Arg and Lys at its C terminus (Fig. 6b), and contains some nuclear targeting sequences, leading to the speculation that the ORF3 protein may be a nuclear protein involved in transcriptional regulation (Tanaka et al., 2001 ). Another possibility is that the ORF3 protein may play an important role in virus replication, since it is homologous with DNA topoisomerase I of D. melanogaster (Takahashi et al., 2000b ). The role of the fourth ORF (ORF4) as well as its function and virological significance in TTVs and TLMVs isolated from humans and non-human primates remains unknown.

There is no consensus on how TTVs and TLMVs should be classified. The TTV in tupaias has a genomic length that is clearly smaller than those of TLMVs (2·8–3·0 kb). Phylogenetic analysis revealed that the TTV in tupaias is closest to TLMVs isolated from humans and chimpanzees rather than to TTVs isolated from tamarins and douroucoulis. In this regard, it seems that it would be better to designate ‘tupaia TTV’ as ‘tupaia TLMV’, although tupaia TTV was identified by PCR with primers derived from the human prototype TTV (TA278). At present, there are no precise criteria that distinguish TLMVs from TTVs. To avoid confusion, taxonomic nomenclature for existing TTVs and TLMVs as well as those that will emerge in extended studies should be determined by the International Committee on Taxonomy of Viruses. In the interim, we would like to designate the virus identified from tupaia in the present study as ‘TTV’.

In conclusion, the data obtained in the present study indicate a very high degree of sequence divergence as well as a common genomic organization among the TTVs and TLMVs infecting humans and non-human primates as well as tupaia TTV, suggesting that hosts as divergent as tupaias and primates are infected by related viruses. It may be tempting to speculate about the possibility that the common ancestor of these hosts may have been infected by a TTV, although there is no clear evidence to judge whether the distribution of TTVs reflects host-dependent evolution over millions of years. Whether this is the case, or whether TTVs have originated and diversified over a much shorter time scale than their many hosts, is an interesting question that should be answered in future work.


   Acknowledgments
 
This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Ministry of Health, Labour and Welfare of Japan. The authors are grateful to Professor M. Mayumi for his advice and encouragement during this study.


   Footnotes
 
The nucleotide sequence data reported in this paper have been assigned DDBJ/EMBL/GenBank accession no. AB057358 for the entire sequence of the Tbc-TTV14 isolate and AB057359–AB057363 for the partial sequences of the five isolates.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
Abe, K., Inami, T., Ishikawa, K., Nakamura, S. & Goto, S. (2000). TT virus infection in nonhuman primates and characterization of the viral genome: identification of simian TT virus isolates. Journal of Virology 74, 1549-1553.[Abstract/Free Full Text]

Bassami, M. R., Berryman, D., Wilcox, G. E. & Raidal, S. R. (1998). Psittacine beak and feather disease virus nucleotide sequence analysis and its relationship to porcine circovirus, plant circovirus, and chicken anaemia virus. Virology 249, 453-459.[Medline]

Biagini, P., Attoui, H., Gallian, P., Touinssi, M., Cantaloube, J. F., de Micco, P. & de Lamballerie, X. (2000). Complete sequence of two highly divergent European isolates of TT virus. Biochemical and Biophysical Research Communications 271, 837-841.[Medline]

Biagini, P., Gallian, P., Attoui, H., Touinssi, M., Cantaloube, J. F., de Micco, P. & de Lamballerie, X. (2001). Genetic analysis of full-length genomes and subgenomic sequences of TT virus-like mini virus human isolates. Journal of General Virology 82, 379-383.[Abstract/Free Full Text]

Breathnach, R., Benoist, C., O’Hare, K., Gannon, F. & Chambon, P. (1978). Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequence at the exon–intron boundaries. Proceedings of the National Academy of Sciences, USA 75, 4853-4857.[Abstract]

Darai, G., Schwaier, A., Komitowski, D. & Munk, K. (1978). Experimental infection of Tupaia belangeri (tree shrews) with herpes simplex virus type I and II. Journal of Infectious Diseases 137, 221-226.[Medline]

Erker, J. C., Leary, T. P., Desai, S. M., Chalmers, M. L. & Mushahwar, I. K. (1999). Analyses of TT virus full-length genomic sequences. Journal of General Virology 80, 1743-1750.[Abstract]

Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783-791.

Hallett, R. L., Clewley, J. P., Bobet, F., McKiernan, P. J. & Teo, C. G. (2000). Characterization of a highly divergent TT virus genome. Journal of General Virology 81, 2273-2279.[Abstract/Free Full Text]

Hijikata, M., Takahashi, K. & Mishiro, S. (1999). Complete circular DNA genomes 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.[Medline]

Ina, Y. (1994). ODEN: a program package for molecular evolutionary analysis and database search of DNA and amino acid sequences. Computer Applications in the Biosciences 10, 11-12.[Medline]

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.[Medline]

Itoh, Y., Takahashi, M., Fukuda, M., Shibayama, T., Ishikawa, T., Tsuda, F., Tanaka, T., Nishizawa, T. & Okamoto, H. (2000). Visualization of TT virus particles recovered from the sera and feces of infected humans. Biochemical and Biophysical Research Communications 279, 718-724.[Medline]

Kamahora, T., Hino, S. & Miyata, H. (2000). Three spliced mRNAs of TT virus transcribed from a plasmid containing the entire genome in COS1 cells. Journal of Virology 74, 9980-9986.[Abstract/Free Full Text]

Khudyakov, Y. E., Cong, M. E., Nichols, B., Reed, D., Dou, X. G., Viazov, S. O., Chang, J., Fried, M. W., Williams, I., Bower, W., Lambert, S., Purdy, M., Roggendorf, M. & Fields, H. A. (2000). Sequence heterogeneity of TT virus and closely related viruses. Journal of Virology 74, 2990-3000.[Abstract/Free Full Text]

Leary, T. P., Erker, J. C., Chalmers, M. L., Desai, S. M. & Mushahwar, I. K. (1999). Improved detection systems for TT virus reveal high prevalence in humans, non-human primates and farm animals. Journal of General Virology 80, 2115-2120.[Abstract/Free Full Text]

Mankertz, A., Mankertz, J., Wolf, K. & Buhk, H. J. (1998). Identification of a protein essential for replication of porcine circovirus. Journal of General Virology 79, 381-384.[Abstract]

Martin, R. D. (1990). Are tree-shrews primates? In Primate Origins and Evolution: A Phylogenetic Reconstruction , pp. 191-213. Edited by R. D. Martin. London:Chapman & Hall.

Miyata, H., Tsunoda, H., Kazi, A., Yamada, A., Khan, M. A., 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. Journal of Virology 73, 3582-3586.[Abstract/Free Full Text]

Mount, S. M. (1982). A catalogue of splice junction sequences. Nucleic Acids Research 10, 459-472.[Abstract]

Muljono, D. H., Nishizawa, T., Tsuda, F., Takahashi, M. & Okamoto, H. (2001). Molecular epidemiology of TT virus (TTV) and characterization of two novel TTV genotypes in Indonesia. Archives of Virology (in press).

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. & Desai, S. M. (1999). Molecular and biophysical characterization of TT virus: evidence for a new virus family infecting humans. Proceedings of the National Academy of Sciences, USA 96, 3177-3182.[Abstract/Free Full Text]

Mushahwar, I. K. (2001). Recently discovered blood-borne viruses: are they hepatitis viruses or merely endosymbionts? Journal of Medical Virology 62, 399-404.

Niagro, F. D., Forstoefel, A. N., Lawther, R. P., Kamalanathan, L., Ritchie, B. W., Latimer, K. S. & Lukert, P. D. (1998). Beak and feather disease virus and porcine circovirus genomes: intermediates between the geminiviruses and plant circoviruses. Archives of Virology 143, 1723-1744.[Medline]

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. Biochemical and Biophysical Research Communications 241, 92-97.[Medline]

Noteborn, M. H., de Boer, G. F., van Roozelaar, D. J., Karreman, C., Kranenburg, O., Vos, J. G., Jeurissen, S. H., Hoeben, R. C., Zantema, A., Koch, G., van Ormondt, H. & van der Eb, A. J. (1991). Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle. Journal of Virology 65, 3131-3139.[Medline]

Noteborn, M. H. M. & Koch, G. (1995). Chicken anaemia virus infection: molecular basis of pathogenicity. Avian Pathology 24, 11-31.

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. Hepatology Research 10, 1-16.

Okamoto, H., Nishizawa, T., Ukita, M., Takahashi, M., Fukuda, M., Iizuka, H., Miyakawa, Y. & Mayumi, M. (1999a). The entire nucleotide sequence of a TT virus isolate from the United States (TUS01): comparison with reported isolates and phylogenetic analysis. Virology 259, 437-448.[Medline]

Okamoto, H., Takahashi, M., Nishizawa, T., Ukita, M., Fukuda, M., Tsuda, F., Miyakawa, Y. & Mayumi, M. (1999b). Marked genomic heterogeneity and frequent mixed infection of TT virus demonstrated by PCR with primers from coding and nonocoding regions. Virology 259, 428-436.[Medline]

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. Journal of Virology 74, 1132-1139.[Abstract/Free Full Text]

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.[Medline]

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. Biochemical and Biophysical Research Communications 279, 700-707.[Medline]

Okamoto, H., Takahashi, M., Nishizawa, T., Tawara, A., Sugai, Y., Sai, T., Tanaka, T. & Tsuda, F. (2000d). Replicative forms of TT virus DNA in bone marrow cells. Biochemical and Biophysical Research Communications 270, 657-662.[Medline]

Okamoto, H., Ukita, M., Nishizawa, T., Kishimoto, J., Hoshi, Y., Mizuo, H., Tanaka, T., Miyakawa, Y. & Mayumi, M. (2000e). Circular double-stranded forms of TT virus DNA in the liver. Journal of Virology 74, 5161-5167.[Abstract/Free Full Text]

Pringle, C. R. (1999). Virus taxonomy at the XIth International Congress of Virology, Sydney, Australia, 1999. Archives of Virology 144, 2065-2070.[Medline]

Romeo, R., Hegerich, P., Emerson, S. U., Colombo, M., Purcell, R. H. & Bukh, J. (2000). High prevalence of TT virus (TTV) in naïve chimpanzees and in hepatitis C virus-infected humans: frequent mixed infections and identification of new TTV genotypes in chimpanzees. Journal of General Virology 81, 1001-1007.[Abstract/Free Full Text]

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406-425.[Abstract]

Tanaka, Y., Primi, D., Wang, R. Y. H., Umemura, T., Yeo, A. E. T., Mizokami, M., Alter, H. J. & Shih, J. W. K. (2001). Genomic and molecular evolutionary analysis of a newly identified infectious agent (SEN virus) and its relationship to the TT virus family. Journal of Infectious Diseases 183, 359-367.[Medline]

Takahashi, K., Hijikata, M., Samokhvalov, E. I. & Mishiro, S. (2000a). Full or near full length nucleotide sequences of TT virus variants (types SANBAN and YONBAN) and the TT virus-like mini virus. Intervirology 43, 119-123.[Medline]

Takahashi, K., Iwasa, Y., Hijikata, M. & Mishiro, S. (2000b). Identification of a new human DNA virus (TTV-like mini virus, TLMV) intermediately related to TT virus and chicken anemia virus. Archives of Virology 145, 979-993.[Medline]

Todd, D., McNulty, M. S., Mankertz, A., Lukert, P. D., Randles, J. W. & Dale, J. L. (2000). Family Circoviridae. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses , pp. 299-303. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego:Academic Press.

Ukita, M., Okamoto, H., Nishizawa, T., Tawara, A., Takahashi, M., Iizuka, H., Miyakawa, Y. & Mayumi, M. (2000). The entire nucleotide sequences of two distinct TT virus (TTV) isolates (TJN01 and TJN02) remotely related to the original TTV isolates. Archives of Virology 145, 1543-1559.[Medline]

Verschoor, E. J., Langenhuijzen, S. & Heeney, J. L. (1999). TT viruses (TTV) of non-human primates and their relationship to the human TTV genotypes. Journal of General Virology 80, 2491-2499.[Abstract/Free Full Text]

Walter, E., Keist, R., Niederost, B., Pult, I. & Blum, H. E. (1996). Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo. Hepatology 24, 1-5.[Medline]

Xie, Z. C., Riezu-Boj, J. I., Lasarte, J. J., Guillen, J., Su, J. H., Civeira, M. P. & Prietor, J. (1998). Transmission of hepatitis C virus infection to tree shrews. Virology 244, 513-520.[Medline]

Zan, M. Y., Liu, C. B., Li, C. M., Zhang, W. Y., Zhu, C., Pang, Q. F., Zhao, T. X., Wang, G. & Wang, J. L. (1981). A preliminary study of hepatitis A virus infection in Chinese tupaia. Acta Academiae Medicinae Sinicae 3, 148-152.[Medline]

Received 23 March 2001; accepted 5 June 2001.