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, Peoples Hospital, Peking University, Beijing 100044, Peoples Republic of China4
Author for correspondence: Hiroaki Okamoto. Fax +81 285 44 1557. e-mail hokamoto{at}jichi.ac.jp
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Abstract |
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Introduction |
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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 3032 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 2·93·0, 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.
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Methods |
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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 213300), and that of the second-round PCR was 66 bp (nt 221286): the nucleotide positions are numbered with reference to the Tbc-TTV14 isolate of 2199 nt determined in the present study (see Results).
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
).
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.
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Results |
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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 4321937) and 59 aa (nt 343519), 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 (3·43·9 kb) as well as the TLMV genomes (2·83·0 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 (2·93·0, 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 (ORF1ORF4) 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 (ORF1ORF3). 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. 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 83111 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
(189299 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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Discussion |
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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 1·82·3 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 2·93·0, 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 260286 aa and 249289 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 (2·83·0 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.
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Acknowledgments |
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Footnotes |
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References |
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Received 23 March 2001;
accepted 5 June 2001.