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1 Division of Virology, Faculty of Medicine, Tottori University, Yonago 683-8503, Japan
2 Division of Immunology, Faculty of Medicine, Tottori University, Yonago 683-8503, Japan
3 National Institute of Animal Health, Tsukuba 305-0856, Japan
Correspondence
Shigeo Hino
hino{at}grape.med.tottori-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers of the CAV mRNA sequences reported in this paper are AB248708–AB248711.
Present address: Department of Virology, Faculty of Proteomics Medical Science, The University of Tokushima Graduate School, Kuramoto 3-18-15, Tokushima 770-8503, Japan. 
Present address: Institute of Virology, Slovak Academy of Sciences, Dúbravská Cesta 9, 842 46 Bratislava, Slovak Republic, and Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University, Mlynská Dolina B2, 842 15 Bratislava, Slovak Republic.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Todd et al., 1990). All three major ORFs are in the antisense orientation and, while partially overlapping, are in three different frames (Noteborn et al., 1991). CAV was previously placed within the genus Circovirus, but has recently been reclassified into Gyrovirus as the genomes of all Circovirus members are ambisense (possessing their ORFs in both sense and antisense orientations) (Niagro et al., 1998). Furthermore, CAV lacks a nanonucleotide stem–loop structure at the replication origin, a feature common to the circoviruses (Niagro et al., 1998).
Torque teno virus (TTV) was discovered in 1997 in a serum sample originating from a non-A–G hepatitis patient (Nishizawa et al., 1997). Like CAV, TTV has a single-stranded DNA genome in an antisense orientation (Okamoto et al., 1998). Comparison of the genomes of TTV and CAV revealed that a 36-nt stretch of the TTV genome showed >80 % sequence similarity to CAV (there were no significant similarities in other regions). This led to the discovery that TTV has a circular genome (Miyata et al., 1999). The genome sizes of CAV and TTV are quite different: 3.8 kb for TTV and 2.3 kb for CAV. Furthermore, there is much diversity between TTV isolates, resulting in an enormous spread of terminal branches in TTV phylogenies (Okamoto et al., 2000). However, diversity between CAV isolates has been noted to be less than 10 % (Islam et al., 2002). In light of these differences, TTV is currently classified into another genus (Anellovirus within the Family Circoviridae) rather than with CAV (Biagini et al., 2004; Hino, 2002).
As with CAV, TTV has several ORFs in the antigenomic orientation and lacks a significant ORF in the genomic orientation (Miyata et al., 1999). The arrangement of ORFs distributed across the three different frames in these two viruses is also similar (Miyata et al., 1999). Analysis of viral mRNA in cells transfected with a plasmid carrying full-length TTV revealed that this virus transcribes three different species of mRNA (3.0, 1.2 and 1.0 kb) (Kamahora et al., 2000). Each mRNA species has one or two splice sites, such that coding regions may shift from one frame to another (Kamahora et al., 2000; Miyata et al., 1999; Qiu et al., 2005).
At least three proteins (a capsid/polymerase polyprotein in frame 1, phosphatase in frame 2 and apoptin in frame 3), have been identified as translational products of CAV (Noteborn et al., 1994). Nevertheless, CAV has long been believed to produce a single, 2.0 kb mRNA, which is consistent with the presence of its single promoter, TATA-box and poly(A) signal (Noteborn et al., 1991, 1992; Phenix et al., 1994). The question of how CAV manages to control the expression of three proteins derived from one class of mRNA has so far not been elucidated and is investigated here.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), were cultured at 39 °C in a 100 mm dish with RPMI1640 medium supplemented with 10 % fetal bovine serum (IBL), 2 mM glutamine, 0.01 % penicillin and 0.01 % streptomycin. COS1 cells, constitutionally expressing the SV40 T antigen, were cultured at 37 °C with Dulbecco's minimum essential medium supplemented with 10 % fetal bovine serum (IBL) and antibiotics (as above). Both cell lines were subcultured twice weekly.
Preparation of molecularly cloned CAV.
A CAV plasmid, pA2-C15, was derived from the replicative form of A2 strain CAV (AB031296
[GenBank]
) (Yamaguchi et al., 2001). This plasmid contains permutated (at XbaI site) full-length CAV (Fig. 1a). To obtain infectious CAV DNA, the plasmid was digested using XbaI, recircularized by T4 DNA ligase (Gibco-BRL) and transfected into MDCC-MSB1 cells. The recovered infectious CAV was titrated using a 96-well microplate method described by Imai & Yuasa (1990).
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). With its circular genome, the origin of transcription (tsp in Fig. 1) and putative promoter are located in the middle of the CAV DNA when cloned into a plasmid (pA2-C15) using XbaI restriction sites. To create a construct with the promoter/transcription start site in the correct position relative to the rest of the viral DNA, an approximately 0.3-genome-length fragment of CAV DNA (spanning the regulatory sequences) was prepared by double digestion of pA2-C15 with PstI and MluI. A clone, designated pCAV0.3G, was generated by insertion of this fragment into the PstI/SmaI site of pBK* (a cloning vector lacking the CMV promoter of pBK-CMV) (Kamahora et al., 2000). Subsequent insertion of an XbaI digest of permutated A2-C15 CAV DNA into the XbaI site of clone pCAV0.3G thus yielded plasmid pCAV1.3G (Fig. 1b), a 1.3-genome-length, non-permutated CAV construct.
Infection and DNA transfection of CAV.
MDCC-MSB1 cells, plated at 4.0x106 cells per 60 mm dish, were infected with CAV at a multiplicity of 1.0 TCID50 per cell. MDCC-MSB1 cells plated at 9.0x106 cells per 100 mm dish were transfected with 15 µg pCAV1.3G DNA construct in the presence of 50 µl trans-IT LT1 (Takara) and 1 ml serum-free medium. COS1 cells plated at 2.0x106 cells per 100 mm dish were transfected with 20 µg pCAV1.3G DNA using the calcium phosphate method (Chen & Okayama, 1987).
RNA extraction and Northern blotting.
Total RNA was extracted from transfected or infected cells using the guanidinium thiocyanate and phenol/chloroform methods (Chomczynski & Sacchi, 1987). After poly(A)+ RNA selection by Oligotex-dT30 (Takara), 0.5 µg poly(A)+ RNA was denatured with 50 % (v/v) formamide and electrophoresed on a 1 % SeaKem ME agarose gel (FMC BioProducts) containing 6.2 % formaldehyde. RNA was then transferred onto a Hybond-N+ membrane (Amersham Pharmacia Biotech) and fixed by UV irradiation (Sambrook & Russell, 2001). Full-length CAV RNA in each orientation was transcribed using T7 or T3 promoters (located at each terminus of the CAV genome) carried by the A2-C15 molecular clone. Full-length transcripts were labelled with digoxigenin (DIG), using a DIG RNA labelling kit (Roche Diagnostics). The filter was prehybridized at 68 °C for 1 h in DIG Easy Hyb hybridization buffer (Roche Diagnostics) and subsequently hybridized at 68 °C for 16 h in prehybridization solution with 200 ng CAV probe. The filter was then washed once in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)/0.1 % SDS at 25 °C for 30 min, and twice in 0.2x SSC/0.1 % SDS at 68 °C for 30 min. The washed filter was incubated with anti-DIG antibody labelled with alkaline phosphatase and mRNA bands were visualized using a DIG luminescent detection kit (Roche). The amount and quality of loaded RNAs were confirmed by constitutionally expressing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.
Detection and cloning of CAV-specific transcripts using reverse transcription PCR (RT-PCR).
To look for splice sites in the RNA transcripts, a series of RT-PCRs were performed using a single sense primer in combination with one of ten antisense primers (Fig. 2). Poly(A)+ RNAs (0.5 µg) were reverse-transcribed using Superscript II RNaseH– reverse transcriptase (Gibco-BRL) at 42 °C for 1 h in 50 mM Tris/HCl (pH 8.3), 75 mM KCl, and in 3 mM MgCl2 at 58 °C for 30 min, using an oligo-dT12–18 primer. The resultant product was subjected to PCR in the presence of 10 mM Tris/HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 and 0.001 % gelatin containing 250 µM each dNTP, 0.2 µM each primer and 2.5 units AmpliTaq Gold DNA polymerase (Perkin-Elmer). After incubation at 95 °C for 10 min, 35 cycles of PCR were performed (95 °C for 30 s, 58 °C for 30 s and 72 °C for 2 min, with a final incubation at 72 °C for 10 min) using a GeneAmp PCR system 9700 (Perkin-Elmer). For cloning of transcripts, PfuTurbo DNA polymerase (Stratagene) was used to minimize misreadings. The PCR product was analysed by 1 % agarose gel electrophoresis, then cloned into pPCR-Script Cam SK(+) vector using a PCR Script-Cam Cloning Kit (Stratagene). The insert was sequenced using the dideoxy method for both strands, employing a SequiTherm EXCEL II Long-Read DNA sequencing kit-ALF (Epicenter).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The intensity of this band increased up to 48 h p.i. (there was no detectable increase from 48–72 h p.i.). A similar result was achieved in MDCC-MSB1 or COS1 cells transfected with pCAV1.3G (data not shown). In both infected and transfected cells, additional bands of 1.6 and 1.3–1.2 kb appeared at 48 and 72 h p.i. (Fig. 3b). The intensities of these additional bands were significantly lower than that of the major 2.0 kb band. Recovery of the 1.6 kb band was more marked in infected or transfected MDCC-MSB-1 cells, while the 1.3–1.2 kb band was more intense in transfected COS1 cells. None of these bands were detected in mock-infected or mock-transfected cells (Fig. 3, lanes Mock). No distinct bands were observed when the same blots were rehybridized with probes in the sense orientation (data not shown).
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) in combination with one of ten CAV-specific antisense primers (A2L1–A2L10 in Fig. 2). The major band (1.7 kb) recovered was observed after RT-PCR using the sense A2U and antisense A2L1 primers (Fig. 4, lane L1). This was consistent with the expected product size, compared to the CAV genomic template run as a positive control. This species of product was also visible in PCRs using antisense primers A2L2–A2L10, with band sizes corresponding to the location of each antisense primer along the CAV genome. This suggested that the major species of poly(A)+ RNA was unlikely to undergo substantial splicing (Fig. 4, lanes L2–L10). This 1.7 kb band was thus proposed to correspond to the 2.0 kb mRNA previously recognized from Northern blotting experiments.
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, lane L1). The sizes of the first two were consistent with the 1.6 kb RNA, and the 1.3–1.2 kb RNA species visualized in Northern blotting (Fig. 3), taking into account the truncation of both 3' and 5' termini by the primers used in RT-PCR. However, a product corresponding to the 0.6 kb band visible in lane L1 (Fig. 4) could not be confirmed by Northern analysis, even after overexposure. The 1.3 kb band (Fig. 4, lane L1), proposed here to correspond to the 1.6 kb band in the Northern blot, was detectable using primers A2L1–A2L7 (Fig. 4, lanes L1–L7), suggesting the loss of sequence upstream of nt 1519 in the 1.6 kb RNA species. The 1.1–1.0 kb band in lane L1 (proposed to correspond to the 1.3–1.2 kb RNA species detected in the Northern blot) was visible only in lanes L1–L3, suggesting the loss of sequence upstream of nt 1915. The 0.6 kb band was also detected by RT-PCR (Fig. 4 lanes L1–L4, although the band in L4 is extremely faint), which suggests a deletion upstream of nt 1800. This band was predicted to correspond to an approximately 0.8 kb RNA species, in line with the differences in size seen in other bands visualized by RT-PCR and Northern analysis (due to truncation by RT-PCR primers), even though this species could not actually be seen using Northern blotting. The COS-1 cells transfected with pCAV1.3G gave the same profile (data not shown).
The structure of splicing donor/acceptor sites
To analyse the splicing donor and acceptor site of these RNA transcripts, DNA from each band in Fig. 4 lane L1 was recovered from the gel, amplified by PCR using the A2U and A2L2 primers, and cloned. Clones, corresponding to each of the 2.0, 1.3 to 1.2 and putative 0.8 kb RNA species, were readily obtained. However, not a single clone corresponding to the 1.6 kb RNA species was obtained. Indeed, after several failed attempts, we finally abandoned trying to clone this species.
Clones corresponding to 2.0, 1.3 to 1.2 and 0.8 kb poly(A)+ RNA species were sequenced. The sequence of the 2.0 kb clone (CAVRt2.0-1) was entirely concordant with the CAV genome, without deletions or mutations (Fig. 5a). The clones corresponding to the 1.3–1.2 kb RNA species (CAVRt1.3-4 and CAVRt1.2-2, respectively) revealed that they are composed of two different species and both had undergone splicing. The 1.3 kb clone had one splice site (SP2), joining nt 1222 with nt 1814. This was consistent with the RT-PCR result (Fig. 4), which had anticipated a splice site upstream of nt 1915. The sequence at the donor site had a single base deviation from the splicing consensus sequence (Aebi et al., 1986), but was conserved at the acceptor site (Fig. 5b).
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). Compared to the splicing consensus sequence, SP1 had two mismatched bases at the donor site and six at the acceptor site (Fig. 5b). Both SP1 and SP2 showed sequence structures compatible with the GT–AG splicing rule (Aebi et al., 1986).
A clone (CAVRt0.8-1) corresponding to the 0.8 kb RNA species had a large deletion resulting in the juxtaposition of nt 639 and nt 1719 (DEL in Fig. 5a). The site of this deletion was consistent with the RT-PCR result (Fig. 4) that suggested a deletion upstream of nt 1800. The donor and acceptor sequences of the deletion in this clone were not concordant with the splicing consensus sequence (Fig. 5b). In addition, the deletion did not follow the GT–AG splicing rule. Moreover, the sequences at the deletion boundary – C640TGCA and C1719TGCA – were identical, suggesting homologous recombination.
ORFs on CAV transcripts
The distribution of CAV ORFs was analysed, based on these newly identified CAV mRNAs (Fig. 6). The three large ORFs, designated ORF1 (for VP1, capsid and polymerase), ORF2 (for VP2, phosphatase) and ORF3 (for VP3, apoptin), were all readable from non-spliced 2.0 kb mRNAs (Fig. 6b). The 1.3 kb mRNA also encoded both VP2 and VP3 (Fig. 6c), but translational products of frame 1 in this species would lack the central 197 aa of VP1 (VP1_1). While the large portion spliced out from SP2 was not found to cause a frameshift, the smaller SP1 site (seen in 1.2 kb mRNA species) did induce a frameshift, dramatically changing the ORF context along the transcript (Fig. 6d). In the 1.2 kb mRNA, ORF1 (initiating at nt 832) was truncated at nt 994 and connected to a newly created ORF4 (nt 1095–1150) in frame 2 to produce VP1_2. SP1 also affected ORF2 on the 1.2 kb mRNA: ORF2, starting at nt 356, was interrupted at nt 994 and connected to a new ORF5 (nt 1095–1222) in frame 3, and then to another new ORF (ORF6, nt 1814–1829) in the same frame to produce VP2_3.
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). ORF2, starting at nt 359 in frame 2, was interrupted at nt 639 and connected to the middle of ORF1 (nt 1719–2178) to produce VP2_1. Additionally, ORF3, starting from nt 465 in frame 3, was truncated at nt 639 and linked to nt 1719 in frame 2, although this reading frame terminated almost immediately at nt 1723 to produce VP3_2. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Phenix et al., 1994). This inferred that a single 2.0 kb transcript encoded all three viral proteins, although the regulatory mechanism for the expression of each protein was not understood. Given that TTV, which shares a similar ORF configuration to CAV, transcribes at least three different mRNA species, the transcriptional properties of CAV were re-examined.
We found that the previously reported 2.0 kb transcript (Noteborn et al., 1992; Phenix et al., 1994) was readily detectable in both cells infected with CAV and in cells transfected with pCAV1.3G. Sequencing of this RNA species revealed no deletions or frameshift mutations in the genomic sequence, confirming the absence of splicing events. In addition, we detected at least two other species of transcripts, of 1.3 and 1.2 kb, not only in chicken cells infected with CAV, but also in transfected chicken (MDCC-MSB1) and monkey (COS1) cells.
Cloning of these transcripts, and subsequent sequence analyses, revealed a single splice site in the 1.3 kb mRNA species and two consecutive splice sites in the 1.2 kb species. The larger splice site (SP2 in Fig. 5), common to both 1.3 and 1.2 kb mRNAs, displayed well conserved donor and acceptor sites, observing the GT–AG splicing consensus rule (Aebi et al., 1986). In contrast, the smaller splice site (SP1 in Fig. 5), which is specific to the 1.2 kb mRNA, was less well conserved at the splice acceptor, although the GT–AG rule was still maintained. The fact that the 1.2 kb RNA species was more abundant in transfected COS1 cells may suggest that SP1 splicing events take place preferentially in artificial conditions, or in a host-dependent manner.
Although two smaller mRNAs (1.3 and 1.2 kb) have been identified by this study, their importance in the CAV life cycle is still not understood. The unspliced 2.0 kb mRNA clearly encodes all three viral proteins (VP1, VP2 and VP3) by itself, and is the major transcript throughout the infectious cycle, being detectable from 3 h p.i. Several newly identified, putative proteins can be created via splicing events in the smaller mRNAs. These smaller mRNAs could be visualized only in the later stages of the viral cycle of infection, and it is unknown if they are actually translated. The conserved nature of the splice donor and acceptor motifs within the CAV genome suggests that they do have roles in the life cycle of this virus. The regulation of the mechanism of differential expression of these putative CAV proteins also remains to be elucidated. The CAV splice products are visible late during CAV infection, which seems to be at the time point that the infected cells are undergoing/underwent apoptosis (Noteborn et al., 1994). Therefore, the apoptotic process of infected cells and these putative spliced proteins might interact with each other.
During RT-PCR and cloning, an additional 0.8 kb species was isolated. Sequence analysis revealed that this species has a large deletion. No conserved splice donor or acceptor sequence motifs could be identified at the boundaries of this deletion. With identical 5' and 3' flanking sequences (5', C640TGCA; 3', C1719TGCA), the deletion may well be the result of homologous recombination rather than a splicing event. Since this species could not be visualized in Northern analyses, the possibility that it may be produced as an artefact of PCR cannot be discounted.
The 1.6 kb species of poly(A)+ RNA detected in Northern blotting analyses (Fig. 3) also remains unexplained. It was detected in both infected and transfected cells by Northern analysis using a full-length CAV probe, and could be reverse-transcribed by oligo-dT12–18 and amplified by PCR using primers A2U and A2L1. However, subcloning of this species of transcript was not successful, even after repeated trials. It is possible that this band may be due to non-specific hybridization, but it was absent in both mock-infected and mock-transfected cells. The nature of this band thus remains to be clarified.
In terms of viral taxonomy, CAV and TTV have historically has been classified into the family circoviridae, because their DNA genomes were both single-stranded and circular. However, CAV has more recently been removed from the genus Circovirus and used to establish a new genus, Gyrovirus, because it has antisense DNA in contrast to the ambisense DNA common to the circoviruses (such as Porcinecircovirus, PCV). Furthermore, a 9-nt sequence conserved at the replication origin in circoviruses is absent in CAV. In this sense, CAV and TTV might better be classified in a different family from the circoviruses.
CAV and TTV differ both in their genome size (2.3 and 3.8 kb, respectively) and in the degree of sequence diversity noted between their isolates. Recently, Jones et al. (2005) found new 2.2 and 2.6 kb viruses related to TTV: the size of 2.2 kb is close to that of CAV. Sequence diversity between CAV isolates is quite limited, while that of TTV is enormous. TTV isolates may share less than 70 % similarity. The genome sequences of these two viruses are also not similar to each other, with the exception of a 38 nt GC-rich region (Miyata et al., 1999). However, CAV and TTV do display several common features in cellular tropisms and genetic structures (Kamada et al., 2004; Kamahora et al., 2000). While CAV was previously thought to express a single 2.0 kb mRNA species, this study has further underlined the similarity between CAV and TTV in the presence of spliced mRNAs.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Akiyama, Y. & Kato, S. (1974). Two cell lines from lymphomas of Marek's disease. Biken J 17, 105–116.[Medline]
Biagini, P., Todd, D., Bendinelli, M. & 8 other authors (2004). Anellovirus. In Virus Taxonomy: Eighth Report 455 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. London: Elsevier/Academic Press.
Chen, C. & Okayama, H. (1987). High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7, 2745–2752.
Chomczynski, P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156–159.[Medline]
Hino, S. (2002). TTV, a new human virus with single stranded circular DNA genome. Rev Med Virol 12, 151–158.[CrossRef][Medline]
Imai, K. & Yuasa, N. (1990). Development of a microtest method for serological and virological examinations of chicken anemia agent. Nippon Juigaku Zasshi 52, 873–875.[Medline]
Islam, M. R., Johne, R., Raue, R., Todd, D. & Muller, H. (2002). Sequence analysis of the full-length cloned DNA of a chicken anaemia virus (CAV) strain from Bangladesh: evidence for genetic grouping of CAV strains based on the deduced VP1 amino acid sequences. J Vet Med B Infect Dis Vet Public Health 49, 332–337.[Medline]
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.
Kamada, K., Kamahora, T., Kabat, P. & Hino, S. (2004). Transcriptional regulation of TT virus: promoter and enhancer regions in the 1.2-kb noncoding region. Virology 321, 341–348.[CrossRef][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. J Virol 74, 9980–9986.
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. J Virol 73, 3582–3586.
Niagro, F. D., Forsthoefel, 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. Arch Virol 143, 1723–1744.[CrossRef][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. Biochem Biophys Res Commun 241, 92–97.[CrossRef][Medline]
Noteborn, M. H., de Boer, G. F., van Roozelaar, D. J. & 7 other authors (1991). Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle. J Virol 65, 3131–3139.
Noteborn, M. H., Kranenburg, O., Zantema, A., Koch, G., de Boer, G. F. & van der Eb, A. J. (1992). Transcription of the chicken anemia virus (CAV) genome and synthesis of its 52-kDa protein. Gene 118, 267–271.[CrossRef][Medline]
Noteborn, M. H., Todd, D., Verschueren, C. A. & 7 other authors (1994). A single chicken anemia virus protein induces apoptosis. J Virol 68, 346–351.
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.[Medline]
Okamoto, H., Takahashi, M., Kato, N., Fukuda, M., Tawara, A., Fukuda, S., Tanaka, T., Miyakawa, Y. & Mayumi, M. (2000). Sequestration of TT virus of restricted genotypes in peripheral blood mononuclear cells. J Virol 74, 10236–10239.
Phenix, K. V., Meehan, B. M., Todd, D. & McNulty, M. S. (1994). Transcriptional analysis and genome expression of chicken anaemia virus. J Gen Virol 75, 905–909.
Qiu, J., Kakkola, L., Cheng, F., Ye, C., Soderlund-Venermo, M., Hedman, K. & Pintel, D. J. (2005). Human circovirus TT virus genotype 6 expresses six proteins following transfection of a full-length clone. J Virol 79, 6505–6510.
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Todd, D., Creelan, J. L., Mackie, D. P., Rixon, F. & McNulty, M. S. (1990). Purification and biochemical characterization of chicken anaemia agent. J Gen Virol 71, 819–823.
Yamaguchi, S., Imada, T., Kaji, N., Mase, M., Tsukamoto, K., Tanimura, N. & Yuasa, N. (2001). Identification of a genetic determinant of pathogenicity in chicken anaemia virus. J Gen Virol 82, 1233–1238.
Received 13 February 2006;
accepted 8 April 2006.
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