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Short Communication |
1 Unité des Virus Emergents EA3292, Etablissement Français du Sang Alpes-Méditerranée, Service de Virologie Moléculaire, 13005 Marseille, France
2 Unité des Virus Emergents EA3292, Faculté de Médecine, 13005 Marseille, France
3 Institute for Animal Health, Pirbright Laboratory, Department of Arbovirology, Pirbright GU24 0NF, UK
Correspondence
Philippe Biagini
pbiagini-ets-ap{at}gulliver.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are EF538875–EF538883.
A figure showing the predicted genomic structure of isolates LIL-y3 and PRA4 and a table showing the selected DNA fragments and oligonucleotide primers used for sequence extensions are available with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). The short nucleotide sequence obtained initially (
500 nt) was first extended to 3700 nt, and the ultimate resolution of an additional GC-rich region of about 120 nt permitted us to complete the TTV prototype sequence (Miyata et al., 1999). During a study of TTV prevalence in blood donors, in 1999, some unexpected amplification products were sequenced and identified as highly divergent when compared with known TTV sequences. A circular genome of about 2900 nt was further characterized and originally designated TTV-like mini virus (TLMV) by analogy with TTV (Takahashi et al., 2000). Progressive determination of partial and full-length genomic sequences highlighted the considerable genetic heterogeneity of this new class of viruses. Extremely divergent sequences were also characterized in non-human primates and in several animal species by using a conserved amplification system (Okamoto et al., 2000, 2002). In 2005, the International Committee on Taxonomy of Viruses officially created the genus for classification of these viruses (Biagini et al., 2005). This floating genus Anellovirus initially included the type species TTV, one tentative species, Torque teno mini virus (TTMV, initially TLMV) and unclassified animal viruses.
Recently, a putative third member of the genus Anellovirus, the ‘small anellovirus’, was identified following the characterization of two highly divergent circular sequences (2200 and 2600 nt) from human plasma samples (Jones et al., 2005). The identification of these sequences was not performed by using the conserved PCR system but rather with the help of a method designated sequence-independent single primer amplification (SISPA). This approach is based on the use of endonuclease restriction of target sequences previously converted to double-stranded DNA, followed by non-specific linker ligation and PCR amplification (Reyes & Kim, 1991; Allander et al., 2001).
Very recently, circular genomes were also successfully amplified by using the technique of multiply primed rolling-circle amplification (RCA) (Esteban et al., 1993; Dean et al., 2001). This approach permitted the amplification and cloning of anellovirus (Niel et al., 2005), circovirus (Johne et al., 2006), geminivirus (Haible et al., 2006) and papillomavirus (Rector et al., 2004) genomes. In this sequence-independent technique, a random hexamer primer anneals to multiple sites of a DNA template; these sites are isothermally extended by the phi29 DNA polymerase, therefore producing multiple copies of the complete genome.
We have combined the potential of both methodologies and used an RCA–SISPA approach for the detection of circular genomes in human and animal biological samples. RCA–SISPA permitted the characterization of nine anelloviruses from human plasma and cat saliva samples. Molecular and phylogenetic analyses revealed that most of these sequences were highly divergent when compared with those previously identified. These results confirmed the potential of the RCA–SISPA approach in the detection of circular (or circularized) target genomes.
Four blood samples were used, originating from blood donor samples excluded from use in blood transfusions, because of a positive serology for hepatitis C virus or human immunodeficiency virus. Plasma samples (300 µl each) were clarified by centrifugation at 10 000 g for 5 min at 20 °C and filtered through a 0.22 µM filter (Millipore). Filtrates were treated with 26 U Benzonase (Novagen) at 37 °C for 30 min in order to remove all non-encapsidated nucleic acid materials. Viral nucleic acids were then extracted using the High Pure Viral Nucleic Acid kit (Roche), resuspended in 30 µl nuclease-free water and stored at –80 °C until use.
One saliva sample from a 5-year-old family cat (Felis catus) was collected by using a sterile oral swab. The swab cotton end was cut and placed in 300 µl sterile water for 10 min under constant shaking. The aqueous phase was recovered and treated with Benzonase as described above. The nucleic acids were ultimately extracted using the High Pure Viral Nucleic Acid kit.
Fourteen microlitres of nucleic acid extract was used for multiply primed RCA. Briefly, the nucleic acids were denatured at 94 °C for 3 min, then immediately quenched in a moistened ice bath. Six microlitres of a previously prepared solution [containing 2 mM each dNTPs (Invitrogen), 25 µM random exo-resistant hexanucleotides (Fermentas), 10 U phi29 DNA polymerase and its corresponding buffer (Fermentas)] was added. The mixture was incubated for 16 h at 30 °C.
For the SISPA procedure, an equal amount (10 µl) of the resulting linear double-stranded templates was treated separately by one of two restriction enzymes (RE). The RE digestion was performed for 2 h, either at 37 °C using 40 U Csp6I (Fermentas) or at 65 °C using 40 U TaqI (New England Biolabs) under manufacturer conditions (final volume 20 µl). Both samples were then purified using the MinElute Reaction Cleanup kit (Qiagen) and resuspended in 10 µl elution buffer (EB; Tris/HCl 10 mM, pH 8.5). The NCsp adaptor or the NTaq adaptor (200 pmol each) (Allander et al., 2001) was ligated to the corresponding digested material in a reaction mixture containing 4 U T4 DNA ligase (Roche) and its corresponding buffer (final volume 20 µl) for 16 h at 4 °C. Samples were subsequently purified (MinElute Reaction Cleanup kit) and resuspended in 10 µl EB. Samples were PCR amplified using the appropriate primer (NBam24 or NTaq24) (2.5 µM) (Allander et al., 2001) in a 50 µl mix containing dNTPs (0.2 mM each) (Roche) and 4 U Takara Ex Taq polymerase (Takara Shuzo) with its corresponding buffer, as described previously (Jones et al., 2005).
One tenth of each PCR mixture was run on 2 % agarose gels for amplification profile analysis. The remaining amplification products were directly purified from the PCR reaction mixture (MinElute Reaction Cleanup kit), cloned into the pGEM-T vector (Promega) and transfected into Escherichia coli XL-Blue competent cells (Biagini et al., 2007). The final PCR screening of DNA clones permitted the selection of amplicons representative of each amplification product; these were subsequently sequenced using universal M13 primers.
Sequences were compared with those deposited in the GenBank database using NCBI's online BLAST2 program (http://www.ncbi.nlm.nih.gov/BLAST/).
Sequence alignments, pairwise distance calculations and phylogenetic analyses were performed using the CLUSTAL W (Thompson et al., 1994) and MEGA3 programs (Kumar et al., 2004).
Secondary structures formed by the GC-rich region of characterized isolates were predicted by using the online MFOLD program (http://frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/dna-form1.cgi) (Zuker, 2003).
The use of the combined RCA–SISPA procedure with plasma and saliva samples permitted us to characterize systematically amplification DNA products. Restriction-amplification fragments of Csp6I and TaqI with sizes ranging between 150 and 1500 bp were visible on agarose gels for each of the plasma samples tested (Fig. 1a) and for the cat saliva sample as well (Fig. 1b).
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The final full-length or nearly full-length sequences obtained from human plasma and animal saliva sources showed a genetic organization characteristic of the members of the genus Anellovirus [i.e. a coding region containing the major open reading frame (ORF) 1, an overlapping ORF2 and several complementary ORFs, an untranslated region of variable size depending on the isolates and a TATA box].
The isolate CSC5 (a partial genome sequence that is 2912 nt long, which contains the complete coding sequences) was identified as related to the species TTV according to the analysis of the entire ORF1, which shared 93 % identity at the nucleotide level with the closest isolate, tth5 (Jelcic et al., 2004). Four isolates could be classified into species TTMV, for which only 12 complete sequences have been described to date (Biagini et al., 2001, 2006b; Takahashi et al., 2000). When ORF1 was analysed, the isolates LIL-y1 (complete genome, 2887 nt) and LIL-y2 (complete genome, 2871 nt) (Fig. 2a) shared only 53 % identity with the closest isolate NLC026; the isolates LIL-y3 (complete genome, 2912 nt) (Supplementary Fig. S1) and LIL-y4 (partial, 2797 nt, complete coding sequence) shared 60 and 67 % sequence identity with their closest isolate (CBD203), respectively. Two isolates, 2PoSMA (complete genome, 2002 nt) and 6PoSMA (complete genome, 2454 nt) (Fig. 2b) were related to the third tentative species ‘small anellovirus’ (Jones et al., 2005; Ninomiya et al., 2007). They shared 64 and 60 % identity, respectively, with their closest isolate (small anellovirus 1). Two full-length genomes were obtained from the cat saliva sample: PRA1 (2019 nt) (Fig. 2c) and PRA4 (2065 nt) (Supplementary Fig. S1). They shared 54 % nucleotide sequence identity (ORF1). By comparison with the unique full-length sequence identified until now in cats (isolate Fc-TTV4, 2064 nt) (Okamoto et al., 2002), the isolate PRA1 shared only 53 % sequence identity; the other isolate, PRA4, exhibited 94 % sequence identity with the reference sequence. Finally, putative secondary structures were identified within the GC-rich region of isolates LIL-y1 and LIL-y2 (Fig. 2d).
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) was constructed on the basis of the entire nucleotide sequence of ORF1 of the isolates described here, along with those of previously described members of the genus Anellovirus (Biagini et al., 2001, 2005, 2006a, b; Ninomiya et al., 2007; Okamoto et al., 2002; Peng et al., 2002; Jones et al., 2005). Analysis of the phylogenetic grouping showed that isolate CSC5 clustered within the group 5 reference sequence JT34F of the species TTV. Study of the new sequences belonging to the species TTMV revealed that, whereas isolates LIL-y3 and LIL-y4 appeared to be related to isolates CBD203 and CLC156, isolates LIL-y1 and LIL-y2 were grouped together, within a new and separate phylogenetic branch. Finally, isolates 2PoSMA and 6PoSMA segregated into the cluster formed by the sequences small anellovirus 1 and 2, and MD1-032, MD1-073 and MD2-013. The fact that this cluster was clearly distinct from the other TTV and TTMV clusters confirmed that the ‘small anellovirus’ species is an independent species of the genus Anellovirus (Andreoli et al., 2006; Jones et al., 2005). In the case of the cat saliva sample, isolates PRA1 and PRA4 clustered within the group formed by the unique TTV species identified so far for cats. These two isolates were identified as either highly divergent and describing a new phylogenetic branch, or very close to the Japanese isolate Fc-TTV4 and confirming the absence of geographical cluster, at least for pets, as previously demonstrated for human anelloviruses.
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). This is why the conventional SISPA technique was only occasionally used to identify anelloviruses (Jones et al., 2005), since virus titres in plasma are frequently below the detection limit of the technique (Pistello et al., 2001).
By combining the power of RCA with a SISPA strategy, we detected numerous sequences in each plasma and saliva sample tested. These were found to belong to various species of the genus Anellovirus and are highly diverse in terms of length and position in the viral genome. These sequences were exploited to design primers for a successful full-length sequence extension. Such an approach opens up new perspectives since, until this date, the ‘gold standard’ for anellovirus DNA investigation has been based on the use of highly conserved primers for a short PCR amplification of about 120 bp (Okamoto et al., 2002). The use of the RCA–SISPA approach will most probably help in completing the analysis of the genetically diverse anelloviruses, which will provide new insights about their taxonomy. By applying this method to a few plasma samples, we were able to identify: (i) several highly divergent strains that were classified into the various species within the genetically diverse genus Anellovirus, (ii) the smallest anellovirus (2002 nt) described up to now in humans and (iii) a new TTMV genogroup. It is noteworthy that the results obtained from the cat saliva sample led to the identification of two genomes that are separated by a genetic distance of 46 %, confirming the existence of co-infections in pets. Moreover the data confirmed the fact that the genetic variability among anelloviruses in infected animal species is probably very high, as already suggested for pigs (Bigarré et al., 2005; Niel et al., 2005), and potentially of the same order of magnitude as that already identified in humans.
Application of the RCA–SISPA approach is however not restricted to this sole class of viruses, since many other viruses identified in humans, animals or plants may have a circular genome. It is further appealing to envisage that the RCA–SISPA protocol, in line with the concept of ‘viral metagenomics’ (Ambrose & Clewley, 2006), could be modified in order to investigate a whole spectrum of viral genomes of DNA and RNA origin in a biological sample, whether they are circular or not (for instance by the use of additional steps aiming to circularize target sequences).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 10 April 2007;
accepted 13 June 2007.
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