Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia1
Department of Veterinary Science, The Queens University of Belfast, Stormont, Belfast BT4 3SD, Northern Ireland2
Electron Microscopy Laboratory, Istituto Zooprofilattico Sperimentale della Lombardia e dellEmilia Romagna, Via Bianchi 9, 25124 Brescia, Italy3
Department of Agriculture and Rural Development for Northern Ireland, Stormont, Belfast BT4 3SD, Northern Ireland4
Author for correspondence: Shane Raidal. Fax +61 93104144. e-mail raidal{at}numbat.murdoch.edu.au
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Abstract |
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Circovirus infections are commonly associated with immunodeficiency-related diseases that are potentially fatal. A condition known as black spot of neonatal canaries, characterized by abdominal enlargement, gall bladder congestion and failure to thrive, has been described for many years in Europe and was reported to have been caused by a circovirus (Goldsmith, 1995 ). More recently, a circovirus-like infection of canaries was identified in adult birds that had died following a short illness characterized by dullness, anorexia, lethargy and feather disorder (Todd et al., 2001b
). Large numbers of circovirus-like, spherical virus particles were detected by electron microscopy in the organ homogenates of diseased birds. A degenerate primer-based PCR technique using degenerate primers specific to conserved amino acid (aa) sequences in the circovirus Rep (replication-associated) protein successfully amplified a circovirus (CaCV)-specific DNA fragment from infected birds (Todd et al., 2001
b). Nucleotide (nt) and predicted aa sequence analysis of the 510 bp genomic fragment indicated that the CaCV sequence was more closely related to CoCV than to BFDV, and more distantly related to PCV1 and PCV2. Here we report the complete genome sequence of CaCV and discuss its relationship with other members of the Circoviridae.
The full-length circular CaCV genome was amplified by inverse PCR (IPPCR) from the original CaCV clone described previously (Todd et al., 2001b ) using the primers CaCV1 5' ATGATTGGACAGAAACCCCGTGAC 3' and CaCV2 5' GTCCAATCATCAGGGCCAAATCCC 3'. PCR was carried out at 94 °C for 30 s, 60 °C for 30 s, 72 °C for 2 min for 35 cycles using the Expand Long Template PCR system (Roche Diagnostics) as described by the manufacturer. The resulting IPPCR product (approximately 2 kb) was cloned into the pCR2.1-TOPO vector (Invitrogen) and two clones derived from the same IPPCR product were selected for sequencing. Clones were sequenced in triplicate in both directions. The nt sequence of CaCV was edited and assembled using SeqEd version 1.0.3 (Applied Biosystems) and analysed using a range of programs provided by the Australian National Genomic Information Services (ANGIS). For multiple sequence alignment and phylogenetic analysis of sequences the programs Global Pair Alignment, Gap, PileUp and ClusTree were used. Sequence data were edited and aligned by the PileUp program and analysed phylogenetically using the ClusTree program with 1000 bootstrap cycles. ClusTree computes a phylogenetic tree according to the neighbour-joining method of Saitou & Nei (1987)
. Database searches were performed in non-redundant nucleic and protein databases with the BLAST program (Altschul et al., 1990
). The GenBank accession number for the CaCV sequence is AF346618.
Sequence analysis revealed that the genome of CaCV was circular and had a size of 1952 nt. The nt numbering and open reading frame (ORF) nomenclature used was similar to that adopted previously for animal circoviruses (Todd et al., 2000 ); nt position 1 is given as the A residue at position 8 of a nonamer sequence (CAGTATTAC) present at the apex of a potential stemloop structure. This stemloop/nonamer element is conserved in circoviruses, with the exception of CAV in which only the nonamer is present, and is thought to represent the origin of rolling circle replication. In CaCV, the nonamer sequence differs slightly from that of other circoviruses in that the first residue of the nonamer is a C, rather than the T residue found in other members of the genus. Such variations in the nonamer sequence (Meehan et al., 1997
, 1998
; Bassami et al., 1998
) and also in the number of base-pair residues comprising the stemloop structure (Fig. 1A
, B
) have previously been reported (Todd et al., 2001
a). In addition, three base differences at nt positions 1825 (C or T), 1931 (C or T) and 1945 (A or G) were found between the two clones sequenced.
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Genome analysis indicated that CaCV displays an ambisense genome organization, with one large ORF being encoded on the viral strand and one major and one minor ORF on the complementary strand (Fig. 1C). The largest ORF, V1 (290 aa), encodes the putative Rep protein, with a predicted molecular mass of 33·4 kDa. Multiple alignment of the deduced aa sequence of the Rep protein of CaCV with those encoded by other circoviruses including PCV1, PCV2, BFDV, CoCV and GCV, identified conserved aa motifs typical for rolling circle replication and also the dNTP-binding domain. Three additional conserved motifs, WWDGY, DDFYGWLP and DRYP, previously reported to be present in CoCV and GCV (Todd et al., 2001a
), were also identified within the Rep protein of CaCV (Fig. 2
). A putative poly(A) signal (AATAAA) was also identified on the viral strand, 39 nt (nt position 943948) downstream from the ORF V1 stop codon.
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In terms of the relationship of CaCV with other circoviruses, nt and aa sequence comparisons indicate that CaCV is more closely related to CoCV and BFDV than to the recently characterized avian circovirus GCV and the two porcine circoviruses, PCV1 and PCV2. Pairwise comparisons indicated that CaCV showed greatest homology to CoCV (58·3% nt identity) and BFDV (55·7% nt identity) and less homology to GCV (49·5% nt identity), PCV1 (50·9% nt identity) and PCV2 (46·8% nt identity).
As expected, highest levels of aa identity were shared between the Rep-coding regions of circoviruses. Pairwise comparisons showed that the level of aa identity of the V1 ORF shared by CaCV with CoCV (63·4%) and BFDV (62·9%) was substantially higher than that shared by CaCV with GCV (45·8%), PCV1 (42·5%) and PCV2 (40·4%). Overall, less homology was observed between the capsid protein-coding regions, with CaCV ORF C1 showing greatest aa identity with the C1 ORFs of BFDV (44%) and CoCV (39%). This level of aa identity was considerably reduced when the C1 ORFs of CaCV were compared with PCV1 (28·5%), PCV2 (29·8%) and GCV (25·7%). Phylogenetic trees derived from the predicted aa sequences of the Rep and capsid proteins of the six members of the Circovirus genus demonstrate these relationships (Fig. 3).
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Received 15 May 2001;
accepted 7 August 2001.