Abstract
Nested PCRs with circovirus/cyclovirus pan-rep (replicase gene) primers detected eukaryotic circular Rep-encoding single-stranded DNA (CRESS DNA) viruses in three (samples CN9E, CN16E and CN34) of 18 canine parvovirus-2-positive fecal samples from household dogs with hemorrhagic gastroenteritis on the Caribbean island of Nevis. The complete genomes of CRESS DNA virus CN9E, CN16E and CN34 were determined by inverse nested PCRs. Based on (i) genome organization, (ii) location of the putative origin of replication, (iii) pairwise genome-wide sequence identities, (iv) the presence of conserved motifs in the putative replication-associated protein (Rep) and the arginine-rich region in the amino terminus of the putative capsid protein (Cp) and (v) a phylogenetic analysis, CN9E, CN16E and CN34 were classified as cycloviruses. Canine-associated cycloviruses CN16E and CN34 were closely related to each other and shared low genome-wide nucleotide (59.642–59.704%), deduced Rep (35.018–35.379%) and Cp (26.601%) amino acid sequence identities with CN9E. All the three canine-associated cycloviruses shared < 80% genome-wide pairwise nucleotide sequence identities with cycloviruses from other animals/environmental samples, constituting two novel species (CN9E and CN16E/34) within the genus Cyclovirus. Considering the feeding habits of dogs, we could not determine whether the cycloviruses were of dietary origin or infected the host. Interestingly, the CN9E putative Rep-encoding open reading frame was found to use the invertebrate mitochondrial genetic code with an alternative initiation codon (ATA) for translation, corroborating the hypothesis that cycloviruses are actually arthropod-infecting viruses. To our knowledge, this is the first report on the detection and complete genome analysis of cycloviruses from domestic dogs.
Highlights
Cycloviruses, members of the genus Cyclovirus within the family Circoviridae, possess a circular, covalently closed, single-stranded DNA genome (~1.7–1.9 kb in length) [1,2].The cyclovirus genome contains at least two major putative open reading frames (ORFs) that are transcribed bidirectionally [1,2]
We report here the complete genome analysis of cycloviruses from three canine parvovirus-2 (CPV-2)-positive household dogs with hemorrhagic gastroenteritis
5) with an alternative initiation codon (ATA), we identified putative replicationassociated protein (Rep) coding sequences that were separated by a putative intron with a canonical splice donor site (GT) and splice acceptor site (AG) (Figure 1 and Supplementary Figure S7)
Summary
The cyclovirus genome contains at least two major putative open reading frames (ORFs) that are transcribed bidirectionally [1,2]. Replication of the cycloviral genome remains to be experimentally investigated, the putative Rep contains conserved sequence motifs that are associated with rolling cycle replication (RCR) [1,2]. The 50 -intergenic region (50 -IR, region between the initiation codons of the rep and cp genes) contains a conserved origin of replication (ori), which is marked by the presence of a canonical nonanucleotide motif ‘NAGTATTAC’ (where ‘N’ represents any nucleotide (nt)) located at the apex of a stem–loop structure [1,2,6,7]. The presence of putative introns has been observed within ORFs of various cyclovirus genomes [1,2]
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