Novel circular DNA viruses in stool samples of wild-living chimpanzees

Abstract

Viral particles in stool samples from wild-living chimpanzees were analysed using random PCR amplification and sequencing. Sequences encoding proteins distantly related to the replicase protein of single-stranded circular DNA viruses were identified. Inverse PCR was used to amplify and sequence multiple small circular DNA viral genomes. The viral genomes were related in size and genome organization to vertebrate circoviruses and plant geminiviruses but with a different location for the stem-loop structure involved in rolling circle DNA replication. The replicase genes of these viruses were most closely related to those of the much smaller (approximately 1 kb) plant nanovirus circular DNA chromosomes. Because the viruses have characteristics of both animal and plant viruses, we named them chimpanzee stool-associated circular viruses (ChiSCV). Further metagenomic studies of animal samples will greatly increase our knowledge of viral diversity and evolution.

Fig. 1.

Genomic organization of ChiSCVs and representative members of the families Circoviridae, Geminiviridae and Nanoviridae. Circoviridae: goose circovirus (GCV) GenBank accession no. AAN37984, porcine circovirus 2 (PCV2) ABY82498 and canary circovirus (CaCV) AF246618. Geminiviridae: tomato yellow leaf curl virus (TYLCV) AF071228, horseradish curly top virus (HrCTV) U49907. Nanoviridae: faba bean necrotic yellows virus (FBNYV) DNA1, NC_005558; DNA2, NC_003560; DNA3, NC_003561; DNA4, NC_003562; DNA5, NC_003563; DNA6, NC_003564; DNA7, NC_003565; DNA8, NC_003566; DNA9, NC_003567; DNA10, NC_003559. The genome organization of GCV, PCV2, BFDV, TYLCV and HrCTV has been redrawn based on GenBank annotations and as described by Klute et al. (1996), Morilla et al. (2005), Todd et al. (2001a, b) and Phenix et al. (2001).

Fig. 2.

Alignment of the ORF1 replicase protein of ChiSCVs and representative members of the families Nanoviridae, Circoviridae and Geminiviridae. GenBank accession nos are given on Fig. 5(a). Amino acid motifs conserved between rolling-circle replication Rep proteins of different viruses are labelled 1–5 (boxed). Amino acid conservation of 100, >80 and >60 % is highlighted in black, dark grey and light grey, respectively.

Fig. 3.

Alignment of partial ChiSCV replicase sequences amplified from consecutively collected stools samples from a single chimpanzee (Ch-065).

Fig. 4.

Putative stem–loop and hairpin structures of (a) ChiSCV-GM415, -GM476, -GM488, -GM495, -GM510, (b) ChiSCV-DP152 and (c) ChiSCV-GT306. (d) Alignment of loop nonanucleotide sequences from ChiSCVs, circoviruses, nanoviruses and a geminivirus. Nucleotides that are 100% conserved between ChiSCV and other circular ssDNA viruses are highlighted in black. Boxed regions indicate loop sequences.

Fig. 5.

Phylogenetic analyses of (a) the ORF1 replicase genes of ChiSCVs and members of the families Geminiviridae, Nanoviridae and Circoviridae and (b) the ORF2 (putative capsid gene) of ChiSCVs. The GenBank accession no. of each sequence is shown on the figure.