Viruses with circular ssDNA genomes that encode a replication initiator protein (Rep) are among the smallest viruses known to infect both eukaryotic and prokaryotic organisms. In the past few years an overwhelming diversity of novel circular Rep-encoding ssDNA (CRESS-DNA) viruses has been unearthed from various hosts and environmental sources. Since there is limited information regarding CRESS-DNA viruses in invertebrates, this study explored the diversity of CRESS-DNA viruses circulating among insect populations by targeting dragonflies (Epiprocta), top insect predators that accumulate viruses from their insect prey over space and time. Using degenerate PCR and rolling circle amplification coupled with restriction digestion, 17 CRESS-DNA viral genomes were recovered from eight different dragonfly species collected in tropical and temperate regions. Nine of the genomes are similar to cycloviruses and represent five species within this genus, suggesting that cycloviruses are commonly associated with insects. Three of the CRESS-DNA viruses share conserved genomic features with recently described viruses similar to the mycovirus Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, leading to the proposal of the genus Gemycircularvirus. The remaining viruses are divergent species representing four novel CRESS-DNA viral genera, including a gokushovirus-like prokaryotic virus (microphage) and three eukaryotic viruses with Reps similar to circoviruses. The novelty of CRESS-DNA viruses identified in dragonflies using simple molecular techniques indicates that there is an unprecedented diversity of ssDNA viruses among insect populations. The GenBank/EMBL/DDBJ accession numbers for the unique cyclovirus genomes of DfCyVs 1-5 are JX185419-JX185427.

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Dragonfly cyclovirus (DfCyV), a new species of ssDNA virus discovered using viral metagenomics in dragonflies (family Libellulidae) from the Kingdom of Tonga. Metagenomic sequences of DfCyV were similar to viruses of the recently proposed genus Cyclovirus within the family Circoviridae. Specific PCRs resulted in the recovery of 21 DfCyV genomes from three dragonfly species (Pantala flavescens, Tholymis tillarga and Diplacodes bipunctata). The 1741 nt DfCyV genomes share .95 % nucleotide identity and are classified into 11 subtypes representing a single strain. The DfCyV genomes share 48-63 % genome-wide nucleotide identity with cycloviruses identified in human faecal samples. Recombination analysis revealed three recombinant DfCyV genomes, suggesting that recombination plays an important role in cyclovirus evolution. To our knowledge, this is the first report of a circular ssDNA virus identified in insects, and the data may help elucidate evolutionary links among novel Circoviridae recently identified in animals and environmental samples.

Members of the family Circoviridae, specifically the genus Circovirus, were thought to infect only vertebrates; however, members of a sister group under the same family, the proposed genus Cyclovirus, have been detected recently in insects. In an effort to explore the diversity of cycloviruses and better understand the evolution of these novel ssDNA viruses, here we present five cycloviruses isolated from three dragonfly species (Orthetrum sabina, Xanthocnemis zealandica and Rhionaeschna multicolor) collected in Australia, New Zealand and the USA, respectively. The genomes of these five viruses share similar genome structure to other cycloviruses, with a circular 1.7 kb genome and two major bidirectionally transcribed ORFs. The genomic sequence data gathered during this study were combined with all cyclovirus genomes available in public databases to identify conserved motifs and regulatory elements in the intergenic regions, as well as determine diversity and recombinant regions within their genomes. The genomes reported here represent four different cyclovirus species, three of which are novel. Our results confirm that cycloviruses circulate widely in winged-insect populations; in eight different cyclovirus species identified in dragonflies to date, some of these exhibit a broad geographical distribution. Recombination analysis revealed both intra-and inter-species recombination events amongst cycloviruses, including genomes recovered from disparate sources (e.g. goat meat and human faeces). Similar to other well-characterized circular ssDNA viruses, recombination may play an important role in cyclovirus evolution. The GenBank/EMBL/DDBJ accession numbers for the genome sequences of dragonfly cycloviruses identified in this study are KC512916-KC512920. One supplementary figure and two supplementary tables are available with the online version of this paper.

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