Characterisation of a diverse range of circular replication-associated protein encoding DNA viruses recovered from a sewage treatment oxidation pond

• Used high-throughput sequencing to identify Rep encoding ssDNA viruses.
• Recovered 50 putatively complete novel circular ssDNA viral genomes.
• 13 viral genomes encode Reps similar to those of gemycircularviruses.
• 37 viral genomes share very low degrees of Rep to those encoded by known viruses.

Our knowledge of circular replication-associated protein encoding single-stranded (CRESS) DNA virus diversity has increased dramatically in recent years, largely due to advances in high-throughput sequencing technologies. These viruses are apparently major virome components in most terrestrial and aquatic environments and it is therefore of interest to determine their diversity at the interfaces between these environments. Treated sewage water is a particularly interesting interface between terrestrial and aquatic viromes in that it is directly pumped into waterways and is likely to contain virus populations that have been strongly impacted by humans. We used a combination of high-throughput sequencing, full genome PCR amplification, cloning and Sanger sequencing to investigate the diversity of CRESS DNA viruses present in a sewage oxidation pond. Using this approach, we recovered 50 putatively complete novel CRESS viral genomes (it remains possible that some are components of multipartite viral genomes) and 11 putatively sub-genome-length circular DNA molecules which may be either defective genomes or components of multipartite genomes. Thirteen of the genomes have bidirectional genome organisations and share similar conserved replication-associated protein (Rep) motifs to those of the gemycircularviruses: a group that in turn is most closely related to the geminiviruses. The remaining 37 viral genomes share very low degrees of Rep similarity to those of all other known CRESS DNA viruses. This number of highly divergent CRESS DNA virus genomes within a single sewage treatment pond further reinforces the notion that there likely exist hundreds of completely unknown genus/family level CRESS DNA virus groupings.