This paper presents standards and best practices for reporting genome sequences of uncultivated viruses.Supplementary informationThe online version of this article (doi:10.1038/nbt.4306) contains supplementary material, which is available to authorized users.

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The family Circoviridae contains viruses with covalently closed, circular, single-stranded DNA (ssDNA) genomes, including the smallest known autonomously replicating, capsid-encoding animal pathogens.

Accurate indicators of fecal pollution are needed in order to minimize public health risks associated with wastewater contamination in recreational waters. However, the bacterial indicators currently used for monitoring water quality do not correlate with the presence of pathogens. Here we demonstrate that the plant pathogen Pepper mild mottle virus (PMMoV) is widespread and abundant in wastewater from the United States, suggesting the utility of this virus as an indicator of human fecal pollution. Quantitative PCR was used to determine the abundance of PMMoV in raw sewage, treated wastewater, seawater exposed to wastewater, and fecal samples and/or intestinal homogenates from a wide variety of animals. PMMoV was present in all wastewater samples at concentrations greater than 1 million copies per milliliter of raw sewage. Despite the ubiquity of PMMoV in human feces, this virus was not detected in the majority of animal fecal samples tested, with the exception of chicken and seagull samples. PMMoV was detected in four out of six seawater samples collected near point sources of secondary treated wastewater off southeastern Florida, where it co-occurred with several other pathogens and indicators of fecal pollution. Since PMMoV was not found in nonpolluted seawater samples and could be detected in surface seawater for approximately 1 week after its initial introduction, the presence of PMMoV in the marine environment reflects a recent contamination event. Together, these data demonstrate that PMMoV is a promising new indicator of fecal pollution in coastal environments.

SummaryReclaimed water use is an important component of sustainable water resource management. However, there are concerns regarding pathogen transport through this alternative water supply. This study characterized the viral community found in reclaimed water and compared it with viruses in potable water. Reclaimed water contained 1000-fold more viruslike particles than potable water, having approximately 10 8 VLPs per millilitre. Metagenomic analyses revealed that most of the viruses in both reclaimed and potable water were novel. Bacteriophages dominated the DNA viral community in both reclaimed and potable water, but reclaimed water had a distinct phage community based on phage family distributions and host representation within each family. Eukaryotic viruses similar to plant pathogens and invertebrate picornaviruses dominated RNA metagenomic libraries. Established human pathogens were not detected in reclaimed water viral metagenomes, which contained a wealth of novel single-stranded DNA and RNA viruses related to plant, animal and insect viruses. Therefore, reclaimed water may play a role in the dissemination of highly stable viruses. Information regarding viruses present in reclaimed water but not in potable water can be used to identify new bioindicators of water quality. Future studies will need to investigate the infectivity and host range of these viruses to evaluate the impacts of reclaimed water use on human and ecosystem health.

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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.

Single-stranded DNA (ssDNA) viruses with circular genomes are the smallest viruses known to infect eukaryotes. The present study identified 10 novel genomes similar to ssDNA circoviruses through data-mining of public viral metagenomes. The metagenomic libraries included samples from reclaimed water and three different marine environments (Chesapeake Bay, British Columbia coastal waters and Sargasso Sea). All the genomes have similarities to the replication (Rep) protein of circoviruses; however, only half have genomic features consistent with known circoviruses. Some of the genomes exhibit a mixture of genomic features associated with different families of ssDNA viruses (i.e. circoviruses, geminiviruses and parvoviruses). Unique genome architectures and phylogenetic analysis of the Rep protein suggest that these viruses belong to novel genera and/or families. Investigating the complex community of ssDNA viruses in the environment can lead to the discovery of divergent species and help elucidate evolutionary links between ssDNA viruses.