Antiviral discovery in toxic cyanobacteria: Low hanging fruit in the age of pandemics.

The power of novel vaccination technologies and their rapid development were elucidated clearly during the COVID-19 pandemic. At the same time, it also became clear that there is an urgent need to discover and manufacture new antivirals that target emerging viral threats. Toxic species of cyanobacteria produce a range of bioactive compounds that makes them good candidates for drug discovery. Nevertheless, few studies demonstrate the antiviral potential of cyanobacteria. This is partly due to the lack of specific and simple protocols designed for the rapid detection of antiviral activity in cyanobacteria and partly because specialized facilities for work with pathogenic viruses are few and far between. We therefore developed an easy method for the screening of cyanobacterial cultures for antiviral activity and used our private culture collection of non-pathogenic virus isolates to show that antiviral activity is a prominent feature in the cyanobacterium Microcystis aeruginosa. In this proof-of-concept study, we show that M. aeruginosa extracts from three different cyanobacterial strains delay infection of diatom-infecting single-stranded DNA and single-stranded RNA viruses by up to 2 days. Our work shows the ease with which cyanobacteria from culture collections can be screened for antiviral activity and highlights the potential of cyanobacteria as an excellent source for the discovery of novel antiviral compounds, warranting further investigation.

Molecular characterization of a tetra segmented ssDNA virus infecting Botrytis cinerea worldwide.

BACKGROUND: Family Genomoviridae was recently established, and only a few mycoviruses have been described and characterized, and almost all of them (Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, Fusarium graminearum gemyptripvirus 1 and Botrytis cinerea gemydayirivirus 1) induced hypovirulence in their host. Botrytis cinerea ssDNA virus 1 (BcssDV1), a tetrasegmented single-stranded DNA virus infecting the fungus Botrytis cinerea, has been molecularly characterized in this work. METHODS: BcssDV1 was detected in Spanish and Italian B. cinerea field isolates obtained from grapevine. BcssDV1 variants genomes were molecularly characterized via NGS and Sanger sequencing. Nucleotide and amino acid sequences were used for diversity and phylogenetic analysis. Prediction of protein tertiary structures and putative associated functions were performed by AlphaFold2 and DALI. RESULTS: BcssDV1 is a tetrasegmented single-stranded DNA virus. The mycovirus was composed by four genomic segments of approximately 1.7 Kb each, which are DNA-A, DNA-B, and DNA-C and DNA-D, that coded, respectively, for the rolling-circle replication initiation protein (Rep), capsid protein (CP) and two hypothetical proteins. BcssDV1 was present in several Italian and Spanish regions with high incidence and low variability among the different viral variants. DNA-A and DNA-D were found to be the more conserved genomic segments among variants, while DNA-B and DNA-C segments were shown to be the most variable ones. Tertiary structures of the proteins encoded by each segment suggested specific functions associated with each of them. CONCLUSIONS: This study presented the first complete sequencing and characterization of a tetrasegmented ssDNA mycovirus, its incidence in Spain and Italy, its presence in other countries and its high conservation among regions.

Diversity of Sea Star-Associated Densoviruses and Transcribed Endogenous Viral Elements of Densovirus Origin.

A viral etiology of sea star wasting syndrome (SSWS) was originally explored with virus-sized material challenge experiments, field surveys, and metagenomics, leading to the conclusion that a densovirus is the predominant DNA virus associated with this syndrome and, thus, the most promising viral candidate pathogen. Single-stranded DNA viruses are, however, highly diverse and pervasive among eukaryotic organisms, which we hypothesize may confound the association between densoviruses and SSWS. To test this hypothesis and assess the association of densoviruses with SSWS, we compiled past metagenomic data with new metagenomic-derived viral genomes from sea stars collected from Antarctica, California, Washington, and Alaska. We used 179 publicly available sea star transcriptomes to complement our approaches for densovirus discovery. Lastly, we focus the study on sea star-associated densovirus (SSaDV), the first sea star densovirus discovered, by documenting its biogeography and putative tissue tropism. Transcriptomes contained only endogenized densovirus elements similar to the NS1 gene, while numerous extant densoviral genomes were recovered from viral metagenomes. SSaDV was associated with nearly all tested species from southern California to Alaska, and in contrast to previous work, we show that SSaDV is one genotype among a high diversity of densoviruses present in sea stars across the West Coast of the United States and globally that are commonly associated with grossly normal (i.e., healthy or asymptomatic) animals. The diversity and ubiquity of these viruses in sea stars confound the original hypothesis that one densovirus is the etiological agent of SSWS.IMPORTANCE The primary interest in sea star densoviruses, specifically SSaDV, has been their association with sea star wasting syndrome (SSWS), a disease that has decimated sea star populations across the West Coast of the United States since 2013. The association of SSaDV with SSWS was originally drawn from metagenomic analysis, which was further studied through field surveys using quantitative PCR (qPCR), with the conclusion that it was the most likely viral candidate in the metagenomic data based on its representation in symptomatic sea stars compared to asymptomatic sea stars. We reexamined the original metagenomic data with additional genomic data sets and found that SSaDV was 1 of 10 densoviruses present in the original data set and was no more represented in symptomatic sea stars than in asymptomatic sea stars. Instead, SSaDV appears to be a widespread, generalist virus that exists among a large diversity of densoviruses present in sea star populations.

Reassortments in single-stranded DNA multipartite viruses: Confronting expectations based on molecular constraints with field observations.

Single-stranded DNA multipartite viruses, which mostly consist of members of the genus Begomovirus, family Geminiviridae, and all members of the family Nanoviridae, partly resolve the cost of genomic integrity maintenance through two remarkable capacities. They are able to systemically infect a host even when their genomic segments are not together in the same host cell, and these segments can be separately transmitted by insect vectors from host to host. These capacities potentially allow such viruses to reassort at a much larger spatial scale, since reassortants could arise from parental genotypes that do not co-infect the same cell or even the same host. To assess the limitations affecting reassortment and their implications in genome integrity maintenance, the objective of this review is to identify putative molecular constraints influencing reassorted segments throughout the infection cycle and to confront expectations based on these constraints with empirical observations. Trans-replication of the reassorted segments emerges as the major constraint, while encapsidation, viral movement, and transmission compatibilities appear more permissive. Confronting the available molecular data and the resulting predictions on reassortments to field population surveys reveals notable discrepancies, particularly a surprising rarity of interspecific natural reassortments within the Nanoviridae family. These apparent discrepancies unveil important knowledge gaps in the biology of ssDNA multipartite viruses and call for further investigation on the role of reassortment in their biology.

Viral Diversity in Benthic Abyssal Ecosystems: Ecological and Methodological Considerations.

Viruses are the most abundant 'biological entities' in the world's oceans. However, technical and methodological constraints limit our understanding of their diversity, particularly in benthic abyssal ecosystems (>4000 m depth). To verify advantages and limitations of analyzing virome DNA subjected either to random amplification or unamplified, we applied shotgun sequencing-by-synthesis to two sample pairs obtained from benthic abyssal sites located in the North-eastern Atlantic Ocean at ca. 4700 m depth. One amplified DNA sample was also subjected to single-molecule long-read sequencing for comparative purposes. Overall, we identified 24,828 viral Operational Taxonomic Units (vOTUs), belonging to 22 viral families. Viral reads were more abundant in the amplified DNA samples (38.5-49.9%) compared to the unamplified ones (4.4-5.8%), with the latter showing a greater viral diversity and 11-16% of dsDNA viruses almost undetectable in the amplified samples. From a procedural point of view, the viromes obtained by direct sequencing (without amplification step) provided a broader overview of both ss and dsDNA viral diversity. Nevertheless, our results suggest that the contextual use of random amplification of the same sample and long-read technology can improve the assessment of viral assemblages by reducing off-target reads.

Gene Overlapping as a Modulator of Begomovirus Evolution.

In RNA viruses, which have high mutation-and fast evolutionary- rates, gene overlapping (i.e., genomic regions that encode more than one protein) is a major factor controlling mutational load and therefore the virus evolvability. Although DNA viruses use host high-fidelity polymerases for their replication, and therefore should have lower mutation rates, it has been shown that some of them have evolutionary rates comparable to those of RNA viruses. Notably, these viruses have large proportions of their genes with at least one overlapping instance. Hence, gene overlapping could be a modulator of virus evolution beyond the RNA world. To test this hypothesis, we use the genus Begomovirus of plant viruses as a model. Through comparative genomic approaches, we show that terminal gene overlapping decreases the rate of virus evolution, which is associated with lower frequency of both synonymous and nonsynonymous mutations. In contrast, terminal overlapping has little effect on the pace of virus evolution. Overall, our analyses support a role for gene overlapping in the evolution of begomoviruses and provide novel information on the factors that shape their genetic diversity.

Discovery of two novel Torque Teno viruses in Callithrix penicillata provides insights on Anelloviridae diversification dynamics.

The development of high-throughput sequencing (HTS) technologies and metagenomics protocols deeply impacted the discovery of viral diversity. Moreover, the characterization of novel viruses in the Neotropical primates (NP) is central for the comprehension of viral evolution dynamics in those hosts, due to their evolutionary proximity to Old World primates, including humans. In the present work, novel anelloviruses were detected and characterized through HTS protocols in the NP Callithrix penicillata, the common black-tufted marmoset. De novo assembly of generated sequences was carried out, and a total of 15 contigs were identified with complete Anelloviridae ORF1 gene, two of them including a flanking GC-rich region, confirming the presence of two whole novel genomes of ~3 kb. The identified viruses were monophyletic within the Epsilontorquevirus genus, a lineage harboring previously reported anelloviruses infecting hosts from the Cebidae family. The genetic divergence found in the new viruses characterized two novel species, named Epsilontorquevirus callithrichensis I and II. The phylogenetic pattern inferred for the Epsilontorquevirus genus was consistent with the topology of their host species tree, echoing a virus-host diversification model observed in other viral groups. This study expands the host span of Anelloviridae and provides insights into their diversification dynamics, highlighting the importance of sampling animal viral genomes to obtain a clearer depiction of their long-term evolutionary processes.

I Am Here: It Took a Global Village.

The saying "It takes a village to raise a child" has never been truer than in my case. This autobiographical article documents my growing up and working on three different continents and my influencers along the way. Born in a village in Nigeria, West Africa, I spent the first 12 years of life with my grandmother living in a mud house and attending a village primary school. I walked barefoot to school every day, learned to read, and wrote on a chalk slate. At the age of 13, I moved to my second "village," London, England. In secondary school my love of science began to blossom. I attained a double major in chemistry and human biology from the University of Hertfordshire and a PhD in biophysics from the University of London, with a research project aimed at designing anticancer agents. I was mentored by Terence Jenkins and Stephen Neidle. For my postdoctoral training, I crossed the ocean again, to the United States, my third "village." In Michael Rossmann's group at Purdue University, my love for viruses was ignited. My independent career in structural virology began at Warwick University, England, working on pathogenic single-stranded DNA packaging viruses. In 2020, I am a full professor at the University of Florida. Most of my research is focused on the adeno-associated viruses, gene delivery vectors. My list of mentors has grown and includes Nick Muzyczka. Here, the mentee has become the mentor, and along the way, we attained a number of firsts in the field of structural virology and contributed to the field at the national and international stages.

Epidemiology and evolutionary analysis of Torque teno sus virus.

Single stranded (ss) DNA viruses are increasingly being discovered due to the ongoing development of modern technologies in exploring the virosphere. Characterized by high rates of recombination and nucleotide substitutions, it could be comparable to RNA virus ones. Torque teno sus virus (TTSuV) is a standard ssDNA virus with a high population diversity, whose evolution is still obscure, further, it is frequently found in co-infections with other viruses threatening the porcine industry and therefore share the same host and epidemiological context. Here, we implement and describe approach to integrate viral nucleotide sequence analysis, surveillance data, and a structural approach to examine the evolution of TTSuVs, we collected samples from pigs displaying respiratory signs in China and revealed a high prevalence of TTSuV1 and TTSuVk2, frequently as part of co-infections with porcine circoviruses (PCVs), especially in spleen and lung. In addition, thirty six strains sequenced were obtained to investigate their genetic diversity in China. The evolutionary history of TTSuVs were unveiled as following: At the nucleotide sequence level, TTSuVs ORF1 was confirmed to be a robust phylogenetic maker to study evolution comparably to full genomes. Additionally, extensive recombination discovered within TTSuVk2a (also 5 out of the 36 sequenced strains in this study revealed to be recombination). Then, pairwise distance, phylogenetic trees, and amino acid analysis confirmed TTSuVs species, and allowed to define circulating genotypes (TTSuV1a-1, 1a-2, 1b-1, 1b-2, 1b-3, and k2a-1, k2a-2, k2b). Selection analysis uncovered seven and six positive selected sites in TTSuV1 and TTSuVk2, respectively. At the protein structure level, mapping of sites onto the three-dimensional structure revealed that several positive selected sites locate into potential epitopes, which might related to the potential escaping from host immune response. Our result could assist future studies on swine ssDNA virus classification, surveillance and control.

Towards optimized viral metagenomes for double-stranded and single-stranded DNA viruses from challenging soils.

Soils impact global carbon cycling and their resident microbes are critical to their biogeochemical processing and ecosystem outputs. Based on studies in marine systems, viruses infecting soil microbes likely modulate host activities via mortality, horizontal gene transfer, and metabolic control. However, their roles remain largely unexplored due to technical challenges with separating, isolating, and extracting DNA from viruses in soils. Some of these challenges have been overcome by using whole genome amplification methods and while these have allowed insights into the identities of soil viruses and their genomes, their inherit biases have prevented meaningful ecological interpretations. Here we experimentally optimized steps for generating quantitatively-amplified viral metagenomes to better capture both ssDNA and dsDNA viruses across three distinct soil habitats along a permafrost thaw gradient. First, we assessed differing DNA extraction methods (PowerSoil, Wizard mini columns, and cetyl trimethylammonium bromide) for quantity and quality of viral DNA. This established PowerSoil as best for yield and quality of DNA from our samples, though ∼1/3 of the viral populations captured by each extraction kit were unique, suggesting appreciable differential biases among DNA extraction kits. Second, we evaluated the impact of purifying viral particles after resuspension (by cesium chloride gradients; CsCl) and of viral lysis method (heat vs bead-beating) on the resultant viromes. DNA yields after CsCl particle-purification were largely non-detectable, while unpurified samples yielded 1-2-fold more DNA after lysis by heat than by bead-beating. Virome quality was assessed by the number and size of metagenome-assembled viral contigs, which showed no increase after CsCl-purification, but did from heat lysis relative to bead-beating. We also evaluated sample preparation protocols for ssDNA virus recovery. In both CsCl-purified and non-purified samples, ssDNA viruses were successfully recovered by using the Accel-NGS 1S Plus Library Kit. While ssDNA viruses were identified in all three soil types, none were identified in the samples that used bead-beating, suggesting this lysis method may impact recovery. Further, 13 ssDNA vOTUs were identified compared to 582 dsDNA vOTUs, and the ssDNA vOTUs only accounted for ∼4% of the assembled reads, implying dsDNA viruses were dominant in these samples. This optimized approach was combined with the previously published viral resuspension protocol into a sample-to-virome protocol for soils now available at protocols.io, where community feedback creates 'living' protocols. This collective approach will be particularly valuable given the high physicochemical variability of soils, which will may require considerable soil type-specific optimization. This optimized protocol provides a starting place for developing quantitatively-amplified viromic datasets and will help enable viral ecogenomic studies on organic-rich soils.

Targeting Plant ssDNA Viruses with Engineered Miniature CRISPR-Cas14a.

CRISPR-Cas14a is a highly compact protein with great potential to be used as a guided genome editing tool for single-stranded (ss) DNA cleavage. Recently isolated from noncultured archaea, its unrestricted and sequence-independent cleavage makes it an ideal tool for engineering resistance against economically important plant ssDNA viruses.

Novel Parvoviruses from Wild and Domestic Animals in Brazil Provide New Insights into Parvovirus Distribution and Diversity.

Parvoviruses (family Parvoviridae) are small, single-stranded DNA viruses. Many parvoviral pathogens of medical, veterinary and ecological importance have been identified. In this study, we used high-throughput sequencing (HTS) to investigate the diversity of parvoviruses infecting wild and domestic animals in Brazil. We identified 21 parvovirus sequences (including twelve nearly complete genomes and nine partial genomes) in samples derived from rodents, bats, opossums, birds and cattle in Pernambuco, São Paulo, Paraná and Rio Grande do Sul states. These sequences were investigated using phylogenetic and distance-based approaches and were thereby classified into eight parvovirus species (six of which have not been described previously), representing six distinct genera in the subfamily Parvovirinae. Our findings extend the known biogeographic range of previously characterized parvovirus species and the known host range of three parvovirus genera (Dependovirus, Aveparvovirus and Tetraparvovirus). Moreover, our investigation provides a window into the ecological dynamics of parvovirus infections in vertebrates, revealing that many parvovirus genera contain well-defined sub-lineages that circulate widely throughout the world within particular taxonomic groups of hosts.

Viral Communities of Shark Bay Modern Stromatolites.

Single stranded DNA viruses have been previously shown to populate the oceans on a global scale, and are endemic in microbialites of both marine and freshwater systems. We undertook for the first time direct viral metagenomic shotgun sequencing to explore the diversity of viruses in the modern stromatolites of Shark Bay Australia. The data indicate that Shark Bay marine stromatolites have similar diversity of ssDNA viruses to that of Highbourne Cay, Bahamas. ssDNA viruses in cluster uniquely in Shark Bay and Highbourne Cay, potentially due to enrichment by phi29-mediated amplification bias. Further, pyrosequencing data was assembled from the Shark Bay systems into two putative viral genomes that are related to Genomoviridae family of ssDNA viruses. In addition, the cellular fraction was shown to be enriched for antiviral defense genes including CRISPR-Cas, BREX (bacteriophage exclusion), and DISARM (defense island system associated with restriction-modification), a potentially novel finding for these systems. This is the first evidence for viruses in the Shark Bay stromatolites, and these viruses may play key roles in modulating microbial diversity as well as potentially impacting ecosystem function through infection and the recycling of key nutrients.

Identification and full-genome characterization of novel circoviruses in masked palm civets (Paguma larvata).

The family Circoviridae comprises a large group of small, circular, single-stranded DNA viruses and is classified into two genera: Circovirus and Cyclovirus. They have marked genetic diversity and a broad host range. In this study, three novel circovirus genomes were identified from wild-caught masked palm civets (Paguma larvata) in Japan and classified as a new species within the genus Circovirus based on the demarcation criteria of the International Committee on the Taxonomy of Viruses. Of note, the presence of two predicted introns at the 5'-terminus of the rep gene was suggested in the Paguma larvata circovirus genomes.

Identification and whole genome characterization of novel anelloviruses in masked palm civets (Paguma larvata): Segregation into four distinct clades.

The members of the family Anelloviridae are small and single-stranded DNA viruses with marked diversity in sequence and length, which ubiquitously infect many vertebrates, including mammals, birds and reptiles. The anelloviruses isolated from mammals are currently classified into 11 assigned and four proposed genera; some anelloviruses remain unassigned. The present study was conducted to identify anelloviruses in wild-caught masked palm civets (Paguma larvata) in Japan using a rolling-circle amplification method. Thirteen novel anellovirus strains were identified from 8 of 10 masked palm civets and their entire genomic sequences (2039-2535 nucleotides) were determined; they were classifiable into four distinct clades. Comparative analyses of all reported anelloviruses for which the entire or near-entire genomic sequences have been determined, including the 13 strains obtained in the present study, revealed that anelloviruses can provisionally be classified into 20 clades, which may correspond to 20 genera (including 11 assigned and four proposed genera) by a >70% amino acid sequence difference in open reading frame 1 (ORF1). This study suggested that novel anelloviruses of marked diversity are circulating in animals worldwide, and that the rolling-circle amplification method would be useful for identifying novel anelloviruses and other viruses with a circular DNA genome.

Discovery of novel anelloviruses in small mammals expands the host range and diversity of the Anelloviridae.

The Anelloviridae comprises single-stranded DNA viruses currently grouped in sixty-eight species classified in twelve genera. They have been found in many vertebrate hosts including primates. In this study, we describe the application of the high-throughput sequencing to examine the frequency and diversity of anelloviruses in rodents, bats and opossums captured in São Paulo State, Brazil. We report a total of twenty-six anelloviruses with sixteen nearly complete genomes and ten partial genomes, which include eleven potential novel species identified in rodents (Cricetidae), bats (Molossidae and Phyllostomidae), and opossums (Didelphidae). We also propose the inclusion of two potential new genera within the Anelloviridae family, provisionally named Omegatorquevirus and Sigmatorquevirus, including six and three novel species of anelloviruses, respectively. In summary, this study expands the diversity and the host range of the known anelloviruses.

Pervasive Chimerism in the Replication-Associated Proteins of Uncultured Single-Stranded DNA Viruses.

Numerous metagenomic studies have uncovered a remarkable diversity of circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses, the majority of which are uncultured and unclassified. Unlike capsid proteins, the Reps show significant similarity across different groups of CRESS DNA viruses and have conserved domain organization with the N-terminal nuclease and the C-terminal helicase domain. Consequently, Rep is widely used as a marker for identification, classification and assessment of the diversity of CRESS DNA viruses. However, it has been shown that in certain viruses the Rep nuclease and helicase domains display incongruent evolutionary histories. Here, we systematically evaluated the co-evolutionary patterns of the two Rep domains across classified and unclassified CRESS DNA viruses. Our analysis indicates that the Reps encoded by members of the families Bacilladnaviridae, Circoviridae, Geminiviridae, Genomoviridae, Nanoviridae and Smacoviridae display largely congruent evolutionary patterns in the two domains. By contrast, among the unclassified CRESS DNA viruses, 71% appear to have chimeric Reps. Such massive chimerism suggests that unclassified CRESS DNA viruses represent a dynamic population in which exchange of gene fragments encoding the nuclease and helicase domains is extremely common. Furthermore, purging of the chimeric sequences uncovered six monophyletic Rep groups that may represent new families of CRESS DNA viruses.

Evolutionary history of ssDNA bacilladnaviruses features horizontal acquisition of the capsid gene from ssRNA nodaviruses.

Bacilladnaviruses have single-stranded (ss) DNA genomes and infect diatoms, a major group of unicellular algae widespread in aquatic habitats. Despite their ecological importance, the provenance and relationships of bacilladnaviruses to other eukaryotic viruses remain unclear. Accordingly, they are currently classified into the 'floating' genus Bacilladnavirus. Here we present three new bacilladnavirus genomes recovered from a mollusc Amphibola crenata and benthic sediments from the Avon-Heathcote estuary in New Zealand. Our analysis shows that the rolling-circle replication-initiation proteins of bacilladnaviruses display unique conserved motifs and in phylogenetic trees form a monophyletic clade separated from other groups of ssDNA viruses. Unexpectedly, distant homology detection combined with structural modeling indicates that bacilladnavirus capsid proteins are homologous to those of ssRNA viruses from the Nodaviridae family. Considering the sequence diversity within the expanding Bacilladnavirus genus, we argue that classification of these viruses has to be revised and the current genus upgraded to the family level.

Sequence-based taxonomic framework for the classification of uncultured single-stranded DNA viruses of the family Genomoviridae.

With the advent of metagenomics approaches, a large diversity of known and unknown viruses has been identified in various types of environmental, plant, and animal samples. One such widespread virus group is the recently established family Genomoviridae which includes viruses with small (∼2-2.4 kb), circular ssDNA genomes encoding rolling-circle replication initiation proteins (Rep) and unique capsid proteins. Here, we propose a sequence-based taxonomic framework for classification of 121 new virus genomes within this family. Genomoviruses display ∼47% sequence diversity, which is very similar to that within the well-established and extensively studied family Geminiviridae (46% diversity). Based on our analysis, we establish a 78% genome-wide pairwise identity as a species demarcation threshold. Furthermore, using a Rep sequence phylogeny-based analysis coupled with the current knowledge on the classification of geminiviruses, we establish nine genera within the Genomoviridae family. These are Gemycircularvirus (n = 73), Gemyduguivirus (n = 1), Gemygorvirus (n = 9), Gemykibivirus (n = 29), Gemykolovirus (n = 3), Gemykrogvirus (n = 3), Gemykroznavirus (n = 1), Gemytondvirus (n = 1), Gemyvongvirus (n = 1). The presented taxonomic framework offers rational classification of genomoviruses based on the sequence information alone and sets an example for future classification of other groups of uncultured viruses discovered using metagenomics approaches.

Towards quantitative viromics for both double-stranded and single-stranded DNA viruses.

BACKGROUND: Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation). METHODS: Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses. RESULTS: Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample. DISCUSSION: Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.