Diversity and distribution of a prevalent Microviridae group across the global oceans.

Small single-stranded DNA phages of the Microviridae family are diverse and prevalent in oceans. Our understanding of Microviridae phages that infect the ecologically important marine Roseobacter is currently limited, comprising few isolates. Here, we report six roseophages that infect Roseobacter RCA strains. Genomic and phylogenetic analyses revealed that they were new members of the previously identified subfamily Occultatumvirinae. Additionally, 232 marine uncultivated virus genomes (UViGs) affiliated to Occultatumvirinae were obtained from environmental genome datasets. Phylogenomic analysis revealed that marine Occultatumvirinae phages could be further grouped into 11 subgroups. Moreover, meta-omics based read-mapping analysis showed that Occultatumvirinae phages were globally distributed, with two low G + C subgroups showing the most prevalent distribution. Furthermore, one phage in subgroup 2 was found to be extremely ubiquitous. Overall, this study expands our understanding of the diversity and ecology of the Occultatumvirinae microviruses in the ocean and highlights their ecological impacts.

The Structure of Spiroplasma Virus 4: Exploring the Capsid Diversity of the Microviridae.

Spiroplasma virus 4 (SpV4) is a bacteriophage of the Microviridae, which packages circular ssDNA within non-enveloped T = 1 icosahedral capsids. It infects spiroplasmas, which are known pathogens of honeybees. Here, the structure of the SpV4 virion is determined using cryo-electron microscopy to a resolution of 2.5 Å. A striking feature of the SpV4 capsid is the mushroom-like protrusions at the 3-fold axes, which is common among all members of the subfamily Gokushovirinae. While the function of the protrusion is currently unknown, this feature varies widely in this subfamily and is therefore possibly an adaptation for host recognition. Furthermore, on the interior of the SpV4 capsid, the location of DNA-binding protein VP8 was identified and shown to have low structural conservation to the capsids of other viruses in the family. The structural characterization of SpV4 will aid future studies analyzing the virus-host interaction, to understand disease mechanisms at a molecular level. Furthermore, the structural comparisons in this study, including a low-resolution structure of the chlamydia phage 2, provide an overview of the structural repertoire of the viruses in this family that infect various bacterial hosts, which in turn infect a wide range of animals and plants.

Microviruses: A World Beyond phiX174.

Two decades of metagenomic analyses have revealed that in many environments, small (∼5 kb), single-stranded DNA phages of the family Microviridae dominate the virome. Although the emblematic microvirus phiX174 is ubiquitous in the laboratory, most other microviruses, particularly those of the gokushovirus and amoyvirus lineages, have proven to be much more elusive. This puzzling lack of representative isolates has hindered insights into microviral biology. Furthermore, the idiosyncratic size and nature of their genomes have resulted in considerable misjudgments of their actual abundance in nature. Fortunately, recent successes in microvirus isolation and improved metagenomic methodologies can now provide us with more accurate appraisals of their abundance, their hosts, and their interactions. The emerging picture is that phiX174 and its relatives are rather rare and atypical microviruses, and that a tremendous diversity of other microviruses is ready for exploration.

Biological Features and In Planta Transcriptomic Analyses of a Microviridae Phage (CLasMV1) in "Candidatus Liberibacter asiaticus".

"Candidatus Liberibacter asiaticus" (CLas) is the causal agent of citrus Huanglongbing (HLB, also called citrus greening disease), a highly destructive disease threatening citrus production worldwide. A novel Microviridae phage (named CLasMV1) has been found to infect CLas, providing a potential therapeutic strategy for CLas/HLB control. However, little is known about the CLasMV1 biology. In this study, we analyzed the population dynamics of CLasMV1 between the insect vector of CLas, the Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) and the holoparasitic dodder plant (Cuscuta campestris Yunck.); both acquired CLasMV1-infected CLas from an HLB citrus. All CLas-positive dodder samples were CLasMV1-positive, whereas only 32% of CLas-positive ACP samples were identified as CLasMV1-positive. Quantitative analyses showed a similar distribution pattern of CLasMV1 phage and CLas among eight citrus cultivars by presenting at highest abundance in the fruit pith and/or the center axis of the fruit. Transcriptome analyses revealed the possible lytic activity of CLasMV1 on CLas in fruit pith as evidenced by high-level expressions of CLasMV1 genes, and CLas genes related to cell wall biogenesis and remodeling to maintain the CLas cell envelope integrity. The up-regulation of CLas genes were involved in restriction-modification system that could involve possible phage resistance for CLas during CLasMV1 infection. In addition, the regulation of CLas genes involved in cell surface components and Sec pathway by CLasMV1 phage could be beneficial for phage infection. This study expanded our knowledge of CLasMV1 phage that will benefit further CLas phage research and HLB control.

Characterization and Genomic Analysis of ssDNA Vibriophage vB_VpaM_PG19 within Microviridae, Representing a Novel Viral Genus.

Vibrio parahaemolyticus, a widespread marine bacterium, is responsible for a variety of diseases in marine organisms. Consumption of raw or undercooked seafood contaminated with V. parahaemolyticus is also known to cause acute gastroenteritis in humans. While numerous dsDNA vibriophages have been isolated so far, there have been few studies of vibriophages belonging to the ssDNA Microviridae family. In this study, a novel ssDNA phage, vB_VpaM_PG19 infecting V. parahaemolyticus, with a 5,572 bp ssDNA genome with a G+C content of 41.31% and encoded eight open reading frames, was isolated. Genome-wide phylogenetic analysis of the total phage isolates in the GenBank database revealed that vB_VpaM_PG19 was only related to the recently deposited vibriophage vB_VpP_WS1. The genome-wide average nucleotide homology of the two phages was 89.67%. The phylogenetic tree and network analysis showed that vB_VpaM_PG19 was different from other members of the Microviridae family and might represent a novel viral genus, together with vibriophage vB_VpP_WS1, named Vimicrovirus. One-step growth curves showed that vB_VpaM_PG19 has a short incubation period, suggesting its potential as an antimicrobial agent for pathogenic V. parahaemolyticus. IMPORTANCE Vibriophage vB_VpaM_PG19 was distant from other isolated microviruses in the phylogenetic tree and network analysis and represents a novel microviral genus, named Vimicrovirus. Our report describes the genomic and phylogenetic features of vB_VpaM_PG19 and provides a potential antimicrobial candidate for pathogenic V. parahaemolyticus.

Isolation and characterization of SGF3, a novel Microviridae phage infecting Shigella flexneri.

In the context of widespread bacterial contamination and the endless emergence of antibiotic-resistant bacteria, more effective ways to control pathogen infection are urgently needed. Phages become potential bactericidal agents due to their bactericidal specificity and not easy resistance to bacteria. But an important factor limiting its development is the lack of phage species. Therefore, the isolation of more new phages and studying their biological and genomic characteristics is of great significance for subsequent applications. So, in this study, SGF3, a Microviridae phage, which has shown lytic activity against Shigella flexneri, was isolated, purified, and characterized. Morphological and phylogenetic analyses identified it as a phiX174 species belonging to the Microviridae family. The latent period of phage SGF3 was 20 min, with an average burst size of approximately 7.1. Host spectrum experiments indicated its strong host specificity. Furthermore, the biofilm removal efficiency was increased by 20%-25% when SGF3 was coupled with other phages. In conclusion, the phage SGF3 found in this study was a lytic phage belonging to the Microviral family, and could be added as an auxiliary material in the phage cocktail. Studies of its characteristics and bactericidal properties had enriched the germplasm resources of microphages, provided more potential material in fighting against emerging and existing multidrug-resistant bacteria.

Organizing the Global Diversity of Microviruses.

Microviruses encompass an astonishing array of small, single-stranded DNA phages that, due to the surge in metagenomic surveys, are now known to be prevalent in most environments. Current taxonomy concedes the considerable diversity within this lineage to a single family (the Microviridae), which has rendered it difficult to adequately and accurately assess the amount of variation that actually exists within this group. We amassed and curated the largest collection of microviral genomes to date and, through a combination of protein-sharing networks and phylogenetic analysis, discovered at least three meaningful taxonomic levels between the current ranks of family and genus. When considering more than 13,000 microviral genomes from recognized lineages and as-yet-unclassified microviruses in metagenomic samples, microviral diversity is better understood by elevating microviruses to the level of an order that consists of three suborders and at least 19 putative families, each with their respective subfamilies. These revisions enable fine-scale assessment of microviral dynamics: for example, in the human gut, there are considerable differences in the abundances of microviral families both between urban and rural populations and in individuals over time. In addition, our analysis of genome contents and gene exchange shows that microviral families carry no recognizable accessory metabolic genes and rarely, if ever, engage in horizontal gene transfer across microviral families or with their bacterial hosts. These insights bring microviral taxonomy in line with current developments in the taxonomy of other phages and increase the understanding of microvirus biology. IMPORTANCE Microviruses are the most abundant single-stranded DNA phages on the planet and an important component of the human gut virome. And yet, productive research into their biology is hampered by the inadequacies of current taxonomic ordering: microviruses are lumped into a single family and treated as a monolithic group, thereby obscuring the extent of their diversity and resulting in little comparative research. Our investigations into the diversity of microviruses define numerous groups, most lacking any isolated representatives, and point toward high-value targets for future research. To expedite microvirus discovery and comparison, we developed a pipeline that enables the fast and facile sorting of novel microvirus genomes into well-defined taxonomic groups. These improvements provide new insights into the biology of microviruses and emphasize fundamental differences between these miniature phages and their large, double-stranded DNA phage competitors.

Complete genome analysis of the newly isolated Vibrio phage vB_VpP_WS1 of the family Microviridae.

Here, we describe the characterization and genome annotation of the newly isolated lytic Vibrio parahaemolyticus phage vB_VpP_WS1, isolated from sewage samples collected in Qingdao, China. Transmission electron microscopy revealed that vB_VpP_WS1 is about 22 nm in size and that the virions are isometric, likely icosahedral, particles similar to those of members of the Microviridae. The digestion patterns of phage nucleic acids and whole-genome sequencing analysis together revealed that phage vB_VpP_WS1 has a single-stranded DNA genome of 5564 nt. Eight open reading frames were identified, only four of which could be annotated. The proteins of vB_VpP_WS1 displayed low sequence similarity to their homologs encoded by other microviruses. Phylogenetic analysis based on the major capsid protein suggested that vB_VpP_WS1 is a tentative new member of the family Microviridae.

Adaptation of the polony technique to quantify Gokushovirinae, a diverse group of single-stranded DNA phage.

Advances in metagenomics have revealed the ubiquity of single-stranded DNA (ssDNA) phage belonging to the subfamily Gokushovirinae in the oceans; however, the abundance and ecological roles of this group are unknown. Here, we quantify gokushoviruses through adaptation of the polony method, in which viral template DNA is immobilized in a gel, amplified by PCR, and subsequently detected by hybridization. Primers and probes for this assay were designed based on PCR amplicon diversity of gokushovirus major capsid protein gene sequences from a depth profile in the Gulf of Aqaba, Red Sea sampled in September 2015. At ≥95% identity, these 87 gokushovirus sequences formed 14 discrete clusters with the largest clades showing distinct depth distributions. The application of the polony method enabled the first quantification of gokushoviruses in any environment. The gokushoviruses were most abundant in the upper 40 m of the stratified water column, with a subsurface peak in abundance of 1.26 × 105 viruses ml-1 . These findings suggest that discrete gokushovirus genotypes infect bacterial hosts that differentially partition in the water column. Since the designed primers and probe are conserved across marine ecosystems, this polony method can be applied broadly for the quantification of gokushoviruses throughout the global oceans.

Fishing for the Virome of Tropical Tuna.

While planktonic viruses have received much attention in recent decades, knowledge of the virome of marine organisms, especially fish, still remains rudimentary. This is notably the case with tuna, which are among the most consumed fish worldwide and represent considerable economic, social and nutritional value. Yet the composition of the tuna virome and its biological and environmental determinants remain unknown. To begin to address this gap, we investigated the taxonomic diversity of viral communities inhabiting the skin mucus, gut and liver of two major tropical tuna species (skipjack and yellowfin) in individuals fished in the Atlantic and Indian Oceans. While we found significant differences in the virome composition between the organs, this was totally independent of the tuna species or sex. The tuna virome was mainly dominated by eukaryotic viruses in the digestive organs (gut and liver), while bacteriophages were predominant in the mucus. We observed the presence of specific viral families in each organ, some previously identified as fish or human pathogens (e.g., Iridoviridae, Parvoviridae, Alloherpesviridae, Papillomaviridae). Interestingly, we also detected a 'core virome' that was shared by all the organs and was mainly composed of Caudovirales, Microviridae and Circoviridae. These results show that tuna host a mosaic of viral niches, whose establishment, role and circulation remain to be elucidated.

Legionella pneumophila CRISPR-Cas Suggests Recurrent Encounters with One or More Phages in the Family Microviridae.

Legionella pneumophila is a ubiquitous freshwater pathogen and the causative agent of Legionnaires' disease. L. pneumophila growth within protists provides a refuge from desiccation, disinfection, and other remediation strategies. One outstanding question has been whether this protection extends to phages. L. pneumophila isolates are remarkably devoid of prophages and to date no Legionella phages have been identified. Nevertheless, many L. pneumophila isolates maintain active CRISPR-Cas defenses. So far, the only known target of these systems is an episomal element that we previously named Legionella mobile element 1 (LME-1). The continued expansion of publicly available genomic data promises to further our understanding of the role of these systems. We now describe over 150 CRISPR-Cas systems across 600 isolates to establish the clearest picture yet of L. pneumophila's adaptive defenses. By searching for targets of 1,500 unique CRISPR-Cas spacers, LME-1 remains the only identified CRISPR-Cas targeted integrative element. We identified 3 additional LME-1 variants-all targeted by previously and newly identified CRISPR-Cas spacers-but no other similar elements. Notably, we also identified several spacers with significant sequence similarity to microviruses, specifically those within the subfamily Gokushovirinae. These spacers are found across several different CRISPR-Cas arrays isolated from geographically diverse isolates, indicating recurrent encounters with these phages. Our analysis of the extended Legionella CRISPR-Cas spacer catalog leads to two main conclusions: current data argue against CRISPR-Cas targeted integrative elements beyond LME-1, and the heretofore unknown L. pneumophila phages are most likely lytic gokushoviruses. IMPORTANCE Legionnaires' disease is an often-fatal pneumonia caused by Legionella pneumophila, which normally grows inside amoebae and other freshwater protists. L. pneumophila trades diminished access to nutrients for the protection and isolation provided by the host. One outstanding question is whether L. pneumophila is susceptible to phages, given the protection provided by its intracellular lifestyle. In this work, we use Legionella CRISPR spacer sequences as a record of phage infection to predict that the "missing" L. pneumophila phages belong to the microvirus subfamily Gokushovirinae. Gokushoviruses are known to infect another intracellular pathogen, Chlamydia. How do gokushoviruses access L. pneumophila (and Chlamydia) inside their "cozy niches"? Does exposure to phages happen during a transient extracellular period (during cell-to-cell spread) or is it indicative of a more complicated environmental lifestyle? One thing is clear, 100 years after their discovery, phages continue to hold important secrets about the bacteria upon which they prey.

Interaction of bacterial metagenome and virome in patients with cirrhosis and hepatic encephalopathy.

OBJECTIVE: Altered bacterial composition is associated with disease progression in cirrhosis but the role of virome, especially phages, is unclear. DESIGN: Cross-sectional and pre/post rifaximin cohorts were enrolled. Cross-sectional: controls and cirrhotic outpatients (compensated, on lactulose (Cirr-L), on rifaximin (Cirr-LR)) were included and followed for 90-day hospitalisations. Pre/post: compensated cirrhotics underwent stool collection pre/post 8 weeks of rifaximin. Stool metagenomics for bacteria and phages and their correlation networks were analysed in controls versus cirrhosis, within cirrhotics, hospitalised/not and pre/post rifaximin. RESULTS: Cross-sectional: 40 controls and 163 cirrhotics (63 compensated, 43 Cirr-L, 57 Cirr-LR) were enrolled. Cirr-L/LR groups were similar on model for end-stage liver disease (MELD) score but Cirr-L developed greater hospitalisations versus Cirr-LR (56% vs 30%, p=0.008). Bacterial alpha/beta diversity worsened from controls through Cirr-LR. While phage alpha diversity was similar, beta diversity was different between groups. Autochthonous bacteria linked negatively, pathobionts linked positively with MELD but only modest phage-MELD correlations were seen. Phage-bacterial correlation network complexity was highest in controls, lowest in Cirr-L and increased in Cirr-LR. Microviridae and Faecalibacterium phages were linked with autochthonous bacteria in Cirr-LR, but not Cirr-L hospitalised patients had greater pathobionts, lower commensal bacteria and phages focused on Streptococcus, Lactococcus and Myoviridae. Pre/post: No changes in alpha/beta diversity of phages or bacteria were seen postrifaximin. Phage-bacterial linkages centred around urease-producing Streptococcus species collapsed postrifaximin. CONCLUSION: Unlike bacteria, faecal phages are sparsely linked with cirrhosis characteristics and 90-day outcomes. Phage and bacterial linkages centred on urease-producing, ammonia-generating Streptococcus species were affected by disease progression and rifaximin therapy and were altered in patients who experienced 90-day hospitalisations.

Complete genome sequence of the extreme-pH-resistant Salmonella bacteriophage αα of the family Microviridae.

A novel Salmonella bacteriophage (phage), named αα, was the first reported member of the family Microviridae to exhibit tolerance to both extreme acidic and alkaline conditions (pH 2-12 for 1 h). Phage αα has a circular single-stranded DNA genome of 5,387 nt with a G+C content of 44.66%. A total of 11 putative gene products and no tRNA genes are encoded in the phage αα genome. Whole-genome sequence comparisons revealed that phage αα shares 95% identity with coliphage phiX174 and had a close evolutionary relationship to the phages NC1 and NC7. Phylogenetic analysis of the structural proteins of phage αα and 18 other phiX174-like phages showed that a phylogenetic tree based on protein B sequences had a topology similar to that obtained using whole genome sequences. In addition, variable sites in proteins F and G distributed on the surface of the mature capsid and the conserved protein J were probably involved in maintaining the structural integrity of the phage under extreme pH conditions. Our findings could open up new perspectives for identifying more extreme-pH-resistant phages and their structural proteins and understanding the mechanism of phage adaptation and evolution under extreme environmental stress.

Resurrection of a global, metagenomically defined gokushovirus.

Gokushoviruses are single-stranded, circular DNA bacteriophages found in metagenomic datasets from diverse ecosystems worldwide, including human gut microbiomes. Despite their ubiquity and abundance, little is known about their biology or host range: Isolates are exceedingly rare, known only from three obligate intracellular bacterial genera. By synthesizing circularized phage genomes from prophages embedded in diverse enteric bacteria, we produced gokushoviruses in an experimentally tractable model system, allowing us to investigate their features and biology. We demonstrate that virions can reliably infect and lysogenize hosts by hijacking a conserved chromosome-dimer resolution system. Sequence motifs required for lysogeny are detectable in other metagenomically defined gokushoviruses; however, we show that even partial motifs enable phages to persist cytoplasmically without leading to collapse of their host culture. This ability to employ multiple, disparate survival strategies is likely key to the long-term persistence and global distribution of Gokushovirinae.

Virus Discovery in Desert Tortoise Fecal Samples: Novel Circular Single-Stranded DNA Viruses.

The Sonoran Desert tortoise Gopherus morafkai is adapted to the desert, and plays an important ecological role in this environment. There is limited information on the viral diversity associated with tortoises (family Testudinidae), and to date no DNA virus has been identified associated with these animals. This study aimed to assess the diversity of DNA viruses associated with the Sonoran Desert tortoise by sampling their fecal matter. A viral metagenomics approach was used to identify the DNA viruses in fecal samples from wild Sonoran Desert tortoises in Arizona, USA. In total, 156 novel single-stranded DNA viruses were identified from 40 fecal samples. Those belonged to two known viral families, the Genomoviridae (n = 27) and Microviridae (n = 119). In addition, 10 genomes were recovered that belong to the unclassified group of circular-replication associated protein encoding single-stranded (CRESS) DNA virus and five circular molecules encoding viral-like proteins.

Single-Stranded DNA Viruses in Antarctic Cryoconite Holes.

Antarctic cryoconite holes, or small melt-holes in the surfaces of glaciers, create habitable oases for isolated microbial communities with tightly linked microbial population structures. Viruses may influence the dynamics of polar microbial communities, but the viromes of the Antarctic cryoconite holes have yet to be characterized. We characterize single-stranded DNA (ssDNA) viruses from three cryoconite holes in the Taylor Valley, Antarctica, using metagenomics. Half of the assembled metagenomes cluster with those in the viral family Microviridae (n = 7), and the rest with unclassified circular replication associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses (n = 7). An additional 18 virus-like circular molecules encoding either a Rep, a capsid protein gene, or other unidentified but viral-like open reading frames were identified. The samples from which the genomes were identified show a strong gradient in microbial diversity and abundances, and the number of viral genomes detected in each sample mirror that gradient. Additionally, one of the CRESS genomes assembled here shares ~90% genome-wide pairwise identity with a virus identified from a freshwater pond on the McMurdo Ice Shelf (Antarctica). Otherwise, the similarity of these viruses to those previously identified is relatively low. Together, these patterns are consistent with the presence of a unique regional virome present in fresh water host populations of the McMurdo Dry Valley region.

The Human Gut Virome Is Highly Diverse, Stable, and Individual Specific.

The human gut contains a vast array of viruses, mostly bacteriophages. The majority remain uncharacterized, and their roles in shaping the gut microbiome and in impacting on human health remain poorly understood. We performed longitudinal metagenomic analysis of fecal viruses in healthy adults that reveal high temporal stability, individual specificity, and correlation with the bacterial microbiome. Using a database-independent approach that uses most of the sequencing data, we uncovered the existence of a stable, numerically predominant individual-specific persistent personal virome. Clustering of viral genomes and de novo taxonomic annotation identified several groups of crAss-like and Microviridae bacteriophages as the most stable colonizers of the human gut. CRISPR-based host prediction highlighted connections between these stable viral communities and highly predominant gut bacterial taxa such as Bacteroides, Prevotella, and Faecalibacterium. This study provides insights into the structure of the human gut virome and serves as an important baseline for hypothesis-driven research.

Unravelling the Single-Stranded DNA Virome of the New Zealand Blackfly.

Over the last decade, arthropods have been shown to harbour a rich diversity of viruses. Through viral metagenomics a large diversity of single-stranded (ss) DNA viruses have been identified. Here we examine the ssDNA virome of the hematophagous New Zealand blackfly using viral metagenomics. Our investigation reveals a plethora of novel ssDNA viral genomes, some of which cluster in the viral families Genomoviridae (n = 9), Circoviridae (n = 1), and Microviridae (n = 108), others in putative families that, at present, remain unclassified (n = 20) and one DNA molecule that only encodes a replication associated protein. Among these novel viruses, two putative multi-component virus genomes were recovered, and these are most closely related to a Tongan flying fox faeces-associated multi-component virus. Given that the only other known multi-component circular replication-associated (Rep) protein encoding single-stranded (CRESS) DNA viruses infecting plants are in the families Geminiviridae (members of the genus Begomovirus) and Nanoviridae, it appears these are likely a new multi-component virus group which may be associated with animals. This study reiterates the diversity of ssDNA viruses in nature and in particular with the New Zealand blackflies.

Diverse single-stranded DNA viruses associated with honey bees (Apis mellifera).

Honey bees (Apis mellifera) research has increased in light of their progressive global decline over the last decade and the important role they play in pollination. One expanding area of honey bee research is analysis of their microbial community including viruses. Several RNA viruses have been characterized but little is known about DNA viruses associated with bees. Here, using a metagenomics based approach, we reveal the presence of a broad range of novel single-stranded DNA viruses from the hemolymph and brain of nurse and forager (worker divisions of labour) bees belonging to two honey bees subspecies, Italian (Apis mellifera linguistica) and New World Carniolan (Apis mellifera carnica). Genomes of 100 diverse viruses were identified, designated into three groupings; genomoviruses (family Genomoviridae) (n = 4), unclassified replication associated protein encoding single-stranded DNA viruses (n = 28), and microviruses (family Microviridae; subfamily Gokushovirinae) (n = 70). Amongst the viruses identified, it appears that nurses harbour a higher diversity of these viruses comparative to the foragers. Between subspecies, the most striking outcome was the extremely high number of diverse microviruses identified in the Italian bees comparative to the New World Carniolan, likely indicating an association to the diversity of the bacterial community associated with these subspecies.

The smallest ssDNA phage infecting a marine bacterium.

In the marine environment, only a few lytic single-stranded DNA (ssDNA) phages have been isolated and characterized, despite the fact that diverse ssDNA bacteriophages have been discovered via metagenomic studies. In this study, we isolated and characterized a new ssDNA phage, vB_RpoMi-Mini, which infects a marine bacterium Ruegeria pomeroyi DSS-3. With a genome size of 4248 bp and only four putative open reading frames (ORF), vB_RpoMi-Mini becomes the smallest ssDNA phage among the known ssDNA phage isolates and represents the DNA bacteriophage with the least number of ORFs. Genome-wide analysis reveals that bacteriophage Mini is distantly related to the known ssDNA phages and belongs to an unclassified ssDNA phage within the Microviridae family. The presence of peptidase in vB_RpoMi-Mini genome further implies that horizontal gene transfer could be an important driving force in the evolution of ssDNA phages. Bacteriophage Mini seems to have lost the spike protein commonly seen in ssDNA phages, suggesting that ssDNA phage can be more diverse than previously thought. Metagenomic analysis indicates that Mini-like phages are widely distributed in the environments. The discovery of vB_RpoMi-Mini expands our understanding of ssDNA phages in nature, and also indicates our dearth of knowledge regarding of ssDNA phages.