Disenfranchised DNA: biochemical analysis of mutant øX174 DNA-binding proteins may further elucidate the evolutionary significance of the unessential packaging protein A.

Most icosahedral DNA viruses package and condense their genomes into pre-formed, volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded (ss) DNA micro- and parvoviruses. Before packaging, ~120 copies of the øX174 DNA-binding protein J interact with double-stranded DNA. 60 J proteins enter the procapsid with the ssDNA genome, guiding it between 60 icosahedrally ordered DNA-binding pockets formed by the capsid proteins. Although J proteins are small, 28-37 residues in length, they have two domains. The basic, positively charged N-terminus guides the genome between binding pockets, whereas the C-terminus acts as an anchor to the capsid's inner surface. Three C-terminal aromatic residues, W30, Y31, and F37, interact most extensively with the coat protein. Their corresponding codons were mutated, and the resulting strains were biochemically and genetically characterized. Depending on the mutation, the substitutions produced unstable packaging complexes, unstable virions, infectious progeny, or particles packaged with smaller genomes, the latter being a novel phenomenon. The smaller genomes contained internal deletions. The juncture sequences suggest that the unessential A* (A star) protein mediates deletion formation.IMPORTANCEUnessential but strongly conserved gene products are understudied, especially when mutations do not confer discernable phenotypes or the protein's contribution to fitness is too small to reliably determine in laboratory-based assays. Consequently, their functions and evolutionary impact remain obscure. The data presented herein suggest that microvirus A* proteins, discovered over 40 years ago, may hasten the termination of non-productive packaging events. Thus, performing a salvage function by liberating the reusable components of the failed packaging complexes, such as DNA templates and replication enzymes.

Defensive hypervariable regions confer superinfection exclusion in microviruses.

Single-stranded DNA phages of the family Microviridae have fundamentally different evolutionary origins and dynamics than the more frequently studied double-stranded DNA phages. Despite their small size (around 5 kb), which imposes extreme constraints on genomic innovation, they have adapted to become prominent members of viromes in numerous ecosystems and hold a dominant position among viruses in the human gut. We show that multiple, divergent lineages in the family Microviridae have independently become capable of lysogenizing hosts and have convergently developed hypervariable regions in their DNA pilot protein, which is responsible for injecting the phage genome into the host. By creating microviruses with combinations of genomic segments from different phages and infecting Escherichia coli as a model system, we demonstrate that this hypervariable region confers the ability of temperate Microviridae to prevent DNA injection and infection by other microviruses. The DNA pilot protein is present in most microviruses, but has been recruited repeatedly into this additional role as microviruses altered their lifestyle by evolving the ability to integrate in bacterial genomes, which linked their survival to that of their hosts. Our results emphasize that competition between viruses is a considerable and often overlooked source of selective pressure, and by producing similar evolutionary outcomes in distinct lineages, it underlies the prevalence of hypervariable regions in the genomes of microviruses and perhaps beyond.

Low-Temperature Adaptation Targets Genome Packing Reactions in an Icosahedral Single-Stranded DNA Virus.

øX174, G4, and α3 represent the three sister genera of a Microviridae subfamily. α3-like genomes are considerably larger than their sister genera genomes, yet they are packaged into capsids of similar internal volumes. They also contain multiple A* genes, which are nested within the larger A gene reading frame. Although unessential under most conditions, A* proteins mediate the fidelity of packaging reactions. Larger genomes and multiple A* genes may indicate that genome packaging is more problematic for α3-like viruses, especially at lower temperatures, where DNA persistence lengths would be longer. Unlike members of the other genera, which reliably form plaques at 20°C, α3-like phages are naturally cold sensitive below 28°C. To determine whether there was a connection between the uniquely α3-like genome characteristics and the cold-sensitive phenotype, the α3 assembly pathway was characterized at low temperature. Although virions were not detected, particles consistent with off-pathway packaging complexes were observed. In a complementary evolutionary approach, α3 was experimentally evolved to grow at progressively lower temperatures. The two major responses to cold adaptation were genome reduction and elevated A* gene expression. IMPORTANCE The production of enzymes, transcription factors, and viral receptors directly influences the niches viruses can inhabit. Some prokaryotic hosts can thrive in widely differing environments; thus, physical parameters, such as temperature, should also be considered. These variables may directly alter host physiology, preventing viral replication. Alternatively, they could negatively inhibit infection processes in a host-independent manner. The members of three sister Microviridae genera (canonical species øX174, G4 and α3) infect the same host, but α3-like viruses are naturally cold sensitive, which could effectively exclude them from low-temperature environments (<28°C). Exclusion appeared to be independent of host cell physiology. Instead, it could be largely attributed to low-temperature packaging defects. The results presented here demonstrate how physical parameters, such as temperature, can directly influence viral diversification and niche determination in a host-independent manner.

Cryo-EM Structure of Gokushovirus ΦEC6098 Reveals a Novel Capsid Architecture for a Single-Scaffolding Protein, Microvirus Assembly System.

Ubiquitous and abundant in ecosystems and microbiomes, gokushoviruses constitute a Microviridae subfamily, distantly related to bacteriophages ΦX174, α3, and G4. A high-resolution cryo-EM structure of gokushovirus ΦEC6098 was determined, and the atomic model was built de novo. Although gokushoviruses lack external scaffolding and spike proteins, which extensively interact with the ΦX174 capsid protein, the core of the ΦEC6098 coat protein (VP1) displayed a similar structure. There are, however, key differences. At each ΦEC6098 icosahedral 3-fold axis, a long insertion loop formed mushroom-like protrusions, which have been noted in lower-resolution gokushovirus structures. Hydrophobic interfaces at the bottom of these protrusions may confer stability to the capsid shell. In ΦX174, the N-terminus of the capsid protein resides directly atop the 3-fold axes of symmetry; however, the ΦEC6098 N-terminus stretched across the inner surface of the capsid shell, reaching nearly to the 5-fold axis of the neighboring pentamer. Thus, this extended N-terminus interconnected pentamers on the inside of the capsid shell, presumably promoting capsid assembly, a function performed by the ΦX174 external scaffolding protein. There were also key differences between the ΦX174-like DNA-binding J proteins and its ΦEC6098 homologue VP8. As seen with the J proteins, C-terminal VP8 residues were bound into a pocket within the major capsid protein; however, its N-terminal residues were disordered, likely due to flexibility. We show that the combined location and interaction of VP8's C-terminus and a portion of VP1's N-terminus are reminiscent of those seen with the ΦX174 and α3 J proteins. IMPORTANCE There is a dramatic structural and morphogenetic divide within the Microviridae. The well-studied ΦX174-like viruses have prominent spikes at their icosahedral vertices, which are absent in gokushoviruses. Instead, gokushovirus major coat proteins form extensive mushroom-like protrusions at the 3-fold axes of symmetry. In addition, gokushoviruses lack an external scaffolding protein, the more critical of the two ΦX174 assembly proteins, but retain an internal scaffolding protein. The ΦEC6098 virion suggests that key external scaffolding functions are likely performed by coat protein domains unique to gokushoviruses. Thus, within one family, different assembly paths have been taken, demonstrating how a two-scaffolding protein system can evolve into a one-scaffolding protein system, or vice versa.

Dynamic alterations of the mice gut virome after Coxsackievirus B3 infection.

The gut microbiome plays an essential role in the human health and dysbiosis has been implicated in numerous diseases. Coxsackievirus B3 infects millions of humans yearly and yet limited research has explored dynamic alterations of the gut virome after infection. Here, we established the mouse model of Coxsackievirus B3 infection and collected fecal samples at several time points to investigate alterations of the gut virome using viral metagenomic analysis. We found that the mice virome was dominated by Caudovirales and Microviridae, and phylogenetic analyses showed that both Caudovirales and Microviridae had high diversity. The gut virome had significant variations with the increase of Caudovirales and the decrease of Microviridae after infection. We proposed that Caudovirales and Microviridae may be biomarkers for the Coxsackievirus infection process. This study provides a reference for the dynamic changes of the gut virome after human Enterovirus infection, which may help guide the rational drug use in clinical treatment and provide new ideas for preventing Enterovirus infection.

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.

Recessive Host Range Mutants and Unsusceptible Cells That Inactivate Virions without Genome Penetration: Ecological and Technical Implications.

Although microviruses do not possess a visible tail structure, one vertex rearranges after interacting with host lipopolysaccharides. Most examinations of host range, eclipse, and penetration were conducted before this "host-induced" unique vertex was discovered and before DNA sequencing became routine. Consequently, structure-function relationships dictating host range remain undefined. Biochemical and genetic analyses were conducted with two closely related microviruses, α3 and ST-1. Despite ∼90% amino acid identity, the natural host of α3 is Escherichia coli C, whereas ST-1 is a K-12-specific phage. Virions attached and eclipsed to both native and unsusceptible hosts; however, they breached only the native host's cell wall. This suggests that unsusceptible host-phage interactions promote off-pathway reactions that can inactivate viruses without penetration. This phenomenon may have broader ecological implications. To determine which structural proteins conferred host range specificity, chimeric virions were generated by individually interchanging the coat, spike, or DNA pilot proteins. Interchanging the coat protein switched host range. However, host range expansion could be conferred by single point mutations in the coat protein. The expansion phenotype was recessive: genetically mutant progeny from coinfected cells did not display the phenotype. Thus, mutant isolation required populations generated in environments with low multiplicities of infection (MOI), a phenomenon that may have impacted past host range studies in both prokaryotic and eukaryotic systems. The resulting genetic and structural data were consistent enough that host range expansion could be predicted, broadening the classical definition of antireceptors to include interfaces between protein complexes within the capsid.IMPORTANCE To expand host range, viruses must interact with unsusceptible host cell surfaces, which could be detrimental. As observed in this study, virions were inactivated without genome penetration. This may be advantageous to potential new hosts, culling the viral population from which an expanded host range mutant could emerge. When identified, altered host range mutations were recessive. Accordingly, isolation required populations generated in low-MOI environments. However, in laboratory settings, viral propagation includes high-MOI conditions. Typically, infected cultures incubate until all cells produce progeny. Thus, coinfections dominate later replication cycles, masking recessive host range expansion phenotypes. This may have impacted similar studies with other viruses. Last, structural and genetic data could be used to predict site-directed mutant phenotypes, which may broaden the classic antireceptor definition to include interfaces between capsid complexes.

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro.

During ϕX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro In previous studies, ϕX174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. IMPORTANCE: Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ϕX174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity.

Diverse Circular DNA Viral Communities in Blood, Oral, and Fecal Samples of Captive Lemurs.

Few studies have addressed viral diversity in lemurs despite their unique evolutionary history on the island of Madagascar and high risk of extinction. Further, while a large number of studies on animal viromes focus on fecal samples, understanding viral diversity across multiple sample types and seasons can reveal complex viral community structures within and across species. Groups of captive lemurs at the Duke Lemur Center (Durham, NC, USA), a conservation and research center, provide an opportunity to build foundational knowledge on lemur-associated viromes. We sampled individuals from seven lemur species, i.e., collared lemur (Eulemur collaris), crowned lemur (Eulemur coronatus), blue-eyed black lemur (Eulemur flavifrons), ring-tailed lemur (Lemur catta), Coquerel's sifaka (Propithecus coquereli), black-and-white ruffed lemur (Varecia variegata variegata), and red ruffed lemur (Varecia rubra), across two lemur families (Lemuridae, Indriidae). Fecal, blood, and saliva samples were collected from Coquerel's sifaka and black-and-white ruffed lemur individuals across two sampling seasons to diversify virome biogeography and temporal sampling. Using viral metagenomic workflows, the complete genomes of anelloviruses (n = 4), cressdnaviruses (n = 47), caudoviruses (n = 15), inoviruses (n = 34), and microviruses (n = 537) were determined from lemur blood, feces, and saliva. Many virus genomes, especially bacteriophages, identified in this study were present across multiple lemur species. Overall, the work presented here uses a viral metagenomics approach to investigate viral communities inhabiting the blood, oral cavity, and feces of healthy captive lemurs.

A new understanding of somatic coliphages belonging to the Microviridae family in urban wastewater.

Somatic coliphages (SC) and F-specific RNA coliphages (FRNAPH) have been included in regulations or guidelines by several developed countries as a way of monitoring water safety and the microbiological quality of shellfish harvesting waters. SC are highly diverse in their morphology, size and genome. The Microviridae family contains three genera of phages (Alphatrevirus, Gequatrovirus, and Sinsheimervirus), all having a capsid of similar morphology (icosahedral) and size (25-30 nm in diameter) to that of common pathogenic enteric viruses. Three PCR assays specific for each genus of Microviridae were designed to study these phages in raw and treated wastewater (WW) in order to gain knowledge about the diversity and prevalence of Microviridae among SC, as well as their inactivation and removal during WW treatments. Among the four wastewater treatment plants (WWTPs) monitored here, two WWTPs applied disinfection by UV light as tertiary treatment. First, we noticed that Microviridae represented 10 to 30 % of infectious SC in both raw and treated WW. Microviridae appeared to behave in the same way as all SC during these WW treatments. As expected, the highest inactivation, at least 4 log10, was achieved for infectious Microviridae and SC in both WWTPs using UV disinfection. PCR assays showed that the highest removal of Microviridae reached about 4 log10, but the phage removal can vary greatly between WWTPs using similar treatments. This work forms the basis for a broader evaluation of Microviridae as a viral indicator of water treatment efficiency and WW reuse.

Diverse Small Circular DNA Viruses Identified in an American Wigeon Fecal Sample.

American wigeons (Mareca americana) are waterfowls that are widely distributed throughout North America. Research of viruses associated with American wigeons has been limited to orthomyxoviruses, coronaviruses, and circoviruses. To address this poor knowledge of viruses associated with American wigeons, we undertook a pilot study to identify small circular DNA viruses in a fecal sample collected in January 2021 in the city of Tempe, Arizona (USA). We identified 64 diverse circular DNA viral genomes using a viral metagenomic workflow biased towards circular DNA viruses. Of these, 45 belong to the phylum Cressdnaviricota based on their replication-associated protein sequence, with 3 from the Genomoviridae family and the remaining 42 which currently cannot be assigned to any established virus group. It is most likely that these 45 viruses infect various organisms that are associated with their diet or environment. The remaining 19 virus genomes are part of the Microviridae family and likely associated with the gut enterobacteria of American wigeons.

IbpAB small heat shock proteins are not host factors for bacteriophage ϕX174 replication.

Bacteriophage ϕX174 is a small icosahedral virus of the Microviridae with a rapid replication cycle. Previously, we found that in ϕX174 infections of Escherichia coli, the most highly upregulated host proteins are two small heat shock proteins, IbpA and IbpB, belonging to the HSP20 family, which is a universally conserved group of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Heat shock proteins were found to protect against ϕX174 lysis, but IbpA/B have not been studied. In this work, we disrupted the ibpA and ibpB genes and measured the effects on ϕX174 replication. We found that in contrast to other E. coli heat shock proteins, they are not necessary for ϕX174 replication; moreover, their absence has no discernible effect on ϕX174 fecundity. These results suggest IbpA/B upregulation is a response to ϕX174 protein expression but does not play a role in phage replication, and they are not Microviridae host factors.

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.

Using archived and biocollection samples towards deciphering the DNA virus diversity associated with rodent species in the families cricetidae and heteromyidae.

Rodentia is the most speciose order of mammals, and they are known to harbor a wide range of viruses. Although there has been significant research on zoonotic viruses in rodents, research on the diversity of other viruses has been limited, especially for rodents in the families Cricetidae and Heteromyidae. In fecal and liver samples of nine species of rodents, we identify 346 distinct circular DNA viral genomes. Of these, a large portion are circular, single-stranded DNA viruses in the families Anelloviridae (n = 3), Circoviridae (n = 5), Genomoviridae (n = 7), Microviridae (n = 297), Naryaviridae (n = 4), Vilyaviridae (n = 15) and in the phylum Cressdnaviricota (n = 13) that cannot be assigned established families. We also identified two large bacteriophages of 36 and 50 kb that are part of the class Caudoviricetes. Some of these viruses are clearly those that infect rodents, however, most of these likely infect various organisms associated with rodents, their environment or their diet.

Diverse DNA virus genomes identified in fecal samples of Mexican free-tailed bats (Tadarida brasiliensis) captured in Chiricahua Mountains of southeast Arizona (USA).

Bats (order Chiroptera) are some of the most abundant mammals on earth and their species ecology strongly influences zoonotic potential. While substantial research has been conducted on bat-associated viruses, particularly on those that can cause disease in humans and/or livestock, globally, limited research has focused on endemic bats in the USA. The southwest region of the US is of particular interest because of its high diversity of bat species. We identified 39 single-stranded DNA virus genomes in the feces of Mexican free-tailed bats (Tadarida brasiliensis) sampled in the Rucker Canyon (Chiricahua Mountains) of southeast Arizona (USA). Twenty-eight of these belong to the virus families Circoviridae (n = 6), Genomoviridae (n = 17), and Microviridae (n = 5). Eleven viruses cluster with other unclassified cressdnaviruses. Most of the viruses identified represent new species. Further research on identification of novel bat-associated cressdnaviruses and microviruses is needed to provide greater insights regarding their co-evolution and ecology relative to bats.

Characterization of Diverse Anelloviruses, Cressdnaviruses, and Bacteriophages in the Human Oral DNA Virome from North Carolina (USA).

The diversity of viruses identified from the various niches of the human oral cavity-from saliva to dental plaques to the surface of the tongue-has accelerated in the age of metagenomics. This rapid expansion demonstrates that our understanding of oral viral diversity is incomplete, with only a few studies utilizing passive drool collection in conjunction with metagenomic sequencing methods. For this pilot study, we obtained 14 samples from healthy staff members working at the Duke Lemur Center (Durham, NC, USA) to determine the viral diversity that can be identified in passive drool samples from humans. The complete genomes of 3 anelloviruses, 9 cressdnaviruses, 4 Caudoviricetes large bacteriophages, 29 microviruses, and 19 inoviruses were identified in this study using high-throughput sequencing and viral metagenomic workflows. The results presented here expand our understanding of the vertebrate-infecting and microbe-infecting viral diversity of the human oral virome in North Carolina (USA).

Reekeekee- and roodoodooviruses, two different Microviridae clades constituted by the smallest DNA phages.

Small circular single-stranded DNA viruses of the Microviridae family are both prevalent and diverse in all ecosystems. They usually harbor a genome between 4.3 and 6.3 kb, with a microvirus recently isolated from a marine Alphaproteobacteria being the smallest known genome of a DNA phage (4.248 kb). A subfamily, Amoyvirinae, has been proposed to classify this virus and other related small Alphaproteobacteria-infecting phages. Here, we report the discovery, in meta-omics data sets from various aquatic ecosystems, of sixteen complete microvirus genomes significantly smaller (2.991-3.692 kb) than known ones. Phylogenetic analysis reveals that these sixteen genomes represent two related, yet distinct and diverse, novel groups of microviruses-amoyviruses being their closest known relatives. We propose that these small microviruses are members of two tentatively named subfamilies Reekeekeevirinae and Roodoodoovirinae. As known microvirus genomes encode many overlapping and overprinted genes that are not identified by gene prediction software, we developed a new methodology to identify all genes based on protein conservation, amino acid composition, and selection pressure estimations. Surprisingly, only four to five genes could be identified per genome, with the number of overprinted genes lower than that in phiX174. These small genomes thus tend to have both a lower number of genes and a shorter length for each gene, leaving no place for variable gene regions that could harbor overprinted genes. Even more surprisingly, these two Microviridae groups had specific and different gene content, and major differences in their conserved protein sequences, highlighting that these two related groups of small genome microviruses use very different strategies to fulfill their lifecycle with such a small number of genes. The discovery of these genomes and the detailed prediction and annotation of their genome content expand our understanding of ssDNA phages in nature and are further evidence that these viruses have explored a wide range of possibilities during their long evolution.

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.

The Human Gut Virome and Its Relationship with Nontransmissible Chronic Diseases.

The human gastrointestinal tract contains large communities of microorganisms that are in constant interaction with the host, playing an essential role in the regulation of several metabolic processes. Among the gut microbial communities, the gut bacteriome has been most widely studied in recent decades. However, in recent years, there has been increasing interest in studying the influences that other microbial groups can exert on the host. Among them, the gut virome is attracting great interest because viruses can interact with the host immune system and metabolic functions; this is also the case for phages, which interact with the bacterial microbiota. The antecedents of virome-rectification-based therapies among various diseases were also investigated. In the near future, stool metagenomic investigation should include the identification of bacteria and phages, as well as their correlation networks, to better understand gut microbiota activity in metabolic disease progression.

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.