Bacteriophages with RNA genomes are among the simplest biological entities on Earth. Since their discovery in the 1960s, they have been used as important models to understand the principal processes of life, including translation and the genetic code. While RNA phages were generally thought of as rare oddities in nature, meta-omics methods are rapidly changing this simplistic view by studying diverse biomes with unprecedented resolution. Metatranscriptomics dramatically expanded the number of known RNA phages from tens to tens of thousands, revealed their widespread abundance, and discovered several new families of potential RNA phages with largely unknown hosts, biology, and environmental impact. At the same time, (meta)genomic analyses of bacterial hosts are discovering an arsenal of defense systems bacteria employ to protect themselves from predation, whose functions in immunity against RNA phages we are only beginning to understand. Here, I review how meta-omics approaches are advancing the field of RNA phage biology with a focus on the discovery of new RNA phages and how bacteria might fight them.

One Health aims to bring together human, animal, and environmental research to achieve optimal health for all. Bacteriophages (phages) are viruses that kill bacteria and their utilisation as biocontrol agents in the environment and as therapeutics for animal and human medicine will aid in the achievement of One Health objectives. Here, we assess the diversity of phages used in One Health in the last 5 years and place them in the context of global phage diversity. Our review shows that 98% of phages applied in One Health belong to the class Caudoviricetes, compared to 85% of sequenced phages belonging to this class. Only three RNA phages from the realm Riboviria have been used in environmental biocontrol and human therapy to date. This emphasises the lack in diversity of phages used commercially and for phage therapy, which may be due to biases in the methods used to both isolate phages and select them for applications. The future of phages as biocontrol agents and therapeutics will depend on the ability to isolate genetically novel dsDNA phages, as well as in improving efforts to isolate ssDNA and RNA phages, as their potential is currently undervalued. Phages have the potential to reduce the burden of antimicrobial resistance, however, we are underutilising the vast diversity of phages present in nature. More research into phage genomics and alternative culture methods is required to fully understand the complex relationships between phages, their hosts, and other organisms in the environment to achieve optimal health for all.

Theobroma cacao plantations are of significant economic importance worldwide, primarily for chocolate production. During the harvest and processing of cocoa beans, they are subjected to fermentation either by microorganisms present in the environment (spontaneous fermentation) or the addition of starter cultures, with different strains directly contributing distinct flavor and color characteristics to the beans. In addition to fungi and bacteria, viruses are ubiquitous and can affect the quality of the fermentation process by infecting fermenting organisms, destabilizing microbial diversity, and consequently affecting fermentation quality. Therefore, in this study, we explored publicly available metatranscriptomic libraries of cocoa bean fermentation in Limon Province, Costa Rica, looking for viruses associated with fermenting microorganisms. Libraries were derived from the same sample at different time points: 7, 20, and 68 h of fermentation, corresponding to yeast- and lactic acid bacteria-driven phases. Using a comprehensive pipeline, we identified 68 viral sequences that could be assigned to 62 new viral species and 6 known viruses distributed among at least nine families, with particular abundance of elements from the Lenarviricota phylum. Interestingly, 44 of these sequences were specifically associated with ssRNA phages (Fiersviridae) and mostly fungi-infecting viral families (Botourmiaviridae, Narnaviridae, and Mitoviridae). Of note, viruses from those families show a complex evolutionary relationship, transitioning from infecting bacteria to infecting fungi. We also identified 10 and 3 viruses classified within the Totiviridae and Nodaviridae families, respectively. The quantification of the virus-derived RNAs shows a general pattern of decline, similar to the dynamic profile of some microorganism genera during the fermentation process. Unexpectedly, we identified narnavirus-related elements that showed similarity to segmented viral species. By exploring the molecular characteristics of these viral sequences and applying Hidden Markov Models, we were capable of associating these additional segments with a specific taxon. In summary, our study elucidates the complex virome associated with the microbial consortia engaged in cocoa bean fermentation that could contribute to organism/strain selection, altering metabolite production and, consequently, affecting the sensory characteristics of cocoa beans.

Non-tailed icosahedral phages belonging to families Fiersviridae (phages MS2 and Qbeta), Tectiviridae (PRD1) and Microviridae (phiX174) have not been considered in detail so far as potential antibacterial agents. The aim of the study was to examine various aspects of the applicability of these phages as antibacterial agents. Antibacterial potential of four phages was investigated via bacterial growth and biofilm formation inhibition, lytic spectra determination, and phage safety examination. The phage phiX174 was combined with different classes of antibiotics to evaluate potential synergistic interactions. In addition, the incidence of phiX174-insensitive mutants was analyzed. The results showed that only phiX174 out of four phages tested against their corresponding hosts inhibited bacterial growth for  > 90% at different multiplicity of infection and that only this phage considerably prevented biofilm formation. Although all phages show the absence of potentially undesirable genes, they also have extremely narrow lytic spectra. The synergism was determined between phage phiX174 and ceftazidime, ceftriaxone, ciprofloxacin, macrolides, and chloramphenicol. It was shown that the simultaneous application of agents is more effective than successive treatment, where one agent is applied first. The analysis of the appearance of phiX174 bacteriophage-insensitive mutants showed that mutations occur with a frequency of 10-3. The examined non-tailed phages have a limited potential for use as antibacterial agents, primarily due to a very narrow lytic spectrum and the high frequency of resistant mutants appearance, but Microviridae can be considered in the future as biocontrol agents against susceptible strains of E. coli in combinations with conventional antimicrobial agents.

Metatranscriptome sequencing expanded the known diversity of the bacterial RNA virome, suggesting that additional riboviruses infecting bacterial hosts remain to be discovered. Here we employed double-stranded RNA sequencing to recover complete genome sequences of two ribovirus groups from acidic hot springs in Japan. One group, denoted hot spring riboviruses (HsRV), consists of viruses with distinct RNA-directed RNA polymerases (RdRPs) that seem to be intermediates between typical ribovirus RdRPs and viral reverse transcriptases. This group forms a distinct phylum, Artimaviricota, or even kingdom within the realm Riboviria. We identified viruses encoding HsRV-like RdRPs in marine water, river sediments and salt marshes, indicating that this group is widespread beyond extreme ecosystems. The second group, denoted hot spring partiti-like viruses (HsPV), forms a distinct branch within the family Partitiviridae. The genome architectures of HsRV and HsPV and their identification in bacteria-dominated habitats suggest that these viruses infect thermoacidophilic bacteria.

Millipedes are important detritivores harbouring a diverse microbiome. Previous research focused on bacterial and archaeal diversity, while the virome remained neglected. We elucidated the DNA and RNA viral diversity in the hindguts of two model millipede species with distinct microbiomes: the tropical Epibolus pulchripes (methanogenic, dominated by Bacillota) and the temperate Glomeris connexa (non-methanogenic, dominated by Pseudomonadota). Based on metagenomic and metatranscriptomic assembled viral genomes, the viral communities differed markedly and preferentially infected the most abundant prokaryotic taxa. The majority of DNA viruses were Caudoviricetes (dsDNA), Cirlivirales (ssDNA) and Microviridae (ssDNA), while RNA viruses consisted of Leviviricetes (ssRNA), Potyviridae (ssRNA) and Eukaryotic viruses. A high abundance of subtypes I-C, I-B and II-C CRISPR-Cas systems was found, primarily from Pseudomonadota, Bacteroidota and Bacillota. In addition, auxiliary metabolic genes that modulate chitin degradation, vitamins and amino acid biosynthesis and sulphur metabolism were also detected. Lastly, we found low virus-to-microbe-ratios and a prevalence of lysogenic viruses, supporting a Piggyback-the-Winner dynamic in both hosts.

While taxonomy is an often underappreciated branch of science, it serves very important roles. Bacteriophage taxonomy has evolved from a discipline based mainly on morphology, characterized by the work of David Bradley and Hans-Wolfgang Ackermann, to the sequence-based approach that is taken today. The Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) takes a holistic approach to classifying prokaryote viruses by measuring overall DNA and protein similarity and phylogeny before making decisions about the taxonomic position of a new virus. The huge number of complete genomes being deposited with the National Center for Biotechnology Information (NCBI) and other public databases has resulted in a reassessment of the taxonomy of many viruses, and the future will see the introduction of new viral families and higher orders.

Bacteriophage diversity is a relatively unknown frontier that is rapidly being explored, leading to a wealth of new information. New bacteriophages are being discovered at an astounding rate via both phage isolation studies and metagenomic analyses. In addition, a nucleotide sequence-based viral taxonomic system has been developed to better handle this wealth of new information. As a result of these developments, phage scientists are transitioning from knowing that there must be huge numbers of diverse kinds of phage particles in natural environments to identifying the actual abundance and phage diversity that is present in specific environments. This review documents the beginning of this transition, offering a glimpse into the magnitude of change unfolding in the field. It stands as a testament to the expanding frontiers of phage research, illuminating the remarkable progress made in unraveling the intricate world of bacteriophage diversity and advancing our understanding of these enigmatic viral entities.

RNA viruses continue to pose significant threats to global public health, necessitating a profound understanding of their pathogenic mechanisms and the development of effective therapeutic interventions. This manuscript provides a comprehensive overview of emerging perspectives on RNA virus-mediated infections, spanning from the intricate intricacies of viral pathogenesis to the forefront of innovative therapeutic strategies. A critical exploration of antiviral drugs sets the stage, highlighting the diverse classes of compounds that target various stages of the viral life cycle, underscoring the ongoing efforts to combat viral infections. Central to this discussion is the exploration of RNA-based therapeutics, with a spotlight on messenger RNA (mRNA)-based approaches that have revolutionized the landscape of antiviral interventions. Furthermore, the manuscript delves into the intricate world of delivery systems, exploring inno-vative technologies designed to enhance the efficiency and safety of mRNA vaccines. By analyzing the challenges and advancements in delivery mechanisms, this review offers a roadmap for future research and development in this critical area. Beyond conventional infectious diseases, the document explores the expanding applications of mRNA vaccines, including their promising roles in cancer immunotherapy and personalized medicine approaches. This manuscript serves as a valuable resource for researchers, clinicians, and policymakers alike, offering a nuanced perspective on RNA virus pathogenesis and the cutting-edge therapeutic interventions. By synthesizing the latest advancements and challenges, this review contributes significantly to the ongoing discourse in the field, driving the development of novel strategies to combat RNA virus-mediated infections effectively.

RNA and single-stranded DNA (ssDNA) phages make up an understudied subset of bacteriophages that have been rapidly expanding in the last decade thanks to advancements in metaviromics. Since their discovery, applications of genetic engineering to ssDNA and RNA phages have revealed their immense potential for diverse applications in healthcare and biotechnology. In this review, we explore the past and present applications of this underexplored group of phages, particularly their current usage as therapeutic agents against multidrug-resistant bacteria. We also discuss engineering techniques such as recombinant expression, CRISPR/Cas-based genome editing, and synthetic rebooting of phage-like particles for their role in tailoring phages for disease treatment, imaging, biomaterial development, and delivery systems. Recent breakthroughs in RNA phage engineering techniques are especially highlighted. We conclude with a perspective on challenges and future prospects, emphasizing the untapped diversity of ssDNA and RNA phages and their potential to revolutionize biotechnology and medicine.

Viruses impact nearly all organisms on Earth, including microbial communities and their associated biogeochemical processes. In soils, highly diverse viral communities have been identified, with a global distribution seemingly driven by multiple biotic and abiotic factors, especially soil temperature and moisture. However, our current understanding of the stability of soil viral communities across time and their response to strong seasonal changes in environmental parameters remains limited. Here, we investigated the diversity and activity of environmental soil DNA and RNA viruses, focusing especially on bacteriophages, across dynamics' seasonal changes in a snow-dominated mountainous watershed by examining paired metagenomes and metatranscriptomes.

We identified a large number of DNA and RNA viruses taxonomically divergent from existing environmental viruses, including a significant proportion of fungal RNA viruses, and a large and unsuspected diversity of positive single-stranded RNA phages (Leviviricetes), highlighting the under-characterization of the global soil virosphere. Among these, we were able to distinguish subsets of active DNA and RNA phages that changed across seasons, consistent with a "seed-bank" viral community structure in which new phage activity, for example, replication and host lysis, is sequentially triggered by changes in environmental conditions. At the population level, we further identified virus-host dynamics matching two existing ecological models: "Kill-The-Winner" which proposes that lytic phages are actively infecting abundant bacteria, and "Piggyback-The-Persistent" which argues that when the host is growing slowly, it is more beneficial to remain in a dormant state. The former was associated with summer months of high and rapid microbial activity, and the latter with winter months of limited and slow host growth.

Taken together, these results suggest that the high diversity of viruses in soils is likely associated with a broad range of host interaction types each adapted to specific host ecological strategies and environmental conditions. As our understanding of how environmental and host factors drive viral activity in soil ecosystems progresses, integrating these viral impacts in complex natural microbiome models will be key to accurately predict ecosystem biogeochemistry. Video Abstract.

Recent advances in viromics have led to the discovery of a great diversity of RNA viruses and the identification of a large number of viral pathogens. A systematic exploration of viruses in Chinese mitten crab (Eriocheir sinensis), one of the most important aquatic commercial species, is still lacking. Here, we characterized the RNA viromes of asymptomatic, milky disease (MD)-affected, and hepatopancreatic necrosis syndrome (HPNS)-affected Chinese mitten crabs collected from 3 regions in China. In total, we identified 31 RNA viruses belonging to 11 orders, 22 of which were first reported here. By comparing viral composition between samples, we observed high variation in viral communities across regions, with most of the viral species being region-specific. We proposed to establish several novel viral families or genera based on the phylogenetic relationships and genome structures of viruses discovered in this study, expanding our knowledge of viral diversity in brachyuran crustaceans. IMPORTANCE High-throughput sequencing and meta-transcriptomic analysis provide us with an efficient tool to discover unknown viruses and explore the composition of viral communities in specific species. In this study, we investigated viromes in asymptomatic and diseased Chinese mitten crabs collected from three distant locations. We observed high regional variation in the composition of viral species, highlighting the importance of multi-location sampling. In addition, we classified several novel and ICTV-unclassified viruses based on their genome structures and phylogenetic relationships, providing a new perspective on current viral taxa.

Recent massive metatranscriptome mining substantially expanded the diversity of the bacterial RNA virome, suggesting that additional groups of riboviruses infecting bacterial hosts remain to be discovered. We employed full length double-stranded (ds) RNA sequencing for identification of riboviruses associated with microbial consortia dominated by bacteria and archaea in acidic hot springs in Japan. Whole sequences of two groups of multisegmented riboviruses genomes were obtained. One group, which we denoted hot spring riboviruses (HsRV), consists of unusual viruses with distinct RNA-dependent RNA polymerases (RdRPs) that seem to be intermediates between typical ribovirus RdRPs and viral reverse transcriptases. We also identified viruses encoding HsRV-like RdRPs in moderate aquatic environments, including marine water, river sediments and salt marsh, indicating that this previously overlooked ribovirus group is not restricted to the extreme ecosystem. The HsRV-like viruses are candidates for a distinct phylum or even kingdom within the viral realm Riboviria. The second group, denoted hot spring partiti-like viruses (HsPV), is a distinct branch within the family Partitiviridae. All genome segments in both these groups of viruses display the organization typical of bacterial riboviruses, where multiple open reading frames encoding individual proteins are preceded by ribosome-binding sites. Together with the identification in bacteria-dominated habitats, this genome architecture indicates that riboviruses of these distinct groups infect thermoacidophilic bacterial hosts.

This article summarises the activities of the Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses for the period of March 2021-March 2022. We provide an overview of the new taxa proposed in 2021, approved by the Executive Committee, and ratified by vote in 2022. Significant changes to the taxonomy of bacterial viruses were introduced: the paraphyletic morphological families Podoviridae, Siphoviridae, and Myoviridae as well as the order Caudovirales were abolished, and a binomial system of nomenclature for species was established. In addition, one order, 22 families, 30 subfamilies, 321 genera, and 862 species were newly created, promoted, or moved.

RNA viruses are characterised by extremely high genetic variability due to fast replication, large population size, low fidelity, and (usually) a lack of proofreading mechanisms of RNA polymerases leading to high mutation rates. Furthermore, viral recombination and reassortment may act as a significant evolutionary force among viruses contributing to greater genetic diversity than obtainable by mutation alone. The above-mentioned properties allow for the rapid evolution of RNA viruses, which may result in difficulties in viral eradication, changes in virulence and pathogenicity, and lead to events such as cross-species transmissions, which are matters of great interest in the light of current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemics. In this review, we aim to explore the molecular mechanisms of the variability of viral RNA genomes, emphasising the evolutionary trajectory of SARS-CoV-2 and its variants. Furthermore, the causes and consequences of coronavirus variation are explored, along with theories on the origin of human coronaviruses and features of emergent RNA viruses in general. Finally, we summarise the current knowledge on the circulating variants of concern and highlight the many unknowns regarding SARS-CoV-2 pathogenesis.

The International Committee on Taxonomy of Viruses recently adopted, and is gradually implementing, a binomial naming format for virus species. Although full Latinization of these names remains optional, a standardized nomenclature based on Latinized binomials has the advantage of comparability with all other biological taxonomies. As a language without living native speakers, Latin is more culturally neutral than many contemporary languages, and words built from Latin roots are already widely used in the language of science across the world. Conversion of established species names to Latinized binomials or creation of Latinized binomials de novo may seem daunting, but the rules for name creation are straightforward and can be implemented in a formulaic manner. Here, we describe approaches, strategies and steps for creating Latinized binomials for virus species without prior knowledge of Latin. We also discuss a novel approach to the automated generation of large batches of novel genus and species names. Importantly, conversion to a binomial format does not affect virus names, many of which are created from local languages.

Weeds often grow alongside crop plants. In addition to competing with crops for nutrients, water and space, weeds host insect vectors or act as reservoirs for viral diversity. However, little is known about viruses infecting rice weeds. In this work, we used metatranscriptomic deep sequencing to identify RNA viruses from 29 weed samples representing 23 weed species. A total of 224 RNA viruses were identified: 39 newly identified viruses are sufficiently divergent to comprise new families and genera. The newly identified RNA viruses clustered within 18 viral families. Of the identified viruses, 196 are positive-sense single-stranded RNA viruses, 24 are negative-sense single-stranded RNA viruses and 4 are double-stranded RNA viruses. We found that some novel RNA viruses clustered within the families or genera of several plant virus species and have the potential to infect plants. Collectively, these results expand our understanding of viral diversity in rice weeds. Our work will contribute to developing effective strategies with which to manage the spread and epidemiology of plant viruses.

Research on aquatic plant viruses is lagging behind that of their terrestrial counterparts. To address this knowledge gap, here we identified viruses associated with freshwater macrophytes, a taxonomically diverse group of aquatic phototrophs that are visible with the naked eye. We surveyed pooled macrophyte samples collected at four spring sites in Florida, USA through next generation sequencing of RNA extracted from purified viral particles. Sequencing efforts resulted in the detection of 156 freshwater macrophyte associated (FMA) viral contigs, 37 of which approximate complete genomes or segments. FMA viral contigs represent putative members from all five major phyla of the RNA viral kingdom Orthornavirae. Similar to viral types found in land plants, viral sequences identified in macrophytes were dominated by positive-sense RNA viruses. Over half of the FMA viral contigs were most similar to viruses reported from diverse hosts in aquatic environments, including phototrophs, invertebrates, and fungi. The detection of FMA viruses from orders dominated by plant viruses, namely Patatavirales and Tymovirales, indicate that members of these orders may thrive in aquatic hosts. PCR assays confirmed the presence of putative FMA plant viruses in asymptomatic vascular plants, indicating that viruses with persistent lifestyles are widespread in macrophytes. The detection of potato virus Y and oat blue dwarf virus in submerged macrophytes suggests that terrestrial plant viruses infect underwater plants and highlights a potential terrestrial-freshwater plant virus continuum. Defining the virome of unexplored macrophytes will improve our understanding of virus evolution in terrestrial and aquatic primary producers and reveal the potential ecological impacts of viral infection in macrophytes.

High throughput sequencing allowed the discovery of many new viruses and viral organizations increasing our comprehension of virus origin and evolution. Most RNA viruses are currently characterized through similarity searches of annotated virus databases. This approach limits the possibility to detect completely new virus-encoded proteins with no detectable similarities to existing ones, i.e. ORFan proteins. A strong indication of the ORFan viral origin in a metatranscriptome is the lack of DNA corresponding to an assembled RNA sequence in the biological sample. Furthermore, sequence homology among ORFans and evidence of co-occurrence of these ORFans in specific host individuals provides further indication of a viral origin. Here, we use this theoretical framework to report the finding of three conserved clades of protein-coding RNA segments without a corresponding DNA in fungi. Protein sequence and structural alignment suggest these proteins are distantly related to viral RNA-dependent RNA polymerases (RdRP). In these new putative viral RdRP clades, no GDD catalytic triad is present, but the most common putative catalytic triad is NDD and a clade with GDQ, a triad previously unreported at that site. SDD, HDD, and ADD are also represented. For most members of these three clades, we were able to associate a second genomic segment, coding for a protein of unknown function. We provisionally named this new group of viruses ormycovirus. Interestingly, all the members of one of these sub-clades (gammaormycovirus) accumulate more minus sense RNA than plus sense RNA during infection.

Whereas DNA viruses are known to be abundant, diverse, and commonly key ecosystem players, RNA viruses are insufficiently studied outside disease settings. In this study, we analyzed ≈28 terabases of Global Ocean RNA sequences to expand Earth's RNA virus catalogs and their taxonomy, investigate their evolutionary origins, and assess their marine biogeography from pole to pole. Using new approaches to optimize discovery and classification, we identified RNA viruses that necessitate substantive revisions of taxonomy (doubling phyla and adding >50% new classes) and evolutionary understanding. "Species"-rank abundance determination revealed that viruses of the new phyla "Taraviricota," a missing link in early RNA virus evolution, and "Arctiviricota" are widespread and dominant in the oceans. These efforts provide foundational knowledge critical to integrating RNA viruses into ecological and epidemiological models.