Genomic and phylogenetic features of the Picobirnaviridae suggest microbial rather than animal hosts.

The RNA virus family Picobirnaviridae has traditionally been associated with the gastrointestinal systems of terrestrial mammals and birds, with the majority of viruses detected in animal stool samples. Metatranscriptomic studies of vertebrates, invertebrates, microbial communities, and environmental samples have resulted in an enormous expansion of the genomic and phylogenetic diversity of this family. Yet picobirnaviruses remain poorly classified, with only one genus and three species formally ratified by the International Committee of Virus Taxonomy. Additionally, an inability to culture picobirnaviruses in a laboratory setting or isolate them in animal tissue samples, combined with the presence of bacterial genetic motifs in their genomes, suggests that these viruses may represent RNA bacteriophage rather than being associated with animal infection. Utilising a data set of 2,286 picobirnaviruses sourced from mammals, birds, reptiles, fish, invertebrates, microbial communities, and environmental samples, we identified seven consistent phylogenetic clusters likely representing Picobirnavirus genera that we tentatively name 'Alpha-', 'Beta-', 'Gamma-', 'Delta-', 'Epsilon-', 'Zeta-', and 'Etapicobirnavirus'. A statistical analysis of topological congruence between virus-host phylogenies revealed more frequent cross-species transmission than any other RNA virus family. In addition, bacterial ribosomal binding site motifs were more enriched in Picobirnavirus genomes than in the two groups of established RNA bacteriophage-the Leviviricetes and Cystoviridae. Overall, our findings support the hypothesis that the Picobirnaviridae have bacterial hosts and provide a lower-level taxonomic classification for this highly diverse and ubiquitous family of RNA viruses.

Australian terrestrial environments harbour extensive RNA virus diversity.

Australia is home to a diverse range of unique native fauna and flora. To address whether Australian ecosystems also harbour unique viruses, we performed meta-transcriptomic sequencing of 16 farmland and sediment samples taken from the east and west coasts of Australia. We identified 2460 putatively novel RNA viruses across 18 orders, the vast majority of which belonged to the microbe-associated phylum Lenarviricota. In many orders, such as the Nodamuvirales and Ghabrivirales, the novel viruses identified here comprised entirely new clades. Novel viruses also fell between established genera or families, such as in the Cystoviridae and Picornavirales, while highly divergent lineages were identified in the Sobelivirales and Ghabrivirales. Viral read abundance and alpha diversity were influenced by sampling site, soil type and land use, but not by depth from the surface. In sum, Australian soils and sediments are home to remarkable viral diversity, reflecting the biodiversity of local fauna and flora.

A metatranscriptomic analysis of geothermal hot springs reveals diverse RNA viruses including the phylum Lenarviricota.

Little is known about the diversity of RNA viruses in geothermal systems. We generated total RNA sequencing data from two hot springs in Kuirau Park, Rotorua, New Zealand. In one data set, from a 71.8 °C pool, we observed a microbial community that was 98.5% archaea. The second data set, representing a cooler 36.8 °C geothermal hot spring, had a more diverse microbial profile: 58% bacteria, 34.5% eukaryotes and 7.5% archaea. Within this latter pool, we detected sequences likely representing 23 RNA viruses from the families Astroviridae, Tombusviridae, Polycipiviridae, Discistroviridae, Partitiviridae, and Mitoviridae, as well as from unclassified clades of the orders Tolivirales, Picornavirales, and Ghabrivirales. Most viruses had uncertain host associations. Of particular note, we identified four novel RNA viruses from the phylum Lenarviricota, commonly associated with bacteria and fungi, that occupied a divergent phylogenetic position within unclassified clades and may represent an ancient order-level taxon of unknown host association.

RdRp-scan: A bioinformatic resource to identify and annotate divergent RNA viruses in metagenomic sequence data.

Despite a rapid expansion in the number of documented viruses following the advent of metagenomic sequencing, the identification and annotation of highly divergent RNA viruses remain challenging, particularly from poorly characterized hosts and environmental samples. Protein structures are more conserved than primary sequence data, such that structure-based comparisons provide an opportunity to reveal the viral 'dusk matter': viral sequences with low, but detectable, levels of sequence identity to known viruses with available protein structures. Here, we present a new open computational resource-RdRp-scan-that contains a standardized bioinformatic toolkit to identify and annotate divergent RNA viruses in metagenomic sequence data based on the detection of RNA-dependent RNA polymerase (RdRp) sequences. By combining RdRp-specific hidden Markov models (HMMs) and structural comparisons, we show that RdRp-scan can efficiently detect RdRp sequences with identity levels as low as 10 per cent to those from known viruses and not identifiable using standard sequence-to-sequence comparisons. In addition, to facilitate the annotation and placement of newly detected and divergent virus-like sequences into the diversity of RNA viruses, RdRp-scan provides new custom and curated databases of viral RdRp sequences and core motifs, as well as pre-built RdRp multiple sequence alignments. In parallel, our analysis of the sequence diversity detected by the RdRp-scan revealed that while most of the taxonomically unassigned RdRps fell into pre-established clusters, some fell into potentially new orders of RNA viruses related to the Wolframvirales and Tolivirales. Finally, a survey of the conserved A, B, and C RdRp motifs within the RdRp-scan sequence database revealed additional variations of both sequence and position that might provide new insights into the structure, function, and evolution of viral polymerases.

Resolving deep evolutionary relationships within the RNA virus phylum Lenarviricota.

The RNA virus phylum Lenarviricota is composed of the fungi-associated families Narnaviridae and Mitoviridae, the RNA bacteriophage Leviviridae, and the plant and fungi-associated Botourmiaviridae. Members of the Lenarviricota are abundant in most environments and boast remarkable phylogenetic and genomic diversity. As this phylum includes both RNA bacteriophage and fungi- and plant-associated species, the Lenarviricota likely mark a major evolutionary transition between those RNA viruses associated with prokaryotes and eukaryotes. Despite the remarkable expansion of this phylum following metagenomic studies, the phylogenetic relationships among the families within the Lenarviricota remain uncertain. Utilising a large data set of relevant viral sequences, we performed phylogenetic and genomic analyses to resolve the complex evolutionary history within this phylum and identify patterns in the evolution of virus genome organisation. Despite limitations reflecting very high levels of sequence diversity, our phylogenetic analyses suggest that the Leviviridae comprise the basal lineage within the Lenarviricota. Our phylogenetic results also support the construction of a new virus family-the Narliviridae-comprising a set of diverse and phylogenetically distinct species, including a number of uniquely encapsidated viruses. We propose a taxonomic restructuring within the Lenarviricota to better reflect the phylogenetic relationships documented here, with the Botourmiaviridae and Narliviridae combined into the order Ourlivirales, the Narnaviridae remaining in the order Wolframvirales, and these orders combined into the single class, the Amabiliviricetes. In sum, this study provides insights into the complex evolutionary relationships among the diverse families that make up the Lenarviricota.

RNA viromes from terrestrial sites across China expand environmental viral diversity.

Environmental RNA viruses are ubiquitous and diverse, and probably have important ecological and biogeochemical impacts. Understanding the global diversity of RNA viruses is limited by sampling biases, dependence on cell culture and PCR for virus discovery, and a focus on viruses pathogenic to humans or economically important animals and plants. To address this knowledge gap, we generated metatranscriptomic sequence data from 32 diverse environments in 16 provinces and regions of China. We identified 6,624 putatively novel virus operational taxonomic units from soil, sediment and faecal samples, greatly expanding known diversity of the RNA virosphere. These newly identified viruses included positive-sense, negative-sense and double-strand RNA viruses from at least 62 families. Sediments and animal faeces were rich sources of viruses. Virome compositions were affected by local environmental factors, including organic content and eukaryote species abundance. Notably, environmental factors had a greater impact on the abundance and diversity of plant, fungal and bacterial viruses than of animal viromes. Our data confirm that RNA viruses are an integral part of both terrestrial and aquatic ecosystems.