# Limited effect of short- to mid-term storage conditions on an Australian farmland soil RNA virome

## TL;DR

This study shows that storing soil samples at ultra-cold temperatures preserves RNA viruses well, revealing over 1,400 new viruses in Australian farmland soils.

## Contribution

The study demonstrates that snap-freezing soil samples for up to 3 months reliably preserves RNA viromes and identifies 1,475 novel RNA viruses.

## Key findings

- Soil samples stored at −80°C maintained RNA quality and viral diversity for at least 3 months.
- 1,475 putative novel RNA viruses were identified, mostly from the Lenarviricota phylum.
- Some novel viruses formed unique clusters, indicating region-specific evolution in Australian soils.

## Abstract

Soils represent one of the largest and most diverse reservoirs of microbial life on Earth, yet their associated RNA viruses remain underexplored compared to animal and aquatic systems. Viral discovery in soils has been further limited by technical hurdles, particularly difficulties in obtaining sufficient yields of high-quality RNA for sequencing. To address this, we evaluated a range of storage and preservation strategies, including the use of commercial preservative solutions and ultra-cold snap-freezing, followed by standardized RNA extraction, sequencing, and virus discovery pipelines. This work aimed to establish minimum sample storage requirements that maintain RNA integrity, generate sufficient RNA sequencing data, and subsequently enable reliable soil virome characterization. While no preservative solution proved effective, “neat” soil samples were stable at 2°C–8°C and −30°C for at least 2 weeks, and at −80°C for at least 3 months, with no measurable reduction in RNA quality, sequencing data, or viral abundance and diversity. From 32 resulting libraries, we identified 1,475 putative novel RNA viruses, with the majority belonging to the microbe-associated phylum Lenarviricota. Several novel viruses formed divergent clusters with other environmentally derived sequences distantly related to traditionally animal-associated families, such as the Astroviridae and Picornaviridae. Furthermore, unique clusters within the Picobirnaviridae, Alsuvirucetes, Ghabrivirales, and Amabiliviricetes comprised exclusively Australian viruses, suggesting instances of region-specific evolution. Together, these findings highlight soils as rich reservoirs of RNA viral diversity and provide practical minimum standards for storage, expanding opportunities to investigate the ecological and evolutionary roles of RNA viruses in terrestrial systems.

RNA viruses are the most abundant and diverse biological entities on Earth and are likely present in all other organisms and ecosystems, including soil-dwelling invertebrates, microbes, and plants. Despite this, their diversity and role in soil systems remain largely unknown. Methodological challenges in preserving and extracting sufficient quantities of RNA from soils have hindered the study of these communities. Here, we identified 1,475 previously undescribed RNA viruses in Australian soils while systematically testing different preservation strategies. The significance of our research lies in the demonstration that snap-freezing soil is a viable and robust storage strategy for at least 3 months, while also highlighting the extraordinary scale of viral diversity present in terrestrial environments. This work establishes a foundation for reliable exploration of terrestrial RNA viruses, improving the accessibility of more remote environmental viromes and enabling future efforts to integrate them into broader models of microbial ecology and ecosystem function.