RNA Viruses Linked to Eukaryotic Hosts in Thawed Permafrost

Abstract

Arctic permafrost is thawing due to global warming, with unknown consequences on the microbial inhabitants or associated viruses. DNA viruses have previously been shown to be abundant and active in thawing permafrost, but little is known about RNA viruses in these systems. To address this knowledge gap, we assessed the composition of RNA viruses in thawed permafrost samples that were incubated for 97 days at 4°C to simulate thaw conditions. A diverse RNA viral community was assembled from metatranscriptome data including double-stranded RNA viruses, dominated by Reoviridae and Hypoviridae, and negative and positive single-stranded RNA viruses, with relatively high representations of Rhabdoviridae and Leviviridae, respectively. Sequences corresponding to potential plant and human pathogens were also detected. The detected RNA viruses primarily targeted dominant eukaryotic taxa in the samples (e.g., fungi, Metazoa and Viridiplantae) and the viral community structures were significantly associated with predicted host populations. These results indicate that RNA viruses are linked to eukaryotic host dynamics. Several of the RNA viral sequences contained auxiliary metabolic genes encoding proteins involved in carbon utilization (e.g., polygalacturosase), implying their potential roles in carbon cycling in thawed permafrost. IMPORTANCE Permafrost is thawing at a rapid pace in the Arctic with largely unknown consequences on ecological processes that are fundamental to Arctic ecosystems. This is the first study to determine the composition of RNA viruses in thawed permafrost. Other recent studies have characterized DNA viruses in thawing permafrost, but the majority of DNA viruses are bacteriophages that target bacterial hosts. By contrast RNA viruses primarily target eukaryotic hosts and thus represent potential pathogenic threats to humans, animals, and plants. Here, we find that RNA viruses in permafrost are novel and distinct from those in other habitats studied to date. The COVID-19 pandemic has heightened awareness of the importance of potential environmental reservoirs of emerging RNA viral pathogens. We demonstrate that some potential pathogens were detected after an experimental thawing regime. These results are important for understanding critical viral-host interactions and provide a better understanding of the ecological roles that RNA viruses play as permafrost thaws.

Keywords: RNA virus; metatranscriptomics; permafrost thaw; soil virus.

FIG 1

Phylogenetic composition and abundance estimates of RNA viruses detected in thawed permafrost. Phylogenetic trees of the detected double-stranded RNA viruses (a), negative single-stranded RNA viruses (b), and positive single-stranded RNA viruses (c) were reconstructed based on multiple sequence alignments of RNA-dependent RNA polymerase (RdRP) protein sequences and rooted by an RNA directed DNA polymerase (RdDP, APO57079.1) of an Alphaproteobacterium (‘Outgroup’). Due to the high sequence diversity, clades of Tombusviridae, Astroviridae and Picornaviridae were collapsed in panel (c) and displayed in separate panels (d), (e), and (f), respectively. The sum (left column) and average (right column) of the calculated average base-coverage of RNA viral contigs per taxon were used to estimate the relative abundances of the detected RNA viruses.

FIG 2

Comparison of RNA viruses across ecosystems. (a) The number of RdRP clusters that were shared and/or unique to each ecosystem are labeled in the respective sections of the Venn diagram. The percentage of shared clusters compared to the total number of clusters for each ecosystem were calculated (blue, thawed permafrost; brown, California grassland; yellow, Kansas grassland; red, invertebrate). The percentage of the clusters that were specific to each ecosystem are labeled in black. (b) The percentage of RdRP clusters with family or phylogenetic group classifications for each ecosystem. Relative richness was calculated at family (gray) and phylogenetic (black) levels.

FIG 3

Potential RNA viral hosts in thawed permafrost. (a) Pairings of RNA viral groups and predicted host assignments. The left stratum represents the RNA viral phylogenetic groups detected. The middle stratum shows the general names of known hosts of the detected RNA viruses. The right stratum lists the paired scientific names of the predicted hosts that are specified in (b). The pairings were colored by host lineage assigned. (b) Heatmap illustrating the composition of eukaryotic communities that were detected from 33 thawed permafrost samples that were collected across the following previously published permafrost transects (25): ‘t14’, ‘t15’, ‘t16’, and ‘t17’. The 18S rRNA transcript abundances of the detected eukaryotes were log transformed and color-coded with warmer colors representing higher relative abundances. The eukaryotic groups were clustered by similarity in the distribution patterns across samples.

FIG 4

Phylogenetic trees of putative AMG proteins encoded by RNA viruses in thawed permafrost. Phylogenetic trees of (a) polygalacturonase and (b) cell wall hydrolase were constructed using multiple sequence alignments of the corresponding proteins detected from the RNA viral contigs and the reference sequences of non-viral genomes deposited in NCBI NR databases. The leaves of the trees are colored by the taxonomic assignments of the sequences. The sequences detected from RNA viral contigs in this study are colored in red.