The oceanic igneous crust, a vast and hydrologically active environment, is home to diverse microbial life, including bacteria, archaea, and fungi. While viruses are known to play a crucial role in microbial evolution and cycling, their diversity and activity within the marine crustal biome remain largely unexplored. Previous studies have identified diverse viral communities in subsurface crustal fluids, including archaeal-infecting viruses and bacteriophages. However, Nucleocytoviricota (NCLDVs), a group of large DNA viruses infecting eukaryotes, have been less well-characterized in this environment. This study focuses on the characterization of NCLDVs from crustal fluids collected from the Juan de Fuca Ridge, aiming to improve our understanding of their taxonomic affiliations, genomic features, and ecological roles.

Prior research on the oceanic crust's microbial communities has revealed a rich diversity of bacterial, archaeal, and fungal taxa, alongside viruses. Studies on viruses in the marine crust have primarily focused on bacteriophages and archaeal viruses, with limited information on eukaryotic viruses like NCLDVs. While NCLDVs are a diverse group infecting a broad range of eukaryotes, their diversity and ecological roles in deep-sea environments remain largely unknown. Existing metagenomic studies have provided glimpses into the viral communities present in the oceanic crust, but targeted approaches are needed to fully characterize the diversity of viruses, especially large DNA viruses like NCLDVs.

A one-milliliter sample of crustal fluid from the Integrated Ocean Drilling Program (IODP) borehole U1362B was collected and processed for single-virion genome amplification. Virus-like particles were stained with SYBR Green I and sorted using flow cytometry. Individual particles were sorted into a 384-well plate, lysed using KOH, and subjected to Multiple Displacement Amplification (MDA) for genome amplification. The MDA products were screened using PCR for bacterial and archaeal 16S rRNA genes to detect contamination. Eight putative viral single-amplified genomes (vSAGs) were selected for sequencing using Illumina MiSeq. Sequence reads were processed, assembled, and annotated using various bioinformatic tools. Phylogenetic analyses were conducted using concatenated alignments of core NCLDV genes and other genes of interest, such as tRNA Tyr and eukaryotic translation initiation factor 4E (eIF4E). Taxonomic classification of 18S rRNA, 28S rRNA, and internal transcribed spacer (ITS) sequences from the metagenome was also performed using BLAST searches against the SILVA database. Contamination analysis was conducted throughout the process and sequences from known contaminations were removed.

Two related NCLDV draft genomes (vSAG1.JdFr and vSAG8.JdFr) were identified. These vSAGs were not detected in previous metagenomic analyses of the same samples, highlighting the advantage of the single-virion approach. Phylogenetic analysis placed vSAG1.JdFr and vSAG8.JdFr within the Mesomimiviridae family, specifically sharing a lineage with Phycodnaviruses and Chrysochromulina parva viruses B02. The vSAGs contained genes from bacteria, archaea, and eukaryotes, with bacterial and archaeal genes clustered in islands across the genome. The presence of eukaryotic genes, including those encoding posttranslational modification enzymes, a peroxisome biogenesis protein, and a regulatory protein, suggested potential horizontal gene transfer (HGT) events, primarily from fungal origins. Notably, both vSAGs contained a tRNA Tyr gene with a short intron (20 bp) at a canonical position, a characteristic of eukaryotic and archaeal tRNA genes. Phylogenetic analysis of the tRNA Tyr gene further supported its eukaryotic origin. Analysis of the metagenome revealed Ascomycota fungi as the dominant eukaryotes in the crustal fluids, providing a putative host for these viruses. The co-occurrence of these viruses and fungi, along with the presence of fungal genes in the vSAGs, suggests a specific association between these NCLDVs and Ascomycota fungi.

The discovery of these giant fungal viruses in subsurface oceanic crustal fluids significantly expands our understanding of viral diversity in this unique ecosystem. The absence of these viruses in previous metagenomic studies highlights the limitations of traditional metagenomic approaches in detecting less abundant viruses. The presence of bacterial, archaeal, and eukaryotic genes in the vSAGs points towards a complex interaction between the virus, its fungal host, and other microbial members of the community. The horizontal gene transfer of fungal genes into the viruses further suggests a long-term interaction between the virus and its fungal host. The dominance of Ascomycota fungi in this environment strengthens the hypothesis of a fungi-virus association. Further research is needed to investigate the prevalence of NCLDVs in other deep-sea environments and to characterize their impacts on host physiology, microbial community dynamics, and biogeochemical cycling.

This study provides the first evidence for a specific association between Nucleocytoviricota viruses and Ascomycota fungi in the subsurface oceanic crust. The discovery of these giant fungal viruses, using a single-virion approach, highlights the significant untapped diversity of viruses in this environment. Future research should focus on characterizing the prevalence and ecological roles of these viruses in deep-sea ecosystems, and exploring the impacts of viral infections on fungal populations and biogeochemical processes. More comprehensive genomic studies are needed to fully understand the extent of HGT events and their role in viral evolution.

The study's limitations include the relatively small sample size and the partial nature of the viral genomes obtained. Further research is needed to obtain complete genomes and examine a larger number of samples to fully assess the diversity and distribution of these viruses. The potential for undiscovered contamination, despite rigorous screening, remains. The reliance on existing databases for gene annotation could also introduce biases, requiring further validation and refinement.