Widespread and intron-rich mirusviruses are predicted to reproduce in nuclei of unicellular eukaryotes.

Mirusviruses infect unicellular eukaryotes and are related to tailed bacteriophages and herpesviruses. Here we expand the known diversity of mirusviruses by screening diverse metagenomic assemblies and characterizing 1,202 non-redundant environmental genomes. Mirusviricota comprises a highly diversified phylum of large and giant eukaryotic viruses that rivals the evolutionary scope and functional complexity of nucleocytoviruses. Critically, major Mirusviricota lineages lack essential genes encoding components of the replication and transcription machineries and, concomitantly, encompass numerous spliceosomal introns that are enriched in virion morphogenesis genes. These features point to multiple transitions from cytoplasmic to nuclear reproduction during mirusvirus evolution. Many mirusvirus introns encode diverse homing endonucleases, suggestive of a previously undescribed mechanism promoting the horizontal mobility of spliceosomal introns. Available metatranscriptomes reveal long-range trans-splicing in a virion morphogenesis gene. Collectively, our data strongly suggest that nuclei of unicellular eukaryotes across marine and freshwater ecosystems worldwide are a major niche for replication of intron-rich mirusviruses.