A virus associated with the zoonotic pathogen Plasmodium knowlesi causing human malaria is a member of a diverse and unclassified viral taxon

The Apicomplexa are a phylum of single-celled eukaryotes that can infect humans and include the mosquito-borne parasite Plasmodium, the cause of malaria. Viruses that infect non-Plasmodium spp. disease-causing protozoa affect the pathogen life cycle and disease outcomes. However, only one RNA virus (Matryoshka RNA virus 1) has been identified in Plasmodium, and none have been identified in zoonotic Plasmodium species. The rapid expansion of the known RNA virosphere via metagenomic sequencing suggests that this dearth is due to the divergent nature of RNA viruses that infect protozoa. We leveraged newly uncovered data sets to explore the virome of human-infecting Plasmodium species collected in Sabah, east (Borneo) Malaysia. From this, we identified a highly divergent RNA virus in two human-infecting P. knowlesi isolates that is related to the unclassified group 'ormycoviruses'. By characterizing 15 additional ormycoviruses identified in the transcriptomes of arthropods, we show that this group of viruses exhibits a complex ecology as noninfecting passengers at the arthropod-mammal interface. With the addition of viral diversity discovered using the artificial intelligence-based analysis of metagenomic data, we also demonstrate that the ormycoviruses are part of a diverse and unclassified viral taxon. This is the first observation of an RNA virus in a zoonotic Plasmodium species. By linking small-scale experimental data to advances in large-scale virus discovery, we characterize the diversity and confirm the putative genomic architecture of an unclassified viral taxon. This approach can be used to further explore the virome of disease-causing Apicomplexa and better understand how protozoa-infecting viruses may affect parasite fitness, pathobiology, and treatment outcomes.