Novel RNA viruses associated with Plasmodium vivax in human malaria and Leucocytozoon parasites in avian disease.

Eukaryotes of the genus Plasmodium cause malaria, a parasitic disease responsible for substantial morbidity and mortality in humans. Yet, the nature and abundance of any viruses carried by these divergent eukaryotic parasites is unknown. We investigated the Plasmodium virome by performing a meta-transcriptomic analysis of blood samples taken from patients suffering from malaria and infected with P. vivax, P. falciparum or P. knowlesi. This resulted in the identification of a narnavirus-like sequence, encoding an RNA polymerase and restricted to P. vivax samples, as well as an associated viral segment of unknown function. These data, confirmed by PCR, are indicative of a novel RNA virus that we term Matryoshka RNA virus 1 (MaRNAV-1) to reflect its analogy to a "Russian doll": a virus, infecting a parasite, infecting an animal. Additional screening revealed that MaRNAV-1 was abundant in geographically diverse P. vivax derived from humans and mosquitoes, strongly supporting its association with this parasite, and not in any of the other Plasmodium samples analyzed here nor Anopheles mosquitoes in the absence of Plasmodium. Notably, related bi-segmented narnavirus-like sequences (MaRNAV-2) were retrieved from Australian birds infected with a Leucocytozoon-a genus of eukaryotic parasites that group with Plasmodium in the Apicomplexa subclass hematozoa. Together, these data support the establishment of two new phylogenetically divergent and genomically distinct viral species associated with protists, including the first virus likely infecting Plasmodium parasites. As well as broadening our understanding of the diversity and evolutionary history of the eukaryotic virosphere, the restriction to P. vivax may be of importance in understanding P. vivax-specific biology in humans and mosquitoes, and how viral co-infection might alter host responses at each stage of the P. vivax life-cycle. Author summary While parasites are a major cause of human disease, they can themselves be infected by viruses. We asked whether three of the major malaria-causing parasites in humans-Plasmodium vivax, P. falciparum and P. knowlesi-were also infected by viruses. To this end we performed total RNA-Sequencing ("meta-transcriptomics") on human blood samples infected with these Plasmodium species. This resulted in the discovery of an abundant bi-segmented virus-Matryoshka RNA virus 1 (MaRNAV-1)-in all P. vivax samples tested (but no other Plasmodium species) that contains a replicase segment related to those of narnaviruses, arguably the simplest type of RNA viruses discovered to date. By screening for MaRNAV-1 in a larger set of Plasmodium species we revealed a strong specificity between this virus and P. vivax, as well as the presence of a related virus-MaRNAV-2-in avian Leucocytozoon hematozoa parasites. This is the first discovery of a Plasmodium-associated virus and will assist in revealing the deep evolutionary history of RNA viruses and our understanding of Plasmodium biology and disease processes.