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. PLOS Pathogens | https://doi.

High-throughput sequencing (HTS) enables the generation of large amounts of genome sequence data at a reasonable cost. Organisms in mixed microbial communities can now be sequenced and identified in a culture-independent way, usually using amplicon sequencing of a DNA barcode. Bulk RNA-seq (metatranscriptomics) has several advantages over DNA-based amplicon sequencing: it is less susceptible to amplification biases, it captures only living organisms, and it enables a larger set of genes to be used for taxonomic identification. Using a model mock community comprising 17 fungal isolates, we evaluated whether metatranscriptomics can accurately identify fungal species and subspecies in mixed communities. Overall, 72.9% of the RNA transcripts were classified, from which the vast majority (99.5%) were correctly identified at the species level. Of the 15 species sequenced, 13 were retrieved and identified correctly. We also detected strain-level variation within the Cryptococcus species complexes: 99.3% of transcripts assigned to Cryptococcus were classified as one of the four strains used in the mock community. Laboratory contaminants and/or misclassifications were diverse, but represented only 0.44% of the transcripts. Hence, these results show that it is possible to obtain accurate species-and strain-level fungal identification from metatranscriptome data as long as taxa identified at low abundance are discarded to avoid false-positives derived from contamination or misclassifications. This study highlights both the advantages and current challenges in the application of metatranscriptomics in clinical mycology and ecological studies.

Myxoma virus (MYXV) has been evolving in a novel host species—European rabbits—in Australia since 1950. Previous studies of viruses sampled from 1950 to 1999 revealed a remarkably clock-like evolutionary process across all Australian lineages of MYXV. Through an analysis of 49 newly generated MYXV genome sequences isolated in Australia between 2008 and 2017, we show that MYXV evolution in Australia can be characterized by three lineages, one of which exhibited a greatly elevated rate of evolutionary change and a dramatic breakdown of temporal structure. Phylogenetic analysis revealed that this apparently punctuated evolutionary event occurred between 1996 and 2012. The branch leading to the rapidly evolving lineage contained a relatively high number of nonsynonymous substitutions, and viruses in this lineage reversed a mutation found in the progenitor standard laboratory strain (SLS) and all previous sequences that disrupts the reading frame of theM005L/Rgene. Analysis of genes encoding proteins involved in DNA synthesis or RNA transcription did not reveal any mutations likely to cause rapid evolution. Although there was some evidence for recombination across the MYXV phylogeny, this was not associated with the increase in the evolutionary rate. The period from 1996 to 2012 saw significant declines in wild rabbit numbers, due to the introduction of rabbit hemorrhagic disease and prolonged drought in southeastern Australia, followed by the partial recovery of populations. It is therefore possible that a rapidly changing environment for virus transmission changed the selection pressures faced by MYXV, altering the course and pace of virus evolution.IMPORTANCEThe coevolution of myxoma virus (MYXV) and European rabbits in Australia is one of the most important natural experiments in evolutionary biology, providing insights into virus adaptation to new hosts and the evolution of virulence. Previous studies of MYXV evolution have also shown that the virus evolves both relatively rapidly and in a strongly clock-like manner. Using newly acquired MYXV genome sequences from Australia, we show that the virus has experienced a dramatic change in evolutionary behavior over the last 20 years, with a breakdown in clock-like structure, the appearance of a rapidly evolving virus lineage, and the accumulation of multiple nonsynonymous and indel mutations. We suggest that this punctuated evolutionary event may reflect a change in selection pressures as rabbit numbers declined following the introduction of rabbit hemorrhagic disease virus and drought in the geographic regions inhabited by rabbits.