Fig 1.
Host read depletion in RNA-Seq libraries.
Reads were mapped against rRNA SILVAdb (SortMeRNA tool), the human genome, and the genomes of three Plasmodium species. (A) Efficiency of successive read filtering (rRNA and host sorting). (B) Proportion of major host transcripts in each data set. The number of reads mapping to the human genome, Plasmodium sp. genomes and MaRNAV-1 are expressed as the percentage of trimmed reads for each library.
Table 1.
Results of the RdRp BLASTx analysis.
Fig 2.
RT-PCR confirmation of host and virus-like sequences in all Plasmodium-infected and non-infected samples used in this study.
From left to right: RT-PCR of each of the samples using human RPS18 primers, Plasmodium LDHP primers, MaRNAV-1 Segment I (S1) primers and MaRNAV-1 Segment II (S2) primers. Numbers in parentheses correspond to the expected size of the corresponding amplicon. (*) indicate different parts of the same gel that have been cropped for ease of visualization only.
Fig 3.
Genomic organization and sequence polymorphism of MaRNAV-1 in the seven P. vivax-infected blood samples.
(A) RdRp-segment analysis. Left: Nucleotide sequence alignment and ORF prediction (orange boxes). Right: Protein sequence alignment and InterPro domains predicted (green boxes). (B) Nucleotide polymorphism and ORFs predicted from the segment II in P. vivax samples. Sequence polymorphisms are highlighted in black. (C) Distance matrix of segment I (left) and segment II (right) with percentage of identity obtained at the nucleotide level.
Fig 4.
Phylogenetic analysis of MaRNAV-1 associated with Plasmodium vivax.
Boxes refer to the newly-described MaRNAV-1 viral sequences obtained in this study (red) or to RNA viruses classified as members of the Narnaviridae (dark orange), genus Narnavirus (light orange) or currently unclassified (grey). Taxa corresponding to the validated (coloured icons, right) and non-validated (grey icons, right) hosts are reported on the left part of the tree. Bootstrap values are indicated on each branch. The tree is mid-point rooted for clarity only.
Fig 5.
Number of Plasmodium SRA reads aligning with the MaRNAV-1 sequence (RdRp-segment) using BLASTx (cut-off 1e-5).
Fig 6.
Phylogenetic analysis, based on the RdRp, of the MaRNAV-1 documented here and from the P. vivax sequences available on the SRA.
Those viruses obtained in this study are shown in red while those from the SRA are shown in black. Sampling location and host characteristics (i.e. human-infected or mosquito-infected samples) are indicated on the right. Colored boxes indicate the samples collected in Asia (green), in South America (orange) or from unknown location (grey; ND: non-determined). The tree is mid-point rooted for clarity only.
Fig 7.
Comparative abundance of Leucocytozoon and MaRNAV-2 transcripts in Leucocytozoon-infected avian RNA-Seq libraries.
Read counts from each RNA-seq data set that mapped to the Leucocytozoon mitochondrial Cox1 gene (light orange), MaRNAV-2 RdRp-like segment (light grey), and MaRNAV-2 Segment II (dark grey) sequences, determined using Bowtie2.
Fig 8.
Evolutionary relationships of the newly-identified hematozoa viral sequences (MaRNAV-1 and MaRNAV-2).
(A) Phylogeny of all the newly-identified viral sequences. Red box: P. vivax viruses MaRNAV-1 (human or mosquitoes infection stage). Pink box: Leucocytozoon sp. MaRNAV-2 (bird infection stage). MaRNAV-1 viruses identified from P. vivax samples from this study are highlighted in red. Putative protozoan hosts are coloured depending on their belonging to the Alveolates (orange dark), Stramenopiles (light orange) and Euglenozoa (blue) major eukaryotic groups. Numbers indicate the branch support from 1000 bootstrap replicates. The virus tree is mid-point rooted for clarity only. (B) Eukaryotic host evolution and timescale, adapted from [38]. The two major groups Alveolates (red) and Euglenozoa (blue) are basal and their separation potentially occurred approximately two billion years ago [38].
Fig 9.
Hypothetical scenarios for the origin and evolution of MaRNAV-1 and MaRNAV-2 and relatives among parasites belonging to the Alveolates and Euglenozoa eukaryotic groups.
(A) Ancient virus-host co-divergence between the Euglenozoa and Alveolates that may have occurred approximately two billion years ago. (B) Horizontal virus transfer between the Alveolates and Euglenozoa parasites that co-infected the same secondary (likely invertebrate) host.