Genomic characterisation and phylogenetic placement of Matryoshka RNA Virus-1 associated with Plasmodium vivax malaria in Africa

Plasmodium vivax is a major cause of human malaria. It harbours Matryoshka RNA virus 1 (MaRNAV-1), a bi-segmented positive-sense RNA virus. MaRNAV-1 was first described in P. vivax and is now recognised as part of a wider group of Matryoshka viruses. These viruses also infect other haemosporidian parasites such as Leucocytozoon and Haemoproteus. The presence of MaRNAV-1 in African-origin human P. vivax, however, has not been clearly established. This study investigated whether MaRNAV-1 is present in public African-origin P. vivax transcriptomic datasets. Any viral sequences recovered were characterised using comparative genomic and phylogenetic analyses. A secondary in silico analysis targeted African-origin P. vivax RNA-seq runs from public repositories. Although the search covered Africa, only Ethiopian datasets could be confidently identified, retrieved, and compiled at the time. After quality control and screening for MaRNAV-1 RNA-dependent RNA polymerase (RdRp) signals, three high-confidence runs were selected for further analysis. Reference-guided reconstruction, open reading frame (ORF) prediction, BLAST-based validation, and RdRp phylogenetic analysis were performed. MaRNAV-1 was identified in three Ethiopian P. vivax malaria transcriptomes. This was supported by strong segment-level mapping, near-complete coverage, high mean depth, and minimal low-depth masking. The recovered genomes showed the expected bi-segmented organisation of MaRNAV-1. Segment I was highly conserved and encoded the canonical RdRp in all three consensus sequences. Segment II showed greater structural variability, with two overlapping hypothetical ORFs in two sequences and a reduced, atypical profile in the third. BLAST analyses confirmed close similarity to MaRNAV-1 reference sequences. Phylogenetic inference grouped the Ethiopian sequences within the broader P. vivax-associated MaRNAV-1 lineage, alongside other recognised MaRNAV lineages distinct from more divergent narna-like viruses. These findings provide initial evidence for MaRNAV-1 in available African-origin P. vivax transcriptomic data and extend its known geographic range to the African malaria context