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

Malaria remains a major global health challenge, with the highest burden in sub-Saharan Africa. While Plasmodium falciparum causes the most severe malaria, Plasmodium vivax is now recognised as widespread and genetically diverse across Africa, and can cause significant disease. Beyond genetic diversity, protozoan pathogens may also harbour intracellular RNA viruses that influence parasite fitness, virulence, host immune responses, and clinical outcomes. This introduces an additional, largely unexplored dimension to malaria biology. Matryoshka RNA virus 1 (MaRNAV-1) is a bi-segmented positive-sense RNA virus associated with P. vivax. Although MaRNAV-1 has been reported outside Africa and related viruses of African origin have been found in avian haemosporidian parasites, its presence in African human malaria has not been previously resolved. We analysed publicly available P. vivax blood RNA-sequencing datasets from Ethiopia to detect and characterise MaRNAV-1, and to place African sequences within global viral diversity. Four datasets with strong viral signals enabled consensus genome reconstruction, open reading frame identification, sequence validation, and phylogenetic analysis. Ethiopian MaRNAV-1 sequences maintained the canonical two-segment genome and clustered within the RNA-dependent RNA polymerase lineage, forming a distinct African group. In contrast, a Papua New Guinea comparator showed geographically structured divergence. Despite considerable distance, African sequences clustered closely with Malaysian P. vivax–associated references, suggesting conservation of the viral marker rather than recent interregional transmission. These findings provide genomic evidence for MaRNAV-1 in African P. vivax infections and expand the known distribution of parasite-associated RNA viruses in human malaria. By placing MaRNAV-1 within the context of protozoan virus–parasite interactions, this study establishes a basis for future research into the influence of parasite-resident viruses on malaria evolution, pathogenesis, and epidemiology. It also supports the need for expanded metatranscriptomic surveillance to assess their potential significance.