The Alphavirus genus (Family Togaviridae) are small, enveloped viruses with positive-sense RNA genomes of 10–12 kb. Most alphaviruses are mosquito-borne and pathogenic in vertebrates, including significant human and veterinary pathogens like Ross River virus and chikungunya. Despite their importance, their evolutionary origins and timescales remain poorly understood.
To explore Alphaviruses' host range and evolution, we bioinformaticically screened RNA sequencing datasets and transcriptomes from the NCBI Sequence Read Archive for Alphavirus RNA-dependent RNA polymerases. Libraries with hits were de novo assembled, discovering eleven novel alphaviruses across diverse metazoan species, including marine vertebrates, cnidarians, early-branching metazoans, and tunicates.
These novel alphaviruses formed two distinct clades. Clade One was identified in cephaloid and ray-finned fishes, with one Alphavirus identified in both lionfish and cuttlefish datasets, indicating vertebrate-invertebrate transmission. These alphaviruses have bisegmented genomes with polyadenylated segments. The first segment contains non-structural protein domains similar to conventional alphaviruses, while the second segment encoded the Alphavirus Capsid and Envelope (E1-3) genes. This clade also featured novel RNA-associated domains, such as dsRNA binding domains and an RNA phosphotransferase. Structural modelling of trimeric, E2-E1 dimers was predicted with AlphaFold3, folding into prototypical alphavirus configuration.
Clade Two Alphaviruses were monosegmented and found in basal metazoans, including multiple coral species. These viruses had the largest genomes (up to ~18kb). Conventional protein annotation revealed that they share typical Alphavirus genomic organization and unknown protein sequences without homology to known virus domains. To improve annotation, we used ab initio structural modelling and guided homology searches with Phyre2, which revealed that clade two alphaviruses share confident homology to crystal structures of Alphavirus E1 proteins.
Phylogenetically, these alphaviruses form discrete basal lineages to the Alphavirus genus and are most closely related to aquatic vertebrate alphaviruses. This suggests an ancient origin of Alphaviruses, likely coinciding with the emergence of metazoans ~800 million years ago.