Identification of novel Zybavirus genome sequences and analysis of programmed ribosomal frameshifting motifs in the family Amalgaviridae

The family Amalgaviridae comprises monopartite double-stranded RNA viruses that encode two overlapping open reading frames (ORFs), ORF1 and ORF2. A programmed ribosomal frameshifting (PRF) mechanism facilitates the translation of an ORF1+2p fusion protein. Among the three recognized genera ( Amalgavirus , Unirnavirus , and Zybavirus ), Zybavirus remains poorly characterized, with only one approved species, Zygosaccharomyces bailii virus Z (ZbV-Z), and a few unclassified proposed members. In this study, we identified four novel zybavirus-like viral genome sequences, tentatively named Zygosaccharomyces bailii virus Z2 (ZbV-Z2), Cryptops hortensis-associated virus Z1 (ChaV-Z1), Drosophila suzukii-associated virus Z1 (DsaV-Z1), and Sand Creek Marshes virus Z1 (SCMV-Z1), from publicly available transcriptome datasets. Phylogenetic analysis placed ZbV-Z2, ChaV-Z1, and DsaV-Z1 in a well-supported clade with ZbV-Z and Xisha Islands zybavirus, supporting their classification within Zybavirus . SCMV-Z1 clustered with seven known viruses in a distinct lineage, which may represent a novel genus within the family Amalgaviridae . Comparative analysis of PRF sites in members of Zybavirus , Amalgavirus , and related clades revealed that UUU_CNN may represent a broader and ancestral consensus +1 PRF motif in this group of viruses. Our study highlights the utility of mining public transcriptome data for novel viral genome discovery and contributes to the refinement of both taxonomic classification and conserved genomic features within this viral family.