Transcriptome mining expands the diversity of Antonospora locustae virus 1-like viruses in the family Amalgaviridae.

The family Amalgaviridae comprises double-stranded RNA viruses with two partially overlapping open reading frames (ORFs), in which the RNA-dependent RNA polymerase is expressed through programmed ribosomal frameshifting. Antonospora locustae virus 1 (AnloV1), the founding member of the proposed genus "Anlovirus", represents a poorly characterized viral lineage because only a limited number of genome sequences have been reported. Here, we systematically mined publicly available transcriptome datasets and identified 13 AnloV1-like genome sequences from four insect species and one mammalian fecal sample. Pairwise sequence comparisons indicated that these genomes represent nine putative viral species, substantially expanding the diversity of this lineage. Comparative genomic analyses showed that all newly identified viruses possess two overlapping ORFs and a highly conserved UUU_CNN motif at the ORF1/ORF2 overlap, further supporting this sequence as the consensus +1 programmed ribosomal frameshifting motif of the proposed genus "Anlovirus". Phylogenetic analysis recovered a well-supported AnloV1-like clade distinct from the recognized genera of the family Amalgaviridae, providing additional evidence for the proposed genus "Anlovirus". Transcriptome datasets from all four insect species also contained transcripts with high similarity to microsporidian proteins, suggesting that at least some AnloV1-like viruses may infect microsporidian parasites rather than the sampled animals. These findings substantially expand the genomic resources available for AnloV1-like viruses and provide an evolutionary framework that may facilitate the future establishment and classification of the proposed genus "Anlovirus".