Continuous in silico screening of fish, amphibians, reptiles, and birds expands the diversity of bornavirids and reveals unexpected genomic architectures.

Using an in silico data mining approach, we previously expanded the known diversity within the family Bornaviridae by identifying novel bornavirids in publicly available sequence datasets from bony and cartilaginous fish. Building on this discovery, we systematically screened numerous additional sequence datasets from fish, amphibians, reptiles, and birds in the Sequence Read Archive and a new RNA-seq dataset to further explore bornaviral diversity and evolutionary history. Our analysis identified nearly complete and partial bornaviral genomes across multiple host taxa, that represented either new viral variants, previously known viruses detected in so far unreported hosts/sources, or novel viruses. Several novel orthobornaviral genomes were identified in snake and lizard datasets. Sequences of the species Orthobornavirus alphapsittaciforme and Orthobornavirus serini were identified in avian datasets. We also found sequences of both known and possibly new species in the genus Cultervirus, which were detected in bony fish and, for the first time, in reptile and amphibian datasets. The identification of culterviruses in reptiles and amphibians suggests that this group of viruses, which was previously only known to be associated with fish, has a much wider host range. A genetically divergent bornaviral genome was discovered in the gold-striped salamander (Chioglossa lusitanica), possessing the genomic architecture of orthobornaviruses but likely representing a new genus within the family Bornaviridae. Interestingly, multiple distinct viral genomes identified in passerine birds were classified as carboviruses based on sequence similarities, but they exhibited distinct genomic features and lacked the genes that code for glycoprotein (G) and, in some cases, the accessory protein (X) and/or matrix protein (M). Subsequent screening of samples from passerine birds further confirmed these findings and showed that these carboviral sequences likely represent genuine viruses rather than endogenous bornaviral elements. Interestingly, these carboviruses appear to co-occur with enveloped viruses in potential co-infection or helper virus scenarios. These findings contribute substantially to our understanding of Bornaviridae diversity, their evolutionary adaptation as well as modes of transcriptional regulation.