Viral Discovery in the Invasive Australian Cane Toad (Rhinella marina) Using Metatranscriptomic and Genomic Approaches.

Cane toads are a notorious invasive species, inhabiting over 1.2 million km2 of Australia and threatening native biodiversity. The release of pathogenic cane toad viruses is one possible biocontrol strategy yet is currently hindered by the poorly described cane toad virome. Metatranscriptomic analysis of 16 cane toad livers revealed the presence of a novel and full-length picornavirus, Rhimavirus A (RhiV-A), a member of a reptile- and amphibian-specific cluster of the Picornaviridae basal to the Kobuvirus -like group. In the combined liver transcriptome, we also identified a complete genome sequence of a distinct epsilonretrovirus, Rhinella marina endogenous retrovirus (RMERV). The recently sequenced cane toad genome contains 8 complete RMERV proviruses as well as 21 additional truncated insertions. The oldest full-length RMERV provirus was estimated to have inserted 1.9 million years ago (MYA). To screen for these viral sequences in additional toads, we analyzed publicly available transcriptomes from six diverse Australian locations. RhiV-A transcripts were identified in toads sampled from three locations across 1,000 km of Australia, stretching to the current Western Australia (WA) invasion front, while RMERV transcripts were observed at all six sites. Finally, we scanned the cane toad genome for nonretroviral endogenous viral elements, finding three sequences related to small DNA viruses in the family Circoviridae This shows ancestral circoviral infection with subsequent genomic integration. The identification of these current and past viral infections enriches our knowledge of the cane toad virome, an understanding of which will facilitate future work on infection and disease in this important invasive species. IMPORTANCE Cane toads are poisonous amphibians that were introduced to Australia in 1935 for insect control. Since then, their population has increased dramatically, and they now threaten many native Australian species. One potential method to control the population is to release a cane toad virus with high mortality rates, yet few cane toad viruses have been characterized. This study samples cane toads from different Australian locations and uses an RNA sequencing and computational approach to find new viruses. We report novel complete picornavirus and retrovirus sequences that were genetically similar to viruses infecting frogs, reptiles, and fish. Using data generated in other studies, we show that these viral sequences are present in cane toads from distinct Australian locations. Three sequences related to circoviruses were also found in the toad genome. The identification of new viral sequences will aid future studies that investigate their prevalence and potential as agents for biocontrol.