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

Abstract

Cane toads are a notorious invasive species, inhabiting over 1.2 million km 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 basal to the-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 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. 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.

Divergent Influenza-Like Viruses of Amphibians and Fish Support an Ancient Evolutionary Association

“…We did not identify any virus reads in any adult tissue samples taken in Australia nor from libraries constructed from cane toad samples from Macapa city, Brazil, from the same study [50]. Similarly, we did not recover any cane toad influenza-like virus reads in any of the 16 liver tissue samples from Australia reported in another metatranscriptomic study, which aimed to reveal the virome of the cane toad [53].…”

Immune and environment‐driven gene expression during invasion: An eco‐immunological application of RNA‐Seq

“…Conversely, the Australian soil bacterium Brucella (Ochrobactrum) anthropi causes spinal spondylosis in toads primarily at the invasion front (Brown, Shilton, Phillips, & Shine, 2007), which may represent a novel infection that forces invaders to remain immunocompetent. Furthermore, Rhimavirus A has only been detected in transcriptomes of toads from areas relatively close to the invasion front (Russo et al, 2018). Invasion history has complex effects on toad immunity (Brown, Phillips, Dubey, & Shine, 2015c;Brown & Shine, 2014;Selechnik, West, et al, 2017b).…”

Abundant and Diverse RNA Viruses in Insects Revealed by RNA-Seq Analysis: Ecological and Evolutionary Implications

“…In addition, some viruses were found to be evolutionarily related to vertebrate-associated species. For example, we observed a new picornavirus in Diplonychus rusticus that best matched Rhimavirus A (39.5% aa identity), a member of the proposed reptile- and amphibian-specific genus Rafivirus within the Picornaviridae ( 36 ). Another virus that was found in Hydrochara caraboides appeared to be closely related to Ampivirus A1 (83.0% aa identity), previously identified in a smooth newt ( 37 ).…”