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Genomic viral integrations, termed endogenous viral elements (EVEs), are fragments of viruses in host chromosomes that provide information about viral evolution and could even help protect the host from infection. In the present study we examined EVEs in thirteen different Australian marsupial species to identify trends in their integration, commonality and to investigate their possible cellular function. We found that marsupial EVEs are commonly derived from viruses of the Bornaviridae, Filoviridae and Parvoviridae families, and circulated up to 160 million years ago. We also show the EVEs are actively transcribed into both long and short RNA molecules in marsupials, and propose they are involved in a cellular defence mechanism to protect the germline from viral genomic invasion.

Marsupial viruses are understudied compared to their eutherian mammal counterparts, although they may pose severe threats to vulnerable marsupial populations. Genomic viral integrations, termed ‘endogenous viral elements’ (EVEs), could protect the host from infection. It is widely known past viral infections and EVEs play an active role in antiviral defence in invertebrates and plants. This study aimed to characterise actively transcribed EVEs in Australian marsupial species, because they may play an integral role in cellular defence against viruses. This study screened publicly available RNA sequencing data sets (n = 35) and characterised 200 viral transcripts from thirteen Australian marsupial species. Of the 200 transcripts, 188 originated from either Bornaviridae, Filoviridae, or Parvoviridae EVEs. The other twelve transcripts were from putative active infections from members of the Herpesviridae and Anelloviridae, and Hepadnaviridae. EVE transcripts (n = 188) were mapped to marsupial genomes (where available, n = 5/13) to identify the genomic insertion sites. Of the 188 transcripts, 117 mapped to 39 EVEs within the koala, bare-nosed wombat, tammar wallaby, brushtail possum, and Tasmanian devil genomes. The remaining eight animals had no available genome (transcripts n = 71). Every marsupial has Bornaviridae, Filoviridae, and Parvoviridae EVEs, a trend widely observed in eutherian mammals. Whilst eutherian bornavirus EVEs are predominantly nucleoprotein-derived, marsupial bornavirus EVEs demonstrate a surprising replicase gene bias. We predicted these widely distributed EVEs were conserved within marsupials from ancient germline integrations, as many were over 65 million years old. One bornavirus replicase EVE, present in six marsupial genomes, was estimated to be 160 million years old, predating the American–Australian marsupial split. We considered transcription of these EVEs through small non-coding RNA as an ancient viral defence. Consistent with this, in koala small RNA sequence data sets, we detected Bornaviridae replicase and Filoviridae nucleoprotein produced small RNA. These were enriched in testis tissue, suggesting they could protect marsupials from vertically transmitted viral integrations.

Cane toads (Rhinella marina) are notoriously successful invaders: from 101 individuals brought to Australia in 1935, poisonous toads now cover an area >1.2 million km2 with adverse effects on native fauna. Despite extensive research on the role of macroparasites in cane toad invasion, viral research is lagging. We compared viral prevalence and diversity between toads in their native range (French Guiana, n=25) and two introduced ranges: Australia (n=151) and Hawai’i (n=10) with a metatranscriptomic and metagenomic approach combined with PCR screening. Australian toads almost exclusively harbor one of seven viruses detected globally. Rhimavirus-A (Picornaviridae) exhibited low genetic diversity and likely actively infected 9% of sampled Australian toads extending across ~2,000km of Northern Australia and up to the current invasion front. In native range cane toads, we identified multiple phylogenetically distinct viruses (Iridoviridae, Picornaviridae, Papillomaviridae, and Nackedna-like virus). None of the same viruses was detected in both ranges, suggesting that Australian cane toads have largely escaped the viral infection experienced by their native range counterparts. The novel native range viruses described here are potential biocontrol agents, as Australian toads likely lack prior immunological exposure to these viruses. Overall, our evidence suggests that there may be differences between viruses infecting cane toads in their native vs. introduced ranges, which lays the groundwork for further studies on how these viruses have influenced the toads’ invasion history.

Feline calicivirus (FCV) is a major cause of upper respiratory tract disease in cats, with widespread distribution in the feline population. Recently, virulent systemic diseases caused by FCV infection has been associated with mortality rates up to 50%. Currently, there are no direct-acting antivirals approved for the treatment of FCV infection. Here, we tested 15 compounds from different antiviral classes against FCV using in vitro protein and cell culture assays. After the expression of FCV protease-polymerase protein, we established two in vitro assays to assess the inhibitory activity of compounds directly against the FCV protease or polymerase. Using this recombinant enzyme, we identified quercetagetin and PPNDS as inhibitors of FCV polymerase activity (IC50 values of 2.8 μM and 2.7 μM, respectively). We also demonstrate the inhibition of FCV protease activity by GC376 (IC50 of 18 µM). Using cell culture assays, PPNDS, quercetagetin and GC376 did not display antivirals effects, however, we identified nitazoxanide and 2′-C-methylcytidine (2CMC) as potent inhibitors of FCV replication, with EC50 values in the low micromolar range (0.6 μM and 2.5 μM, respectively). In conclusion, we established two in vitro assays that will accelerate the research for FCV antivirals and can be used for the high-throughput screening of direct-acting antivirals.