Revealing the uncharacterised diversity of amphibian and reptile viruses.

Amphibians and non-avian reptiles represent a significant proportion of terrestrial vertebrates, however knowledge of their viruses is not proportional to their abundance. Many amphibians and reptiles have strict habitual environments and localised populations and are vulnerable to viral outbreaks and potential elimination as a result. We sought to identify viruses that were hidden in amphibian and reptile metatranscriptomic data by screening 235 RNA-sequencing datasets from a 122 species covering 25 countries. We identified 26 novel viruses and eight previously characterised viruses from fifteen different viral families. Twenty-five viruses had RNA genomes with identity to Arteriviridae, Tobaniviridae, Hantaviridae, Rhabdoviridae, Astroviridae, Arenaviridae, Hepeviridae, Picornaviridae, Orthomyxoviridae, Reoviridae, Flaviviridae and Caliciviridae. In addition to RNA viruses, we also screened datasets for DNA viral transcripts, which are commonly excluded from transcriptomic analysis. We identified ten DNA viruses with identity to Papillomaviridae, Parvoviridae, Circoviridae and Adomaviridae. With the addition of these viruses, we expand the global amphibian and reptile virome and identify new potentially pathogenic viruses that could challenge populations. We speculate that amphibian viruses often have simpler genomes than those in amniotes, as in the case of the Secondpapillomavirinae and Orthomyxoviridae viruses identified in this study. In addition, we find evidence of inter-family recombination in RNA viruses, and we also identify new members of the recombinant Adomaviridae family. Overall, we provide insights into the uncharacterised diversity of amphibian and reptile viruses with the aim of improving population management, treatment and conservation into the future.

Ancient viral integrations in marsupials: a potential antiviral defence.

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.

Discovery of Novel Viruses Associated With the Invasive Cane Toad (Rhinella marina) in Its Native and Introduced Ranges.

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.

Recombinant GII.P16/GII.4 Sydney 2012 Was the Dominant Norovirus Identified in Australia and New Zealand in 2017.

For the past two decades, norovirus pandemic variants have emerged every 3⁻5 years, and dominate until they are replaced by alternate strains. However, this scenario changed in 2016 with the co-circulation of six prevalent viruses, three of which possessed the pandemic GII.4 Sydney 2012 capsid. An increased number of institutional gastroenteritis outbreaks were reported within the Oceania region in mid-2017. This study identified emerging noroviruses circulating in Australia and New Zealand in 2017 to assess the changing dynamics of the virus infection. RT-PCR-based methods, next generation sequencing, and phylogenetic analyses were used to genotype noroviruses from both clinical and wastewater samples. Antigenic changes were observed between the capsid of pandemic Sydney 2012 variant and the two new Sydney recombinant viruses. The combination of these antigenic changes and the acquisition of a new ORF1 through recombination could both facilitate their ongoing persistence in the population. Overall, an increased prevalence of GII.P16/GII.4 Sydney 2012 viruses was observed in 2017, replacing the GII.P16/GII.2 recombinant that dominated in the region at the end of 2016. This shift in strain dominance was also observed in wastewater samples, demonstrating the reliability of wastewater as a molecular surveillance tool.

Potential Therapeutic Agents for Feline Calicivirus Infection.

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.