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Organoids emulate many aspects of their parental tissue and are therefore used to study pathogen-host interactions and other complex biological processes. Here, we report a robust protocol for the isolation, maintenance and differentiation of rabbit small intestinal organoids and organoid-derived cell monolayers. Our rabbit intestinal spheroid and monolayer cultures grew most efficiently in L-WRN-conditioned medium that contained Wnt, R-spondin and Noggin, and that had been supplemented with ROCK and TGF-β inhibitors. Organoid and monolayer differentiation was initiated by reducing the concentration of the L-WRN-conditioned medium and by adding ROCK and Notch signalling inhibitors. Immunofluorescence staining and RT-qPCR demonstrated that our organoids contained enterocytes, enteroendocrine cells, goblet cells and Paneth cells. Finally, we infected rabbit organoids with Rabbit calicivirus Australia-1 , an enterotropic lagovirus that—like many other caliciviruses—does not grow in conventional cell culture. Despite testing various conditions for inoculation, we did not detect any evidence of virus replication, suggesting either that our organoids do not contain suitable host cell types or that additional co-factors are required for a productive infection of rabbit organoids with Rabbit calicivirus Australia-1 .

In the 1950s the myxoma virus was released into European rabbit populations in Australia and Europe, decimating populations and resulting in the rapid evolution of resistance. We investigated the genetic basis of resistance by comparing the exomes of rabbits collected before and after the pandemic. We found a strong pattern of parallel evolution, with selection on standing genetic variation favoring the same alleles in Australia, France, and the United Kingdom. Many of these changes occurred in immunity-related genes, supporting a polygenic basis of resistance. We experimentally validated the role of several genes in viral replication and showed that selection acting on an interferon protein has increased the protein’s antiviral effect.

Lagovirus europaeus GI.2, also known as RHDV2 or RHDVb, is an emerging virus that causes rabbit haemorrhagic disease (RHD) in European rabbits ( Oryctolagus cuniculus ). In contrast to L. europaeus GI.1 (or RHDV/RHDVa) viruses that are only pathogenic for adults, GI.2 causes clinical disease in both adults and kittens. However, detailed descriptions of the pathology of this virus that may provide insight into its pathogenicity and emergence are lacking. Using an Australian GI.2 field strain isolated in 2015, we provide the first detailed description of pathology, viral antigen distribution and tissue load of GI.2 in adult and 5-week old New Zealand white rabbits using histology, immunohistochemistry and RT-qPCR. Liver was the target organ, but in contrast to GI.1 viruses, lesions and inflammatory responses did not differ between adults and kittens. Lymphocytic inflammation, proposed to be protective in kittens infected with GI.1, was notably absent. We also present the first descriptions of bone marrow changes in RHD, including decreased myeloid-to-erythroid ratio. Consistent with other pathogenic lagoviruses, intracellular viral antigen was demonstrated in hepatocytes and cells of the mononuclear phagocytic system. In terminal stages of disease, viral loads were highest in liver, serum and spleen. Despite the small sample size, our data suggest that unlike early European GI.2 strains, the pathogenicity of the Australian GI.2 virus is similar to GI.1 viruses. Additionally, GI.2 was fatal for all ( n  = 5) inoculated kittens in this study. This may significantly alter RHD epidemiology in the field, and may impact biocontrol programs for invasive rabbits in Australia where GI.1 viruses are intentionally released.

Mx proteins are interferon-induced GTPases that inhibit a wide range of viruses. The loss of functional Mx genes in mice and other model species is associated with inferior innate immune responses and increased virus susceptibility. Here, we describe genetic variations in the Mx genes of sheep ( Ovis aries ). More than 700 single nucleotide polymorphisms within or adjacent to MX1 and MX2 were identified by analysing whole genome sequence data from 68 sheep, representing 43 breeds from 19 countries. Amongst those are two biologically significant variations in the ovine MX2 gene: a guanosine-to-adenosine transition that generates a stop at codon 166 (c.497G > A; p.W166*) and a single nucleotide deletion in codon 329 that creates a frameshift and a premature stop of translation eight codons later (c.985del; p.Q329Sfs7*). A subsequent genotyping of Australian Merino sheep identified animals with a stop codon at position 166 in two research flocks that have been kept as closed flocks since the 1970s. Immunoblotting, immunofluorescence and mass spectrometry assays show that animals homozygous for the defect do not express detectable amounts of Mx2 proteins, and quantitative PCR suggests that the premature stop codon destabilises Mx2 mRNA. Furthermore, we found that Mx2-negative ewes and rams are fertile and that Mx2-negative lambs are indistinguishable from heterozygotes and wild-type animals in appearance, birth weight and growth rate.

The extremely pathogenic Rabbit haemorrhagic disease virus (RHDV) and the completely benign Rabbit calicivirus (RCV) are closely related members of the genus Lagovirus (family Caliciviridae). The molecular mechanisms that determine the dramatic difference in virulence are unknown, but indirect evidence suggests that different properties of their RNA-dependent RNA polymerases (RdRps) may at least partially be responsible for the contrasting phenotypes. Here we report that the unusual ability of the RHDV RdRp to induce a striking rearrangement of the Golgi network is not specific to RHDV, but a common feature of virulent and benign rabbit caliciviruses alike. Expression of rabbit calicivirus RdRps induced a redistribution of both cis/medial and medial/trans Golgi membrane markers, but not that of an endoplasmic reticulum membrane marker. Inactivating mutations in the conserved GDD motif did not abolish the ability of RHDV RdRp to rearrange the Golgi network, suggesting that polymerase activity and metal co-factors are not required for this function. Finally, we discuss possible implications of RdRp-induced membrane rearrangements on virus replication and host immune responses.

Our best hope of developing innovative methods to combat invasive species is likely to come from the study of high-profile invaders that have attracted intensive research not only into control, but also basic biology. Here we illustrate that point by reviewing current thinking about novel ways to control one of the world’s most well-studied invasions: that of the cane toad in Australia. Recently developed methods for population suppression include more effective traps based on the toad’s acoustic and pheromonal biology. New tools for containing spread include surveillance technologies (e.g., eDNA sampling and automated call detectors), as well as landscape-level barriers that exploit the toad’s vulnerability to desiccation— a strategy that could be significantly enhanced through the introduction of sedentary, rangecore genotypes ahead of the invasion front. New methods to reduce the ecological impacts of toads include conditioned taste aversion in free-ranging predators, gene banking, and targeted gene flow. Lastly, recent advances in gene editing and gene drive technology hold the promise of modifying toad phenotypes in ways that may facilitate control or buffer impact. Synergies between these approaches hold great promise for novel and more effective means to combat the toad invasion and its consequent impacts on biodiversity.

Rabbit haemorrhage disease virus 2 (RHDV2) is a highly pathogenic lagovirus that causes lethal disease in rabbits and hares (lagomorphs). Since its first detection in Europe in 2010, RHDV2 has spread worldwide and has been detected in over 35 countries so far. Here, we provide the first detailed report of the detection and subsequent circulation of RHDV2 in New Zealand. RHDV2 was first detected in New Zealand in 2018, with positive samples retrospectively identified in December 2017. Subsequent time-resolved phylogenetic analysis suggested a single introduction into the North Island between March and November 2016. Genetic analysis identified a GI.3P-GI.2 variant supporting a non-Australian origin for the incursion; however, more accurate identification of the source of the incursion remains challenging due to the wide global distribution of the GI.3P-GI.2 variant. Furthermore, our analysis suggests the spread of the virus between the North and South Islands of New Zealand at least twice, dated to mid-2017 and around 2018. Further phylogenetic analysis also revealed a strong phylogeographic pattern. So far, no recombination events with endemic benign New Zealand rabbit caliciviruses have been identified. This study highlights the need for further research and surveillance to monitor the distribution and diversity of lagoviruses in New Zealand and to detect incursions of novel variants.

Rabbit haemorrhagic disease virus 2 (RHDV2 or GI.2, referring to any virus with lagovirus GI.2 structural genes) is a recently emerged calicivirus that causes generalised hepatic necrosis and disseminated intravascular coagulation leading to death in susceptible lagomorphs (rabbits and hares). Previous studies investigating the virulence of RHDV2 have reported conflicting results, with case fatality rates ranging from 0% to 100% even within a single study. Lagoviruses are of particular importance in Australia and New Zealand where they are used as biocontrol agents to manage wild rabbit populations, which threaten over 300 native species and result in economic impacts in excess of $200 million AUD annually to Australian agricultural industries. It is critically important that any pest control method is both highly effective (i.e., virulent, in the context of viral biocontrols) and has minimal animal welfare impacts. To determine whether RHDV2 might be a suitable candidate biocontrol agent, we investigated the virulence and disease progression of a naturally occurring Australian recombinant RHDV2 in both 5-week-old and 11-week-old New Zealand White laboratory rabbits after either high or low dose oral infection. Objective measures of disease progression were recorded through continuous body temperature monitoring collars, continuous activity monitors, and twice daily observations. We observed a 100% case fatality rate in both infected kittens and adult rabbits after either high dose or low dose infection. Clinical signs of disease, such as pyrexia, weight loss, and reduced activity, were evident in the late stages of infection. Clinical disease, i.e., welfare impacts, were limited to the period after the onset of pyrexia, lasting on average 12 h and increasing in severity as disease progressed. These findings confirm the high virulence of this RHDV2 variant in naïve rabbits. While age and infectious dose significantly affected disease progression, the case fatality rate was consistently 100% under all conditions tested.

Following the arrival of rabbit haemorrhagic disease virus 2 (RHDV2) in Australia, average rabbit population abundances were reduced by 60% between 2014 and 2018 based on monitoring data acquired from 18 sites across Australia. During this period, as the seropositivity to RHDV2 increased, concurrent decreases were observed in the seroprevalence of both the previously circulating RHDV1 and RCVA, a benign endemic rabbit calicivirus. However, the detection of substantial RHDV1 seropositivity in juvenile rabbits suggested that infections were continuing to occur, ruling out the rapid extinction of this variant. Here we investigate whether the co-circulation of two pathogenic RHDV variants was sustained after 2018 and whether the initially observed impact on rabbit abundance was still maintained. We monitored rabbit abundance and seropositivity to RHDV2, RHDV1 and RCVA at six of the initial eighteen sites until the summer of 2022. We observed sustained suppression of rabbit abundance at five of the six sites, with the average population reduction across all six sites being 64%. Across all sites, average RHDV2 seroprevalence remained high, reaching 60–70% in adult rabbits and 30–40% in juvenile rabbits. In contrast, average RHDV1 seroprevalence declined to <3% in adult rabbits and 5–6% in juvenile rabbits. Although seropositivity continued to be detected in a low number of juvenile rabbits, it is unlikely that RHDV1 strains now play a major role in the regulation of rabbit abundance. In contrast, RCVA seropositivity appears to be reaching an equilibrium with that of RHDV2, with RCVA seroprevalence in the preceding quarter having a strong negative effect on RHDV2 seroprevalence and vice versa, suggesting ongoing co-circulation of these variants. These findings highlight the complex interactions between different calicivirus variants in free-living rabbit populations and demonstrate the changes in interactions over the course of the RHDV2 epizootic as it has moved towards endemicity. While it is encouraging from an Australian perspective to see sustained suppression of rabbit populations in the eight years following the arrival of RHDV2, it is likely that rabbit populations will eventually recover, as has been observed with previous rabbit pathogens.

European brown hares ( ) and European rabbits ( ) are invasive pest species in Australia, with rabbits having a substantially larger environmental impact than hares. As their spatial distribution in Australia partially overlaps, we conducted a comparative microbiome study to determine how the composition of gastrointestinal microbiota varies between these species, since this may indicate species differences in diet, physiology, and other internal and external factors. We analysed the faecal microbiome of nine wild hares and twelve wild rabbits from a sympatric periurban reserve in Canberra, Australia, using a 16S rRNA amplicon-based sequencing approach. Additionally, we compared the concordance between results from Illumina and Nanopore sequencing platforms. We identified significantly more variation in faecal microbiome composition between individual rabbits compared to hares, despite both species occupying a similar habitat. The faecal microbiome in both species was dominated by the phyla and , typical of many vertebrates. Many phyla, including , and , were shared between rabbits and hares. In contrast, bacteria from phylum were present only in rabbits, while phyla and were represented only in hares. We did not identify phylum in Australian hares; this phylum was previously shown to be present at high relative abundance in European hare faecal samples. These differences in the composition of faecal microbiota may be indicative of less discriminate foraging behaviour in rabbits, which in turn may enable them to adapt quicker to new environments, and may reflect the severe environmental impacts that this species has in Australia.