Biology subjects

Hall, R. N.

Publications and source records attributed to Hall, R. N..

10 recordsLinked to original sources

bioRxiv2024

A genetic and microscopy toolkit for live imaging of whole-body regeneration in Macrostomum lignano

Live imaging of regenerative processes can reveal how animals restore their bodies after injury through a cascade of dynamic cellular events. Here, we present a comprehensive toolkit for live imaging of whole-body regeneration in the flatworm Macrostomum lignano, including a high throughput cloning pipeline, targeted cellular ablation, and advanced microscopy solutions. Using tissue-specific reporter expression, we examine how various structures regenerate. Enabled by a custom, low-cost luminescence/fluorescence microscope, we overcome intense stress-induced autofluorescence to demonstrate the first application of genetic cellular ablation in flatworms to reveal the limited regenerative capacity of neurons and their essential role during wound-healing. Finally, we build a novel open-source tracking microscope to continuously image moving animals throughout the week-long process of regeneration, quantifying kinetics of wound healing, nerve cord repair, body regeneration, growth, and behavioral recovery. Our findings suggest that nerve cord reconnection operates independently from primary body axis re-establishment and other downstream regenerative processes.

bioengineering↗

bioRxiv2023

DNA polymerase diversity reveals multiple incursions of Polintons during nematode evolution

Polintons are dsDNA, virus-like self-synthesizing transposons widely found in eukaryotic genomes. Recent metagenomic discoveries of Polinton-like viruses are consistent with the hypothesis that Polintons invade eukaryotic host genomes through infectious viral particles. Nematode genomes contain multiple copies of Polintons and provide an opportunity to explore the natural distribution and evolution of Polintons during this process. We performed an extensive search of Polintons across nematode genomes, identifying multiple full-length Polinton copies in several species. We provide evidence of both ancient Polinton integrations and recent mobility in strains of the same nematode species. In addition to the major nematode Polinton family, we identified a group of Polintons that are overall closely related to the major family, but encode a distinct protein-primed B family DNA polymerase (pPolB) that is related to homologs from a different group of Polintons present outside of the Nematoda. Phylogenetic analyses on the pPolBs support the evolutionary scenarios in which these extrinsic pPolBs that seem to derive from Polinton families present in oomycetes and molluscs replaced the canonical pPolB in subsets of Polintons found in terrestrial and marine nematodes, respectively, suggesting inter-phylum horizontal gene transfers. The pPolBs of the terrestrial nematode and oomycete Polintons share a unique feature, an insertion of a HNH nuclease domain, whereas the pPolBs in the marine nematode Polintons share an insertion of a VSR nuclease domain with marine mollusc pPolBs. We hypothesize that horizontal gene transfer occurs among Polintons from widely different but cohabiting hosts.

evolutionary biology↗

bioRxiv2022

Hepatobiliary organoids derived from leporids support the replication of hepatotropic lagoviruses

Lagoviruses, family Caliciviridae, are some of the most virulent vertebrate viruses known. They infect leporids, i.e., rabbits (Oryctolagus spp.), hares (Lepus spp.) and cottontails (Sylvilagus spp.) and rapidly kill over 95% of susceptible animals. Pathogenic lagoviruses are hepatotropic and induce a fulminant hepatitis that typically leads to disseminated intravascular coagulation within 24-72 hours of infection. However, the pathophysiological mechanisms governing this extreme phenotype and other aspects of the fundamental biology of these viruses are poorly understood due to a lack of cell culture systems. Here, we report on a robust and reliable ex vivo model for the cultivation of hepatotropic lagoviruses. We show that three rabbit haemorrhagic disease virus (RHDV) variants, RHDV1, RHDV2 and RHDVa-K5, replicate in monolayer cultures derived from rabbit hepatobiliary organoids. Viral replication was demonstrated by a (i) increase in viral RNA levels of greater than one log10 over a 23-hour period, (ii) detection of viral structural and non-structural proteins, and (iii) detection of double-stranded RNA viral replication intermediates. Furthermore, we generated hepatobiliary organoids from a feral cat (Felis domesticus), a wild mouse (Mus musculus) and a European brown hare (Lepus europaeus) and showed that monolayer cultures derived from the cat and mouse organoids were not permissive for lagovirus infection, while those derived from the hare organoids only supported replication of RHDV2, recapitulating the species tropism that has been observed with these viruses. Our organoid culture system will facilitate future studies into the molecular biology of lagoviruses, which will have considerable import for the conservation of endangered leporid species in Europe and North America and the biocontrol of overabundant rabbit populations in Australia and New Zealand.

microbiology↗

bioRxiv2022

Pathogen profiling of Australian rabbits by metatranscriptomic sequencing

Australia is known for its long history of using biocontrol agents, like myxoma virus and rabbit haemorrhagic disease virus (RHDV), to manage wild European rabbit populations. Interestingly, while undertaking RHDV surveillance of rabbits that were found dead we observed that approximately 40% of samples were negative for RHDV. To investigate whether other infectious agents are responsible for killing rabbits in Australia we subjected a subset of these RHDV-negative liver samples to metatranscriptomic sequencing. In addition, we investigated whether the host transcriptome data could provide additional differentiation between likely infectious versus non-infectious causes of death. We identified transcripts from several Clostridia species, Pasteurella multocida, Pseudomonas, and Eimeria stiedae in liver samples of several rabbits that had died suddenly, all of which are known to infect rabbits and are capable of causing fulminant disease. In addition, we identified Hepatitis E virus and Cyniclomyces yeast in some samples, both of which are not usually associated with severe disease. In one third of the sequenced liver samples, no infectious agent could be identified. While metatranscriptomic sequencing cannot provide definitive evidence of causation, additional host transcriptome analysis provided further insights to distinguish between pathogenic microbes and commensals or environmental contaminants. Interestingly, three samples where no pathogen could be identified showed evidence of upregulated host immune responses, while immune response pathways were not upregulated when E. stiedae, Pseudomonas, or yeast were detected. In summary, although no new putative rabbit pathogens were identified, this study provides a robust workflow for future investigations into rabbit mortality events. ImportanceWe have observed that approximately 40% of rabbit liver samples submitted for RHDV testing (from rabbits that had died suddenly without obvious cause) are RHDV-negative. Interestingly, a similar finding was reported in pet rabbits in the United Kingdom. This raises the intriguing question of what else is killing rabbits, both in Australia and internationally? Using a metatranscriptomic sequencing approach, we found that Clostridiaceae, Pasteurella multocida, and Eimeria are frequently detected in cases of sudden rabbit death in Australia. While we did not identify any potential new pathogens that could be explored in the context of wild rabbit management, we have validated an approach to explore future mortality events of lagomorphs that may identify candidate novel biocontrols. Furthermore, our findings reaffirm the recommendation to follow good hygiene practices when handling rabbits, since domestic rabbits harboured several pathogens of potential public health significance, including Escherichia, Pasteurella multocida, and Hepatitis E virus.

microbiology↗

bioRxiv2021

Age and infectious dose significantly affect disease progression after RHDV2 infection in naive domestic rabbits

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; wild rabbits threaten over 300 native species and result in economic impacts in excess of $200 million AUD 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 hours and increasing in severity as disease progressed. These findings confirm the high virulence of this RHDV2 variant in naive rabbits. While age and infectious dose significantly affected disease progression, the case fatality rate was consistently 100% under all conditions tested.

microbiology↗

bioRxiv2021

Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection

Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence background of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating measurement of expression kinetics both in isolated cells and in whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a strong foundation for the development and expansion of transgenic techniques in this unique model system. MotivationThe study of planarians has contributed to advances in our understanding of regeneration, stem cell dynamics, and many other fundamental biological processes. However, the persistent challenge of expressing transgenes in planarians has led to the speculation that they may be resistant to transfection. In this work, we develop methods to express exogenous mRNAs in both isolated planarian cells and whole animals by optimizing delivery techniques, genetic constructs, and detection methods. These methods allow us to study transfection kinetics and post-transcriptional regulation of gene expression in a quantitative manner. Beyond planarian research, this work should also provide a broadly applicable strategy to develop similar tools for animals that are also challenging to modify genetically.

bioengineering↗

bioRxiv2020

Isolation, culture and maintenance of rabbit intestinal organoids, and organoid-derived cell monolayers

Organoids emulate many aspects of their parental tissue and have been used to study pathogen-host interactions, tissue development and regeneration, metabolic diseases, 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. We also report conditions that sustain an intestinal stem cell population in spheroid culture. Rabbit intestinal spheroids and monolayer cultures propagated and expanded most efficiently in L-WRN-conditioned medium that contained the signalling factors Wnt, R-spondin and Noggin, and that had been supplemented with ROCK and TGF-{beta} inhibitors. Organoid and monolayer differentiation was initiated by switching to a medium that contained less of the L-WRN-conditioned medium and was supplemented with ROCK and Notch signalling inhibitors. Using immunofluorescence staining and RT-qPCR, we demonstrate that organoids contained enterocytes, enteroendocrine cells, goblet cells and Paneth cells. These findings demonstrate that our rabbit intestinal organoids have many of the multi-cellular characteristics of, and closely resemble, an intestinal epithelium. This newly established organoid culture system will provide a useful tool to study rabbit gastrointestinal physiology and disease. For example, organoids and organoid-derived cells may be used to propagate and study caliciviruses and other enterotropic pathogens that cannot be grown in conventional cell culture systems.

cell biology↗

bioRxiv2019

Carrion flies as sentinels for monitoring rabbit calicivirus activity in Australia

Rabbit caliciviruses are an essential tool for managing wild rabbit populations in Australia. Our understanding of rabbit calicivirus epidemiology in Australia currently depends on members of the public submitting liver samples from dead rabbits through a monitoring program called Rabbitscan. However, many wild rabbits die in inaccessible locations or are scavenged before sampling can occur, leading to considerable sampling bias. In this study we screened field-caught carrion flies for the presence of rabbit caliciviruses to monitor virus circulation patterns in the landscape, with an aim to establish a less biased epidemiological surveillance tool. Carrion flies were collected from two study sites over a 22 month period and these samples were used to optimise and validate molecular testing methods in this sample type for the currently circulating rabbit calicivirus variants. Virus was clearly detectable in field-caught carrion flies using optimised SYBR-green RT-qPCR and RT-PCR assays. However, variant identification was frequently hindered by the low virus loads present in carrion fly samples and spurious RT-PCR amplification. This was overcome by frequent sampling, which effectively acts as replicate sampling to verify inconclusive results. There was good correlation between virus detections in carrion flies and in samples recovered from wild rabbits, both temporally and for virus variant identification. The methods reported here provide a robust and efficient additional surveillance tool to monitor rabbit calicivirus activity at a landscape scale, which in turn can help to guide more effective rabbit management programs.

microbiology↗

bioRxiv2018

The discovery of three new hare lagoviruses reveals unexplored viral diversity in this genus

Our knowledge of mammalian viruses has been strongly skewed toward those that cause disease in humans and animals. However, recent metagenomic studies indicate that most apparently healthy organisms carry viruses, and that these seemingly benign viruses may comprise the bulk of virus diversity. The bias toward studying viruses associated with overt disease is apparent in the lagoviruses (family Caliciviridae) that infect rabbits and hares: although most attention has been directed toward the highly pathogenic members of this genus - the rabbit haemorrhagic disease virus and European brown hare syndrome virus - a number of benign lagoviruses have also been identified. To determine whether wild European brown hares in Australia might also carry undetected benign viruses, we used a meta-transcriptomics approach to explore the gut and liver RNA viromes of these invasive animals. This led to the discovery of three new lagoviruses. While one of the three viruses was only detected in a single hare, the other two viruses were detected in 20% of all hares tested. All three viruses were most closely related to other hare lagoviruses, but were highly distinct from both known viruses and each other. We also found evidence for complex recombination events in these viruses, which, combined with their phylogenetic distribution, suggests that there is likely extensive unsampled diversity in this genus. Additional metagenomic studies of hares and other species are clearly needed to fill gaps in the lagovirus phylogeny and hence better understand the evolutionary history of this important group of mammalian viruses.

microbiology↗

bioRxiv2017

A strain-specific multiplex RT-PCR for Australian rabbit haemorrhagic disease viruses uncovers a new recombinant virus variant in rabbits and hares

Rabbit haemorrhagic disease virus (RHDV, or GI.1), is a calicivirus in the genus Lagovirus that has been widely utilised in Australia as a biological control agent for the management of overabundant wild European rabbit (Oryctolagus cuniculus) populations since 1996. Recently, two exotic incursions of pathogenic lagoviruses have been reported in Australia; GI.1a-Aus, previously called RHDVa-Aus, is a GI.1a virus detected in January 2014, and the novel lagovirus GI.2 (previously known as RHDV2). Furthermore, an additional GI.1a strain, GI.1a-K5 (also known as 08Q712), was released nationwide in March 2017 as a supplementary tool for wild rabbit management. To discriminate between these lagoviruses, a highly sensitive strain-specific multiplex RT-PCR assay was developed, which allows fast, cost-effective, and sensitive detection of the four pathogenic lagoviruses currently known to be circulating in Australia. In addition, we developed a universal qRT-PCR assay to be used in conjunction with the multiplex assay that broadly detects all four viruses and facilitates quantification of viral RNA load in samples. These assays enable rapid detection, identification, and quantification of pathogenic lagoviruses in the Australian context. Using these assays, a novel recombinant lagovirus was detected in rabbit tissues samples, which contained the non-structural genes of GI.1a-Aus and the structural genes of GI.2. This variant was also recovered from the liver of a European brown hare (Lepus europaeus). The impact of this novel recombinant on Australian wild lagomorph populations and its competitiveness in relation to circulating field strains, particularly GI.2, requires further studies.

microbiology↗