Norovirus antiviral discovery: host-modulators and direct-acting antivirals

Human norovirus is a leading cause of acute gastroenteritis (AGE) worldwide and is estimated to be responsible for over 200,000 deaths each year. Norovirus infections are estimated to cost $60 billion in societal costs globally each year. Yet despite the substantial health and economic burden of norovirus, there is no vaccine or norovirus-specific antiviral approved for clinical use. Effective norovirus therapies are highly desired, particularly for the treatment of chronic norovirus infections, or for prophylaxis to limit outbreaks and protect high-risk groups, including the immunocompromised. Several direct-acting antivirals (DAAs) and host-targeted therapies have demonstrated inhibitory activity against noroviruses in vitro and in vivo, however none of these compounds have progressed through clinical trials. Therefore, this thesis aimed to discover new antivirals and expand the repertoire of compounds with the potential to be further developed for the treatment of norovirus infections. In the first study, RNA-sequencing was performed to provide insights into norovirus pathogenesis and to help identify new host targets that could be explored antiviral development. We found that norovirus infection dampens the transcriptional profile of several genes involved in MHC class I antigen presentation, likely for immune evasion. We also observed a significant reduction in TLR7 expression which could represent a mechanism to avoid recognition by the host. To explore this further, we screened several Toll-like receptor (TLR) agonists, currently in clinical trials for antiviral therapy, against norovirus and found that these compounds potently inhibit infection in vitro. Here we discovered a new target for norovirus antivirals. Lastly using in vitro antiviral assays against several caliciviruses, we discovered a new nucleoside analogue (NA), called NITD008, which represents the most potent NA described in the literature to date and a strong candidate for continued development. The new anti- norovirus compounds described in this thesis could be used as scaffolds for the generation of derivatives with improved drug properties or be used in combination with other compounds for improved efficacy. Overall, this thesis involved a multi-faceted approach to antiviral discovery which has increased the available compounds in the pre-clinical pipeline for norovirus therapy

Bacillus cereus: Epidemiology, Virulence Factors, and Host-Pathogen Interactions

Bacillus cereus is an important human pathogen, and new findings have expanded our understanding of how this bacterium causes disease. B. cereus Hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE) induce membrane pore formation, leading to activation of the NLRP3 inflammasome, systemic inflammation, and death. Lipopolysaccharide-induced tumor necrosis factor (TNF)-α factor (LITAF) and cell death-inducing P53 target 1 (CDIP1) are bona fide mammalian surface receptors of HBL. These newly identified toxin receptors and the NLRP3 inflammasome represent unique targets for potential future therapies against severe B. cereus infections. The toxin-producing bacterium Bacillus cereus is an important and neglected human pathogen and a common cause of food poisoning. Several toxins have been implicated in disease, including the pore-forming toxins hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE). Recent work revealed that HBL binds to the mammalian surface receptors LITAF and CDIP1 and that both HBL and NHE induce potassium efflux and activate the NLRP3 inflammasome, leading to pyroptosis. These mammalian receptors, in part, contribute to inflammation and pathology. Other putative virulence factors of B. cereus include cytotoxin K, cereulide, metalloproteases, sphingomyelinase, and phospholipases. In this review, we highlight the latest progress in our understanding of B. cereus biology, epidemiology, and pathogenesis, and discuss potential new directions for research in this field.S.M.M. is supported by the Australian National University and the National Health and Medical Research Council of Australia under Project Grants (APP1141504, APP1146864, APP1162103 and APP1163358) and the R.D. Wright Career Development Fellowship (APP1162025). D.E.T. and S.M.M. are supported by Therapeutic Innovation Australia. D.E.T and A.M. are supported by The Gretel and Gordon Bootes Medical Research Foundation. A.M. is supported by a John Curtin School of Medical Research International PhD scholarshi

In silico screening for human norovirus antivirals reveals a novel non-nucleoside inhibitor of the viral polymerase

Human norovirus causes approximately 219,000 deaths annually, yet there are currently no antivirals available. A virtual screening of commercially available drug-like compounds (~300,000) was performed on the suramin and PPNDS binding-sites of the norovirus RNA-dependent RNA polymerase (RdRp). Selected compounds (n = 62) were examined for inhibition of norovirus RdRp activity using an in vitro transcription assay. Eight candidates demonstrated RdRp inhibition (>25% inhibition at 10 μM), which was confirmed using a gel-shift RdRp assay for two of them. The two molecules were identified as initial hits and selected for structure-activity relationship studies, which resulted in the synthesis of novel compounds that were examined for inhibitory activity. Five compounds inhibited human norovirus RdRp activity (>50% at 10 μM), with the best candidate, 54, demonstrating an IC50 of 5.6 μM against the RdRp and a CC50 of 62.8 μM. Combinational treatment of 54 and the known RdRp site-B inhibitor PPNDS revealed antagonism, indicating that 54 binds in the same binding pocket. Two RdRps with mutations (Q414A and R419A) previously shown to be critical for the binding of site-B compounds had no effect on inhibition, suggesting 54 interacts with distinct site-B residues. This study revealed the novel scaffold 54 for further development as a norovirus antiviral

In silico screening for human norovirus antivirals reveals a novel non-nucleoside inhibitor of the viral polymerase

Human norovirus causes approximately 219,000 deaths annually, yet there are currently no antivirals available. A virtual screening of commercially available drug-like compounds (~300,000) was performed on the suramin and PPNDS binding-sites of the norovirus RNA-dependent RNA polymerase (RdRp). Selected compounds (n = 62) were examined for inhibition of norovirus RdRp activity using an in vitro transcription assay. Eight candidates demonstrated RdRp inhibition (>25% inhibition at 10 μM), which was confirmed using a gel-shift RdRp assay for two of them. The two molecules were identified as initial hits and selected for structure-activity relationship studies, which resulted in the synthesis of novel compounds that were examined for inhibitory activity. Five compounds inhibited human norovirus RdRp activity (>50% at 10 μM), with the best candidate, 54, demonstrating an IC50 of 5.6 μM against the RdRp and a CC50 of 62.8 μM. Combinational treatment of 54 and the known RdRp site-B inhibitor PPNDS revealed antagonism, indicating that 54 binds in the same binding pocket. Two RdRps with mutations (Q414A and R419A) previously shown to be critical for the binding of site-B compounds had no effect on inhibition, suggesting 54 interacts with distinct site-B residues. This study revealed the novel scaffold 54 for further development as a norovirus antiviral

Horizontal transposon transfer and its implications for the ancestral ecology of hydrophiine snakes

Transposable elements (TEs), also known as jumping genes, are sequences able to move or copy themselves within a genome. As TEs move throughout genomes they often act as a source of genetic novelty, hence understanding TE evolution within lineages may help in understanding environmental adaptation. Studies into the TE content of lineages of mammals such as bats have uncovered horizontal transposon transfer (HTT) into these lineages, with squamates often also containing the same TEs. Despite the repeated finding of HTT into squamates, little comparative research has examined the evolution of TEs within squamates. Here we examine a diverse family of Australo–Melanesian snakes (Hydrophiinae) to examine if the previously identified, order-wide pattern of variable TE content and activity holds true on a smaller scale. Hydrophiinae diverged from Asian elapids ~30 Mya and have since rapidly diversified into six amphibious, ~60 marine and ~100 terrestrial species that fill a broad range of ecological niches. We find TE diversity and expansion differs between hydrophiines and their Asian relatives and identify multiple HTTs into Hydrophiinae, including three likely transferred into the ancestral hydrophiine from fish. These HTT events provide the first tangible evidence that Hydrophiinae reached Australia from Asia via a marine route

Canfam GSD: De novo chromosome-length genome assembly of the German Shepherd Dog (Canis lupus familiaris) using a combination of long reads, optical mapping, and Hi-C

Background: The German Shepherd Dog (GSD) is one of the most common breeds on earth and has been bred for its utility and intelligence. It is often first choice for police and military work, as well as protection, disability assistance, and search-and-rescue. Yet, GSDs are well known to be susceptible to a range of genetic diseases that can interfere with their training. Such diseases are of particular concern when they occur later in life, and fully trained animals are not able to continue their duties. Findings: Here, we provide the draft genome sequence of a healthy German Shepherd female as a reference for future disease and evolutionary studies. We generated this improved canid reference genome (CanFam GSD) utilizing a combination of Pacific Bioscience, Oxford Nanopore, 10X Genomics, Bionano, and Hi-C technologies. The GSD assembly is ∼80 times as contiguous as the current canid reference genome (20.9 vs 0.267 Mb contig N50), containing far fewer gaps (306 vs 23,876) and fewer scaffolds (429 vs 3,310) than the current canid reference genome CanFamv3.1. Two chromosomes (4 and 35) are assembled into single scaffolds with no gaps. BUSCO analyses of the genome assembly results show that 93.0% of the conserved single-copy genes are complete in the GSD assembly compared with 92.2% for CanFam v3.1. Homology-based gene annotation increases this value to ∼99%. Detailed examination of the evolutionarily important pancreatic amylase region reveals that there are most likely 7 copies of the gene, indicative of a duplication of 4 ancestral copies and the disruption of 1 copy. Conclusions: GSD genome assembly and annotation were produced with major improvement in completeness, continuity, and quality over the existing canid reference. This resource will enable further research related to canine diseases, the evolutionary relationships of canids, and other aspects of canid biology

Emerging activators and regulators of inflammasomes and pyroptosis

The inflammasome is a cytosolic immune signaling complex that induces inflammation and pyroptosis. Inflammasome complexes respond to a variety of pathogens, as well as danger or homeostasis-altering signals; they can play critical roles in the development of autoinflammatory conditions and cancer. Studies have now provided additional insights into the activation mechanisms and regulation of established inflammasome complexes, including NLRP1b, NLRP3, NOD-like receptor family apoptosis inhibitory protein (NAIP)-NLRC4, absent in melanoma (AIM)2, caspase-11, and pyrin. New activators and regulators of emerging NLRP6 and NLRP9b inflammasome complexes have also been described. We highlight the latest progress in our understanding of the molecular mechanisms governing inflammasome activation and pyroptosis, including the discovery of the pore-forming protein gasdermin D (GSDMD). We also discuss the importance of inflammasome activators and regulators in health and disease.W.H.T. is supported by a Terrell International Undergraduate Scholarship from the Australian National University. C.K. is supported by a Joyce Fildes Honours Scholarship from The John Curtin School of Medical Research. S.M.M. is supported by the Australian National University, The Gretel and Gordon Bootes Medical Research Foundation, and the National Health and Medical Research Council of Australia under Project Grants (APP1141504, APP1146864, APP1162103 and APP1163358) and the R.D. Wright Career Development Fellowship (APP1162025)

RNA Sequencing of Murine Norovirus-Infected Cells Reveals Transcriptional Alteration of Genes Important to Viral Recognition and Antigen Presentation

Viruses inherently exploit normal cellular functions to promote replication and survival. One mechanism involves transcriptional control of the host, and knowledge of the genes modified and their molecular function can aid in understanding viral-host interactions. Norovirus pathogenesis, despite the recent advances in cell cultivation, remains largely uncharacterized. Several studies have utilized the related murine norovirus (MNV) to identify innate response, antigen presentation, and cellular recognition components that are activated during infection. In this study, we have used next-generation sequencing to probe the transcriptomic changes of MNV-infected mouse macrophages. Our in-depth analysis has revealed that MNV is a potent stimulator of the innate response including genes involved in interferon and cytokine production pathways. We observed that genes involved in viral recognition, namely IFIH1, DDX58, and DHX58 were significantly upregulated with infection, whereas we observed significant downregulation of cytokine receptors (Il17rc, Il1rl1, Cxcr3, and Cxcr5) and TLR7. Furthermore, we identified that pathways involved in protein degradation (including genes Psmb3, Psmb4, Psmb5, Psmb9, and Psme2), antigen presentation, and lymphocyte activation are downregulated by MNV infection. Thus, our findings illustrate that MNV induces perturbations in the innate immune transcriptome, particularly in MHC maturation and viral recognition that can contribute to disease pathogenesis

The Adenosine Analogue NITD008 has Potent Antiviral Activity against Human and Animal Caliciviruses

The widespread nature of calicivirus infections globally has a substantial impact on the health and well-being of humans and animals alike. Currently, the only vaccines approved against caliciviruses are for feline and rabbit-specific members of this group, and thus there is a growing effort towards the development of broad-spectrum antivirals for calicivirus infections. In this study, we evaluated the antiviral activity of the adenosine analogue NITD008 in vitro using three calicivirus model systems namely; feline calicivirus (FCV), murine norovirus (MNV), and the human norovirus replicon. We show that the nucleoside analogue (NA), NITD008, has limited toxicity and inhibits calicivirus replication in all three model systems with EC50 values of 0.94 μM, 0.91 µM, and 0.21 µM for MNV, FCV, and the Norwalk replicon, respectively. NITD008 has a similar level of potency to the most well-studied NA 2′-C-methylcytidine in vitro. Significantly, we also show that continual NITD008 treatment effectively cleared the Norwalk replicon from cells and treatment with 5 µM NITD008 was sufficient to completely prevent rebound. Given the potency displayed by NITD008 against several caliciviruses, we propose that this compound should be interrogated further to assess its effectiveness in vivo. In summary, we have added a potent NA to the current suite of antiviral compounds and provide a NA scaffold that could be further modified for therapeutic use against calicivirus infections

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