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The repurposing of licenced drugs for use against COVID-19 is one of the most rapid ways to develop new and alternative therapeutic options to manage the ongoing pandemic. Given circa 7817 licenced compounds available from Compounds Australia that can be screened, this paper demonstrates the utility of commercially available ex vivo/3D airway and alveolar tissue models. These models are a closer representation of in vivo studies than in vitro models, but retain the benefits of rapid in vitro screening for drug efficacy. We demonstrate that several existing drugs appear to show anti-SARS-CoV-2 activity against both SARS-CoV-2 Delta and Omicron Variants of Concern in the airway model. In particular, fluvoxamine, as well as aprepitant, everolimus, and sirolimus, has virus reduction efficacy comparable to the current standard of care (remdesivir, molnupiravir, nirmatrelvir). Whilst these results are encouraging, further testing and efficacy studies are required before clinical use can be considered. [ABSTRACT FROM AUTHOR]

Despite being vaccine preventable, rabies (lyssavirus) still has a significant impact on global mortality, disproportionally affecting children under 15 years of age. This neurotropic virus is deft at avoiding the immune system while travelling through neurons to the brain. Until recently, research efforts into the role of non-coding RNAs in rabies pathogenicity and detection have been hampered by a lack of human in vitro neuronal models. Here, we utilized our previously described human stem cell-derived neural model to investigate the effect of lyssavirus infection on microRNA (miRNA) expression in human neural cells and their secreted exosomes. Conventional differential expression analysis identified 25 cellular and 16 exosomal miRNAs that were significantly altered (FDR adjusted P-value <0.05) in response to different lyssavirus strains. Supervised machine learning algorithms determined 6 cellular miRNAs (miR-99b-5p, miR-346, miR-5701, miR-138-2-3p, miR-651-5p, and miR-7977) were indicative of lyssavirus infection (100% accuracy), with the first four miRNAs having previously established roles in neuronal function, or panic and impulsivity-related behaviors. Another 4-miRNA signatures in exosomes (miR-25-3p, miR-26b-5p, miR-218-5p, miR-598-3p) can independently predict lyssavirus infected cells with >99% accuracy. Identification of these robust lyssavirus miRNA signatures offers further insight into neural lineage responses to infection and provides a foundation for utilizing exosome miRNAs in the development of next-generation molecular diagnostics for rabies. [ABSTRACT FROM AUTHOR]

Neurotropic viral infections continue to pose a serious threat to human and animal wellbeing. Host responses combatting the invading virus in these infections often cause irreversible damage to the nervous system, resulting in poor prognosis. Rabies is the most lethal neurotropic virus, which specifically infects neurons and spreads through the host nervous system by retrograde axonal transport. The key pathogenic mechanisms associated with rabies infection and axonal transmission in neurons remains unclear. Here we studied the pathogenesis of different field isolates of lyssavirus including rabies using ex-vivo model systems generated with mouse primary neurons derived from the peripheral and central nervous systems. In this study, we show that neurons activate selective and compartmentalized degeneration of its axons and dendrites in response to infection with different field strains of lyssavirus. We further show that this axonal degeneration is mediated by the loss of NAD and calpain-mediated digestion of key structural proteins such as MAP2 and neurofilament. We then analysed the role of SARM1 gene in rabies infection, which has been shown to mediate axonal self-destruction during injury. We show that SARM1 is required for the accelerated execution of rabies induced axonal degeneration and the deletion of SARM1 gene significantly delayed axonal degeneration in rabies infected neurons. Using a microfluidic-based ex-vivo neuronal model, we show that SARM1-mediated axonal degeneration impedes the spread of rabies virus among interconnected neurons. However, this neuronal defense mechanism also results in the pathological loss of axons and dendrites. This study therefore identifies a potential host-directed mechanism behind neurological dysfunction in rabies infection. This study also implicates a novel role of SARM1 mediated axonal degeneration in neurotropic viral infection. Author summary: Lyssavirus including rabies still causes devastating loss of human life every year and many of its victims are children under the age of 15. Rabies infection causes severe neurological dysfunction in the host resulting in paralysis, cognitive deficits and behavioural abnormalities. The mechanism of how rabies infection induces neurological dysfunction in the host remains unclear. This is because unlike other microbial infections, rabies infection rarely causes neuronal cell death and loss of neurons in the host nervous system. In this study, we show that neurons activate specific axonal self-destruction mechanism during rabies infection to prevent the spread of virus. However, this neuronal self-defense mechanism results in the loss of axons and dendrites, the structural components essential for the functioning of neurons. We further show that axonal degeneration in rabies infection is mediated by SARM1 gene, which has been previously shown to mediate defensive self-destruction of axons and dendrites in the event of neuronal injury. In summary, this study identifies a novel molecular mechanism behind neuronal dysfunction in rabies infection. This study also describes a novel intrinsic anti-viral defence mechanism in neurons, which could influence the pathogenesis of neurotropic viral infections. [ABSTRACT FROM AUTHOR]

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. S tem C ells 2017;35:626-640 [ABSTRACT FROM AUTHOR]

Rabies is a zoonotic neurological infection caused by lyssavirus that continues to result in devastating loss of human life. Many aspects of rabies pathogenesis in human neurons are not well understood. Lack of appropriate ex-vivo models for studying rabies infection in human neurons has contributed to this knowledge gap. In this study, we utilize advances in stem cell technology to characterize rabies infection in human stem cell-derived neurons. We show key cellular features of rabies infection in our human neural cultures, including upregulation of inflammatory chemokines, lack of neuronal apoptosis, and axonal transmission of viruses in neuronal networks. In addition, we highlight specific differences in cellular pathogenesis between laboratory-adapted and field strain lyssavirus. This study therefore defines the first stem cell-derived ex-vivo model system to study rabies pathogenesis in human neurons. This new model system demonstrates the potential for enabling an increased understanding of molecular mechanisms in human rabies, which could lead to improved control methods. [ABSTRACT FROM AUTHOR]

Many studies have suggested the significance of glycosyltransferase-mediated macromolecule glycosylation in the regulation of pluripotent states in human pluripotent stem cells (hPSCs). Here, we observed that the sialyltransferase ST6GAL1 was preferentially expressed in undifferentiated hPSCs compared to non-pluripotent cells. A lectin which preferentially recognizes α-2,6 sialylated galactosides showed strong binding reactivity with undifferentiated hPSCs and their glycoproteins, and did so to a much lesser extent with differentiated cells. In addition, downregulation of ST6GAL1 in undifferentiated hPSCs led to a decrease in POU5F1 (also known as OCT4) protein and significantly altered the expression of many genes that orchestrate cell morphogenesis during differentiation. The induction of cellular pluripotency in somatic cells was substantially impeded by the shRNA-mediated suppression of ST6GAL1, partially through interference with the expression of endogenous POU5F1 and SOX2. Targeting ST6GAL1 activity with a sialyltransferase inhibitor during cell reprogramming resulted in a dose-dependent reduction in the generation of human induced pluripotent stem cells (hiPSCs). Collectively, our data indicate that ST6GAL1 plays an important role in the regulation of pluripotency and differentiation in hPSCs, and the pluripotent state in human cells can be modulated using pharmacological tools to target sialyltransferase activity. [ABSTRACT FROM AUTHOR]