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Voorbereiden op de volgende pandemie: Strategieën ter ontwikkeling van pan-bunya antivirale middelen

Year: 2025
Type: dissertation
Abstract: The order of Bunyavirales includes numerous (re)emerging viruses that are able to cause disease in humans and collectively have a major impact on human health worldwide. These viruses have a segmented negative sense single‐stranded [(–)ss]RNA genome and depending on the viral family are spread through either mosquitos, sandflies, or arthropods such as ticks. Despite many similarities in viral structure, genome organization, and replication cycle, the different bunyavirus families cause a broad range of clinical manifestations. Many of which can be life-threatening, such as hemorrhagic fever or encephalitis. Collectively the pathogens of the Bunyavirales form a serious threat to global human health. Especially, since for humans there are no specific prevention or treatment options available. Acquiring a deeper understanding of the replication cycle and involved proteins from the different bunyavirus species may provide insight on how best to target these viruses and facilitate antiviral drug discovery. One of the most well suited and conserved viral targets among bunyaviruses based on literature study, supplemented with phylogenetic analyses (Chapter 3), was found to be the endonuclease domain of the L-protein. This protein acts as RNA dependent RNA polymerase (RdRp) and is essential in the replication cycle of all bunyaviruses. The endonuclease domain specifically is responsible for cap-snatching', the process of cleaving the 5'-end of host mRNAs to use these as primer to initiate transcription of viral RNA. The amino acid similarity of the endonuclease domain of species between the four studied bunyaviral families ranges from 30% to 45%. However, within the active site of the domain there are motifs that are close to perfectly conserved. A similar endonuclease domain can be found in influenza viruses, where it already has been successfully exploited as druggable target to treat infection. To allow fast and reliable analyses of prospective drug candidates a next-generation antiviral assay was developed, making use of virus strains that express a fluorescent reporter protein. One major benefit of using our fluorescence-based assay over classical CPE-based assays is the fact that the antiviral effect of the tested compounds and their effect on the cell viability can be determined within the same assay plate. The first bunyavirus for which a protocol using this technique was developed is the Bunyamwera orthobunyavirus (BUNV) (Chapter 4). The protocol is easily adapted to a 384-well plate format, and therefore employable in high-throughput screening efforts. In Chapter 5, we investigate a class of molecules, the diketo acids, for their potential anti-bunyaviral properties. Molecules from this class have the capacity to chelate the two metal ions at the heart of the active site of the endonuclease domain, thereby rendering it unfunctional. We show the biochemical inhibition of several bunyaviral endonucleases translating to a modest antiviral activity in our fluorescent reporter-BUNV based assay. Our results confirm that the endonuclease is a suitable target for the development of broader acting antivirals against bunyaviruses and that the diketo acid class of compounds is worthy of further exploring in the design of novel inhibitors. Since a diketo-acid class molecule, baloxavir, has already been developed against influenza and successfully brought to clinical use we have evaluated whether this compound would also target the endonuclease domain of orthobunyaviruses in Chapter 6. We demonstrate that targeting the endonuclease of orthobunyavirus using the influenza inhibitor baloxavir yields a more potent antiviral effect than that brought about by ribavirin, which is currently the only last resort treatment given in case of severe bunyavirus infections. Hence, the design of potent and selective endonuclease inhibitors against bunyaviruses as drug candidates holds great promise. In Chapter 7 we describe how we utilize zebrafish larvae as model for Rift Valley fever virus (RVFV) infections and implement that model to study various aspects of the viral replication and dissemination, and the hosts antiviral response. Pericardial injection of RVFV in zebrafish larvae resulted in infection and robust viral replication. The optical transparency of the larvae allowed detection of the reporter virus RVFVeGFP in the liver and sensory nervous system, including the optic tectum and retina. Thereby demonstrating that RVFV-induced blindness is a result of damage to the eye and peripheral neurons, rather than the central nervous system. We show that viral replication in the larvae can be inhibited by adding the polymerase inhibitor 2'-fluoro-2'-deoxycytidine (2'-FdC) to their swimming water, proving the models viability for antiviral evaluation studies. Moreover, treatment of the larvae with JAK-inhibitor ruxolitinib, as well as knock-out of stat1a, strongly enhanced RVFV replication, but without dissemination to sensory neurons or the eye, confirming the defining role of stat1 in RVFV pathogenesis. In conclusion, the endonuclease domain of bunyaviruses is one of the most promising antiviral targets throughout this viral order. The here described enhanced methodology for antiviral analyses in vitro was developed allowing for the simultaneous evaluation of antiviral potency and cell toxicity in a single assay. Additionally, a new animal model was established using zebrafish larvae in combination with a RVFV reporter virus, and validated this model for the use in antiviral testing.
Source: Lirias
Author: ter Horst, Sebastiaan
Language: English
Field: Medicine
Open Access status: green
OpenAlex ID: W7112307677
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