A robust human norovirus replication model in zebrafish larvae.

Human noroviruses (HuNoVs) are the most common cause of foodborne illness, with a societal cost of $60 billion and 219,000 deaths/year. The lack of robust small animal models has significantly hindered the understanding of norovirus biology and the development of effective therapeutics. Here we report that HuNoV GI and GII replicate to high titers in zebrafish (Danio rerio) larvae; replication peaks at day 2 post infection and is detectable for at least 6 days. The virus (HuNoV GII.4) could be passaged from larva to larva two consecutive times. HuNoV is detected in cells of the hematopoietic lineage and the intestine, supporting the notion of a dual tropism. Antiviral treatment reduces HuNoV replication by >2 log10, showing that this model is suited for antiviral studies. Zebrafish larvae constitute a simple and robust replication model that will largely facilitate studies of HuNoV biology and the development of antiviral strategies.

A Single Nucleoside Viral Polymerase Inhibitor Against Norovirus, Rotavirus, and Sapovirus-Induced Diarrhea.

A safe and highly efficient antiviral is needed for the prophylaxis and/or treatment of viral diarrhea. We here demonstrate the in vitro antiviral activity of four 2'-C-methyl nucleoside analogues against noro-, rota-, and sapoviruses. The most potent nucleoside analogue, 7-deaza-2'-C-methyladenosine, inhibits replication of these viruses with a 50% effective concentration < 5 µM. Mechanistically, we demonstrate that the 2'-C-methyl nucleoside analogues act by inhibiting transcription of the rotavirus genome. This provides the first evidence that a single viral-diarrhea-targeted treatment can be developed through a viral-polymerase-targeting small molecule.

Treatment with a Nucleoside Polymerase Inhibitor Reduces Shedding of Murine Norovirus in Stool to Undetectable Levels without Emergence of Drug-Resistant Variants.

Prolonged norovirus shedding may occur in certain patients, such as organ transplant recipients. We established a mouse model for persistent norovirus infection (using the mouse norovirus MNV.CR6 strain). The nucleoside viral polymerase inhibitor 2'-C-methylcytidine (2CMC), but not favipiravir (T-705), reduced viral shedding to undetectable levels. Viral rebound was observed after stopping treatment, which was again effectively controlled by treatment with 2CMC. No drug-resistant variants emerged.

Molnupiravir inhibits human norovirus and rotavirus replication in 3D human intestinal enteroids.

Human norovirus (HuNoV) and human rotavirus (HRV) are the leading causes of gastrointestinal diarrhea. There are no approved antivirals and rotavirus vaccines are insufficient to cease HRV associated mortality. Furthermore, treatment of chronically infected immunocompromised patients is limited to off-label compassionate use of repurposed antivirals with limited efficacy, highlighting the urgent need of potent and specific antivirals for HuNoV and HRV. Recently, a major breakthrough in the in vitro cultivation of HuNoV and HRV derived from the use of human intestinal enteroids (HIEs). The replication of multiple circulating HuNoV and HRV genotypes can finally be studied and both in the same non-transformed and physiologically relevant model. Activity of previously described anti-norovirus or anti-rotavirus drugs, such as 2'-C-methylcytidine (2CMC), 7-deaza-2'-C-methyladenosine (7DMA), nitazoxanide, favipiravir and dasabuvir, was assessed against clinically relevant human genotypes using 3D-HIEs. 2CMC showed the best activity against HuNoV GII.4, while 7DMA was the most potent antiviral against HRV. We identified the anti-norovirus and -rotavirus activity of molnupiravir and its active metabolite, N4-hydroxycytidine (NHC), a broad-spectrum antiviral used to treat coronavirus disease 2019 (COVID-19). Molnupiravir and NHC inhibit HuNoV GII.4, HRV G1P[8], G2P[4] and G4P[6] in 3D-HIEs with high selectivity and show a potency comparable to 2CMC against HuNoV. Moreover, molnupiravir and NHC block HRV viroplasm formation, but do not alter its size or subcellular localization. Taken together, molnupiravir inhibits both HuNoV and HRV replication, suggesting that the drug could be a candidate for the treatment of patients chronically infected with either one of these diarrhea causing viruses.

Structural Investigations on Novel Non-Nucleoside Inhibitors of Human Norovirus Polymerase.

Human norovirus is the first cause of foodborne disease worldwide, leading to extensive outbreaks of acute gastroenteritis, and causing around 200,000 children to die annually in developing countries. No specific vaccines or antiviral agents are currently available, with therapeutic options limited to supportive care to prevent dehydration. The infection can become severe and lead to life-threatening complications in young children, the elderly and immunocompromised individuals, leading to a clear need for antiviral agents, to be used as treatments and as prophylactic measures in case of outbreaks. Due to the key role played by the viral RNA-dependent RNA polymerase (RdRp) in the virus life cycle, this enzyme is a promising target for antiviral drug discovery. In previous studies, following in silico investigations, we identified different small-molecule inhibitors of this enzyme. In this study, we rationally modified five identified scaffolds, to further explore structure-activity relationships, and to enhance binding to the RdRp. The newly designed compounds were synthesized according to multiple-step synthetic routes and evaluated for their inhibition of the enzyme in vitro. New inhibitors with low micromolar inhibitory activity of the RdRp were identified, which provide a promising basis for further hit-to-lead optimization.

Assessment of the anti-norovirus activity in cell culture using the mouse norovirus: Identification of active compounds.

Human norovirus is the main cause of viral gastroenteritis, resulting annually in ∼ 700 million infections and 200,000 deaths, of whom most are children <5 years. Mouse norovirus-infected macrophages are the most widely used in vitro system to screen and characterize the antiviral effect of norovirus-targeting small molecules. We have previously established antiviral assays using this system, identified novel inhibitors and performed additional studies in order to have a first insight into their mechanism of action. As potent and safe anti-norovirus small molecules are urgently needed, we here describe the detailed protocol for a set of assays that will allow the identification of novel norovirus inhibitors.

Current and Future Antiviral Strategies to Tackle Gastrointestinal Viral Infections.

Acute gastroenteritis caused by virus has a major impact on public health worldwide in terms of morbidity, mortality, and economic burden. The main culprits are rotaviruses, noroviruses, sapoviruses, astroviruses, and enteric adenoviruses. Currently, there are no antiviral drugs available for the prevention or treatment of viral gastroenteritis. Here, we describe the antivirals that were identified as having in vitro and/or in vivo activity against these viruses, originating from in silico design or library screening, natural sources or being repurposed drugs. We also highlight recent advances in model systems available for this (hard to cultivate) group of viruses, such as organoid technologies, and that will facilitate antiviral studies as well as fill some of current knowledge gaps that hamper the development of highly efficient therapies against gastroenteric viruses.

Infection of zebrafish larvae with human norovirus and evaluation of the in vivo efficacy of small-molecule inhibitors.

We have recently established that human norovirus (HuNoV) replicates efficiently in zebrafish larvae after inoculation of a clinical sample into the yolk, providing a simple and robust in vivo system in which to study HuNoV. In this Protocol Extension, we present a detailed description of virus inoculation by microinjection, subsequent daily monitoring and harvesting of larvae, followed by viral RNA quantification. This protocol can be used to study viral replication of genogroup (G)I and GII HuNoVs in vivo within 3-4 d. Additionally, we describe how to evaluate the in vivo antiviral effect and toxicity of small molecules using HuNoV-infected zebrafish larvae, in multi-well plates and without the need for specific formulations. This constitutes a great advantage for drug discovery efforts, as no specific antivirals or vaccines currently exist to treat or prevent norovirus gastroenteritis.

Discovery of a Novel Class of Norovirus Inhibitors with High Barrier of Resistance.

Human noroviruses (HuNoVs) are the most common cause of viral gastroenteritis resulting in ~219,000 deaths annually and a societal cost of ~USD60 billion. There are no antivirals or vaccines available to treat and/or prevent HuNoV. In this study, we performed a large-scale phenotypical antiviral screening using the mouse norovirus (MNV), which included ~1000 drug-like small molecules from the Drug Design and Synthesis Centre (Sapienza University, Rome). Compound 3-((3,5-dimethylphenyl)sulfonyl)-5-chloroindole-N-(phenylmethanol-4-yl)-2.carboxamide (compound 1) was identified as an inhibitor of MNV replication with an EC50 of 0.5 ± 0.1 µM. A series of 10 analogs were synthesized of which compound 6 showed an improved potency/selectivity (EC50 0.2 ± 0.1 µM) against MNV; good activity was also observed against the HuNoV GI replicon (EC50 1.2 ± 0.6 µM). Time-of-drug-addition studies revealed that analog 6 acts at a time point that coincides with the onset of viral RNA replication. After six months of selective pressure, two compound 6res variants were independently selected, both harboring one mutation in VPg and three mutations in the RdRp. After reverse engineering S131T and Y154F as single mutations into the MNV backbone, we did not find a markedly compound 6res phenotype. In this study, we present a class of novel norovirus inhibitors with a high barrier to resistance and in vitro antiviral activity.

A Novel Class of Norovirus Inhibitors Targeting the Viral Protease with Potent Antiviral Activity In Vitro and In Vivo.

Human noroviruses (HuNoVs) are the most common cause of viral gastroenteritis resulting annually in ~219,000 deaths and a societal cost of ~USD 60 billion, and no antivirals or vaccines are available. Here, we assess the anti-norovirus activity of new peptidomimetic aldehydes related to the protease inhibitor rupintrivir. The early hit compound 4 inhibited the replication of murine norovirus (MNV) and the HuNoV GI.1 replicon in vitro (EC50 ~1 µM) and swiftly cleared the HuNoV GI.1 replicon from the cells. Compound 4 still inhibits the proteolytic activity. We selected a resistant GI.1 replicon, with a mutation (I109V) in a highly conserved region of the viral protease, conferring a low yield of resistance against compound 4 and rupintrivir. After testing new derivatives, compound 10d was the most potent (EC50 nanomolar range). Molecular docking indicated that the aldehyde group of compounds 4 and 10d bind with Cys139 in the HuNoV 3CL protease by a covalent linkage. Finally, compound 10d inhibited the replication of HuNoV GII.4 in infected zebrafish larvae, and PK studies in mice showed an adequate profile.

Structure-Activity Relationship Studies on Novel Antiviral Agents for Norovirus Infections.

Human norovirus is the leading cause of acute gastroenteritis worldwide, affecting every year 685 million people. Norovirus outbreaks are associated with very significant economic losses, with an estimated societal cost of 60 billion USD per year. Despite this, no therapeutic options or vaccines are currently available to treat or prevent this infection. An antiviral therapy that can be used as treatment and as a prophylactic measure in the case of outbreaks is urgently needed. We previously described the computer-aided design and synthesis of novel small-molecule agents able to inhibit the replication of human norovirus in cell-based systems. These compounds are non-nucleoside inhibitors of the viral polymerase and are characterized by a terminal para-substituted phenyl group connected to a central phenyl ring by an amide-thioamide linker, and a terminal thiophene ring. Here we describe new modifications of these scaffolds focused on exploring the role of the substituent at the para position of the terminal phenyl ring and on removing the thioamide portion of the amide-thioamide linker, to further explore structure-activity relationships (SARs) and improve antiviral properties. According to three to four-step synthetic routes, we prepared thirty novel compounds, which were then evaluated against the replication of both murine (MNV) and human (HuNoV) norovirus in cells. Derivatives in which the terminal phenyl group has been replaced by an unsubstituted benzoxazole or indole, and the thioamide component of the amide-thioamide linker has been removed, showed promising results in inhibiting HuNoV replication at low micromolar concentrations. Particularly, compound 28 was found to have an EC50 against HuNoV of 0.9 µM. Although the most active novel derivatives were also associated with an increased cytotoxicity in the human cell line, these compounds represent a very promising starting point for the development of new analogues with reduced cytotoxicity and improved selectivity indexes. In addition, the experimental biological data have been used to create an initial 3D quantitative structure-activity relationship model, which could be used to guide the future design of novel potential anti-norovirus agents.

Targeting the Viral Polymerase of Diarrhea-Causing Viruses as a Strategy to Develop a Single Broad-Spectrum Antiviral Therapy.

Viral gastroenteritis is an important cause of morbidity and mortality worldwide, being particularly severe for children under the age of five. The most common viral agents of gastroenteritis are noroviruses, rotaviruses, sapoviruses, astroviruses and adenoviruses, however, no specific antiviral treatment exists today against any of these pathogens. We here discuss the feasibility of developing a broad-spectrum antiviral treatment against these diarrhea-causing viruses. This review focuses on the viral polymerase as an antiviral target, as this is the most conserved viral protein among the diverse viral families to which these viruses belong to. We describe the functional and structural similarities of the different viral polymerases, the antiviral effect of reported polymerase inhibitors and highlight common features that might be exploited in an attempt of designing such pan-polymerase inhibitor.

Assessing the Efficacy of Small Molecule Inhibitors in aMouse Model of Persistent Norovirus Infection.

Human norovirus is the most common cause of acute gastroenteritis worldwide, resulting in estimated mortality of ~210,000 each year, of whom most are children under the age of five. However, norovirus can infect people of all age groups. There is a risk of prolonged infection in children, the elderly and patients who are immunocompromised. To study the inhibition of persistent norovirus replication by small molecule antivirals in vivo, we used a murine norovirus CR6 strain (MNV.CR6). We demonstrated earlier that efficient small molecules can reduce viral shedding in the stool of infected mice. Here we present how to generate the MNV.CR6 virus stock, infect type I and II interferon receptor knockout AG129 mice via oral gavage, administer antivirals to mice, and quantify viral genome copies in the stool of these mice.

Norovirus genetic diversity and evolution: implications for antiviral therapy.

Human noroviruses are the leading cause of foodborne illness causing both acute and chronic gastroenteritis. In recent years, a number of vaccine candidates entered (pre-) clinical development and the first efforts to develop antiviral therapy have been made. We here discuss aspects of norovirus genetic evolution, persistence in immunocompromised patients as well as the risk and potential consequences of resistance development toward future antiviral drugs.