Animal models for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19), an emerging respiratory infection caused by the introduction of a novel coronavirus into humans late in 2019 (first detected in Hubei province, China). As of 18 September 2020, SARS-CoV-2 has spread to 215 countries, has infected more than 30 million people and has caused more than 950,000 deaths. As humans do not have pre-existing immunity to SARS-CoV-2, there is an urgent need to develop therapeutic agents and vaccines to mitigate the current pandemic and to prevent the re-emergence of COVID-19. In February 2020, the World Health Organization (WHO) assembled an international panel to develop animal models for COVID-19 to accelerate the testing of vaccines and therapeutic agents. Here we summarize the findings to date and provides relevant information for preclinical testing of vaccine candidates and therapeutic agents for COVID-19.

Favipiravir induces HuNoV viral mutagenesis and infectivity loss with clinical improvement in immunocompromised patients.

Chronic human norovirus (HuNoV) infections in immunocompromised patients result in severe disease, yet approved antivirals are lacking. RNA-dependent RNA polymerase (RdRp) inhibitors inducing viral mutagenesis display broad-spectrum in vitro antiviral activity, but clinical efficacy in HuNoV infections is anecdotal and the potential emergence of drug-resistant variants is concerning. Upon favipiravir (and nitazoxanide) treatment of four immunocompromised patients with life-threatening HuNoV infections, viral whole-genome sequencing showed accumulation of favipiravir-induced mutations which coincided with clinical improvement although treatment failed to clear HuNoV. Infection of zebrafish larvae demonstrated drug-associated loss of viral infectivity and favipiravir treatment showed efficacy despite occurrence of RdRp variants potentially causing favipiravir resistance. This indicates that within-host resistance evolution did not reverse loss of viral fitness caused by genome-wide accumulation of sequence changes. This off-label approach supports the use of mutagenic antivirals for treating prolonged RNA viral infections and further informs the debate surrounding their impact on virus evolution.

Human Norovirus Efficiently Replicates in Differentiated 3D-Human Intestinal Enteroids.

Human norovirus (HNoV) accounts for one-fifth of all acute viral gastroenteritis worldwide and an economic burden of ~$60 billion globally. The lack of treatment options against HNoV is in part due to the lack of cultivation systems. Recently, a model of infection in biopsy-derived human intestinal enteroids (HIE) has been described: 3D-HIE are first dispersed in 2D-monolayers and differentiated prior to infection, resulting in a labor-intensive, time-consuming procedure. Here, we present an alternative protocol for HNoV infection of 3D-HIE. We found that 3D-HIE differentiated as efficiently as 2D-monolayers. In addition, immunofluorescence-based quantification of UEA-1, a lectin that stains the villus brush border, revealed that ~80% of differentiated 3D-HIE spontaneously undergo polarity inversion, allowing for viral infection without the need for microinjection. Infection with HNoV GII.4-positive stool samples attained a fold-increase over inoculum of ~2 Log10 at 2 days postinfection or up to 3.5 Log10 when ruxolitinib, a JAK1/2-inhibitor, was added. Treatment of GII.4-infected 3D-HIE with the polymerase inhibitor 2'-C-Methylcytidine (2CMC) and other antivirals showed a reduction in viral infection, suggesting that 3D-HIE are an excellent platform to test anti-infectives. The transcriptional host response to HNoV was then investigated by RNA sequencing in infected versus uninfected 3D-HIE in the presence of ruxolitinib to focus on virus-associated signatures while limiting interferon-stimulated gene signatures. The analysis revealed upregulated hormone and neurotransmitter signal transduction pathways and downregulated glycolysis and hypoxia-response pathways upon HNoV infection. Overall, 3D-HIE have proven to be a highly robust model to study HNoV infection, screen antivirals, and to investigate the host response to HNoV infection. IMPORTANCE The human norovirus (HNoV) clinical and socio-economic impact calls for immediate action in the development of anti-infectives. Physiologically relevant in vitro models are hence needed to study HNoV biology, tropism, and mechanisms of viral-associated disease, and also as a platform to identify antiviral agents. Biopsy-derived human intestinal enteroids are a biomimetic of the intestinal epithelium and were recently described as a model that supports HNoV infection. However, the established protocol is time-consuming and labor-intensive. Therefore, we sought to develop a simplified and robust alternative model of infection in 3D enteroids that undergoes differentiation and spontaneous polarity inversion. Advantages of this model are the shorter experimental time, better infection yield, and spatial integrity of the intestinal epithelium. This model is potentially suitable for the study of other pathogens that infect intestinal cells from the apical surface but also for unraveling the interactions between intestinal epithelium and indigenous bacteria of the human microbiome.

Restriction of Viral Replication, Rather than T Cell Immunopathology, Drives Lethality in Murine Norovirus CR6-Infected STAT1-Deficient Mice.

Recent evidence indicates that viral components of the microbiota can contribute to intestinal homeostasis and protection from local inflammatory or infectious insults. However, host-derived mechanisms that regulate the virome remain largely unknown. In this study, we used colonization with the model commensal murine norovirus (MNV; strain CR6) to interrogate host-directed mechanisms of viral regulation, and we show that STAT1 is a central coordinator of both viral replication and antiviral T cell responses. In addition to restricting CR6 replication to the intestinal tract, we show that STAT1 regulates antiviral CD4+ and CD8+ T cell responses and prevents systemic viral-induced tissue damage and disease. Despite altered T cell responses that resemble those that mediate lethal immunopathology in systemic viral infections in STAT1-deficient mice, depletion of adaptive immune cells and their associated effector functions had no effect on CR6-induced disease. However, therapeutic administration of an antiviral compound limited viral replication, preventing virus-induced tissue damage and death without impacting the generation of inflammatory antiviral T cell responses. Collectively, our data show that STAT1 restricts MNV CR6 replication within the intestinal mucosa and that uncontrolled viral replication mediates disease rather than the concomitant development of dysregulated antiviral T cell responses in STAT1-deficient mice. IMPORTANCE The intestinal microbiota is a collection of bacteria, archaea, fungi, and viruses that colonize the mammalian gut. Coevolution of the host and microbiota has required development of immunological tolerance to prevent ongoing inflammatory responses against intestinal microbes. Breakdown of tolerance to bacterial components of the microbiota can contribute to immune activation and inflammatory disease. However, the mechanisms that are necessary to maintain tolerance to viral components of the microbiome, and the consequences of loss of tolerance, are less well understood. Here, we show that STAT1 is integral for preventing escape of a commensal-like virus, murine norovirus CR6 (MNV CR6), from the gut and that in the absence of STAT1, mice succumb to infection-induced disease. In contrast to the case with other systemic viral infections, mortality of STAT1-deficient mice is not driven by immune-mediated pathology. Our data demonstrate the importance of host-mediated geographical restriction of commensal-like viruses.

Favipiravir at high doses has potent antiviral activity in SARS-CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus has infected millions of people of which more than half a million succumbed to the viral disease, COVID-19. The urgent need for an effective treatment together with a lack of small animal infection models has led to clinical trials using repurposed drugs without preclinical evidence of their in vivo efficacy. We established an infection model in Syrian hamsters to evaluate the efficacy of small molecules on both infection and transmission. Treatment of SARS-CoV-2-infected hamsters with a low dose of favipiravir or hydroxychloroquine with(out) azithromycin resulted in, respectively, a mild or no reduction in virus levels. However, high doses of favipiravir significantly reduced infectious virus titers in the lungs and markedly improved lung histopathology. Moreover, a high dose of favipiravir decreased virus transmission by direct contact, whereas hydroxychloroquine failed as prophylaxis. Pharmacokinetic modeling of hydroxychloroquine suggested that the total lung exposure to the drug did not cause the failure. Our data on hydroxychloroquine (together with previous reports in macaques and ferrets) thus provide no scientific basis for the use of this drug in COVID-19 patients. In contrast, the results with favipiravir demonstrate that an antiviral drug at nontoxic doses exhibits a marked protective effect against SARS-CoV-2 in a small animal model. Clinical studies are required to assess whether a similar antiviral effect is achievable in humans without toxic effects.

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.

Enhanced efficacy of endonuclease inhibitor baloxavir acid against orthobunyaviruses when used in combination with ribavirin.

OBJECTIVES: Baloxavir acid is an endonuclease inhibitor approved for use against influenza. We evaluated whether this compound also targets the endonuclease domain of orthobunyaviruses and therefore could potentially be used against orthobunyavirus infections. METHODS: We performed a thermal shift assay and a fluorescence resonance energy transfer (FRET)-based nuclease monitoring assay using the La Crosse virus (LACV) endonuclease and baloxavir acid to prove their interaction and identify an inhibitory effect. Their interaction was further studied in a docking simulation using Glide SP. We show that baloxavir acid inhibits the viral replication of Bunyamwera virus (BUNV)-mCherry in vitro using high-content imaging and virus yield assay. Lastly, we investigated the use of baloxavir acid in combination with ribavirin in vitro by implementing the Zero Interaction Potency response surface model. RESULTS: We show that baloxavir acid augments LACV enzyme's melting temperature with ΔTm 9.5 ± 0.4°C and inhibited substrate cleavage with IC50 0.39 ± 0.03 µM. Moreover, our docking simulation suggests that baloxavir acid is able to establish an efficient binding with the LACV endonuclease. In the cell-based assay, we observed that baloxavir acid and ribavirin inhibited BUNV-mCherry with an EC50 of 0.7 ± 0.2 µM and 26.6 ± 8.9 µM, respectively. When used in combination, we found a maximum synergistic effect of 8.64. CONCLUSIONS: The influenza endonuclease inhibitor baloxavir acid is able to bind to and interfere with the endonuclease domain of orthobunyaviruses and yields a more potent antiviral effect than ribavirin against BUNV-mCherry. The combination of both compounds results in a more potent antiviral effect, suggesting that these molecules could potentially be combined to treat orthobunyavirus-infected patients.

Structural and functional similarities in bunyaviruses: Perspectives for pan-bunya antivirals.

The order of Bunyavirales includes numerous (re)emerging viruses that collectively have a major impact on human and animal health worldwide. There are no vaccines for human use or antiviral drugs available to prevent or treat infections with any of these viruses. The development of efficacious and safe drugs and vaccines is a pressing matter. Ideally, such antivirals possess pan-bunyavirus antiviral activity, allowing the containment of every bunya-related threat. The fact that many bunyaviruses need to be handled in laboratories with biosafety level 3 or 4, the great variety of species and the frequent emergence of novel species complicate such efforts. We here examined the potential druggable targets of bunyaviruses, together with the level of conservation of their biological functions, structure, and genetic similarity by means of heatmap analysis. In the light of this, we revised the available models and tools currently available, pointing out directions for antiviral drug discovery.

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.

Synthesis and in vitro antiviral evaluation of 4-substituted 3,4-dihydropyrimidinones.

A series of 4-substituted 3,4-dihydropyrimidine-2-ones (DHPM) was synthesized, characterized by IR, 1H NMR, 13C NMR and HRMS spectra. The compounds were evaluated in vitro for their antiviral activity against a broad range of DNA and RNA viruses, along with assessment for potential cytotoxicity in diverse mammalian cell lines. Compound 4m, which possesses a long lipophilic side chain, was found to be a potent and selective inhibitor of Punta Toro virus, a member of the Bunyaviridae. For Rift Valley fever virus, which is another Bunyavirus, the activity of 4m was negligible. DHPMs with a C-4 aryl moiety bearing halogen substitution (4b, 4c and 4d) were found to be cytotoxic in MT4 cells.

Molecular chaperone Hsp90 is a therapeutic target for noroviruses.

UNLABELLED: Human noroviruses (HuNoV) are a significant cause of acute gastroenteritis in the developed world, and yet our understanding of the molecular pathways involved in norovirus replication and pathogenesis has been limited by the inability to efficiently culture these viruses in the laboratory. Using the murine norovirus (MNV) model, we have recently identified a network of host factors that interact with the 5' and 3' extremities of the norovirus RNA genome. In addition to a number of well-known cellular RNA binding proteins, the molecular chaperone Hsp90 was identified as a component of the ribonucleoprotein complex. Here, we show that the inhibition of Hsp90 activity negatively impacts norovirus replication in cell culture. Small-molecule-mediated inhibition of Hsp90 activity using 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) revealed that Hsp90 plays a pleiotropic role in the norovirus life cycle but that the stability of the viral capsid protein is integrally linked to Hsp90 activity. Furthermore, we demonstrate that both the MNV-1 and the HuNoV capsid proteins require Hsp90 activity for their stability and that targeting Hsp90 in vivo can significantly reduce virus replication. In summary, we demonstrate that targeting cellular proteostasis can inhibit norovirus replication, identifying a potential novel therapeutic target for the treatment of norovirus infections. IMPORTANCE: HuNoV are a major cause of acute gastroenteritis around the world. RNA viruses, including noroviruses, rely heavily on host cell proteins and pathways for all aspects of their life cycle. Here, we identify one such protein, the molecular chaperone Hsp90, as an important factor required during the norovirus life cycle. We demonstrate that both murine and human noroviruses require the activity of Hsp90 for the stability of their capsid proteins. Furthermore, we demonstrate that targeting Hsp90 activity in vivo using small molecule inhibitors also reduces infectious virus production. Given the considerable interest in the development of Hsp90 inhibitors for use in cancer therapeutics, we identify here a new target that could be explored for the development of antiviral strategies to control norovirus outbreaks and treat chronic norovirus infection in immunosuppressed patients.

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.

The viral polymerase inhibitor 2'-C-methylcytidine inhibits Norwalk virus replication and protects against norovirus-induced diarrhea and mortality in a mouse model.

Human noroviruses are a major cause of food-borne illness, accountable for 50% of all-etiologies outbreaks of acute gastroenteritis (in both developing and developed countries). There is no vaccine or antiviral drug for the prophylaxis or treatment of norovirus-induced gastroenteritis. We recently reported the inhibitory effect of 2'-C-methylcytidine (2CMC), a hepatitis C virus polymerase inhibitor, on the in vitro replication of murine norovirus (MNV). Here we evaluated the inhibitory effect of 2CMC on in vitro human norovirus replication through a Norwalk virus replicon model and in a mouse model by using AG129 mice orally infected with MNV. Survival, weight, and fecal consistency were monitored, and viral loads in stool samples and organs were quantified. Intestines were examined histologically. 2CMC reduced Norwalk virus replicon replication in a dose-dependent manner and was able to clear cells of the replicon. Treatment of MNV-infected AG129 mice with 2CMC (i) prevented norovirus-induced diarrhea; (ii) markedly delayed the appearance of viral RNA and reduced viral RNA titers in the intestine, mesenteric lymph nodes, spleen, lungs, and stool; (iii) completely prevented virus-induced mortality; and (iv) resulted in protective immunity against a rechallenge. We demonstrate for the first time that a small-molecule inhibitor of norovirus replication protects from virus-induced disease and mortality in a relevant animal model. These findings pave the way for the development of potent and safe antivirals as prophylaxis and therapy of norovirus infection.

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.

Multiplexed multicolor antiviral assay amenable for high-throughput research.

To curb viral epidemics and pandemics, antiviral drugs are needed with activity against entire genera or families of viruses. Here, we develop a cell-based multiplex antiviral assay for high-throughput screening against multiple viruses at once, as demonstrated by using three distantly related orthoflaviviruses: dengue, Japanese encephalitis and yellow fever virus. Each virus is tagged with a distinct fluorescent protein, enabling individual monitoring in cell culture through high-content imaging. Specific antisera and small-molecule inhibitors are employed to validate that multiplexing approach yields comparable inhibition profiles to single-virus infection assays. To facilitate downstream analysis, a kernel is developed to deconvolute and reduce the multidimensional quantitative data to three cartesian coordinates. The methodology is applicable to viruses from different families as exemplified by co-infections with chikungunya, parainfluenza and Bunyamwera viruses. The multiplex approach is expected to facilitate the discovery of broader-spectrum antivirals, as shown in a pilot screen of approximately 1200 drug-like small-molecules.

Lower respiratory tract single-cell RNA sequencing and neutrophil extracellular trap profiling of COVID-19-associated pulmonary aspergillosis: a single centre, retrospective, observational study.

BACKGROUND: COVID-19-associated pulmonary aspergillosis (CAPA) is a severe superinfection with the fungus Aspergillus affecting patients who are critically ill with COVID-19. The pathophysiology and the role of neutrophil extracellular traps (NETs) in this infection are largely unknown. We aimed to characterise the immune profile, with a focus on neutrophils and NET concentrations, of critically ill patients with COVID-19, with or without CAPA. METHODS: We conducted a single-centre, retrospective, observational study in two patient cohorts, both recruited at University Hospitals Leuven, Belgium. We included adults aged 18 years or older who were admitted to the intensive care unit because of COVID-19 between March 31, 2020, and May 18, 2021, and who were included in the previous Contagious trial (NCT04327570). We investigated the immune cellular landscape of CAPA versus COVID-19 only by performing single-cell RNA sequencing (scRNA-seq) on bronchoalveolar lavage fluid. Bronchoalveolar lavage immune cell fractions were compared between patients with CAPA and patients with COVID-19 only. Additionally, we determined lower respiratory tract NET concentrations using biochemical assays in patients aged 18 years and older who were admitted to the intensive care unit because of severe COVID-19 between March 15, 2020, and Dec 31, 2021, for whom bronchoalveolar lavage was available in the hospital biobank. Bronchoalveolar lavage NET concentrations were compared between patients with CAPA and patients with COVID-19 only and integrated with existing data on immune mediators in bronchoalveolar lavage and 90-day mortality. FINDINGS: We performed scRNA-seq of bronchoalveolar lavage on 43 samples from 39 patients, of whom 36 patients (30 male and six female; 14 with CAPA) were included in downstream analyses. We performed bronchoalveolar lavage NET analyses in 59 patients (46 male and 13 female), of whom 26 had CAPA. By scRNA-seq, patients with CAPA had significantly lower neutrophil fractions than patients with COVID-19 only (16% vs 33%; p=0·0020). The remaining neutrophils in patients with CAPA preferentially followed a hybrid maturation trajectory characterised by expression of genes linked to antigen presentation, with enhanced transcription of antifungal effector pathways. Patients with CAPA also showed depletion of mucosal-associated invariant T cells, reduced T helper 1 and T helper 17 differentiation, and transcriptional defects in specific aspects of antifungal immunity in macrophages and monocytes. We observed increased formation of NETs in patients with CAPA compared with patients with COVID-19 only (DNA complexed with citrullinated histone H3 median 15 898 ng/mL [IQR 4588-86 419] vs 7062 ng/mL [775-14 088]; p=0·042), thereby explaining decreased neutrophil fractions by scRNA-seq. Low bronchoalveolar lavage NET concentrations were associated with increased 90-day mortality in patients with CAPA. INTERPRETATION: Qualitative and quantitative disturbances in monocyte, macrophage, B-cell, and T-cell populations could predispose patients with severe COVID-19 to develop CAPA. Hybrid neutrophils form a specialised response to CAPA, and an adequate neutrophil response to CAPA is a major determinant for survival in these patients. Therefore, measuring bronchoalveolar lavage NETs could have diagnostic and prognostic value in patients with CAPA. Clinicians should be wary of aspergillosis when using immunomodulatory therapy that might inhibit NETosis to treat patients with severe COVID-19. FUNDING: Research Foundation Flanders, KU Leuven, UZ Leuven, VIB, the Fundação para a Ciência e a Tecnologia, the European Regional Development Fund, la Caixa Foundation, the Flemish Government, and Horizon 2020.

Evaluation of 3D Human Intestinal Organoids as a Platform for EV-A71 Antiviral Drug Discovery.

Enteroviruses are a leading cause of upper respiratory tract, gastrointestinal, and neurological infections. Management of enterovirus-related diseases has been hindered by the lack of specific antiviral treatment. The pre-clinical and clinical development of such antivirals has been challenging, calling for novel model systems and strategies to identify suitable pre-clinical candidates. Organoids represent a new and outstanding opportunity to test antiviral agents in a more physiologically relevant system. However, dedicated studies addressing the validation and direct comparison of organoids versus commonly used cell lines are lacking. Here, we described the use of human small intestinal organoids (HIOs) as a model to study antiviral treatment against human enterovirus 71 (EV-A71) infection and compared this model to EV-A71-infected RD cells. We used reference antiviral compounds such as enviroxime, rupintrivir, and 2'-C-methylcytidine (2'CMC) to assess their effects on cell viability, virus-induced cytopathic effect, and viral RNA yield in EV-A71-infected HIOs and cell line. The results indicated a difference in the activity of the tested compounds between the two models, with HIOs being more sensitive to infection and drug treatment. In conclusion, the outcome reveals the value added by using the organoid model in virus and antiviral studies.

Ex vivo gut cultures of Aedes aegypti are efficiently infected by mosquito-borne alpha- and flaviviruses.

Aedes aegypti mosquitoes can transmit several arboviruses, including chikungunya virus (CHIKV), dengue virus (DENV), and Zika virus (ZIKV). When blood-feeding on a virus-infected human, the mosquito ingests the virus into the midgut (stomach), where it replicates and must overcome the midgut barrier to disseminate to other organs and ultimately be transmitted via the saliva. Current tools to study mosquito-borne viruses (MBVs) include 2D-cell culture systems and in vivo mosquito infection models, which offer great advantages, yet have some limitations. Here, we describe a long-term ex vivo culture of Ae. aegypti guts. Cultured guts were metabolically active for 7 d in a 96-well plate at 28°C and were permissive to ZIKV, DENV, Ross River virus, and CHIKV. Ex vivo guts from Culex pipiens mosquitoes were found to be permissive to Usutu virus. Immunofluorescence staining confirmed viral protein synthesis in CHIKV-infected guts of Ae. aegypti. Furthermore, fluorescence microscopy revealed replication and spread of a reporter DENV in specific regions of the midgut. In addition, two known antiviral molecules, ß-d-N4-hydroxycytidine and 7-deaza-2'-C-methyladenosine, were able to inhibit CHIKV and ZIKV replication, respectively, in the ex vivo model. Together, our results show that ex vivo guts can be efficiently infected with mosquito-borne alpha- and flaviviruses and employed to evaluate antiviral drugs. Furthermore, the setup can be extended to other mosquito species. Ex vivo gut cultures could thus be a new model to study MBVs, offering the advantage of reduced biosafety measures compared to infecting living mosquitoes. IMPORTANCE Mosquito-borne viruses (MBVs) are a significant global health threat since they can cause severe diseases in humans, such as hemorrhagic fever, encephalitis, and chronic arthritis. MBVs rely on the mosquito vector to infect new hosts and perpetuate virus transmission. No therapeutics are currently available. The study of arbovirus infection in the mosquito vector can greatly contribute to elucidating strategies for controlling arbovirus transmission. This work investigated the infection of guts from Aedes aegypti mosquitoes in an ex vivo platform. We found several MBVs capable of replicating in the gut tissue, including viruses of major health importance, such as dengue, chikungunya, and Zika viruses. In addition, antiviral compounds reduced arbovirus infection in the cultured gut tissue. Overall, the gut model emerges as a useful tool for diverse applications such as studying tissue-specific responses to virus infection and screening potential anti-arboviral molecules.

Meeting report: 36th International Conference on Antiviral Research in Lyon, France - March 13-17, 2023.

The 36th International Conference on Antiviral Research (ICAR), sponsored by the International Society for Antiviral Research (ISAR), was held March 13-17, 2023, in Lyon, France, and concurrently through an interactive remote meeting platform. Here we provide a report summarizing the presentations at the 36th ICAR, including the ISAR speaker awards. We also detail special events, sessions, and additional awards conferred at the meeting. ICAR returned to in-person meetings in 2022, convening in Seattle, WA, USA. The 36th ICAR is the first in-person meeting of the society in Europe since the beginning of the COVID-19 pandemic, which restricted most events to virtual attendance to help mitigate the spread and subsequent public health impact of SARS-CoV-2. An exceptionally high number of registrants and record attendance at this year's ICAR, along with a vast array of demonstrable expertise in a variety of antiviral research-related fields, reflected a strong and growing antiviral research community committed to improving health outcomes from viral diseases, including SARS-CoV-2, and to future pandemic preparedness. This report highlights the breadth of expertise, quality of research, and notable advancements that were contributed by members of ISAR and other participants at the meeting. ICAR aims to continue to provide a platform for sharing information, fostering collaborations, and supporting trainees in the field of antiviral research. The 37th ICAR will be held in Gold Coast, Australia, May 20-24, 2024.

The Role of Histo-Blood Group Antigens and Microbiota in Human Norovirus Replication in Zebrafish Larvae.

Human norovirus (HuNoV) is the major agent for viral gastroenteritis, causing >700 million infections yearly. Fucose-containing carbohydrates named histo-blood group antigens (HBGAs) are known (co)receptors for HuNoV. Moreover, bacteria of the gut microbiota expressing HBGA-like structures have shown an enhancing effect on HuNoV replication in an in vitro model. Here, we studied the role of HBGAs and the host microbiota during HuNoV infection in zebrafish larvae. Using whole-mount immunohistochemistry, we visualized the fucose expression in the zebrafish gut for the HBGA Lewis X [LeX, α(1,3)-fucose] and core fucose [α(1,6)-fucose]. Costaining of HuNoV-infected larvae proved colocalization of LeX and to a lower extent core fucose with the viral capsid protein VP1, indicating the presence of fucose residues on infected cells. Upon blocking of fucose expression by a fluorinated fucose analogue, HuNoV replication was strongly reduced. Furthermore, by comparing HuNoV replication in conventional and germfree zebrafish larvae, we found that the natural zebrafish microbiome does not have an effect on HuNoV replication, contrary to earlier reports about the human gut microbiome. Interestingly, monoassociation with the HBGA-expressing Enterobacter cloacae resulted in a minor decrease in HuNoV replication, which was not triggered by a stronger innate immune response. Overall, we show here that fucose has an essential role for HuNoV infection in zebrafish larvae, as in the human host, but their natural gut microbiome does not affect viral replication. IMPORTANCE Despite causing over 700 million infections yearly, many gaps remain in the knowledge of human norovirus (HuNoV) biology due to an historical lack of efficient cultivation systems. Fucose-containing carbohydrate structures, named histo-blood group antigens, are known to be important (co)receptors for viral entry in humans, while the natural gut microbiota is suggested to enhance viral replication. This study shows a conserved mechanism of entry for HuNoV in the novel zebrafish infection model, highlighting the pivotal opportunity this model represents to study entry mechanisms and identify the cellular receptor of HuNoV. Our results shed light on the interaction of HuNoV with the zebrafish microbiota, contributing to the understanding of the interplay between gut microbiota and enteric viruses. The ease of generating germfree animals that can be colonized with human gut bacteria is an additional advantage of using zebrafish larvae in virology. This small animal model constitutes an innovative alternative to high-severity animal models.