Evaluation of anti-noroviral compounds on human norovirus replication using 2D monolayers of human intestinal organoids

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Evaluation of anti-noroviral compounds on human norovirus replication using 2D monolayers of human intestinal organoids Tsuyoshi Hayashi , Yoshiki Fujii , Junki Hirano , Sakura Kobayashi , Kosuke Murakami doi: https://doi.org/10.1101/2025.04.25.650600 Tsuyoshi Hayashi 1 Department of Virology II, National Institute of Infectious Diseases , Gakuen 4-7-1, Musashimurayama-shi, Tokyo 208-0011, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: hayashit{at}niid.go.jp Yoshiki Fujii 1 Department of Virology II, National Institute of Infectious Diseases , Gakuen 4-7-1, Musashimurayama-shi, Tokyo 208-0011, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Junki Hirano 2 Laboratory of Viral Control, Research Institute for Microbial Diseases, University of Osaka , Osaka, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sakura Kobayashi 3 Center for Emergency Preparedness and Response, National Institute of Infectious Diseases , Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kosuke Murakami 1 Department of Virology II, National Institute of Infectious Diseases , Gakuen 4-7-1, Musashimurayama-shi, Tokyo 208-0011, Japan 3 Center for Emergency Preparedness and Response, National Institute of Infectious Diseases , Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Preview PDF Abstract We evaluated the anti-noroviral effects of compounds including molnupiravir, on human norovirus (HuNoV) replication using 2D monolayers of human intestinal organoids (HIOs), which have been widely used in HuNoV infection studies. We found that these compounds exhibited lower antiviral activity in 2D HIO monolayers compared with that in apical-out 3D HIOs, a recently developed culture system for HuNoV. This suggests that the anti-noroviral efficacy of these compounds may vary depending on the assay system used. Main text Human norovirus (HuNoV) is a major cause of acute gastroenteritis worldwide, affecting individuals across all age groups ( 1 ). HuNoV infection results in symptoms, including nausea, vomiting, and diarrhea. Although typically self-limiting and resolving within a few days to a week in healthy individuals, HuNoV is capable of causing chronic infection in vulnerable populations such as immunocompromised patients. In such cases, it may lead to prolonged viral shedding and gastroenteritis with severe complications ( 2 , 3 ). Despite its clinical relevance, no antiviral therapies or vaccines are currently available. Efforts to develop anti-HuNoV agents have been limited, partly due to the lack of a reproducible laboratory model for HuNoV replication until recent developments. In 2016, Ettayebi et al . developed a novel HuNoV cultivation system using human intestinal organoids (HIOs) ( 4 ). By culturing HIOs as 2D monolayers (See Protocol A in Figure 1 ), they demonstrated that HuNoVs with diverse genotypes (e.g., GII.3, GII.4, and GII.17) can efficiently replicate in these cells, achieving up to approximately a 3 log 10 -fold increase in viral RNA ( 4 , 5 ). This cultivation system and infection protocol using 2D HIO monolayers have been widely adopted in norovirus laboratories and are currently used for various HuNoV studies, including those on infection mechanisms, antiviral screening, and virus inactivation (reviewed in references ( 6 , 7 )). In antiviral studies, several anti-HuNoV drug candidates have been reported; however, their effects are generally weak to moderate (at μ M scale) ( Table 1 ) ( 7 ). View this table: View inline View popup Download powerpoint Table 1. Summary of anti-noroviral compound’s effect assayed by means of 2D HIO monolayers or apical-out 3D HIOs. Download figure Open in new tab Figure 1. HuNoV infection using 2D HIO monolayers and apical-out 3D HIOs It is assumed that HuNoV infects host cells via the apical side, which is located inside 3D HIOs. To enhance HuNoV infection efficiency, two protocols have been developed: Protocol (A): 3D HIOs are dissociated into single cells, seeded onto a 96-well plate, and cultured as 2D monolayers. Consequently, the apical cell surface is located on the upper side, allowing HuNoV to efficiently bind and infect the cells ( 5 , 14 ). Protocol (B); Mirabelli et al . recently reported that during differentiation, 3D HIOs can spontaneously adapt an apical-out orientation, which enables efficient HuNoV infection ( 8 ). More recently, Mirabelli et al . developed an alternative HuNoV infection protocol using apical-out 3D HIOs, which reportedly support efficient HuNoV replication (See Protocol B in Figure 1 ) ( 8 ). Using this system, Santos-Ferreira et al . demonstrated that molnupiravir, an antiviral known to show inhibitory effects on several RNA viruses (e.g., SARS-CoV-2 and influenza virus) through mutagenesis, strongly inhibits HuNoV replication in 3D HIOs ( 9 ). Due to its low 50% effective concentration (EC 50 ) (1.0 μ M), molnupiravir is considered a promising candidate for anti-HuNoV therapy ( 9 ). In this study, the efficacy of other previously reported anti-noroviral compounds, including 2′-C-methylcytidine (2-CMC), nitazoxanide, and dasabuvir, was also tested ( 9 ). Interestingly, 2-CMC and nitazoxanide demonstrated over 20-fold greater antiviral activity (lower EC 50 values) in the 3D HIO system compared with prior results from 2D monolayer assays ( Table 1 ) ( 10 , 11 ). This discrepancy prompted us to re-evaluate the anti-noroviral effects of these compounds, including molnupiravir, using 2D HIO monolayers. Differentiated 2D HIO monolayers, derived from 3D HIOs (J2 line), were inoculated with genotype GII.4 or GII.3 HuNoV-positive stool samples in the presence of anti-noroviral compounds in a 100 μ L volume. The infection was maintained for 24 h post-inoculation. Subsequently, the cells and 75 μ L of the culture medium were harvested for RNA extraction, followed by reverse transcription quantitative PCR (RT-qPCR) analysis to assess viral replication, as previously described ( 12 ). The remaining 25 μ L of medium was used to evaluate cytotoxicity. The EC 50 and the half-maximal cytotoxic concentration (CC 50 ) for each compound were calculated using GraphPad Prism 9 software. Consistent with previous findings using apical-out 3D HIOs, molnupiravir and its active metabolite, β-d-N4-hydroxycytidine (NHC), showed dose-dependent inhibition of GII.3 and GII.4 HuNoV replication without compromising cell viability ( Figure 2 and Table 1 ) ( 9 ). However, their EC 50 values were 45.4- to 238.4-fold higher than those reported in 3D HIOs, indicating reduced antiviral efficacy ( Table 1 ). Similar trends were observed for 2-CMC and nitazoxanide ( Table 1 ). In contrast, the difference in EC 50 for 7-deaza-2′-C-methyladenosine (7-DMA) was relatively small (approximately 6-fold), almost comparable to that of dasabuvir ( Table 1 ). Although its antiviral effect was modest, favipiravir inhibited HuNoV replication in apical-out 3D HIOs (EC 50 = 82.1 μ M; SI > 12.2), whereas its effect was minimal in 2D monolayers (EC 50 = 801.2 μ M; SI > 1.0) ( Figure 2 , Table 1 ). These findings suggest that compounds showing weak inhibitory effects in 3D HIOs may have negligible efficacy in 2D monolayer systems. Download figure Open in new tab Figure 2. Effect of anti-noroviral compounds on HuNoV replication in 2D HIO monolayers 2D HIO monolayers were infected with GII.4 or GII.3 HuNoV in the presence of the indicated compounds for 24 h. The cells and supernatants were harvested for RNA extraction, followed by RT-qPCR to quantify HuNoV RNA copies. Experiments were conducted in at least three independent replicates with two or three technical replicates each. Results are normalized to the DMSO control and presented as the mean ± standard deviation (n ≧ 3). EC 50 , 50% effective concentration; CC 50 , 50% cytotoxic concentration. The reason for the weaker inhibitory effects of the compounds in the 2D monolayers compared with those in 3D HIOs remains unclear. One possibility is that differences in viral replication efficiency may contribute to the variation in antiviral effects. HuNoV replicates more rapidly in 2D monolayers than in 3D HIOs; in 2D monolayers, viral RNA levels increased by 100- to 1,000-fold at 24 h post-infection relative to the input (Figure S1), whereas viral infection in 3D HIOs resulted in approximately 100-fold increases at 48 h ( 9 ). Therefore, compounds may have more time to exert inhibitory effects in the context of slower viral replication in 3D HIOs. Additional studies, such as head-to-head comparisons, are needed to elucidate the mechanisms underlying these differences. A previous study identified molnupiravir as a promising anti-HuNoV candidate (EC 50 = 1.0 μ M in 3D HIOs), while our result indicates that its antiviral efficacy may be overestimated in 3D HIO system. Meanwhile, it should be noted that none of the current HuNoV culture systems, including 2D and 3D HIOs, perfectly reflect acute or persistent HuNoV infections in real-world clinical settings where antiviral treatment would be required. Although significant improvements in the HIO culture system have been achieved through optimized culture media conditions and genetic modifications ( 5 , 13 ), HuNoV replication efficiency remains lower than that observed during acute infection in humans. In addition, no relevant in vitro system mimicking chronic HuNoV infection in humans has been developed, highlighting the need for further optimization and/or development of culture models to more accurately assess anti-noroviral efficacy. Supplemental material Figure S1. Growth kinetics of HuNoV in 2D HIO monolayers 2D HIO monolayers were infected with GII.4 or GII.3 HuNoV. At the indicated time points, cells and supernatants were collected to determine viral RNA copy numbers, as shown in Figure 2 . Experiments were performed thrice with four technical replicates. Results are presented as the mean ± standard deviation (n =12). Author Contributions Conceptualization, T.H.; methodology, T.H.; software, T.H.; validation, T.H.; formal analysis, T.H.; investigation, T.H., Y.F., S.K., and K.M.; resources, T.H., Y.F., J.H., and K.M.; data curation, T.H.; writing—original draft preparation, T.H.; writing—review and editing, Y.F., J.H., S.K., and K.M.; visualization, T.H. and J.H.; supervision, T.H.; project administration, T.H.; funding acquisition, T.H., Y.F., and K.M. All authors have read and agreed to the published version of the manuscript. Funding This study was financially supported by the Japan Society for the Promotion of Science (KAKENHI grant JP23K18381 to T.H. and K.M., and grant JP21H02743 to K.M.) and the Japan Agency for Medical Research and Development (AMED) (grant JP23fk0108669 to T.H., and grant JP23fk0108667 to Y.F., and K.M.). Declaration of competing interest The authors declare no conflict of interest. Acknowledgments We thank Chizuko Hirano (National Institute of Infectious Diseases, Japan) for technical assistance and Enago ( www.enago.jp ) for the English language review. References 1. ↵ Robilotti E , Deresinski S , Pinsky BA . 2015 . Norovirus . Clin Microbiol Rev 28 : 134 – 64 . OpenUrl Abstract / FREE Full Text 2. ↵ Petrignani M , Verhoef L , de Graaf M , Richardus JH , Koopmans M. 2018 . Chronic sequelae and severe complications of norovirus infection: A systematic review of literature . J Clin Virol 105 : 1 – 10 . OpenUrl CrossRef PubMed 3. ↵ Bok K , Green KY . 2012 . Norovirus gastroenteritis in immunocompromised patients . N Engl J Med 367 : 2126 – 32 . OpenUrl CrossRef PubMed Web of Science 4. ↵ Ettayebi K , Crawford SE , Murakami K , Broughman JR , Karandikar U , Tenge VR , Neill FH , Blutt SE , Zeng XL , Qu L , Kou B , Opekun AR , Burrin D , Graham DY , Ramani S , Atmar RL , Estes MK . 2016 . 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