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 biopsies-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 differentiate as efficiently as 2D-monolayers. In addition, immunofluorescence-based quantification of UEA-1, a lectin that stains the villus brush border, revealed that over 90% 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 post infection 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), other antivirals, or with a HNoV-neutralizing antibody showed a reduction in viral infection, suggesting that 3D-HIE are an excellent platform to test anti-infectives. The host response to HNoV was then investigated by RNA sequencing in infected versus uninfected 3D-HIE, in the presence of ruxolitinib to focus on viral-associated signatures. The analysis revealed upregulated hormones and neurotransmitter signal transduction pathways and downregulated inflammatory pathways upon HNoV infection. Overall, 3D-HIE have proven to be a more robust model to study HNoV infection, screen antivirals and investigate host response to HNoV infection. Importance Human norovirus (HNoV) clinical and socio-economic impact calls for immediate actions in the development of anti-infectives. Physiologically-relevant in vitro models are hence needed to study HNoV biology, tropism and mechanism of viral-associated disease but also as a platform to identify antiviral agents. Biopsy-derived human intestinal enteroids are a biomimetic of the intestine and recently described as a model that supports HNoV infection. 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 undergo 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 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.


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Diarrheal diseases are the fourth cause of death worldwide and the second cause of morbidity in 57 children less than 5 years old(1). In particular, human norovirus (HNoV) is the main causative agent 58 of viral gastroenteritis worldwide, with a clinical burden of nearly 200,000 hospitalizations in 59 Europe(2) and an economic burden of 65 billion US dollars, worldwide(1). The virus has been poorly 60 characterized in terms of its viral life cycle, tropism, and pathophysiology due to the lack of easy-to-61 use cell culture systems that support robust viral replication and ensure the production of cell culture- reversal. The mechanism is dependent on extracellular matrix protein (ECM) concentration, because 80 addition of ECM to HIE in the absence of BME blocks polarity reversion(5). Apical-Out HIE thus 81 provide a potential solution to ensure greater accessibility of 3D-HIE to pathogens.

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The induction of the host innate response plays an essential role in the suppression of pathogen 83 infection. In the case of enteric pathogens, interferon responses are highly upregulated in vivo and in 84 vitro(6, 7). We previously reported that human astrovirus VA1, another enteric virus and causative 85 agent of viral gastroenteritis, induces interferon and interferon-stimulated genes (ISG) in the HIE 86 system but not in an immortalized cell line, the Caco2 cells(8). This also suggest that, intrinsically, the 87 HIE culture restricts enteric virus infection more efficiently than immortalized cell lines. For this 88 reason, a JAK inhibitor, ruxolitinib, has been successfully added to the infection protocol to prevent 89 stimulating the innate immune response and thus increase the yield of HNoV in HIE(9).
Here, we describe the establishment of a HNoV infection protocol in 3D-HIE. We characterized the 91 differentiation state of 3D-HIE compared to 2D-HIE and the degree of polarity inversion in 3D-HIE 92 upon differentiation. We demonstrate that 3D-HIE are amenable to infection with HNoV with a 93 reduction of experimental time (9 vs 15 days) and increased yield (fold increase over inculum) and 94 reproducibility. We also characterized the host response of 3D-HIE to HNoV infection in culture 95 treated with ruxolitinib by RNA sequencing. Altogether, we describe an adapted protocol for HNoV 96 infection in 3D-HIE that is amenable to antiviral discovery and virus-host interaction studies. In

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Differentiation of HIE is achieved by withdrawal of Wnt3a from the maintenance media, that triggers 104 the development of a heterogeneous, terminally differentiated epithelium by day 6(8). We sought to 105 determine if HIE derived from fetal ileum HT124 undergo terminal differentiation when maintained 106 in 3D in basal membrane extract (BME). Towards that end, we monitored the mRNA levels of 107 differentiation markers by RT-qPCR. As a control, 2D-monolayers of HT124 HIE were also prepared 108 and differentiated in collagen-coated plates. At 6 days post differentiation, comparison of mRNA 109 levels of Lgr5, lysozyme, mucin 2 (MUC2) and sucrase isomaltase (SI) as markers of stem-cells,

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Paneth cells, goblet cells and mature enterocytes, respectively, revealed similar transcript levels in 111 differentiated 2D versus 3D-HIE ( Figure 1A). This suggests that the withdrawal of Wnt3a triggers 112 terminal differentiation of 3D-HIE in BME to the same extent and in the same time frame as 2D-HIE.

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Representative images of differentiated 2D-and 3D-HIE are shown in Figure 1B. To address whether 114 differentiated 3D-HIE could support HNoV infection, GII.4-positive stool samples were used to infect 115 the 3D-HIE, after removal from BME. After a 2 h incubation at 37°C in the presence of the bile acid 116 GCDCA (500µM), the inoculum was washed off, one set of 3D-HIE was harvested in TRI Reagent (2 117 h), while another set was re-embedded in BME and kept at 37°C for 2 days (2 d) in differentiation media supplemented with GCDCA (500µM) and a JAK inhibitor ruxolitinib (2µM). Differentiated 119 2D-HIE were infected in parallel according to the published protocol, also in the presence of GCDCA 120 (500µM) and ruxolitinib (2µM). Quantification of HNoV yields by RT-qPCR revealed a 2.5 Log 10 121 versus 1 Log 10 of fold increase in 3D-HIE versus 2D-HIE, that was achieved over only 2 d post 122 infection (dpi) ( Figure 1C). These data suggest that 3D-HIE differentiation is sufficient to support 123 HNoV infection, and that in our system, the protocol of infection of 3D-HIE results in better yield 124 (fold increase over inoculum) as compared with the established 2D-HIE protocol.

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Efficient infection of 3D-HIE by HNoV suggest that the virus has access to the apical surface of 129 enterocytes. Interestingly, 3D-HIE have been described to revert polarity spontaneously after removal 130 of BME. The inversion is dependent on the percentage of extracellular matrix (ECM) proteins in the 131 media(5). Because our differentiation protocol starts 3-4 days after passaging, resulting in 3D-HIE 132 being embedded in BME for ~9-10 days, we hypothesized that the consequent reduction in % ECM 133 proteins (caused by medium changes) could trigger spontaneous inversion. We also sought to 134 determine whether the process of terminal differentiation, with Wnt3a withdrawal, may further 135 enhance the polarity inversion. To this end, we differentiated 3D-HIE for 6 days and as a control, we 136 kept in parallel 3D-HIE in proliferation media ( Figure 2A). Next, differentiated (-Wnt3A) and 137 undifferentiated (+Wnt3a) 3D-HIE were removed from BME, fixed with paraformaldehyde (4% in 138 PBS-/-) and stained with DAPI for nuclear detection and Ulex Europeus Agglutinin-1 (UEA-1), a 139 lectin that binds the α-L-fucosyl residues of glycoproteins and glycolipids at the apical surface of the 140 intestinal epithelium(8). For ease of analysis, 10-20 3D-HIE were transferred into a well of a poly-141 lysin-coated black 96-well plate and subjected to confocal microscopy imaging with the CellVoyager 142 CQ1 high-content microscope (Yokogawa). Image segmentation by Cell Profiler and analysis of the 143 UEA-1 positive 3D-HIE revealed that after 10 days of culturing in proliferation media, about 50% of 144 3D-HIE spontaneously inverted polarity. In differentiation media, the proportion of UEA-1-positive 145 spheroids was ~ 90% ( Figure 2B). Because a protocol of polarity inversion was recently published(5), we next wanted to compare the efficacy of replication in 3D-HIE generated with the published 147 protocol (3D-HIE without BME, Apical-Out) or with our protocol (3D-HIE embedded in BME).

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Interestingly, the efficacy of replication at 2 dpi was >2 Log 10 higher in ECM 3D-HIE than in HIE 149 generated with the Apical-Out protocol ( Figure 3C). These data suggest that the apical surface of 150 differentiated 3D-HIE is exposed and explain why these cultures are amenable to infection with

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HNoV without the burden of microinjection. In addition, BME appears to support infection by HNoV 152 as Apical-Out HIE did not reach the same yield of infection.

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We observed a 1 Log 10 increase at 2 dpi versus 2 hpi ( Figure 3B), suggesting that infection occurs in 164 this adult line also in the 3D-format but the permissiveness of infection is reduced as compared to the 165 fetal line HT124. We next infected 3D-HIE with increasing doses of HNoV-positive stool samples 166 ( Figure 3C). A dose-dependent increase of replication was observed at 2 dpi. However, the fold 167 increase between 2 dpi and 2 hpi remained constant at ~2.5 Log 10 , suggesting that the system supports 168 a defined amount of infection regardless of the initial infectious dose.

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To test whether the infection model could be used to test antivirals, we lastly infected 3D-HIE in the 170 presence of compounds that were previously described as active against norovirus: Nitazoxanide, and 171 its active metabolite TZX(10), the protease inhibitor Rupintrivir(11); and the polymerase inhibitor 172 Favipiravir ( Figure 3D). We also performed a neutralization assay with an antibody raised against nitazoxanide, TZX and Rupintrivir. Higher concentrations of Favipiravir could potentially show an 175 antiviral effect, as no toxicity was observed at the concentration tested. Altogether, these data clearly 176 demonstrate that 3D-HIE are amenable to infection with HNoV and to medium-throughput screening 177 of anti-infectives. infection. The data presented herein demonstrates that HIE dispersion is not necessary for a successful 220 HIE differentiation and that 3D-HIE spontaneously revert polarity upon differentiation, even if they 221 are kept in BME. This finding was puzzling as a recent report suggests that HIE polarity is dependent 222 on % ECM proteins(5). We reasoned that in our protocol, the 3D-HIE are kept in BME for more than 223 the canonical 6 days and with every medium change, the concentration of ECM proteins could hence 224 decrease. However, we also observed that in our model, terminal differentiation exacerbates the that the BME provides an environment that is beneficial for a sustained viral infection.

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We further demonstrated that this model is amenable to antiviral studies as we successfully reported 237 activity of compounds that are endowed with antiviral activity against HNoV, such as Nitazoxanide 238 and its prodrug TZX (10) consequence of viral replication, we included ruxolitinib, an inhibitor of JAK 1/2 and the downstream 247 STAT-1 signaling, that limits interferon signaling. As a consequence, genes under the control of 248 STAT-1 that might be upregulated upon infection will be reduced in expression. To our surprise, 249 amongst downregulated pathways, we found HIF-1 pathways and glycolysis, that we previously

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Our RNA sequencing analysis could also shed some light into norovirus mechanisms of pathogenesis.