PMID- 36342297 OWN - NLM STAT- MEDLINE DCOM- 20221125 LR - 20230508 IS - 1098-5514 (Electronic) IS - 0022-538X (Print) IS - 0022-538X (Linking) VI - 96 IP - 22 DP - 2022 Nov 23 TI - Human Norovirus Efficiently Replicates in Differentiated 3D-Human Intestinal Enteroids. PG - e0085522 LID - 10.1128/jvi.00855-22 [doi] LID - e00855-22 AB - 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 Log(10) at 2 days postinfection or up to 3.5 Log(10) 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. FAU - Mirabelli, Carmen AU - Mirabelli C AUID- ORCID: 0000-0002-4785-5482 AD - Institute for Virology and Cell Biology, University of Lubeck, Germany. AD - Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA. FAU - Santos-Ferreira, Nanci AU - Santos-Ferreira N AD - KU Leuven, Department of Microbiology, Immunology & Transplantation, Rega Institute, Laboratory of Virology & Chemotherapy, Leuven, Belgium. FAU - Gillilland, Merritt G 3rd AU - Gillilland MG 3rd AD - Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA. FAU - Cieza, Roberto J AU - Cieza RJ AUID- ORCID: 0000-0001-7400-722X AD - Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA. FAU - Colacino, Justin A AU - Colacino JA AD - Department of Environmental Health Sciences, University of Michigan, School of Public Health, Ann Arbor, Michigan, USA. FAU - Sexton, Jonathan Z AU - Sexton JZ AUID- ORCID: 0000-0002-9244-5888 AD - College of Pharmacology, University of Michigan, Ann Arbor, Michigan, USA. FAU - Neyts, Johan AU - Neyts J AUID- ORCID: 0000-0002-0033-7514 AD - KU Leuven, Department of Microbiology, Immunology & Transplantation, Rega Institute, Laboratory of Virology & Chemotherapy, Leuven, Belgium. FAU - Taube, Stefan AU - Taube S AD - Institute for Virology and Cell Biology, University of Lubeck, Germany. FAU - Rocha-Pereira, Joana AU - Rocha-Pereira J AUID- ORCID: 0000-0001-8299-3787 AD - KU Leuven, Department of Microbiology, Immunology & Transplantation, Rega Institute, Laboratory of Virology & Chemotherapy, Leuven, Belgium. FAU - Wobus, Christiane E AU - Wobus CE AUID- ORCID: 0000-0001-5286-0924 AD - Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA. LA - eng GR - UL1TR002240/UM | Michigan Institute for Clinical and Health Research (MICHR)/ GR - P30 ES017885/ES/NIEHS NIH HHS/United States GR - P30ES017885/HHS | NIH | OSC | Common Fund (NIH Common Fund)/ GR - R01 ES028802/ES/NIEHS NIH HHS/United States GR - P30DK034933/HHS | NIH | OSC | Common Fund (NIH Common Fund)/ PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20221107 PL - United States TA - J Virol JT - Journal of virology JID - 0113724 RN - 82S8X8XX8H (ruxolitinib) RN - 0 (Pyrazoles) RN - 0 (Antiviral Agents) SB - IM MH - Humans MH - *Norovirus/physiology MH - Pyrazoles MH - *Caliciviridae Infections MH - *Gastroenteritis MH - Antiviral Agents/pharmacology PMC - PMC9683019 OTO - NOTNLM OT - 3D-human intestinal enteroids OT - RNA sequencing OT - human norovirus OT - polarity inversion COIS- The authors declare no conflict of interest. EDAT- 2022/11/08 06:00 MHDA- 2022/11/26 06:00 PMCR- 2023/05/07 CRDT- 2022/11/07 09:05 PHST- 2022/11/08 06:00 [pubmed] PHST- 2022/11/26 06:00 [medline] PHST- 2022/11/07 09:05 [entrez] PHST- 2023/05/07 00:00 [pmc-release] AID - 00855-22 [pii] AID - jvi.00855-22 [pii] AID - 10.1128/jvi.00855-22 [doi] PST - ppublish SO - J Virol. 2022 Nov 23;96(22):e0085522. doi: 10.1128/jvi.00855-22. Epub 2022 Nov 7.