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

Human norovirus (HNoV) accounts for one-fifth of all acute viral gastroenteri- tis 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-mono- layers. 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 29-C- Methylcytidine (2CMC) and other antivirals showed a reduction in viral infection, suggest- ing 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 ro- bust model to study HNoV infection, screen antivirals, and to investigate the host response to HNoV infection.