The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the leading cause of gastroenteritis worldwide. We report successful cultivation of multiple HuNoV strains in enterocytes in stem cell-derived, nontransformed human intestinal enteroid monolayer cultures. Bile, a critical factor of the intestinal milieu, is required for strain-dependent HuNoV replication. Lack of appropriate histoblood group antigen expression in intestinal cells restricts virus replication, and infectivity is abrogated by inactivation (e.g., irradiation, heating) and serum neutralization. This culture system recapitulates the human intestinal epithelium, permits human host-pathogen studies of previously noncultivatable pathogens, and allows the assessment of methods to prevent and treat HuNoV infections.

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The performance of a norovirus antigen detection assay was assessed using monoclonal antibody NV23 and single-chain antibody HJT-R3-A9 to identify both virus-like particles and virus-containing fecal samples. The detection of 25 different norovirus genotypes as recombinant virus-like particles or in clinical samples was dependent on virus or antigen concentration. N oroviruses are a major cause of acute nonbacterial gastroenteritis in humans (1). Noroviruses are genetically highly diverse and have been classified into seven genogroups, three of which contain human strains. These genogroups are further subdivided into at least 41 genotypes based upon the amino acid sequence of the major capsid protein, VP1 (2). The diversity of genotypes has been a barrier to the development of sensitive, broadly reactive antigen detection assays. The development of monoclonal (3) and human single-chain (4) antibodies (MAbs and single-chain variable fragment [scFv], respectively) that recognize many different norovirus strains led us to assess a combination of these reagents for identification of noroviruses in fecal samples.Recombinant norovirus virus-like particles (VLPs) served as the antigen in initial studies and were generated using a baculovirus expression system, as previously described (3). Recombinant clones were generated either by amplification of viral genomes from clinical samples or by in vitro synthesis of viral genes from GenBank sequences (Epoch Life Science, Ltd., Missouri City, TX). Expressed VLPs were purified using cesium chloride gradients. Adequacy of the VLP structure was confirmed by electron microscopy, and VLP protein concentrations were determined with a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Rockford, IL) using bovine serum albumin as a reference standard. Fecal samples collected in previous clinical studies (5, 6) were characterized for the presence of noroviruses by conventional reverse transcription (RT)-PCR and were used in the current study under protocols approved by the Baylor College of Medicine Institutional Review Board. Each sample was from a unique individual; samples were selected to represent an array of different genotypes from available specimens. Genotype was assigned by sequencing of the 5= end of the VP1 gene, and virus concentration was determined semiquantitatively by real-time RT-quantitative PCR (qPCR) with a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA) using Cog1F/Cog1R/Ring1C and Cog2F/Cog2R/Ring2 primers and probes for genogroup I (GI) and GII strains, respectively (7, 8). Viral RNA was isolated with a QIAamp viral RNA minikit (Qiagen, Mainz, Germany). A sample with a cycle threshold (C T ) value under 40 was considered positive. Negative samples were assigned a C T value of 40 for graphic display.We previously generated and characterized a panel of MAbs and scFvs that recognize norovirus antigens (3-5). For this study, we selected MAb NV23, which recognizes an epitope on the VP1 protruding domain of GI, GII, and GIV n...