Abstract
Human noroviruses cause severe, self-limiting gastroenteritis that typically lasts 24-48 hours. Because of the lack of suitable tissue culture or animal models, the true nature of norovirus pathogenesis remains unknown. We show, for the first time, that noroviruses can infect and replicate in a physiologically relevant 3-dimensional (3-D), organoid model of human small intestinal epithelium. This level of cellular differentiation was achieved by growing the cells on porous collagen-I coated microcarrier beads under conditions of physiological fluid shear in rotating wall vessel bioreactors. Microscopy, PCR, and fluorescent in situ hybridization provided evidence of norovirus infection. Cytopathic effect and norovirus RNA were detected at each of the 5 cell passages for genogroup I and II viruses. Our results demonstrate that the highly differentiated 3-D cell culture model can support the natural growth of human noroviruses, whereas previous attempts that used differentiated monolayer cultures failed.
Highlights
Human noroviruses cause severe, self-limiting gastroenteritis that typically lasts 24–48 hours
We present the results of our first attempts to infect a physiologically relevant 3-D small intestinal epithelium model (INT-407) with genogroup I and II human noroviruses
First Infection Trial (March 2005) This first attempt was performed with a cocktail of norovirus strains 149, 155, and flag2
Summary
Self-limiting gastroenteritis that typically lasts 24–48 hours. Noroviruses are nonenveloped, positive-sense, singlestranded RNA viruses ≈27–35 nm in diameter [6,7] They belong to the genus Norovirus in the family Caliciviridae and consist of 3 genogroups (I, II, and IV) that infect humans [8,9,10,11]. Asanaka et al [20] reported production of Norwalk virus particles (norovirus GI., the prototype strain) after transfection of cultured kidney cells. These models do not answer the fundamental questions of human norovirus attachment to, entry into, and replication within cells of the human gastrointestinal tract, and the resulting symptoms. In vitro differentiation of small intestinal epithelium that approaches physiologic functionality of the in vivo host may allow for the development of a pathogenesis model for norovirus
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