In Vitro Cell Culture Infectivity Assay for Human Noroviruses

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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 that noroviruses can infect and replicate in a 3-dimensional (3-D), organoid model of human small intestinal epithelium. Cells grown on porous collage-coated beads under fluid shear conditions in rotating wall vessel bioreactors differentiate into 3-D architectures resembling both the morphologic and physiologic function of in vivo tissues. 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.