title: In vitro cell culture infectivity assay for human noroviruses.
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.
authors.affiliation: Pacific Northwest National Laboratory, Chemical and Biological Sciences Group, Richland, Washington 99352, USA. timothy.straub@pnl.gov
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Straub
authors.forename: Kerstin
authors.initials: K
authors.lastname: Höner zu Bentrup
authors.forename: Patricia
authors.initials: P
authors.lastname: Orosz-Coghlan
authors.forename: Alice
authors.initials: A
authors.lastname: Dohnalkova
authors.forename: Brooke K
authors.initials: BK
authors.lastname: Mayer
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Bartholomew
authors.forename: Catherine O
authors.initials: CO
authors.lastname: Valdez
authors.forename: Cynthia J
authors.initials: CJ
authors.lastname: Bruckner-Lea
authors.forename: Charles P
authors.initials: CP
authors.lastname: Gerba
authors.forename: Morteza
authors.initials: M
authors.lastname: Abbaszadegan
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Nickerson
mesh_terms: D017250:Caliciviridae Infections / Q000821:virology*; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D024042:Collagen Type I; D003588:Cytopathogenic Effect, Viral; D006801:Humans; D007413:Intestinal Mucosa; D008863:Microspheres; D029322:Norovirus / Q000235:genetics / Q000254:growth & development*; D012367:RNA, Viral / Q000235:genetics
chemical_list: D024042:Collagen Type I; D012367:RNA, Viral