In vitro cell culture infectivity assay for human noroviruses.
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.
Straub
Timothy M
TM
Pacific Northwest National Laboratory, Chemical and Biological Sciences Group, Richland, Washington 99352, USA. timothy.straub@pnl.gov
Höner zu Bentrup
Kerstin
K
Orosz-Coghlan
Patricia
P
Dohnalkova
Alice
A
Mayer
Brooke K
BK
Bartholomew
Rachel A
RA
Valdez
Catherine O
CO
Bruckner-Lea
Cynthia J
CJ
Gerba
Charles P
CP
Abbaszadegan
Morteza
M
Nickerson
Cheryl A
CA
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
D024042:Collagen Type I; D012367:RNA, Viral