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Title: In Vitro Cell Culture Infectivity Assay for Human Noroviruses

Abstract

Human noroviruses (NoV) cause severe, self-limiting gastroenteritis that typically lasts 24 - 48 hours. The true nature of NoV pathogenesis remains unknown due to the lack of suitable tissue culture or animal models. Here we show, for the first time, that NoV can infect and replicate in an organoid, three-dimensional (3-D) model of human small intestinal epithelium (INT-407). Cellular differentiation for this model was achieved by growing the cells in 3-D 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 were employed to provide evidence of NoV infection. CPE and norovirus RNA was detected at each of the five cell passages for both 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 using differentiated monolayer cultures failed.

Authors:
; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
907937
Report Number(s):
PNNL-SA-53115
16297; 16297a; TRN: US200721%%507
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Emerging Infectious Diseases, 13(3):396-403; Journal Volume: 13; Journal Issue: 3
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; ANIMALS; BIOREACTORS; CELL CULTURES; COLLAGEN; EPITHELIUM; IN VITRO; INFECTIVITY; IN-SITU HYBRIDIZATION; MICROSCOPY; PATHOGENESIS; RNA; SHEAR; TISSUE CULTURES; VIRUSES; Norovirus; Cytopathogenic Effect, Viral (CPE); Polymerase Chain Reaction; In Situ Hybridization, Fluorescence; Microscopy, Electron; three-dimensional cell culture; Organoids; RWV bioreactor; Environmental Molecular Sciences Laboratory

Citation Formats

Straub, Tim M., Honer Zu Bentrup, Kerstin A., Orosz Coghlan, Patricia A., Dohnalkova, Alice, Mayer, Brooke K., Bartholomew, Rachel A., Valdez, Catherine O., Bruckner-Lea, Cindy J., Gerba, Charles P., Abbaszadegan, Morteza, and Nickerson, Cheryl A. In Vitro Cell Culture Infectivity Assay for Human Noroviruses. United States: N. p., 2007. Web. doi:10.3201/eid1303.060549.
Straub, Tim M., Honer Zu Bentrup, Kerstin A., Orosz Coghlan, Patricia A., Dohnalkova, Alice, Mayer, Brooke K., Bartholomew, Rachel A., Valdez, Catherine O., Bruckner-Lea, Cindy J., Gerba, Charles P., Abbaszadegan, Morteza, & Nickerson, Cheryl A. In Vitro Cell Culture Infectivity Assay for Human Noroviruses. United States. doi:10.3201/eid1303.060549.
Straub, Tim M., Honer Zu Bentrup, Kerstin A., Orosz Coghlan, Patricia A., Dohnalkova, Alice, Mayer, Brooke K., Bartholomew, Rachel A., Valdez, Catherine O., Bruckner-Lea, Cindy J., Gerba, Charles P., Abbaszadegan, Morteza, and Nickerson, Cheryl A. Tue . "In Vitro Cell Culture Infectivity Assay for Human Noroviruses". United States. doi:10.3201/eid1303.060549.
@article{osti_907937,
title = {In Vitro Cell Culture Infectivity Assay for Human Noroviruses},
author = {Straub, Tim M. and Honer Zu Bentrup, Kerstin A. and Orosz Coghlan, Patricia A. and Dohnalkova, Alice and Mayer, Brooke K. and Bartholomew, Rachel A. and Valdez, Catherine O. and Bruckner-Lea, Cindy J. and Gerba, Charles P. and Abbaszadegan, Morteza and Nickerson, Cheryl A.},
abstractNote = {Human noroviruses (NoV) cause severe, self-limiting gastroenteritis that typically lasts 24 - 48 hours. The true nature of NoV pathogenesis remains unknown due to the lack of suitable tissue culture or animal models. Here we show, for the first time, that NoV can infect and replicate in an organoid, three-dimensional (3-D) model of human small intestinal epithelium (INT-407). Cellular differentiation for this model was achieved by growing the cells in 3-D 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 were employed to provide evidence of NoV infection. CPE and norovirus RNA was detected at each of the five cell passages for both 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 using differentiated monolayer cultures failed.},
doi = {10.3201/eid1303.060549},
journal = {Emerging Infectious Diseases, 13(3):396-403},
number = 3,
volume = 13,
place = {United States},
year = {Tue Jan 30 00:00:00 EST 2007},
month = {Tue Jan 30 00:00:00 EST 2007}
}