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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}
}
  • Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that leads to more reproducible hNoV infectivity in vitro requires that the cell line be 1) of human gastrointestinal origin, 2) expresses apical microvilli, and 3) be a positive secretor cell line. The C 2BBe1 cell line, which is a brush-border producing clone of Caco-2, meetsmore » these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log10 increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both reverse transcription quantitative PCR (qRT-PCR) and microscopy. Using 3-D C 2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using quantitative reverse transcription PCR (qRT-PCR) that measures all RNA vs. plaque assays that measure infectious virus.« less
  • We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay willmore » significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools« less
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