Human norovirus (hNoV) infectivity was studied using a three-dimensional model of large intestinal epithelium. Large intestine Caco-2 cells were grown in rotating wall vessel bioreactors for 18–21 days at 37°C and then transferred to 24-well tissue culture plates where they were infected with GI.1 and GII.4 human noroviruses collected from human challenge trials and various outbreak settings, respectively. Compared with uninfected cells, transmission micrographs of norovirus-infected cells displayed evidence of shortening or total loss of apical microvilli, and vacuolization. Quantitative reverse transcription real-time PCR (qRT-PCR) indicated an approximate 2–3 log10 increase in viral RNA copies for the infected cells. A passage experiment examined both the ability for continued viral RNA and viral antigen detection. In the passaged samples 1.01 × 106 copies ml−1 were detected by qRT-PCR. Immune electron microscopy using primary antibody to hNoV GI.1 capsids in conjunction with 6 nm gold-labelled secondary antibodies was performed on crude cellular lysates. Localization of antibody was observed in infected but not for uninfected cells. Our present findings, coupled with earlier work with the three-dimensional small intestinal INT407 model, demonstrate the utility of 3-D cell culture methods to develop infectivity assays for enteric viruses that do not readily infect mammalian cell cultures.
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Research Article|
December 23 2010
Human norovirus infection of Caco-2 cells grown as a three-dimensional tissue structure
Timothy M. Straub;
1Pacific Northwest National Laboratory, Chemical and Biological Signature Sciences Group, National Security Directorate, PO Box 999 MS P7-50, Richland, WA 99354, USA
Tel.: +1 (509) 371-6961; E-mail: [email protected]
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Rachel A. Bartholomew;
Rachel A. Bartholomew
1Pacific Northwest National Laboratory, Chemical and Biological Signature Sciences Group, National Security Directorate, PO Box 999 MS P7-50, Richland, WA 99354, USA
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Catherine O. Valdez;
Catherine O. Valdez
1Pacific Northwest National Laboratory, Chemical and Biological Signature Sciences Group, National Security Directorate, PO Box 999 MS P7-50, Richland, WA 99354, USA
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Nancy B. Valentine;
Nancy B. Valentine
1Pacific Northwest National Laboratory, Chemical and Biological Signature Sciences Group, National Security Directorate, PO Box 999 MS P7-50, Richland, WA 99354, USA
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Alice Dohnalkova;
Alice Dohnalkova
1Pacific Northwest National Laboratory, Chemical and Biological Signature Sciences Group, National Security Directorate, PO Box 999 MS P7-50, Richland, WA 99354, USA
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Richard M. Ozanich;
Richard M. Ozanich
1Pacific Northwest National Laboratory, Chemical and Biological Signature Sciences Group, National Security Directorate, PO Box 999 MS P7-50, Richland, WA 99354, USA
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Cynthia J. Bruckner-Lea;
Cynthia J. Bruckner-Lea
1Pacific Northwest National Laboratory, Chemical and Biological Signature Sciences Group, National Security Directorate, PO Box 999 MS P7-50, Richland, WA 99354, USA
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Douglas R. Call
Douglas R. Call
2Washington State University, Veterinary Microbiology and Pathology, 402 Bustad Hall, PO Box 647040, Pullman, WA 99164-7040, USA
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J Water Health (2011) 9 (2): 225–240.
Article history
Received:
June 01 2010
Revision Received:
September 19 2010
Citation
Timothy M. Straub, Rachel A. Bartholomew, Catherine O. Valdez, Nancy B. Valentine, Alice Dohnalkova, Richard M. Ozanich, Cynthia J. Bruckner-Lea, Douglas R. Call; Human norovirus infection of Caco-2 cells grown as a three-dimensional tissue structure. J Water Health 1 June 2011; 9 (2): 225–240. doi: https://doi.org/10.2166/wh.2010.106
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