期刊
JOURNAL OF WATER AND HEALTH
卷 9, 期 2, 页码 225-240出版社
IWA PUBLISHING
DOI: 10.2166/wh.2010.106
关键词
cell culture; infectivity; in vitro organoids; noroviruses; quantitative PCR
资金
- National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services [NO1-AI-30055]
- United States Environmental Protection Agency [R833831010]
- Department of Energy's Office of Biological and Environmental Research located at Pacific Northwest National Laboratory
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [N01AI030055] Funding Source: NIH RePORTER
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 degrees 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 log(10) 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 x 10(6) 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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