Publication DetailsHuman Norovirus Infection of Caco-2 Cells Grown as a Three-Dimensional Tissue Structure.
Straub TM, RA Bartholomew, CO Valdez, NB Valentine, A Dohnalkova, RM Ozanich, CJ Bruckner-Lea, and DR Call.2011."Human Norovirus Infection of Caco-2 Cells Grown as a Three-Dimensional Tissue Structure."Journal of Water and Health 9(2):225-240.
Human norovirus (hNoV) infectivity was studied using a 3-dimensional model of large intestinal epithelium. Large intestine Caco-2 cells were grown in rotating wall vessel bioreactors for 18-21 days and 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 to 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 that a 2-3 Log10 increase in viral RNA copies for infected cells and no amplification in uninfected cells. A passage experiment that examined both RNA amplification and presence of viral antigen by immune electron microscopy demonstrated that hNoV amplification in this model produces infectious virions. Viral titer measured by qRT-PCR was 1-2 x 106 copies/mL. Immune electron microscopy using primary mouse monoclonal antibody to hNoV GI.1 and 6 nm gold secondary antibodies (goat anti-mouse) was performed on crude cellular lysates. These results showed significant localization of the gold secondary antibodies for the infected cells, but not for uninfected cells or infected cells where primary antibody was omitted. Our present findings coupled with earlier work with small intestinal INT407 cells support the hypothesis that the large intestine serves as a reservoir for viral replication and long-term asymptomatic shedding after acute symptoms of hNoV infection have subsided.