Abstract
Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional (3-D) 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 lead 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 C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log(10) increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microscopy. In our hands, using 3-D C2BBe1 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 qRT-PCR that measures all RNA vs. plaque assays that measure infectious virus.
Highlights
The human noroviruses continue to be one of the most difficult viruses to study due to the challenge of developing a relevant and robust in vitro model for their study (Duizer et al ; Asanaka et al ; Guix et al ; Straub et al, )
Based on the literature and our observations, secretor status is important. Combining these factors of 3-D organotypic gastrointestinal cell cultures, apical expression of microvilli, and positive secretor status, we present new data, not discussed in Straub et al ( ), on improvements in reproducibility for the in vitro infectivity assay for human noroviruses (hNoV)
We present the results of three independent trials where NoV GGII outbreak isolate 8G was used to infect the cells. Two of these studies are passage 0 (P0) trials: Diluted stool sample was applied to the cells and allowed to infect the cells (Table 1)
Summary
The human noroviruses (hNoV) continue to be one of the most difficult viruses to study due to the challenge of developing a relevant and robust in vitro model for their study (Duizer et al ; Asanaka et al ; Guix et al ; Straub et al , ). There are several conflicting reports regarding the actual cell type in the gastrointestinal tract that hNoV infect. Wobus et al ( ) reported that macrophages are the primary cell type. Lay et al ( ) found that hNoV does not infect human macrophages. Two studies have reported localization of noroviruses in the cells of the lamina propria and Brunner’s glands (Bok et al ; Chan et al ). Questions regarding what cell types should comprise a relevant in vitro cell culture system for the hNoV remains in question
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