NoV VLPs specifically bound to undifferentiated and differentiated Caco-2 cells.

<p>VLPs of Ueno 7k strain were prepared in a baculovirus expression system. Purified VLPs were subjected to SDS-PAGE and examined by CBB staining (A, left) or western blotting with rabbit anti-Ueno 7k VLP serum (A, right). An arrow indicates the capsid protein, VP1. Purified VLPs were further examined by negative-stain electron microscopy (B). Scale bar in panel B = 100 nm. Caco-2 cells were incubated with (+) and without (−) 2.5 µg/ml of NoV VLPs at 4°C and fixed with paraformaldehyde. Cells were blocked and permeabilized with NETG and stained with rabbit anti-Ueno 7k VLP serum and mouse anti-ZO-1 antibody and subsequently with Alexa Fluor 568 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG, and DAPI. Immunofluorescence micrographs were obtained by confocal laser-scanning microscopy (C). Red, NoV VLPs; green, ZO-1; blue, nuclei. Scale bars in panel C = 50 µm.</p

NoV VLPs specifically bound and internalized into human small intestinal epithelium.

<p>Fresh human ileum biopsy specimens from a single individual (individual A) were incubated with 2.5 µg/ml of VLPs in PBS(-) for 1 h at 4°C, and washed three times with PBS(-). After fixation with periodate lysine paraformaldehyde, specimens were dehydrated stepwise in 10, 15, and 20% sucrose/PBS, and embedded in OCT compound. Cryostat sections at 6 µm were incubated with primary antibodies specific for VLPs and stained with the Alexa dye–conjugated secondary antibody and DAPI, and subjected to confocal laser-scanning microscopy. The results of experiment with rabbit anti-Ueno 7k VLP serum and the 5B18 mouse monoclonal antibody as primary antibody are depicted in the panels A and B, respectively. Panels b–d are high magnification views of the area in boxes in panel a. Green, NoV VLPs; blue, nuclei. Scale bars in panel a = 200 µm, and in panel d = 100 µm.</p

Binding of NoV VLPs to Caco-2 cells depended on the state of cell differentiation.

<p>Caco-2 cells were incubated with VLPs at 4°C and subjected to immunofluorescence microscopy (A). Areas of VLP binding on undifferentiated and differentiated Caco-2 cells were quantified with Adobe Photoshop software. Quantifications were performed with nine images, and Student's <i>t</i>-test was used for statistical comparisons. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066534#s3" target="_blank">Results</a> represent the means of ± S.D. of nine determinations (B). Caco-2 cells were lysed, and lysates were analyzed by SDS-PAGE/western blot with rabbit antibody against sucrase-isomaltase (SI), a differentiation marker for Caco-2 cells, with ß-actin as an internal control (ß-ACT) (C).</p

Antibodies used in this study.

1<p>rabbit serum immunized with Ueno 7k VLPs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066534#pone.0066534-Hansman1" target="_blank">[19]</a>.</p>2<p>rabbit serum immunized with 485 VLPs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066534#pone.0066534-Hansman1" target="_blank">[19]</a>.</p

NoV VLPs of 485 strain also bound and internalized to intestinal epithelium.

<p>Fresh human ileum biopsy specimens from a single individual (individual B) were incubated with 2.5 µg/ml of Ueno 7k VLPs or 485 VLPs in PBS(-) for 1 h at 4°C and subjected to immunofluorescence microscopy. Cryostat sections were incubated with rabbit anti-Ueno 7k VLP serum or rabbit anti-485 VLP serum, and stained with Alexa dye–conjugated secondary antibody. Immunofluorescence images for Ueno 7k VLPs and 485 VLPs were depicted in left and right panels, respectively. Green, NoV VLPs. Scale bar = 100 µm.</p

NoV VLPs were colocalized with each HBGA on the surface of epithelial and goblet cells.

<p>Fresh human ileum biopsy specimens from a single individual (individual B) were incubated with 2.5 µg/ml of NoV VLPs in PBS(-) for 1 h at 4°C and subjected to immunofluorescence microscopy. Cryostat sections were incubated with rabbit anti-Ueno 7k VLP serum and mouse anti-type H1, H2 or Le^b HBGA antibody and stained with Alexa dye–conjugated secondary antibodies and DAPI. Immunofluorescence images for type H1, H2 or Le^b HBGA are shown in panels A, B and C, respectively. Panels d–f and g–i are high magnification views of areas in boxes in panels a–c, respectively. Green, NoV VLPs; red, type H1 HBGA, type H2 HBGA or type Le^b HBGA. Scale bars in panel c = 100 µm, and in panel f and i = 20 µm.</p