No evidence of Norovirus replication in 3D Int-407 cells.

<p>The RT-qPCR data are expressed as the average log RNA copies/ml of at least two replicates ± standard deviation.</p>*<p>cells were obtained from Arizona State University; ** indicates a mixed infection; nd: not determined.</p

No evidence of Norovirus replication in 3D Caco-2 cells.

<p>No significant increase of RNA copy number was detected in supernatants of Caco-2 cells harvested at 0, 24 and 48 hours post inoculation. Different norovirus strains and different virus concentrations were evaluated. Viral RNA was detected by quantitative reverse transcription-realtime RT-PCR. Inoculation of 3 different concentrations (MOI 1, 10, 100) of: A) GII.4 (#19 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063485#pone-0063485-t001" target="_blank">table 1</a>) on 3D Caco-2 cultures (Lab B) and B) GI.3 strain (#18 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063485#pone-0063485-t001" target="_blank">table 1</a>) on 3D Caco-2 cultures (Lab B). C) Comparison of 2D and 3D Caco-2 cells by GI.3 (#7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063485#pone-0063485-t001" target="_blank">table 1</a>) and GII.4 (#6 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063485#pone-0063485-t001" target="_blank">table 1</a>) (Lab A).</p

Morphology of 3D Int-407 cells after inoculation with norovirus.

<p>Images A and B: control Int-407 cells (left) and (right) Int-407 cells inoculated with GII.4 norovirus at 48 h post inoculation (light microscopy, 1∶20 magnification).</p

Immunohistochemical imaging of differentiation markers in Int-407 and Caco-2 cells.

<p>Image A. β-catenin localization in three-dimensional structures of Caco-2 cells after 3 weeks in culture in STLV (1∶40). Image B. Localization of ZO-1 in 3D of Int-407 cells after 4 weeks of culture in STLV and HARV (1∶40). Image C. Distribution of claudin in 3D of Caco-2 cells after 3 weeks in culture in STLV (cell nuclei were stained with DAPI, 1∶40 magnification).</p

Electron micrographs of microvilli development in Caco-2 cells during a 5-week growth period.

<p>Caco-2 cells were cultured in GTSF-2 medium for up to 5 weeks at 37°C. At 2, 3 and 5 weeks of culture, under either 2D, or 3D conditions in a rotating wall vessel, cells were removed and processed for visualization under electron microscope. Top Panel [A (1200x), B (440x), C (1400x)]: 3D structures Lower Panel [D (690x), E (890x), F (690x)]: 2D structures.</p

Virolysis patterns of GI.1, GI.3b and GI.6 norovirus strains after exposure to ethanol (E) and isopropyl alcohol (I) at 3 different concentrations (50%, 70%, and 90%).

<p>3 GI.1, 4 GI.3b and 4 GI.4 strains with three replicate per each were consolidated by genotype and were expressed as a box plot. The upper, lower ends of the box and the horizontal line in the box indicate the first (Q1), third quantiles (Q3) and median value of all data, respectively. The lower and higher ends of whiskers indicate the minimum and maximum value of all data, respectively. Isolated data points are outliers.</p

P2 subdomain (amino acids (aa) 279–405) sequence alignment of GII.4 Den Haag, GII.4 New Orleans, and GII.4 Sydney viruses.

<p>Key amino acid changes, S310N, andN341D, and P396H are indicated with black boxes.</p

Percent of enteric virus co-infections with norovirus or sapovirus.

<p>Percent of enteric virus co-infections with norovirus or sapovirus.</p

Virolysis patterns of GII norovirus strains after exposure to ethanol and isopropanol.

<p>Virolysis patterns of GII norovirus strains after exposure to ethanol and isopropanol.</p

Virolysis patterns of GII.4 variants including GII.4 Den Haag, GII.4 New Orleans and GII.4 Sydney viruses after exposure to ethanol (E) and isopropyl alcohol (I) at 3 different concentrations (50%, 70%, and 90%).

<p>2 GII.4 Den Haag, 3 GII.4 New Orleans and 4 GII.4 Sydney strains with three replicates were consolidated by genotype and were expressed as a box plot. The upper, lower ends of the box and the horizontal line in the box indicate the first (Q1), third quantiles (Q3) and median value of all data, respectively. The lower and higher ends of whiskers indicate the minimum and maximum value of all data, respectively. Isolated points are outliers.</p