Fold changes in HuNoV and HuSaV RNA levels at 120 hpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV or HuSaV in medium supplemented with bacteria or the culture supernatants of co-culturing bacteria and immune cells.

<p>Fold changes in HuNoV and HuSaV RNA levels at 120 hpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV or HuSaV in medium supplemented with bacteria or the culture supernatants of co-culturing bacteria and immune cells.</p

Fold changes in HuNoV RNA levels at 6 or 9 dpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV GII.4 mixture in long-term cultured HT29-Cl.16E and Caco-2 cells.

<p>Fold changes in HuNoV RNA levels at 6 or 9 dpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV GII.4 mixture in long-term cultured HT29-Cl.16E and Caco-2 cells.</p

Fold changes in HuNoV RNA levels at 96 hpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV or HuSaV.

<p>Fold changes in HuNoV RNA levels at 96 hpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV or HuSaV.</p

Neither the IRF3 nor NFκB singaling pathway appears to be involved in decreased IFN-α responses induced by simvastatin.

<p>mRNA expression levels of IRF3 (<b>A</b>) and NFκB (<b>B</b>) in simvastatin-treated macrophages at 9 and 24 hours after poly (I:C) stimulation. The mRNA expression levels were measured by qRT-PCR and normalized to the expression levels of β-actin. Each bar represents the mean ± SEM. Data from 2 independent experiments were combined, and each test was performed in triplicate.</p

Simvastatin impairs TLR3-mediated induction of type I IFN and diminishes IFN-α levels from pulmonary macrophages (PAMs) <i>in vitro,</i> possibly due to lowered expression of TLR3 after treatment.

<p>The PAMs (2∼4×10⁶ cells/ml) were first treated with 1 µM simvastatin, and 24 hours later, were treated with either simvastatin (1 µM) or poly (I:C) (25 µg/ml), or treated with both. The cell culture supernatants were harvested to measure IFN-α levels (<b>A</b>) by biological assay at 4 and 24 hours afters poly (I:C) treatment. Each bar represents the mean ± SEM. Different letters denote significant differences among groups at each time-point (ANOVA test, <i>P</i><0.05). (<b>B, C</b>) Flow cytometry of TLR3⁺ cells after simvastatin treatment. PBMC-derived macrophages (2∼5×10⁶ cells/ml) were first treated with 1 µM simvastatin, and 24 hours later, were treated with either simvastatin (1 µM) or poly (I:C) (25 µg/ml), or both. Cells were harvested at 24 hours afters poly (I:C) treatment and stained intracellularly for TLR3. (B) represents cells from poly (I:C) alone treatment and (C) represents cells from simvastatin + poly (I:C) treatment. A total of 5×10⁴ events were analyzed.</p

IFN-α levels (U/ml) released from intestinal and PBMC-derived macrophages or dendritic cells treated with either simvastatin (1 µM) + poly (I:C) (50 µg/ml) or poly (I:C) (50 µg/ml) alone.

a<p>Mean (U/ml) ± SEM (<i>n</i> = 6–12) performed in three separate experiments.</p>b<p>Since IFN-α was not detectable (<20 U/ml) in cells treated with either simvastatin alone or mock, these two treatment groups are not shown in the table.</p>c<p>Detection limit of the IFN-α assay was 20 U/ml.</p>d<p>Different capital letters denote significant differences among groups within a cell type at each time-point (ANOVA test, <i>P</i><0.05).</p

Simvastatin treatment lowers the serum cholesterol level in Gn pigs and results in increased early LDLR gene expression in IPEC-J2 cells.

<p>(<b>A</b>) Total serum cholesterol levels were assessed in all animals of 3 independent trials (<i>n</i> = 6 to 11 pigs at each time-point for Simvastatin + HuNoV and HuNoV alone; n = 2 to 4 pigs at each time-point for Mock and Simvastatin alone) by using an Amplex Red cholesterol assay kit. Pigs were inoculated with HuNoV at 6 days after simvastatin treatment began, i.e. when lower serum cholesterol levels were detected in treated animals compared to pre-treatment levels. Each bar represents the mean ± SEM. **<i>P</i><0.01 for simvastatin treatment groups vs Mock (or HuNoV alone) at each time-point. (<b>B</b>) LDLR gene expression levels in IPEC-J2 cells treated with various concentrations (0, 1 µM, 20 µM, and 80 µM) of simvastatin. LDLR gene expression was analyzed by qRT-PCR at 12 and 24 hours after treatment. Each bar represents the mean ± SEM. Different letters denote significant differences among groups at each time-point (ANOVA test, <i>P</i><0.05). Data from 2 independent experiments were combined, and each test was performed in triplicate.</p

Fecal shedding coincides with IHC evidence for human NoV replication in the intestine.

<p>Jejunum of a Simvastatin + HuNoV pig at PID 3, showing viral antigens (arrows) in the cytoplasm of enterocytes. An IHC using guinea pig antiserum against the capsid protein VP1 of GII.4 HS194 HuNoV was used for detection of HuNoV antigen in formalin-fixed, paraffin-embedded tissues. Nuclei were stained with blue-fluorescent DAPI. Original magnification, x400.</p

HuNoV fecal shedding in infected pigs can be reduced or curtailed during oral treatment with natural human IFN-α.

<p>The mean onset and duration of fecal HuNoV shedding are summarized (<b>A, B</b>), and the daily viral titers (mean) as monitored by qRT-PCR are shown (<b>C</b>). Five or six day-old piglets were treated orally with 300 IU of nhIFN-α once a day from PID -1 to PID 4. On day 2 after nhIFN-α treatment, they were inoculated orally with GII.4 HS194 HuNoV, and subsequently treated with nhIFN-α (300 IU) for 5 more days. After nhIFN-α treatment or HuNoV inoculation, clinical signs and fecal virus shedding were monitored daily until shedding terminated. Data from 2 independent animal trials were combined, and the PCR test was performed in duplicate or triplicate. Duration of virus shedding in nhIFN-α-treated and untreated pigs were analyzed based on nhIFN-α treatment period, i.e. during treatment at PIDs 1–4; post-treatment at PIDs 5–18; and overall at PIDs 1–18. Each bar represents the mean ± SEM. *<i>P</i><0.05; **<i>P</i><0.01 for nhIFN-α + HuNoV vs HuNoV alone by the unpaired two-tailed Mann-Whitney test. Animal numbers (<i>n</i>) are indicated at the bottom of each graph. The dotted line indicates the detection limit (4.7 log₁₀ GE/ml) of the qRT-PCR.</p

<i>In situ</i> expression of histo-blood group antigens in Gn pigs.

<p>(<b>A</b>) Jejunum and salivary gland of A⁺ or H⁺ pigs, expressing large amounts of HBGA antigens (arrows). (<b>B</b>) Bronchus of a representative A⁺ pig, expressing large amounts of A antigens on the surface or in the cytoplasm of bronchial epithelial cells. (<b>C</b>) Renal tubule of a representative A⁺ pig, expressing large amounts of A antigens on the surface or in the cytoplasm of renal tubular epithelial cells. Original magnifications, x200 or x400, are indicated at the bottom of each picture.</p