Phylogenic tree for 119 PEDV strains and identified motifs.

<p>(A) Phylogenetic tree based on the genomes of 119 PEDV strains isolated in 2013–2014. Phylogenetic analysis was performed using a maximum-likelihood method with general time reversible nucleotide substitution model and with a bootstrap test using 1000 replicates in the MEGA6 program. Notations on the very left side represent the clades shown by Vlasova et al. (9). (B) The presence of sequence motifs in each strain. (C) Color chart of nucleotides in the sites having inconsistencies within the 119 PEDV strains. Nucleotides in agreement with the sequence of Indiana12.83/USA/2013 are colored with respect to the type of nucleotide (a: red, t: blue, c: green, and g: yellow). To increase the discriminability of motifs, nucleotides in sites with only one inconsistent strain were not colored.</p

Innate immune response of porcine intestinal epithelial cell line (PIE cells) after infection with rotaviruses.

<p>PIE cells were cultured in DMEM media for 10 days, and then infected with OSU or UK rotaviruses. After 0, 3, 6, and 12 hours post-infection the expression level of IFN-β, MxA, RNaseL, RIG-I, TLR3, and cytokines (CXCL10, IL-6, IL-8, and MCP-1) were quantified. The results represent data from three independent experiments and are expressed as mean ± S.D. Asterisks indicate significant differences: <i>p</i> <0.05 (*), <i>p</i> <0.01 (**), and <i>p</i> < 0.001(***).</p

Effect of immunobiotic bifidobacteria on IRF3 activation in porcine intestinal epithelial cell line (PIE cells).

<p>PIE cells were pre-treated with <i>B</i>. <i>infantis</i> MCC12 or <i>B</i>. <i>breve</i> MCC1274, and then challenged with rotaviruses OSU or UK; or poly(I:C). Total proteins were extracted from lysed cells and separated by SDS-PAGE, and then western-blot was performed to analyze phosphorylation of IRF3 after 10, 30, 60 and 120 minutes. The phosphorylated IRF3 specific bands were normalized to that corresponding to total IRF3. Intensities of proteins bands were calculated from peak area of densitogram by using image software. The results represent data from three independent experiments and are expressed as relative index vs 0 min control with mean ± S.D. ^{a, b, c} Different superscripts letters indicate significant difference (p<0.05) among stimulants at the same time point.</p

Effect of immunobiotic bifidobacteria on TRAF3 activation in porcine intestinal epithelial cell line (PIE cells).

<p>PIE cells were pre-treated with <i>B</i>. <i>infantis</i> MCC12 or <i>B</i>. <i>breve</i> MCC1274, and then challenged with rotaviruses OSU or UK; or poly(I:C). Total proteins were extracted from lysed cells and separated by SDS-PAGE, and then western-blot was performed to analyze phosphorylation of TRAF3 after 10, 30, 60 and 120 minutes. The TRAF3 specific bands were normalized to that corresponding to β-actin. Intensities of proteins bands were calculated from peak area of densitogram by using image software. The results represent data from three independent experiments and are expressed as relative index vs 0 min control with mean ± S.D. ^{a, b, c} Different superscripts letters indicate significant difference (p<0.05) among stimulants at the same time point.</p

Infectivity of rotaviruses in porcine intestinal epithelial cell line (PIE cells).

<p>PIE cells cultured for 3 or 10 days. MA104 cells were used for comparison. PIE and MA104 cells were inoculated with porcine OSU or bovine UK rotaviruses and evaluated by immunofluorescence assay. The cells with specific green-fluorescence in the cytoplasm were photographed by confocal laser microscopy after labeling with fluorescence antibody.</p

Effect of immunobiotic bifidobacteria in antiviral immune response of porcine intestinal epithelial cell line (PIE cells).

<p>PIE cells were pre-treated with <i>B</i>. <i>infantis</i> MCC12 or <i>B</i>. <i>breve</i> MCC1274 for 48 hours, and then challenged with rotavirus UK. The expression level of IFN-β, MxA, RNaseL, RIG-I, TLR3, A20, and cytokines (CXCL10, IL-6, IL-8, MCP-1) were quantified by RT-PCR after 6 and 12 hours. The results represent data from three independent experiments and are expressed as mean ± S.D. Asterisks indicate significant differences: <i>p</i> <0.05 (*), <i>p</i> <0.01 (**), and <i>p</i> < 0.001(***).</p

Infectivity of rotaviruses in porcine intestinal epithelial cell line (PIE cells).

<p>PIE cells cultured for 3 or 10 days. MA104 cells were used for comparison. PIE and MA104 cells were inoculated with rotaviruses from different host species including: human Wa, murine EW, bovine UK, and porcine OSU rotaviruses. Numbers of rotavirus antigen positive cells were counted by immunofluorescence assay after 16 hours post-inoculation. The virus titer was expressed as Log₁₀ focus forming units (FFU)/0.1 ml. NI: no infectivity of rotaviruses. The results represent data from three independent experiments. ^{a, b, c} In the comparison within means of the same virus, the difference among means with different superscripts was significant at 5% level.</p

Proposed mechanism.

<p>The possible immunomodulatory activity of <i>B</i>. <i>infantis</i> MCC12, and <i>B</i>. <i>breve</i> MCC1274 in porcine intestinal epithelial cell line (PIE cells) after stimulation with rotaviruses. Arrows indicate up and down-regulation of cytokines/chemokines, and anti-viral factors. (+): upregulation, (--): strong down-regulation, (-): moderate down-regulation.</p