Fig 1.
Microscopic analysis of porcine intestinal epithelial cell line (PIE cells).
PIE cells were grown in DMEM medium for 3 and 10 days. The cell growth and the development of microvilli on the surface of PIE cells were analyzed by light and scanning electron microscopy (SEM) microscopies. Arrows indicate the development of microvilli. Scale bars indicate 10 μm (black) and 1 μm (white).
Fig 2.
Infectivity of rotaviruses in porcine intestinal epithelial cell line (PIE cells).
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.
Fig 3.
Infectivity of rotaviruses in porcine intestinal epithelial cell line (PIE cells).
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.
Fig 4.
Innate immune response of porcine intestinal epithelial cell line (PIE cells) after infection with rotaviruses.
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: p <0.05 (*), p <0.01 (**), and p < 0.001(***).
Fig 5.
Antiviral activities of lactic acid bacteria in porcine intestinal epithelial cell line (PIE cells).
(A) PIE cells were pre-stimulated with different lactic acid bacteria strains for 48 hours: Bifidobacterium longum MCC1, B. infantis MCC12, B. breve MCC167, MCC1274, B. pseudolongum MCC92, Lactobacillus paracasei MCC1375, L. gasseri MCC587, Lactococcus lactis sub sp. lactis MCC866; and then stimulated with Poly(I:C) for 12 hours. The expression level of IFN-β was quantified by RT-PCR. The results represent data from three independent experiments and are expressed as mean ± S.D. Asterisks indicate significant differences: p <0.05 (*). (B) Scanning electron microscopy (SEM) analysis to evaluate the attachment of B. infantis MCC12 and B. breve MCC1274 to the surface of PIE cells. (C) Effect of B. infantis MCC12 and B. breve MCC1274 on rotavirus infectivity evaluated by immunofluorescence after 12 hours of challenge. (D) The protective ability of B. infantis MCC12 and B. breve MCC1274 was examined by comparing them with untreated virus control, and calculating the reduction radio.
Fig 6.
Effect of immunobiotic bifidobacteria in antiviral immune response of porcine intestinal epithelial cell line (PIE cells).
PIE cells were pre-treated with B. infantis MCC12 or B. breve MCC1274 for 48 hours, and then challenged with rotavirus OSU. 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: p <0.05 (*), p <0.01 (**), and p < 0.001(***).
Fig 7.
Effect of immunobiotic bifidobacteria in antiviral immune response of porcine intestinal epithelial cell line (PIE cells).
PIE cells were pre-treated with B. infantis MCC12 or B. breve 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: p <0.05 (*), p <0.01 (**), and p < 0.001(***).
Fig 8.
Effect of immunobiotic bifidobacteria on TRAF3 activation in porcine intestinal epithelial cell line (PIE cells).
PIE cells were pre-treated with B. infantis MCC12 or B. breve 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.
Fig 9.
Effect of immunobiotic bifidobacteria on IRF3 activation in porcine intestinal epithelial cell line (PIE cells).
PIE cells were pre-treated with B. infantis MCC12 or B. breve 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.
Fig 10.
The possible immunomodulatory activity of B. infantis MCC12, and B. breve 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.