| Porcine transmissible gastroenteritis virus (TGEV) is the main pathogen which would cause the transmissible gastroenteritis (TGE) in swine. Virulent TGEV infection leads to severe vomiting, acute villous atrophy, malabsorptive diarrhea and dehydration, which approach 100% mortality in seronegative suckling pigs less than two weeks of age. Even older piglets that recovered from virulent TGEV infections would have impaired immune function and cause the secondary viral or bacterial infection. However, the piglets exposed to attenuated TGEV only had mild clinical signs and low mortality, and older piglets had not been infected. Porcine intestinal epithelial cells are the target cells infected by TGEV. Dendritic cells (DCs), the most powerful antigen-presenting cells, are widely distributed in the mucosal tissue underneath the porcine intestinal epithelial cell and play an important role in preventing the virus invasion and initiating the relative immune responses. In this study, we mainly used the virulent TGEV strain (SHXB) and the attenuated TGEV strain (STC3) to infect the porcine intestinal epithelial cell (IPEC-J2 cells) and the porcine dendritic cells (Mo-DCs), and discuss the differences in the pathogenesis of virulent and attenuated TGEV infection in piglets.1 Effects of different virulence of TGEV strains on the susceptibility of IPEC-J2 cellsIn order to study whether SHXB and STC3 could infect the IPEC-J2 cells, a sucrose gradient centrifugation was firstly used to purify the two strains. Secondly, the direct immunofluorescence, transmission electron microscopy, viral titer and flow cytometry were used to detect the susceptibility of IPEC-J2 cells to SHXB and STC3. Finally, we used the scanning electron microscopy and transmission electron microscopy to observe the pathological changes of IPEC-J2 cells caused by different strains of TGEV. The results showed that there was no significant difference between the virus particles appearance of the purified SHXB and STC3. The virions of the two strains were a round or oval station, the spikes were long and sparse, and virus particles have an electron transparent center; Both SHXB and STC3 could infect the IPEC-J2 cells, but the replication of the virulent strain infection was higher than the attenuated strain (p<0.05); SHXB infection leaded to serious pathological changes in IPEC-J2 cells. The microvilli became scattered and formatted enlargement of mushroom-shape. The microfilaments also became scattered and lost. The bilayer membrane structures of mitochondria, Golgi apparatus, endoplasmic reticulum were lost, and the interior of the cell found the vacuoles or U-shaped single-layer membrane structure. The perinuclear pool of cells became larger, inner and outer the nuclear membranes were separated, and the cell nuclear was condensation. However, STC3 infection only caused the minor lesions of IPEC-J2 cells. These tests showed that both SHXB and STC3 could infect the IPEC-J2 cells, while the replication of the virulent strain infection was higher than the attenuated strain, and the pathological destroy in IPEC-J2 cells caused by the virulent strain were higher than the attenuated strain.2 Effects of different virulence of TGEV strains on the tight junctions and microfilaments of IPEC-J2 cellsThe tight junction is one of the main connections between different intestinal epithelial cells, which play an important role in maintaining the integrity of epithelial cells and preventing different pathogens invasion. The virus and bacterial could make their replication reaching the optimal level by changing the cell microfilaments. In order to study the disruption of tight junctions and microfilaments of IPEC-J2 cells caused by different virulence of TGEV strains, transepithelial electrical resistance (TEER) assay, Dextran permeability test and western blotting were firstly used to study the effects of different virulence of TGEV strains on the tight junctions and microfilaments of IPEC-J2 cells; Secondly, the effect of different virulence of TGEV strains on the microfilaments remodeling of IPEC-J2 cells was observed by immunofluorescence (FITC-phalloidin staining) and transmission electron microscopy. We then treated IPEC-J2 cells with Jas, which promotes stabilisation of the microfilaments, and Cyto D, which induces destabilisation of the microfilaments, and the viral invasion, replication and release were detected by TCID50. At last, this study detected the changes of ERK、JNK and p38 which belong to three important subgroup in the MAPK signalling pathway of IPEC-J2 cells infected by different virulence of TGEV strains, which could further explain that the stimulated MAPK signal pathway of IPEC-J2 cells modulated the disruption of tight junctions and microfilaments remodeling caused by TGEV. The results showed that SHXB and STC3 could decrease transepithelial electrical resistance (TEER) of infected IPEC-J2 cells at early stages of infection, increase the permeability of paracellular pathway, down-regulate expression of Claudin-1 and Occludin. The disruption extent of tight junctions integrity in IPEC-J2 cells caused by SHXB was higher than STC3 (p<0.05). Both SHXB and STC3 could induce the microfilament remodelling, but the disruption extent of microfilaments in IPEC-J2 cells caused by SHXB was higher than STC3 (p<0.05). The two strains replication and release in the IPEC-J2 cells were reduced when we treated IPEC-J2 cells with Jas or Cyto D, and the above treatment could significantly reduce the replication and release of the two TGEV strains. Finally, the ERK1/2 and JNK protein in the MAPK signalling pathways could be activated after infected by SHXB for 1h, and JNK protein could be significantly activated after infected by SHXB for 24h. However, and SHXB infection inhibited the activation of ERK1/2 and p38 protein (p<0.05). All these data suggested that the virulent TGEV strain (SHXB) and the attenuated TGEV strain (STC3) could destroy the barrier integrity of IPEC-J2 cells in their early infection, and they can regulate the microfilament remodelling in favour of their own replication throughout the viral cycle, which might be relative to the activation of MAPK signalling pathways in the IPEC-J2 cells. After infected by the virulent TGEV strain (SHXB), the disruption of tight junctions in IPEC-J2 cells, the effect to the microfilament remodelling, and the activated extent to the protein in the MAPK signalling pathways were significantly higher than the attenuated TGEV strain (STC3).3 Interaction of different virulence of TGEV strains with porcine Mo-DCs in vitroDendritic cells (DCs), the most powerful antigen-presenting cells,:are widely distributed in the mucosal tissue underneath the porcine intestinal epithelial cell and play an important role in preventing the virus invasion and initiating the relative immune responses. In order to study the interaction of different virulence of TGEV strains with porcine Mo-DCs, the monocytes in the peripheral blood of piglets were firstly induced into Mo-DCs. Mo-DC was then co-cultured with SHXB and STC3 infected IPEC-J2 cells to investigate the sample TGEV ability of Mo-DCs across the tight junction of IPEC-J2 cells. The absolute quantitative real-time PCR, viral titer and flow cytometry were used to detect the susceptibility of IPEC-J2 cells to different virulence of TGEV strains. Thereafter, the phenotype markers changes of Mo-DCs were tested by flow cytometric analysis, and the expression of cytokines on SHXB and STC3 infected the Mo-DCs was determine by ELISA. Finally, the presenting-antigen ability of SHXB and STC3 infected the Mo-DCs was detected by mixed lymphocyte reaction. The results showed that the study cultured the high-purified Mo-DCs. Mo-DCs could uptake SHXB and STC3 through helding out the dendrites, and the uptake ability to SHXB was significantly reduced with time (p<0.05). Both SHXB and STC3 could infect the immature and mature Mo-DCs at lower level, and the infectivity in immature Mo-DCs was higher than in mature Mo-DCs, the expression of CDla+, SLA-Ⅱ-DR+ in SHXB infected immature and mature Mo-DCs were significantly lower than the control group (p<0.05), while that in STC3 infected immature Mo-DCs were similar to the control group.The secretion of IL-12 and IFN-y in SHXB infected immature Mo-DCs was significantly lower than STC3 infected group (p<0.05). However, there are no changes on the secretion of IL-10 in SHXB and STC3 infected immature and mature Mo-DCs (p>0.05); SHXB infected immature and mature Mo-DCs did not stimulate T cells proliferation, but STC3 infected immature and mature Mo-DCs could stimulate T cell proliferation to some extent. These results indicated that different virulence of TGEV had different effect on the functions of Mo-DCs, such as the uptake and presenting antigen, cytokine secretion and the ability to stimulate T cell proliferation. TGEV virulent strain could injury these functions of Mo-DCs, TGEV attenuated strain could enhance those of Mo-DCs.4 Effects of different virulence of TGEV strains on porcine DCs in vivoIn order to verify the SHXB could severely impaire the functions of porcine DCs in vitro, while STC3 strains could enhance the above functions. In this study, we carried out the intestinal ligation surgery by injecting the SHXB and the STC3 into the intestinal lumen of piglets, and immunofluorescence and Immunohistochemistry were used to detected the number of CD11b+ CD16+ DCs, SWC3a+SLA-DR-Ⅱ+ DCs and CD3+, CD4+ and CD8+T cells in jejunum villi and lamina propria at different times, which was studied for the uptake and presenting function changes of the TGEV antigen in porcine DCs. The results showed that at the early injection of SHXB (15 min-3 h), the number of CD11b+CD16+ DCs, SWC3a+SLA-DR-Ⅱ+ DCs and CD3+, CD4+ and CD8+ T cells in intestinal villi rapidly increased. As the time increased (48 h), the number of DCs, CD3+, CD4+ and CD8+ T cells in jejunum villi and lamina propria were significantly decreased (p<0.05). Such a response did not occur after STC3 injection. At the early injection of STC3 (15 min-3 h), no significant changes were found on the number of CD11b+CD16+ DCs, SWC3a+SLA-DR-Ⅱ+ DCs and CD3+, CD4+ and CD8+ T cells in intestinal villi (p>0.05). However, as the time increased (48 h), the number of DCs, CD3+, CD4+ and CD8+ T cells in jejunum villi and lamina propria were significantly increased (p<0.05). The above results demonstrated that the ability to uptake and present antigen decreased with the time after piglet infected with SHXB, but the ability to uptake and present antigen decreased with the time after piglet infected with STC3. For further study the effect of different virulence of TGEV strains on antigen-present function of porcine DCs, the test injected inactivate Escherichia coli K88 (model antigen) into the intestinal lumen of piglets by the intestinal ligation surgery 48 h after fed with the SHXB and STC3. Immunofluorescence and Immunohistochemistry were used to detected the number of CD11b+CD16+ DCs, SWC3a+SLA-DR-II+ DCs, CD3+, CD4+ and CD8+ T cells in jejunum villi and lamina propria at different times. The results showed that 15 min-3 h after SHXB-infected piglets injected with inactivate Escherichia Coli K88, no significant changes were found on the number of CD11b+CD16+ DCs and SWC3a+SLA-DR-II+ DCs, CD3+, CD4+ and CD8+ T cells in intestinal villi and lamina propria (p>0.05). However,15 min-3 h after STC3-infected piglets injected with inactivate Escherichia coli K88, the number of CDllb+CD16+DCs, SWC3a+SLA-DR-II+ DCs and CD3+, CD4+ and CD8+ T cells in intestinal villi and lamina propria obviously increased (p<0.05). These results suggest that SHXB significantly impaired the ability of porcine DCs to uptake, process and present inactivate Escherichia coli K88. However, the immune function in porcine DCs infected with STC3 strain was not impaired. The article could provide fundamental basis for studying the pathogenic mechanism of the different virulence of TGEV strains and developing the vaccine. |
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