Gene Expression Of Antiviral Pattern Recognition Receptors In Different Pig Breeds And Vitamin D’s Antiviral Activity And Mechanism

The study was designed to investigate differences of messenger RNA expression for antiviral pattern recognition receptors between Tibetan and DLY pigs, the antiviral activity of vitamin D and the effect of vitamin D on gene expression in order to reveal the differences in disease resistance of different breeds of pigs, and vitamin D’s antiviral activity and mechanism. The present study firstly cloned and characterized Tibetan porcine retinoic acid-inducible gene Ⅰ (RIG-Ⅰ), interferon-beta promoter stimulator1(IPS-1) and Toll-like receptor3(TLR3) via the method of molecular cloning, bioinformatics and fluorescence quantitative PCR, and then investigate the tissue distribution and differences of messenger RNA expression for RIG-Ⅰ, IPS-1and TLR3between Tibetan and DLY pigs. Afterwardswe explored the difference of the innate antiviral immune response to PRRS virus between Tibetan and DLY pigs with PRRS vaccine challenge model. Finally, we explored the antiviral activity of vitamin D and molecular mechanism in vitro and in vivo. This study includes the following four experiments.Experiment1:Expression differences of RIG-Ⅰ, IPS-1and TLR3between DLY and Tibetan pigs.Six Tibetan pigs and six DLY pigs with half male and half female were slaughtered at the same growth stage and similar weight. The tissues, including heart, liver, spleen, lung, kidney, duodenum, jejunum, ileum, bronchial lymph nodes, mesenteric lymph node, inguinal lymph nodes, muscle and adipose tissue, were collected. Total RNA was extracted for cloning of Tibetan pig RIG-Ⅰ, IPS-1and TLR3gene cDNA sequence, and was used to detect RIG-I, IPS-1and TLR3gene expression by real-time quantitative PCR. The results are as follows:(1) The Tibetan RIG-I cDNA was successfully cloned. This sequence contains a2832bp opening reading frame, and encodes943amino acid residues. The nucleotide sequence shares99.58%homology with the known RIG-I sequences of the common pig. The AA sequence has77.67%and71.85%homology with human and mus musculus respectively. Hydrophobicity analysis of the AA sequence suggests no transmembrane region.(2) The Tibetan IPS-1cDNA contains a1575bp opening reading frame, and encodes524amino acid residues. The nucleotide sequence shares99.37%homology with the known IPS-1sequences of the common pig. The AA sequence has55.04%and45.03%homology with human and mus musculus respectively. Hydrophobicity analysis of the AA sequence suggests the presence of1putative transmembrane domain.(3) The Tibetan TLR3cDNA contains a2718bp opening reading frame, and encodes901amino acid residues. The nucleotide sequence shares99.56%homology with the known TLR3sequences of the common pig. The AA sequence has83.43%and77.35%homology with human and mus musculus, respectively. Hydrophobicity analysis of the AA sequence suggests the presence of1putative transmembrane domain.(4) RIG-I, IPS-1and TLR3are ubiquitously expressed in all tissues. The RIG-Ⅰ and IPS-1expression of most tissues of Tibetan pigs were higher than those of DLY pigs (P<0.01). The RIG-Ⅰ and TLR3expression of the liver and duodenum were highest for Tibetan pig. RIG-Ⅰ expression was highest in liver and jejunum of DLY pig. TLR3expression was highest in liver and kidney of DLY pig. The expression of IPS-1in the liver and kidney was highest for Tibetan and DLY pig.Experiment2:RIG-Ⅰ, IPS-1and TLR3gene expression of DLY pigs and Tibetan pigs after challenge with PRRS vaccine.Twelve Tibetan pigs (15.44±0.34kg) and twelve DLY pigs (15.21±0.3kg) were randomly allotted to4treatments with6replicates per treatment, and1pig per replicate. The experiment was designed by2×2factorial arrangement based on the factors of pig breed (Tibetan pigs and DLY pigs) and challenge status (PRRS vaccine challenge and unchallenged). The trial period included7days of preliminary experiment and13days of formal experiment. The results are as follows:(1) The ADG of DLY pigs was significantly higher than that of Tibetan pigs (P<0.01). After challenged with PRRS vaccine, ADG and ADFI were decreased, feed conversion rate was increased in Tibetan pigs and DLY pigs (P<0.01). The effect of PRRS vaccine on growth performance of DLY pigs was greater than that of Tibetan pigs (P<0.01).(2) The IgG and IgM concentrations of serum increased after challenge with PRRS vaccine in both breeds of pigs (P<0.01). But there is no interaction effect between breed and PRRS vaccine.(3) Serum IL-1β (P<0.01), TNF-α (P<0.05), and IFN-β (P<0.01) concentrations increased after challenge with PRRS vaccine. The effect of PRRS vaccine on IL-1β and IFN-β concentrations of DLY pigs were greater than that of Tibetan pigs (P<0.01).(4) The challenge with PRRS vaccine could increase the RIG-Ⅰ expression in liver (P<0.01), spleen (P<0.05), lung (P<0.01), bronchial lymph nodes (P<0.05), mesenteric lymph node (P<0.01), inguinal lymph nodes (P<0.01), jejunum (P<0.01) and ileum (P<0.01), stimulate the IPS-1expression in liver (P<0.01), spleen (P<0.01), lung (P<0.01), mesenteric lymph node (P<0.01), jejunum (P<0.01) and ileum (P<0.05), and enhance the TLR3expression in liver (P<0.01), spleen (P<0.01), lung (P<0.05), mesenteric lymph node (P<0.01), inguinal lymph nodes (P<0.01), jejunum (P<0.01) and ileum (P<0.01).(5) The effect of PRRS vaccine on lung (P<0.01), mesenteric lymph node (P<0.05) and and ileum (P<0.01) RIG-Ⅰ mRNA expression, liver IPS-1mRNA expression (P<0.01), and inguinal lymph nodes (P<0.01). TLR3mRNA expression of DLY pigs was greater than that of Tibetan pigs (P<0.01).Experiment3:The effect of vitamin D on RIG-I, IPS-1, and TLR3gene expression and IFN beta protein expression in RV infected IPEC-J2cellThe study evaluated whether vitamin D could decrease rotavirus replication potentially through RIG-Ⅰ signaling pathway in IPEC-J2cells. Porcine rotavirus OSU strain was used in this study. The experiment was designed by a2×2×2factorial arrangement based on the factors of infect status (RV infected and uninfected),25D3concentration (0and10-7M25D3), and ITRA concentration (0and10-7M ITRA). At24hours after treatment, total RNA was extracted. The expression of RIG-Ⅰ, IPS-1, and TLR3mRNA were determined by real-time PCR, and the protein levels of IFN-(3were measured by ELISA. The results are as follows:(1) The model of RV infected IPEC-J2cell was established. At24h after infected with RV, IPEC-J2cells were shrinked, and the connection among cells disappeared. At72h after infected with RV, IPEC-J2cells showed necrosis and shedding. The RV infection significantly decreased IPEC-J2cell proliferation. RV RNA were detected in RV infected IPEC-J2cells.(2) The CYP27B1mRNA in RV-infected cells was higher than that in control cells (P<0.01). The1,25D3level of the supernatant in RV-infected cells was higher than that in uninfected cells (P<0.01).(3) RV infection alone resulted in the increase in RIG-Ⅰ, IPS-1, ISG15and IFN-β expression (P<0.01), but did not affect TLR3expression (P>0.05). RIG-I, IPS-1, ISG15, IFN-P mRNA expression and IFN-p protein levels in IPEC-J2cells infected with RV and treated with25D3(10-7M) showed a significant increase. The effect of vitamin D on RIG-Ⅰ, IPS-1, ISG15, IFN-β mRNA expression and IFN-P protein levels of RV infected IPEC-J2cell was greater than that of uninfected cell (P<0.01).Experiment4:The effect of vitamin D on RIG-Ⅰ and its downstream signaling molecules, TLR3mRNA expression in RV infected Tibetan and DLY pigsThe study evaluated whether vitamin D could decrease rotavirus replication potentially through RIG-Ⅰ signaling pathway in vivo. The experiment was designed by2×2×2factorial arrangement based on the factors of pig breed (Tibetan pigs and DLY pigs), challenge status (RV challenged and unchallenged), VD concentration (200IU and5000IU). Sixteen Tibetan pigs (13.73±0.95kg) and twenty four DLY pigs (26.46±2.07kg) were randomly allotted to8treatments with6replicates per treatment for DLY pigs, and4replicate per treatment for Tibetan pigs. The trial period included7days of preliminary experiment and6days of formal experiment. The results are as follows: (1) ADG and ADFI were decreased, and F/G was elevated by RV challenge (P<0.01). Dietary supplementation with vitamin D elevated ADG and ADFI (P<0.01). The effect of RV challenge on growth performance of DLY pigs was greater than that of Tibetan pigs (P<0.01).(2) Villus height was decreased by RV challenge (P<0.01). There was not effect on crypt depth and the villus height:crypt depth ratio by RV challenge (P>0.05). Dietary supplementation with vitamin D elevated villus height, whereas there was no significantly difference. The effect of RV challenge on villus height of DLY pigs was greater than that of Tibetan pigs (P<0.01).(3) Serum IFN-(3, IL-6and IL-2concentrations decreased by RV challenge (P<0.01). The effect of RV challenge on serum IFN-β,IL-6and IL-2concentrations of DLY pigs was greater than that of Tibetan pigs (P<0.01). Dietary supplementation with vitamin D elevated IFN-β level, and decreased IL-6and IL-2concentrations in serum (P<0.01).(4) The expression of CYP27B1mRNA in duodenum, jejunum and ileum increased by RV challenge and vitamin D treatment (P<0.01). The effect of dietary vitamin D levels on CYP27B1mRNA expression of DLY pigs was greater than that of Tibetan pigs. Serum1,25D3concentrations decreased by RV challenge (P<0.01), but elevated by dietary supplementation with vitamin D (P<0.01).(5) The expression of RIG-Ⅰ, IPS-1, ISG15and IFN-P mRNA in duodenum, jejunum and ileum increased by RV challenge and dietary supplementation with vitamin D (P<0.01). But there was no difference in TLR3gene expression by RV challenge and dietary supplementation with vitamin D (P>0.05).In summary, the expression distribution of RIG-Ⅰ, IPS-1and TLR3mRNA in Tibetan and DLY pigs was different. The higher expression of RIG-Ⅰ, IPS-1might be the reason that Tibetan pigs has higher immunity and disease resistance. Vitamin D had certain inhibiting ability for rotavirus. The anti-rotavirus effect of vitamin D was mediated at least in part by RIG-Ⅰ signaling pathways in IPEC-J2cells.