Vitamin D3 supplementation alleviates rotavirus infection in pigs and IPEC-J2 cells via regulating the autophagy signaling pathway.

Vitamin D had an anti-infection effect and benefited to the intestinal health. Autophagy signaling pathway was regulated by vitamin D3 to inhibit the infection of human immunodeficiency virus type-1. Rotavirus (RV) was a major cause of the severe diarrheal disease in young children and young animals. Although evidence suggested that vitamin D3 attenuates the negative effects of RV infection via the retinoic acid-inducible gene I signaling pathway, little is known of its antiviral effect whether through the regulation of autophagy. The present study was performed to investigate whether vitamin D3 alleviates RV infection in pig and porcine small intestinal epithelial cell line (IPEC-J2) models via regulating the autophagy signaling pathway. RV administration increased the Beclin 1 mRNA abundance in porcine jejunum and ileum. 5000 IU/kg dietary vitamin D3 supplementation greatly up-regulated LC3-II/LC3-I ratios and PR-39 mRNA expression under the condition of RV challenged. The viability of IPEC-J2 was significantly inhibited by RV infection. Incubation with 25-hydroxyvitamin D3 significantly decreased the concentrations of RV antigen and non-structural protein 4 (NSP4), and up-regulated the mRNA expression of Beclin 1 and PR-39 in the RV-infected IPEC-J2 cells. And then, based on the 25-hydroxyvitamin D3 treatment and RV infection, LC3-II mRNA expression in cells was inhibited by an autophagy inhibitor 3-methyladenine (3-MA). Bafilomycin A1 (Baf A1, a class of inhibitors of membrane ATPases, inhibits maturation of autophagic vacuoles) treatment numerically enhanced the LC3-II mRNA abundance, but had no effect on NSP4 concentration. Furthermore, 25-hydroxyvitamin D3 decreased the p62 mRNA expression and increased porcine cathelicidins (PMAP23, PG1-5 and PR-39) mRNA expression in the RV-infected cells. Taken together, these results indicated that vitamin D3 attenuates RV infection through regulating autophagic maturation and porcine cathelicidin genes expression.

Effect of 25-hydroxyvitamin D3 on rotavirus replication and gene expressions of RIG-I signalling molecule in porcine rotavirus-infected IPEC-J2 cells.

The study evaluated whether a 25-hydroxyvitamin D3 (25D3) supplementation decreases the replication of rotavirus by the retinoic acid-inducible gene I (RIG-I) signalling pathway in a porcine small intestinal epithelial cell line (IPEC-J2). The results show that IPEC-J2 cells express high baseline levels of 1α-hydroxylase (CYP27B1), which converts inactive 25D3 to the active 1,25-dihydroxyvitamin D3 (1,25D3). Porcine rotavirus (PRV) infection alone resulted in a significant increase in CYP27B1 mRNA, which augmented the production of active vitamin D. Physiological concentrations of 25D3 were found to decrease PRV replication in IPEC-J2 cells. RIG-I plays an important role in the recognition of double-stranded RNA virus by host cells. Upon recognition, RIG-I triggers a series of signalling molecules such as interferon-ß (IFN-ß) promoter stimulator 1 (IPS-1) leading to the expression of type I interferons (IFN-ß). Active 25D3 that was generated by PRV-infected IPEC-J2 cells led to an increased expression of toll-like receptors 3 (TLR3), RIG-I, IPS-1, IFN-ß and IFN-stimulated genes 15 (ISG15) with important innate immune functions. Inhibiting CYP27B1 also failed to increase RIG-I, IPS-1, IFN-ß and ISG15 mRNA expression. These observations suggest that 25D3 can directly inhibit PRV in IPEC-J2 cells, which requires this active form of vitamin D. The anti-rotavirus effect of 25D3 is mediated at least in part by RIG-I signalling pathways in IPEC-J2 cells.

Dietary vitamin D supplementation attenuates immune responses of pigs challenged with rotavirus potentially through the retinoic acid-inducible gene I signalling pathway.

In the present study, twenty-four Duroc × Landrance × Yorkshire (initial body weight (BW) of 21·82 (sem 2·06) kg) cross-bred pigs were used to determine whether dietary vitamin D supplementation could confer protection against viral infections through the retinoic acid-inducible gene I (RIG-I) signalling pathway in pigs. Experimental treatments were arranged in a 2 × 2 factorial manner with the main effects of immune challenge (control v. porcine rotavirus (PRV) challenge) and dietary concentrations of vitamin D (200 and 5000 IU; where 1 IU of vitamin D is defined as the biological activity of 0.025 mg of cholecalciferol). The pigs were fed a diet containing 200 or 5000 IU vitamin D in the first week of the study period. On day 8, the pigs were orally dosed with 4 ml of Dulbecco's modified Eagle's medium/Ham's F-12 medium containing PRV or essential medium (control). Serum samples were collected on day 8 (pre-challenge), and 6 d after the PRV challenge, the pigs were killed to evaluate intestinal morphology and tissue gene expression following the last blood collection. Pigs challenged with PRV had decreased BW gain (P< 0·01), feed intake (P< 0·01), villus height (P< 0·01), faecal consistency (P< 0·05), and serum 1,25-dihydroxyvitamin D concentration (P< 0·01) and increased (P< 0·01) serum IL-2, IL-6 and interferon (IFN)-ß concentrations. Vitamin D supplementation mitigated these effects. The mRNA expression of RIG-I (P< 0·01), IFN-ß promoter stimulator 1 (P< 0·01), IFN-ß (P< 0·01) and interferon-stimulated gene 15 (ISG 15 ) (P< 0·01) was up-regulated by the PRV challenge and vitamin D supplementation in the intestine. In conclusion, vitamin D supplementation could activate the RIG-I signalling pathway and thus alleviate the negative effects caused by PRV challenge.