ABSTRACT
Porcine epidemic diarrhea virus (PEDV) causes severe infectious diseases in all ages of swine and leads to serious economic losses. Serologic tests are widely accepted and used to detect anti-PEDV antibodies that could indicate PEDV infection or vaccination. In this study, PEDV recombinant S1 protein (rS1) was expressed with the Bac-to-Bac system and purified by nickel-affinity chromatography. An indirect enzyme-linked immunosorbent assay based on rS1 (rS1-ELISA) was then developed and optimized by checkerboard assays with serial dilutions of antigen and serum. Serum samples from 453 domestic pigs and 42 vaccinated pigs were analyzed by the indirect fluorescent antibody (IFA) test and rS1-ELISA. Taking IFA as a gold standard, rS1-ELISA produced a high sensitivity (90.7%) and specificity (94.6%) by a receiver operating characteristic (ROC) curve. In addition, ROC analysis also revealed that rS1-ELISA was consistent with IFA (area under the curve 0.9583 +or- 0.0082). This rS1-ELISA was then applied to antibody detection in inactivated PEDV vaccinated pigs. The antibody could be detected 2-4 weeks after the first inoculation. These results indicated that the rS1-ELISA established in this study provides a promising and reliable tool for serologic detection of anti-PEDV IgG antibodies in infected or vaccinated pigs.
ABSTRACT
Normally, type III interferon (IFN-lambda) was highly expressed in intestinal mucosa and other mucosal systems, with relatively stronger broad-spectrum antiviral ability and immune regulating ability. Porcine epidemic diarrhea virus (PEDV) and porcine deltacorona virus (PDCoV) were two major intestinal pathogens causing diarrhea in piglets, which hindered the swine industry severely. In this study, the recombinant plasmid containing PoIFN-lambda3 fragment was constructed. The recombinant plasmid was then transformed into E.coli Rosaetta cells for recombinant expression. Analyzed by SDS-PAGE and western blotting, the results showed that the recombinant PoIFN-lambda3 (27 ku) was successfully expressed in the inclusion bodies of E. coli cells. After denaturation, purification and renaturation, the active PoIFN-lambda3 recombinant protein was obtained and was used to treat IPEC-J2 cells. Challenged by recombinant PoIFN-lambda3, the immune-stimulating genes (ISGs), such as ISG15-, OAS1, Mx-1, IFIT1, IFITM1 and IFITM3 were all up-regulated significantly and reached to the peak (P 0.001) at 12 h post challenge. For PEDV and PDCoV infection in IPEC-J2, the virus replication was detected after pretreatment, simultaneous treatment and post-infection treatment of recombinant PoIFN-lambda3. Compared with the control, the viral copy numbers of both PEDV and PDCoV were decreased significantly (P 0.05) after treatment of recombinant PoIFN-lambda3. The above results indicated that the purified and renatured PoIFN-lambda3 recombinant protein could have good biological activity. The recombinant PoIFN-lambda3 can induce high expression of different ISGs in IPEC-J2, and can also inhibit PEDV and PDCoV replication in the host cells. In summary, our study provided a basis for preventing and treating viral diarrhea in piglets.
ABSTRACT
Porcine deltacoronavirus (PDCoV) is a novel emerging enteric coronavirus found in pigs. Intestinal enteroids, which partially recreate the structure and function of intestinal villi-crypts, have many physiological similarities to the intestinal tissues in vivo. Enteroids exhibit advantages in studying the interactions between intestines and enteric pathogens. To create a novel infection model for PDCoV, we developed an in vitro system to generate porcine intestinal enteroids from crypts of duodenum, jejunum, and ileum of pigs. Enterocytes, enteroendocrine cells, Paneth cells, stem cells, proliferating cells, and goblet cells were found in the differentiated enteroids. Replication of PDCoV was detected in the cultured enteroids by immunofluorescence and quantitative RT-PCR. Double immunofluorescence labeling demonstrated that PDCoV was present in Sox9-positive intestinal cells and Villin1-positive enterocytes. There were multiple cellular responses shown as changes of transcription of genes related to mucosal immunity, antiviral genes, and marker genes of stem cells and other cells in the enteroids infected with PDCoV. We conclude that the 2-D enteroids derived from porcine jejunum can be used as an in vitro multicellular model for the investigation of pathogenesis and host immune responses to porcine enteric pathogens, such as PDCoV.