Isolation and identification of a variant subtype G 2b porcine epidemic diarrhea virus and S gene sequence characteristic.

Porcine epidemic diarrhea (PED) which is caused by porcine epidemic diarrhea virus (PEDV), is an intestinal communicable disease. In recent years, though pigs have been immunized with the vaccines in pig farms, PED still broke out and caused severe economic losses to the swine industry in the northeast China. In this study, the sample was positive for PEDV variant strains via the nano-nest PCR. The strain was successfully isolated from positive samples and was serially passaged in Vero-E6 cells. In addition, the strain was identified via electron microscopy observation, indirect immunofluorescence assay and infection experiment in newborn piglets and named PEDV CH/JLDH/2016 strain (Accession No. MF346935). Phylogenetic analysis of the S gene showed that the CH/JLDH/2016 strain was clustered into G2b subgroup. Comparing with the CV777 vaccine strain, amino acid sequence analysis of CH/JLDH/2016 strain showed that 15 nucleotides were inserted and 9 were absent in S gene, whose amino acid sequence it educed insertions of 5 amino acids(58NQGX61 and 145N) and absences of 3 amino acids(164RD165 and 1204Y). Our strain, in the SS2 epitope have no amino acid, variant while in SS6 epitope, Y changed into S in 776th amino acid. The results indicated that PEDV G2b variant strains have been emerged in Jilin province. The identification of new types of PEDV variant strains would stimulate the development of effective vaccines for the prevention and control of PED. The novel vaccines that based on these newly identified PEDV variant strains may contribute to the control of PED outbreaks in China.

Adoptive Transfers of CD4+CD25+ Tregs Raise Foxp3 Expression and Alleviate Mouse Enteritis.

CD4+CD25+Foxp3+ Tregs control the immune response and maintain immune homeostasis. This study examined whether Tregs can affect mouse enteritis and the Foxp3 (Forkhead transcription factor) transcriptional pathway. Mouse CD4+CD25+ Treg cells were labelled using CFSE (5,6-carboxyfluorescein diacetate succinimidyl ester) and transferred to enteritis model mice. The mice were randomly divided into an enteritis group, a Treg-infusion group, a Treg-inhibiting group, and a control group. Histopathology, ELISA, flow cytometry, western blot, immunohistochemistry, and immunofluorescence were performed. Our results demonstrated that CD4+CD25+ Tregs were successfully transferred. The disease activity index (DAI) scores in the Tregs-infusion group were lower than those of the enteritis and Tregs-inhibiting groups. The number of goblet cells and inflammatory cells was reduced, and the levels of IL-1ß, TNF-α, NO, and PGE2 were significantly decreased in the Tregs-infusion group compared to those in the enteritis group (p<0.05). The number of CD4+CD25+Foxp3+ Tregs and CD4+IL-17A+ Th17 cells in the mesenteric lymph nodes differed significantly from the enteritis and Tregs-inhibiting groups (p<0.05). There were more Foxp3+ Tregs and Smad3 and NFAT2 infiltrated into the duodenum after adoptive transfer of CD4+CD25+ Tregs, which was a significant difference relative to the enteritis group (p<0.05). This study demonstrated that adoptive transfer of CD4+CD25+ Tregs can decrease mouse enteritis. Foxp3 expression may be improved through the Smad3 and NFAT2 signalling pathways.