Transmissible gastroenteritis virus (TGEV), the etiologic agent of transmissible gastroenteritis, infects swine of all ages causing vomiting and diarrhea, in newborn piglets the mortality rate is near 100%. Intestinal epithelial cells are the primary target cells of TGEV. Transferrin receptor 1 (TfR1), which is highly expressed in piglets with anemia, may play a role in TGEV infection. However, the underlying mechanism of TGEV invasion remains largely unknown. Our study investigated the possibility that TfR1 can serve as a receptor for TGEV infection and enables the invasion and replication of TGEV. We observed that TGEV infection promoted TfR1 internalization, clustering, and co-localization with TfR1 early in infection, while TfR1 expression was significantly down-regulated as TGEV infection proceeded. TGEV infection and replication were inhibited by occluding TfR1 with antibodies or by decreasing TfR1 expression. TGEV infection increased in TGEV-susceptible ST or IPEC-J2 cell lines and TGEV-resistant Caco-2 cells when porcine TfR1 was over-expressed. Finally, we found that the TGEV S1 protein interacts with the extracellular region of TfR1, and that pre-incubating TGEV with a protein fragment containing the extracellular region of TfR1 blocked viral infection. Our results support the hypothesis that TfR1 is an additional receptor for TGEV and assists TGEV invasion and replication.

Background: Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome, and is associated with a number of other diseases. PCV2 is widely distributed in most developed swine industries, and is a severe economic burden. With an eye to developing an effective, safe, and convenient vaccine against PCV2-associated diseases, we have constructed a recombinant Bacillus subtilis strain (B. subtilis-Cap) that expresses the PCV2 capsid protein (Cap). Methods: Electroporation of a plasmid shuttle vector encoding the PCV2 Cap sequence was use to transform Bacillus subtilis. Flow cytometry was used to evaluate in vitro bone marrow derived dendritic cell (BM-DC) maturation and T cell proliferation induced by B. subtilis-Cap. Orally inoculated piglets were used for in vivo experiments; ELISA and western blotting were used to evaluate B. subtilis-Cap induced PCV2-specific IgA and IgG levels, as well as the secretion of cytokines and the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 9 (TLR9). Results: We evaluated the immune response to B. subtilis-Cap in vitro using mouse BM-DCs and in vivo using neonatal piglets orally inoculated with B. subtilis-Cap. Our results showed that the recombinant B. subtilis-Cap activated BM-DCs, significantly increased co-stimulatory molecules (CD40 and CD80) and major histocompatibility complex II, and induced allogenic T cells proliferation. Piglets immunized with B. subtilis-Cap had elevated levels of PCV2-specific IgA in the mucosal tissues of the digestive and respiratory tract, and PCV2-specific IgG in serum (P < 0.05 or P < 0.01). Ileal immunocompetent cells, such as the IgA-secreting cells (P < 0.01), intestinal intraepithelial lymphocytes (IELs) (P < 0.01), CD3⁺ T lymphocytes (P < 0.01) and CD4⁺ T lymphocytes (P < 0.01) increased significantly in the B. subtilis-Cap immunized piglets. Additionally, B. subtilis-Cap inoculation resulted in increased the expression of TLR2 and TLR9 (P < 0.01), and induced the secretion of cytokines IL-1β, IL-6, interferon-γ, and β-defensin 2 (P < 0.01). Conclusions: We constructed a prototype PCV2 vaccine that can be administered orally and elicits a more robust humoral and cellular immunity than inactivated PCV2. B. subtilis-Cap is a promising vaccine candidate that is safe, convenient, and inexpensive. Further in vivo research is needed to determine its full range of efficacy in pigs. Graphical

Bacillus subtilis (B. subtilis), a probiotic bacterium and feeding additive, is widely used for heterologous antigen expression and protective immunisation. Porcine epidemic diarrhoea virus (PEDV) invades swine via mucosal tissue. To enhance the mucosal immune response to PEDV, we modified B. subtilis to express a PEDV antigen and used it as a mucosal vaccine delivery system. Initially, we constructed a recombinant B. subtilis strain (B.s-RCL) that expressed the PEDV spike protein and L-Lectin-β-GF, with the goal of inducing mucosal secretory immunoglobulin A (sIgA) and anti-PEDV serum immunoglobulin G (IgG) production, as well as to increase the number of microfold cells (M cells). Following the oral administration of B.s-RCL to mice, the small intestinal PEDV-specific sIgA expression levels significantly increased, as well as the increased number of B.s-RCL adhered to M cells. Moreover, we found that mice administered B.s-RCL exhibited markedly higher percentages of CD4⁺ and CD8⁺ T cells in the mesenteric lymph nodes and spleen compared to the control mice. Furthermore, we found that intestinal mucosa sIgA and serum anti-PEDV IgG levels were higher in mice orally immunised with B.s-RCL, suggesting that the mice could be more resistant to PEDV. In this study, we developed a novel oral vaccine to prevent porcine diarrhoea epidemics.