Classical swine fever virus (CSFV) is a single‐stranded RNA flavivirus that can cause serious diseases in porcine species, including symptoms of infarction, systemic hemorrhage, high fever, or depression. Viperin is an important interferon‐inducible antiviral gene that has been shown to inhibit CSFV, but the exact mechanisms by which it is able to do so remain poorly characterized. In the present study, we determined that CSFV infection led to viperin upregulation in PK‐15 cells (porcine kidney cell). When viperin was overexpressed in these cells, this markedly attenuated CSFV replication, with clear reductions in viral copy number after 12 to 48 hours postinfection. Immunofluorescence microscopy revealed that the viral NS5A protein colocalized with viperin in infected cells, and this was confirmed via confocal laser scanning microscopy using labeled versions of these proteins, and by co‐immunoprecipitation which confirmed that NS5A directly interacts with viperin. When NS5A was overexpressed, this inhibited the replication of CSFV, and we determined that the radical SAM domain and N‐terminal domain of viperin was critical for its ability to bind to NS5A, with the latter being most important for this interaction. Together, our in vitro results highlight a potential mechanism whereby viperin is able to inhibit CSFV replication. These results have the potential to assist future efforts to prevent or treat systemic CSFV‐induced disease, and may also offer more general insights into the antiviral role of viperin in innate immunity.

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Trefoil factor 3 (TFF3) is involved in cell adhesion, motility and apoptosis, regulates mucosal immunity and maintains the functional integrity of intestinal epithelia. The upregulation of TFF3 expression in the weaning rat intestine attracted our interest. The present study hypothesized that TFF3 may serve a role in preventing diarrhea in weaning piglets, which is an important consideration in the pig farming industry. Previous recombinant TFF3 protein expression yields obtained from Escherichia coli were too low and the bioactivity of the protein was poor. Hence, this expression system was unsuitable for industrial applications. The present study explored the production of recombinant sus scrofa TFF3 in a Brevibacillus choshinensis (B. choshinensis) expression system, aiming to enhance the expression level of bioactive protein. To achieve this, the sus scrofa TFF3-encoding gene fragment was fused into an E. coli-Brevibacillus shuttle vector pNCMO2. High levels of TFF3 (30 mg/l) were produced and secreted into the B. choshinensis culture medium in soluble form with a molecular mass of 13.6 kDa and high immunoreactivity in western blotting. Thus, Brevibacillus may be used to produce useful mucosal factors for biochemical analyses and mucosal protection, and in industrial applications to produce novel inhibitors of diarrhea.