African swine fever (ASF) is a highly lethal hemorrhagic viral disease of domestic pigs caused by African swine fever virus (ASFV). Although a good advance has been made to understand the virus, a safe and effective vaccine against ASFV is still lacking and its eradication solely depends on its early and accurate diagnosis. Thus, improving the available diagnostic assays and adding some validated techniques are useful for a range of serological investigations. The aim of this study was to produce and characterize p54 monoclonal antibodies with an ultimate goal of developing a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for ASFV antibody detection. Five monoclonal antibodies against p54 protein expressed in Escherichia coli was generated and their characterizations were investigated. Furthermore, a competitive enzyme-linked immunosorbent assay (cELISA) based on a monoclonal antibody designated as 2A7 was developed. To evaluate the performance of the assay, a total of 365 pig serum samples (178 negative and 187 positive samples) were tested and a receiver-operating characteristic (ROC) analysis was applied to determine the cut-off value. Based on the ROC analysis, the area under the curve (AUC) was 0.982 (95% confidence interval: 96.9% to 99.4%), besides a sensitivity of 92.5% and a specificity of 98.9% was achieved when the percent inhibition of 20% was selected as a threshold. Moreover, the result showed an excellent agreement when compared to other commercially available blocking ELISA (kappa value = 0.912) and showed no reaction to other swine pathogens. Overall, the newly developed cELISA method offers a promising approach for a rapid and convenient ASFV serodiagnosis, which could be used as alternative to other serological assays for screening possible ASFV infection.

Paraliobacillus zengyii CGMCC1.16464 (P. zengyii) is a novel antiviral probiotic candidate strain. To ensure its safety as a potential probiotic, a safety evaluation was conducted in this study. The safety and functional potential of P. zengyii were systematically assessed through genomic bioinformatics analysis, in vitro experiments, and acute oral toxicity tests in mice. Genomic analysis revealed that P. zengyii is rich in genes related to carbohydrate and amino acid metabolisms and carries genes encoding antimicrobial and antiviral agents (such as ectoine, type III polyketide synthase, and lasso peptides). It also expresses gastrointestinal tolerance-related proteins (ClpC, GroEL, and ClpP). Its resistance to polymyxins is an inherent trait with no risk of plasmid-mediated transfer. In vitro experiments confirmed that P. zengyii is somewhat tolerant to bile salts and acidic environments and does not exhibit hemolytic or gelatinase activity. Importantly, an acute oral toxicity test in mice revealed that after intervention with high, medium, or low doses, no significant abnormalities in the body weight, organ index, or tissue morphology of the mice were observed. In conclusion, P. zengyii exhibited good safety and probiotic potential in terms of genomic safety, metabolic function, and in vitro and in vivo toxicities, providing a theoretical basis for the development of novel functional probiotics.

Enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD), leading to a serious health threat to young children. Probiotics are effective at treating or preventing gastrointestinal infections, especially viral infections. Probiotics against EV71 are mainly traditional lactic acid-producing bacteria, and most of them have been proven to be effective only in vitro. Here, we report that the marine bacterium Paraliobacillus zengyii X-1125 (P. zengyii) has promising anti-EV71 activity. The antiviral effect of P. zengyii against EV71 was assessed in different cell lines, and the viral RNA levels and titers were obviously reduced after treatment with P. zengyii. Furthermore, we established an EV71-infected mouse model to evaluate its antiviral efficacy in vivo. The oral administration of P. zengyii significantly decreased the viral loads in the hindlimb muscles, spleens, and ileums. Further research revealed that P. zengyii enhances the expression of type I interferon (IFN-I) in EV71-infected cells. Similarly, transcriptome analysis indicated that the expression of interferon-stimulated genes (ISGs) in EV71-infected mice significantly increased after P. zengyii treatment. Taken together, the results of this study indicated that P. zengyii markedly reduces EV71 infection by regulating the IFN response both in vivo and in vitro, providing a potential means to work against EV71 infection.