The African Swine Fever virus (ASFV) causes an infectious viral disease in pigs of all ages. The development of antiviral drugs primarily aimed at inhibition of proteases required for the proteolysis of viral polyproteins. In this study, the conformation of the pS273R protease in physiological states were investigated, virtually screened the multi-protein conformation of pS273R target proteins, combined various molecular docking scoring functions, and identified five potential drugs from the Food and Drug Administration drug library that may inhibit pS273R. Subsequent validation of the dynamic interactions of pS273R with the five putative inhibitors was achieved using molecular dynamics simulations and binding free energy calculations using the molecular mechanics/Poison-Boltzmann (Generalized Born) (MM/PB(GB)SA) surface area. These findings demonstrate that the arm domain and Thr159-Lys167 loop region of pS273R are significantly more flexible compared to the core structural domain, and the Thr159-Lys167 loop region can serve as a “gatekeeper” in the substrate channel. Leucovorin, Carboprost, Protirelin, Flavin Mononucleotide, and Lovastatin Acid all have Gibbs binding free energies with pS273R that were less than −20 Kcal/mol according to the MM/PBSA analyses. In contrast to pS273R in the free energy landscape, the inhibitor and drug complexes of pS273R showed distinct structural group distributions. These five drugs may be used as potential inhibitors of pS273R and may serve as future drug candidates for treating ASFV.

Classical swine fever and porcine reproductive and respiratory syndrome have seriously affected the development of the swine breeding industry in China. Vaccine immunization remains the main way to prevent these infections. The aim of this study was to establish an optimized protocol for vaccine immunization against classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV). Blood samples were collected from the anterior vena cava of pigs after immunization, and blood indices, secreted levels of specific antibodies and neutralizing antibodies associated with humoral immunity, the proliferation capacity of T lymphocytes as a measure of cellular immunity, and secreted levels of IFN-γ and TNF-α were determined. The results showed that simultaneous immunization against CSFV and PRRSV infections induced strong and specific humoral and T-cellular immune responses, high levels of cytokine IFN-γ secretion and delayed secretion of cytokine TNF-α. Moreover, significantly higher lymphocyte percentages and red blood cell and leukocyte counts were found in the group simultaneously immunized against CSFV and PRRSV. However, no statistically significant differences were observed in hemoglobin values, neutrophil counts, and median cell percentages among the S + PRRS, PRRS-S, and S-PRRS groups. This study demonstrated that simultaneous immunization against CSFV and PRRSV had the advantages of inducing a rapid, enhanced, and long-lasting immune response. These findings provide a theoretical basis for the establishment of a reasonable and optimized vaccine immunization protocol against CSFV and PRRSV in combination with a variety of other vaccine inoculations.

Identification of novel drugs for anti-African swine fever (ASF) applications is of utmost urgency, as it negatively affects pig farming and no effective vaccine or treatment is currently available. African swine fever virus (ASFV) encoded pS273R is a cysteine protease that plays an important role in virus replication. E64, acting as an inhibitor of cysteine protease, has been established as exerting an inhibitory effect on pS273R. In order to obtain a better understanding of the interaction between E64 and pS273R, common docking, restriction docking, and covalent docking were employed to analyze the optimal bonding position between pS273R−E64 and its bonding strength. Additionally, three sets of 100 ns molecular dynamics simulations were conducted to examine the conformational dynamics of pS273R and the dynamic interaction of pS273R−E64, based on a variety of analytical methods including root mean square deviation (RMSD), root mean square fluctuation (RMSF), free energy of ligand (FEL), principal component analysis (PCA), and molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) analysis. The results show that E64 and pS273R exhibited close binding degrees at the activity center of ASFV pS273R protease. The data of these simulations indicate that binding of E64 to pS273R results in a reduction in flexibility, particularly in the ARM region, and a change in the conformational space of pS273R. Additionally, the ability of E64 to interact with polar amino acids such as ASN158, SER192, and GLN229, as well as charged amino acids such as LYS167 and HIS168, seems to be an important factor in its inhibitory effect. Finally, Octet biostratigraphy confirmed the binding of E64 and pS273R with a KD value of 903 uM. Overall, these findings could potentially be utilized in the development of novel inhibitors of pS273R to address the challenges posed by ASFV.