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Foot-and-mouth disease virus (FMDV) 2C protein is a conserved non-structural protein and crucial for replication of the virus. In this study, FMDV 2C protein was prepared and the enzymatic activities were investigated in detail. The protein could digest ssDNA or ssRNA into a small fragment at about 10 nt, indicating that the protein has nuclease activity. But it did not show digestion to blunt-end dsDNA or dsRNA. The nuclease activity of 2C protein could be inhibited in 2 mM Zn2+ or Ca2+ while enhanced by Mg2+ or Mn2+. FMDV 2C protein exhibited unwinding activity to all the three kinds of dsDNA and dsRNA (5′ protruded, 3′ protruded, and blunt-end). The unwinding velocity to 5′ protruded dsRNA was higher than to the blunt-end dsRNA. 2C protein only showed unwinding activity in high concentration of Mg2+, but no unwinding activity in physiological concentrations of Mg2+ and Ca2+, as well as in cell lysate. The 2C protein could catalyze two structured ssRNA to form double strand, thus it was proved to have RNA chaperone activity. The Mg2+ and ATP in different concentrations did not show promotion to the RNA chaperone activity. Finally, six mutant proteins (K116A, D160A, D170A, N207A, R226A, and F316A) were constructed and the enzymatic activities were analyzed. All the six mutations reduced the ATPase activity, D170A and F361A could inactivate the nuclease activity, while the N207A and F316A could inactivate the helicase activity. Our study provides a comprehensive understanding of the enzymatic activities of FMDV 2C protein.

The misuse of antibiotics in veterinary medicine presents significant challenges, highlighting the need for alternative therapeutic approaches such as antibody drugs. Therefore, it is necessary to explore the application of antibody drugs in veterinary settings to reduce economic losses and health risks. This study focused on targeting the F4ac subtype of the FaeG protein, a key adhesion factor in enterotoxigenic Escherichia coli (ETEC) infections in piglets. By utilizing formaldehyde-inactivated ETEC and a soluble recombinant FaeG (rFaeG) protein, an antibody library against the FaeG protein was established. The integration of fluorescence-activated cell sorting (FACS) and a eukaryotic expression vector containing murine IgG Fc fragments facilitated the screening of anti-rFaeG IgG monoclonal antibodies (mAbs). The results demonstrate that the variable regions of the screened antibodies could inhibit K88-type ETEC adhesion to IPEC-J2 cells. Furthermore, in vivo neutralization assays in mice showed a significant increase in survival rates and a reduction in intestinal inflammation. This research underscores the potential of antibody-based interventions in veterinary medicine, emphasizing the importance of further exploration in this field to address antibiotic resistance and improve animal health outcomes.