Veterinary Research Forum

Abstract Foot-and-mouth disease virus (FMDV) is a highly transmissible pathogen causing severe economic losses in the global livestock sector. Frequent outbreaks of FMDV type-O in Pakistan highlight the need for continuous genomic and antigenic surveillance to track its evolution. This study aimed to isolate and molecularly characterize FMDV type-O from an outbreak in the Punjab province of Pakistan using in vitro cell culture techniques. Samples were processed for viral isolation on a susceptible cell line, followed by RNA extraction. The VP1 gene, pivotal for antigenicity and immunogenicity, was amplified using a one-step polymerase chain reaction protocol. Purified amplicons underwent sequencing, and the nucleotide sequences were translated into amino acid sequences for further analysis. Protein three-dimensional modeling and in silico comparison were performed against the vaccinal seed strain PanAsia-2. The VP1 sequence analysis revealed notable genetic variability among the isolates, indicating adaptive evolution. Structural and antigenic modeling uncovered key differences between the field isolates and vaccinal strain, suggesting potential antigenic drift, which could undermine vaccine performance. The study underscores the dynamic evolution of FMDV type-O in Pakistan and the critical importance of ongoing genomic monitoring to refine vaccine strategies and enhance outbreak control.

Abstract The non-structural protein (nsp) 8 of the porcine epidemic diarrhea virus (PEDV) is highly stable across different PEDV strains and plays an important role in PEDV virulence. In current study, nsp8 prokaryotic expression vectors were constructed based on parental vectors pMAL-c2x-maltose binding protein (MBP) and pET-28a (+). Subsequently, the optimization of expression conditions in Escherichia coli, including induced temperature, time and isopropyl β-D-thiogalactopyranoside concentration were performed to obtain a stable expression of MBP-nsp8 and nsp8. The nsp8 fused with MBP increased the water solubility of the expressed products. Target proteins were further purified from E. coli culture and their immunogenicities were evaluated in vivo by mice. The antibody titers of serum from nsp8 immunized mice were up to 1:7,750,000 when measured by indirect enzyme-linked immunosorbent assay; meanwhile, the mice immunized with MBP-nsp8 gave an antibody titer reaching 1:1,000,000. In all, the expression and purification system of PEDV nsp8 and MBP-nsp8 were successfully established in this work and a strong immune response was elicited in mice by both purified nsp8 and MBP-nsp8, providing a basis for the study of the structure and function of PEDV nsp8.