Foot‐and‐mouth disease virus (FMDV) escapes host immune surveillance via adaptive evolution driven by vaccine‐mediated selective pressure, leading to persistent breakthrough infections in immunized animals. In this study, the dominant neutralizing epitope VP1 G–H loop (141–160 aa) was analyzed among 46 serotype O FMDV strains belonging to Southeast Asian (SEA) and Middle East–South Asian (ME‐SA) topotypes isolated during 1980–2019. The key amino acid residues at VP1 positions 142 and 153 underwent sequential stepwise evolution across three phases under natural selection. Based on the reverse genetic system of Wt, single‐site mutant (T142P, Q153P) and double‐site mutant (T142P&Q153P) strains were rescued. These mutation sites were further introduced into an efficient FMDV nanoparticle vaccine to construct four vaccine candidates. Mouse immunization verified all vaccines conferred solid protection against Wt and single‐mutant strains, yet protection efficacy was greatly impaired against the double mutant. The double‐mutant vaccine elicited high‐level neutralizing antibodies against the double‐mutant strain (T142P&Q153P) with a titer of 1:426.67, 10–20 folds higher than Wt (1:21.33), T142P (1:32), and Q153P (1:42.67) vaccines. Consistent results were also obtained in pigs immunized with commercial inactivated vaccines. Collectively, combined mutations at VP1 142 and 153 reshape viral antigenicity and act as core drivers of FMDV immune evasion. Integrating such immune escape hotspots into vaccine antigens can broaden neutralizing antibody coverage, offering an experimental basis for clarifying FMDV evolution and developing broad‐spectrum vaccines.
Nan Cao, Yamei Li, Xinghua Chen et al.· Transboundary and Emerging D...· 0 citations
Background Foot-and-mouth disease virus (FMDV) causes substantial economic losses in global livestock production; however, the key host factors supporting its early infection process remain poorly characterized. Methods In this study, we performed an unbiased genome-wide CRISPR/Cas9 knockout screening using porcine cells to screen and identify host factors involved in FMDV infection. Results We identified that the E3 ubiquitin ligase RNF24 supports efficient FMDV entry. RNF24 depletion inhibits viral entry and replication, whereas its overexpression enhances viral infectivity. Mechanistically, RNF24 preferentially promotes K27-linked non-degradative polyubiquitination of leupaxin (LPXN) at lysine 162, driving LPXN’s trafficking to the plasma membrane. At the membrane, LPXN assembles a ternary integrin-LPXN-VP1 complex that strengthens virus-receptor interactions and promotes viral adsorption and entry. Disruption of this ubiquitination event via the LPXN K162R mutation impairs complex formation and compromises viral entry. Conclusion Together, our study reveals a ubiquitin-dependent RNF24-LPXN regulatory axis that supports FMDV entry, highlights the role of non-degradative ubiquitination in viral pathogenesis, and proposes this interface as a potential target for antiviral intervention.
Jinyan Zhang, Hai-Long Liu, Jian Du et al.· Frontiers in Cell and Develo...· 0 citations
Classical swine fever virus (CSFV) and pseudorabies virus (PRV) remain significant threats to the swine industry. Although recombinant PRV vectors represent promising platforms for bivalent vaccine development, their application is frequently limited by insufficient expression of heterologous antigens, which may compromise protective efficacy. To address this limitation, we employed a multi-copy expression strategy to enhance CSFV E2 protein levels in a PRV-based vector. Using CRISPR/Cas9-mediated gene editing, we constructed recombinant PRVs expressing one, two, or three copies of the E2 gene by sequential insertion into the gE/gI, tk, and gG loci. In vitro validation demonstrated increased detectable E2 expression in the multi-copy recombinant viruses. Meanwhile, the recombinant viruses maintained virion morphology and replication kinetics comparable to those of the parental strain PRV-GX. Immunogenicity studies in rabbits showed that PRV-3CE2 elicited stronger E2-specific humoral responses and E2-associated cytokine recall responses than PRV-2CE2, while both recombinant viruses induced detectable neutralizing activity. In challenge experiments, although vaccination did not completely prevent febrile responses following CSFV challenge, both PRV-2CE2 and PRV-3CE2 reduced CSFV RNA loads in blood compared with the mock group, suggesting partial protective efficacy. Additionally, both PRV-2CE2 and PRV-3CE2 provided complete protection against lethal PRV challenge in rabbits. These findings suggest that multi-copy E2 expression enhances E2-associated immunogenicity and supports further optimization and evaluation of PRV-3CE2 as a PRV-CSFV bivalent vaccine candidate.
Xianfei Shang, Hui Zhao, Yufeng He et al.· Veterinary Microbiology· 0 citations
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