A neutralizing epitope within the VP1 protein of duck hepatitis A virus type 3: From single-cell BCR discovery to in vivo protection.

Duck hepatitis A virus type 3 (DHAV-3) continues to circulate, threatening the health of duck flocks and highlighting the urgent need for novel antibody-based interventions. In this study, we systematically profiled the DHAV-3-specific B cell immune repertoire in VP1-immunized mice, using high-throughput single-cell RNA sequencing and BCR sequencing of antigen-enriched B cells. From 27 functionally validated antibody clones, we identified three neutralizing antibodies, A1, A2, and A3. Among these, A2 exhibited the highest neutralizing activity, with an IC₅₀ of 19.48 μg/mL against DHAV-3. Through serial truncation mapping and alanine scanning, the A2 epitope was precisely defined as a linear motif, 98DNLTS102, located within the VP1 hypervariable region. Two residues, N99 and T101, were identified as critical for antibody binding. Homology modeling further revealed that A1, A2, and A3 share highly similar three-dimensional structures (RMSD <3 Å), providing a structural explanation for their recognition of the same epitope. In a DHAV-3-infected duckling model, A2 provided substantial protection when administered either prophylactically or therapeutically. The treatment and prevention groups exhibited survival rates of 75% and 65%, respectively, substantially exceeding the 10% recorded in the challenge control group. Collectively, our findings demonstrate that single-cell B cell sequencing can serve as a powerful platform for rapid discovery of neutralizing antibodies against emerging veterinary pathogens. Moreover, the antibodies and epitopes identified here offer promising candidates for precision control of DHAV-3.