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Porcine epidemic diarrhea virus (PEDV) is the most common diarrhea-causing pathogen in newborn piglets.The clarifications of the overall antibody repertoire and antigen-specific antibody repertoire are essential to provide important insights into the B-cell response and reshape new vaccines.Here,we applied next-generation sequencing (NGS) technology to investigate immunoglobulin (lg) variable (V) gene segment usage of swine B-cells from peripheral blood lymphocytes (PBL) and mesenteric lymph node (MLN) cells following PEDV vaccination.We identified the transcripts of all functional Ig V-genes in antibody repertoire.IgHV1S2,IgKV1-11,and IgLV3-4 were the most prevalent gene segments for heavy,kappa,and lambda chains,respectively,in PBL and MLN.Unlike previous studies,IgKV1,instead of IgKV2,and IgLV3,instead of IgLV8,were the prevalent Ig V-gene families for kappa and lambda light chains,respectively.We further examined the antibody repertoire of PEDV spike-specific B cells by single-cell RT-PCR.In contrast to the overall antibody repertoire,Ig V-gene segments of PEDV spike-specific B cells preferentially adopted IgHV1-4 and IgHV1-14 for heavy chain,IgKV1-11 for kappa chain,and IgLV3-3 for lambda chain.These results represent a comprehensive analysis to characterize the Ig V-gene segment usage in the overall and PEDV spike-specific antibody repertoire in PBL and MLN.