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Identification of a conserved B-cell epitope on duck hepatitis A type 1 virus VP1 protein.

ABSTRACT: The VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized.To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1-positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope.We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.

SUBMITTER: Wu X

PROVIDER: S-EPMC4337900 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Identification of a conserved B-cell epitope on duck hepatitis A type 1 virus VP1 protein.

Wu Xiaoying X Li Xiaojun X Zhang Qingshan Q Wulin Shaozhou S Bai Xiaofei X Zhang Tingting T Wang Yue Y Liu Ming M Zhang Yun Y

PloS one 20150223 2

<h4>Background</h4>The VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized.<h4>Methods and results</h4>To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlappin ...[more]

PMID: 25706372

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Project description:Duck hepatitis A virus (DHAV) causes an infectious disease that mainly affects 1- to 4-week-old ducklings, resulting in considerable loss to the duck industry. Although there have been many studies on DHAV in recent years, the effects on host infection and pathogenesis of DHAV-1 remain largely unknown. This study investigated the effects of the DHAV-1 structural protein VP3 on DHAV-1 virus adsorption and apoptosis to explore the role of VP3 in the viral life cycle. The effects of DHAV-1 VP3 and an antibody against the protein on virion adsorption was analyzed by qRT-PCR. The results showed that the virus copy number for the rabbit anti-VP3 IgG-treated group was significantly lower than that for the negative control group but higher than that for the rabbit anti-DHAV-1 IgG-treated group. This result indicates that VP3 mediates DHAV-1 virus adsorption but that it is not the only protein that involved in this process. In addition, a eukaryotic recombinant plasmid, pCAGGS/VP3, was transfected into duck embryo fibroblasts (DEFs), and the apoptotic rate was determined by DAPI staining, the TUNEL assay and flow cytometry. DAPI staining showed nucleus fragmentation and nuclear edge shifting. TUNEL assay results revealed yellow nuclei, and flow cytometry indicated a significant increase in the apoptotic rate. In addition, qRT-PCR revealed increased in the transcriptional levels of the apoptotic caspase-3, -8 and -9, with the largest increase for caspase-3, followed by caspase-9 and caspase-8. Enzyme activity analysis confirmed these results. Furthermore, the VP3 protein decreased the mitochondrial membrane potential, and the transcriptional levels of the proapoptotic factors Bak, Cyt c and Apaf-1 in the mitochondrial apoptotic pathway were significantly upregulated. These data suggest that expression of VP3 in DEFs induces apoptosis and may primarily activate caspase-3-induced apoptosis through mitochondrion-mediated intrinsic pathways. The findings provide scientific data to clarify DHAV-1 infection and pathogenesis.

Dataset Information

Identification of a conserved B-cell epitope on duck hepatitis A type 1 virus VP1 protein.

Publications

Identification of a conserved B-cell epitope on duck hepatitis A type 1 virus VP1 protein.

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