Identification of three PPV1 VP2 protein-specific B cell linear epitopes using monoclonal antibodies against baculovirus-expressed recombinant VP2 protein

Abstract

Porcine parvovirus type 1 (PPV1) is a major causative agent of embryonic and fetal death in swine. The PPV1 VP2 protein is closely associated with viral immunogenicity for eliciting neutralizing antibodies, but its antigenic structures have been largely unknown. We generated three monoclonal antibodies (MAbs) against baculovirus-expressed recombinant PPV1 VP2 protein. A PEPSCAN analysis identified the minimal B cell linear epitopes of PPV1 VP2 based on these MAbs. Three core epitopes, (228)QQITDA(233), (284)RSLGLPPK(291), and (344)FEYSNGGPFLTPI(356), were defined and mapped onto three-dimensional models of the PPV1 virion and VP2 monomer. The epitope (228)QQITDA(233) is exposed on the virion surface, and the other two are located inside the protein. An alignment of the PPV1 VP2 amino acid sequences showed that (284)RSLGLPPK(291) and (344)FEYSNGGPFLTPI(356) are absolutely conserved, whereas (228)QQITDA(233) has a single substitution at residue 233 in some (S → A or T). We developed a VP2 epitope-based indirect enzyme-linked immunosorbent assay (iELISA) to test for anti-PPV1 antibodies. In a comparative analysis with an immunoperoxidase monolayer assay using 135 guinea pig sera, the VP2-epitope-based iELISA had a concordance rate of 85.19 %, sensitivity of 83.33 %, and specificity of 85.47 %. MAb 8H6 was used to monitor VP2 during the PPV1 replication cycle in vitro with an indirect immunofluorescence assay, which indicated that newly encapsulated virions are released from the nucleus at 24 h postinfection and the PPV1 replication cycle takes less than 24 h. This study provides valuable information clarifying the antigenic structure of PPV1 VP2 and lays the foundations for PPV1 serodiagnosis and antigen detection.

PCR-based detection and genetic characterization of porcine parvoviruses in South Korea in 2018

“…On the VP2 capsid protein, three linear B cell epitopes were experimentally identified [6]. Following the previous publication [24], this study looked for substitutions in the VP2 capsid protein that are responsible for tissue Fig.…”

“…One codes for large nonstructural proteins (NS1), which has low nucleotide substitution, and the other codes for structural viral proteins (VP1 and VP2) [2]. The two structural viral proteins VP1 and VP2 are considered related to the virulence of the virus that mainly targets the neutralization of antibodies against PPV1 [4][5][6][7].…”

Identification of specific B cell linear epitopes of Mycoplasma hyorhinis P37 protein using monoclonal antibodies against baculovirus-expressed P37 protein

“…Based on alignment of P37 amino acids, sequence analysis of different Mhr strains showed that the 206 KIKKAWNDKDWNTFRNF 222 epitope was completely conserved in all Mhr strains. Although antigenic epitopes are usually contained within aa6-10, interestingly this epitope region was in aa17 [23,24].…”

“…were mixed with DMEM medium containing hypoxanthine-aminopurine-thymidine (HAT) (Sigma-Aldrich, New York, NY, USA) and 20% FBS. Cells were cultured together and the media were replaced with selection medium containing hypoxanthine-thymidine (HT) (Sigma-Aldrich) and 10% FBS after 5 d [23]. Hybridoma supernatants were collected after 7 d and screened for the presence of Mhrspecific antibodies by indirect enzyme-linked immunosorbent assay (ELISA) [26].…”

Porcine parvovirus replication is suppressed by activation of the PERK signaling pathway and endoplasmic reticulum stress-mediated apoptosis

“…Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse Ab against IgG and tetramethylrhodamine isothiocyanate-conjugated goat anti-mouse Ab against IgG were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd. (Beijing, China). Anti-PPV VP2 monoclonal Ab (McAb) 8E4, which recognizes the PPV VP2 and VP1 proteins, was prepared in our laboratory (Sun et al, 2015).…”