Analysis of Antigenic Determinants on B/C Domains of E2 Glycoprotein of Classical Swine Fever Virus
Date Issued
2011
Date
2011
Author(s)
Chang, Chia-Yi
Abstract
Classical swine fever (CSF), also known as hog cholera, is caused by classical swine fever virus (CSFV) and is an economically important and highly contagious disease of pigs. Analysis of the E2 sequences of CSFVs from field outbreaks in Taiwan showed that the viruses could be divided into historical subgroup (subgroup 3.4) and exotic subgroup (subgroup 2.1). In the field there has been a switch of CSFV from subgroup 3.4 to 2.1 since and after year 1996. Glycoprotein E2 of CSFV is the major antigenic protein exposed on the outer surface of the virion that induces main neutralizing antibodies during infection in pigs. To investigate whether the antigenicity differences among various CSFVs subgroups were responsible for the replacement of subgroup 3.4 by subgroup 2.1 in the field, antigenicities of E2 of various Taiwanese isolates were analyzed. This study demonstrated the antigenicity differences of E2 between vaccine and field strains of CSFV by their variable reaction patterns with monoclonal antibodies (mAbs). The mAbs against B/C domains were able to differentiate field viruses TD/96/TWN (subgroup 2.1) and 94.4/IL/94/TWN (subgroup 3.4) from the vaccine virus LPC/AHRI (subgroup 1.1). The D/A domains of various CSFVs were relatively conserved and recognized by all mAbs against the A domain. By analyszing the expressed truncated proteins, the epitope(s) on B/C domains were mapped to the N-terminal 90 residues of E2 between amino acids 690 and 779. Site-directed mutagenesis further showed that residues 693C, 737C, 771L, 772L, 773F and 774D were critical for the reactivity of E2 protein with mAbs. Thus, the B/C domains are responsible for antigen specificity among various CSFVs, and the disulfide bond and motif 771LLFD774 are essential for the structural integrity of its conformational recognition. Site-directed mutagenesis of E2 further demonstrated that residues 713E and 729D were critical for antigenic specificity of field strain (94.4/IL/94/TWN), while residues 705D and 761K were specific for vaccine strain (LPC/AHRI). These specific residues likely mediated in determining the topography of mAb binding sites of E2 to allow for differentiation between strains based on the premise that the structural integrity of the conformational epitope is maintained. To mimic the conformational epitopes, a set of synthetic cyclized peptides spanning the B/C domains of E2 were used to react with mAbs against E2 and with swine anti-CSFV polyclonal sera. All antibodies recognized a highest common element, 753RYLASLHKKALPTSV767, on the double-looped peptides. This epitope region has not been revealed previously in the literature. Both substitution-scanning of residues 753RYLASLHKKALPTSV767 on a double-looped peptide and site-directed mutagenesis of expressed E2 demonstrated that residues 761K, 763L and 764P were critical for the reactivity with mAbs. In addition, the up- and downstream residues 753R, 754Y, 755L and 765T were also crucial. Alignment showed that this stretch of amino acids was relatively conserved among various CSFVs. Thus, we identified a motif 753RYLASLHKKALPT765, which may be part of group-specific antigen and important for the structural integrity of conformational epitope recognition. These data significantly further our understanding on the antigenic structure of E2 and the antigenic dterminants on B/C domains.
Subjects
Classical swine fever virus
E2 glycoprotein
Antigenic specificity
Conformational epitope
Monoclonal antibodies
Site-directed mutagenesis
SDGs
Type
thesis
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