Development of Monoclonal Antibody to Specifically Recognize VP0 but Not VP4 and VP2 of Foot-and-Mouth Disease Virus

Abstract

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease that affects cloven-hoofed animals and often causes enormous economic loss in the livestock industry. The capsid of FMD virus (FMDV) consists of four structural proteins. Initially, one copy each of the proteins VP0, VP3, and VP1 are folded together into a protomer, and five copies of the protomer compose a pentamer. Finally, 12 pentamers are assembled into an icosahedral capsid. At the maturation stage during RNA encapsidation, VP0 is cleaved into VP4 and VP2. The mechanism underlying VP0 maturation remains unclear. While monoclonal antibodies (mAbs) against VP2 have been developed in previous studies, a mAb specific to VP0 has not yet been reported. In this study, we generated VP0-specific mAbs by immunizing mice with peptides spanning the C-terminal amino acids of VP4 and N-terminal amino acids of VP2. We verified that these mAbs displayed specificity to VP0 with no reactivity to VP4 or VP2. Therefore, these mAbs could prove useful in identifying the role of VP0 in FMDV replication and elucidating the mechanism underlying VP0 cleavage into VP4 and VP2.

Keywords: VP0; VP2; VP4; foot-and-mouth disease virus; monoclonal antibody.

Figure 1

Alignment of the amino acid sequences in the junction region between VP4 and VP2 of different strains of foot-and-mouth disease virus (FMDV). The amino acid sequences of the VP4 C-terminus and VP2 N-terminus derived from seven local strains and two representative strains (O1 Manisa and A22 IRQ) of FMDV were aligned. The box with solid lines indicates the selected sequence (16-mer) for the production of VP0-specific mAb. The sequences of VP4 and VP2 are separated by the dotted line. Dots represent identical amino acids.

Figure 2

Identification of the recombinant FMDV VP0, VP2, VP4, and VP0 epitope proteins. The glutathione S-transferase (GST)–fused recombinant proteins were each expressed in Escherichia coli and detected using Western blot with (a) anti-GST, (b) anti-VP2, and (c) anti-VP4 antibodies. Lane M, molecular weight protein marker; lane 1, GST-VP0 protein; lane 2, GST-VP2 protein; lane 3, GST-VP4 protein; lane 4, GST-VP0 epitope protein.

Figure 3

Reactivity of the FMDV VP0-specific monoclonal antibodies (mAbs) with the recombinant proteins. The reactivity of five types of VP0-specific antibodies, (a) 8G9, (b) 9A5, (c) 17E6, (d) 23F2, and (e) 24A12, to the GST-fused recombinant VP0, VP2, VP4, and VP0 epitope proteins was evaluated using Western blotting. Lane M, molecular weight protein marker; lane 1, GST-VP0 protein; lane 2, GST-VP2 protein; lane 3, GST-VP4 protein; and lane 4, GST-VP0 epitope protein.