Use of epitope mapping to identify a PCR template for protein amplification and detection by enzyme-linked immunosorbent assay of bovine herpesvirus type 1 glycoprotein D

Abstract

Infection with bovine herpesvirus type 1 (BHV-1) occurs worldwide and causes serious economic losses due to the deaths of animals, abortions, decreased milk production, and loss of body weight. BHV-1 is frequently found in bovine semen and is transmitted through natural service and artificial insemination. The detection of BHV-1 in bovine semen is a long-standing problem in veterinary virology which is important in disease control schemes. In the present study, ordered deletions of the full-length BHV-1 glycoprotein open reading frame were used to identify an epitope recognized by a specific monoclonal antibody (MAb). A glycoprotein D fragment containing this epitope was then amplified using an in vitro protein amplification assay developed previously (J. Zhou, J. Lyaku, R. A. Fredrickson, and F. S. Kibenge, J. Virol. Methods 79:181-189, 1999), and the resulting peptide was detected by indirect enzyme-linked immunosorbent assay (ELISA) with the specific MAb. This method detected 0.0395 50% tissue culture infective dose of BHV-1 in raw bovine semen, which was 1,000-fold more sensitive than traditional PCR. We therefore conclude that this in vitro protein amplification assay combined with ELISA has superior sensitivity for direct virus detection in clinical samples.

FIG. 1.

PCR amplification of gD gene DNA and overlapping fragments of gD ORF. Lane 1, 1-kbp-plus DNA ladder; lane 2, gD gene DNA; lanes 3, 4, and 5, overlapping fragments AB, ED, and CD, respectively.

FIG. 2.

SDS-PAGE analysis of affinity-purified GST-gD and various overlapping protein fragments. Lane 1, protein standards (Bio-Rad), with values indicated on the left; lanes 2, 3, and 4, overlapping protein fragments CD, ED, and AB, respectively; lane 5, full-length gD. In each case, the GST fragment has been cleaved away (as discussed in the text) and is indicated at the low-molecular-weight end of the gel. The positions of gD, CD, ED, AB, and GST are indicated on the right.

FIG. 3.

Schematic representation of BH-1 gD and its segments that were expressed by E. coli as GST fusion proteins. Each protein is fused with a GST tag at the amino end and Streptag at the carboxyl end.

FIG. 4.

Detection of the CD peptide of gD by autoradiography after in vitro transcription and translation. Lane 1, luciferase control reaction product at 61 kDa full length; lane 2, CD peptide; lane 3, in vitro transcription and translation reaction product without the template DNA.

FIG. 5.

PCR amplification of CD fragment of gD gene using purified BHV-1 DNA and infected semen samples. Lane 1, 1-kbp-plus DNA ladder (Invitrogen Life Technologies); lane 2, PCR product from purified BHV-1 DNA; lanes 3 to 8, PCR products from semen samples infected with virus dilutions from 10⁻¹ to 10⁻⁶, respectively.