Improved detection of bovine herpesvirus 1 in artificially infected bovinesemen by protein amplification

Infection with bovine herpesvirus 1 (BHV 1) occurs worldwide and causes ser ious economic losses due to loss of animals, abortions, decreased milk prod uction, and loss of body weight. There is a real need for sensitive diagnos tic procedures for detection of the presence of virus in order to achieve e ffective control of BHV I-induced diseases. BHV 1 is frequently found in bo vine semen and can be widely transmitted through artificial insemination. T hus the detection of BHV 1 in artificial insemination centers and semen ban ks is of crucial importance in the control of its dissemination to the catt le industry, worldwide. In the present study, a protein amplification assay following polymerase chain reaction (PCR) of the highly conserved BHV 1 gl ycoprotein D gene was used in order to improve the sensitivity of direct vi rus detection in bovine semen. This method of BHV 1 detection is at least 2 00 orders of magnitude more sensitive than traditional PCR and would have d irect clinical applications in antigen-based detection tests, In this metho d, amplification of the BHV 1 go gene by PCR is followed by a coupled in vi tro transcription-translation of a small aliquot from the reaction. When th e transcription-translation was carried out in the presence of [S-35]methio nine and the products analyzed by SDS-PAGE and autoradiography, 0.0014 TCID 50 of virus could be detected in raw bovine semen in contrast to 0.28 TCID5 0 of virus detected using traditional PCR. Given the limitations in the met hod used for protein detection, this 'in vitro protein amplification' has t he potential of attaining superior sensitivity for direct virus detection i n clinical samples. (C) 1999 Elsevier Science B.V. All rights reserved.