title: Improved detection of bovine herpesvirus 1 in artificially infected bovine semen by protein amplification.
abstract: Infection with bovine herpesvirus 1 (BHV 1) occurs worldwide and causes serious economic losses due to loss of animals, abortions, decreased milk production, and loss of body weight. There is a real need for sensitive diagnostic procedures for detection of the presence of virus in order to achieve effective control of BHV 1-induced diseases. BHV 1 is frequently found in bovine semen and can be widely transmitted through artificial insemination. Thus the detection of BHV 1 in artificial insemination centers and semen banks is of crucial importance in the control of its dissemination to the cattle industry, worldwide. In the present study, a protein amplification assay following polymerase chain reaction (PCR) of the highly conserved BHV 1 glycoprotein D gene was used in order to improve the sensitivity of direct virus detection in bovine semen. This method of BHV 1 detection is at least 200 orders of magnitude more sensitive than traditional PCR and would have direct clinical applications in antigen-based detection tests. In this method, amplification of the BHV 1 gD gene by PCR is followed by a coupled in vitro transcription translation of a small aliquot from the reaction. When the transcription translation was carried out in the presence of [35S]methionine and the products analyzed by SDS PAGE and autoradiography, 0.0014 TCID50 of virus could be detected in raw bovine semen in contrast to 0.28 TCID50 of virus detected using traditional PCR. Given the limitations in the method used for protein detection, this 'in vitro protein amplification' has the potential of attaining superior sensitivity for direct virus detection in clinical samples.
authors.affiliation: Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Zhou
authors.forename: J
authors.initials: J
authors.lastname: Lyaku
authors.forename: R A
authors.initials: RA
authors.lastname: Fredrickson
authors.forename: F S
authors.initials: FS
authors.lastname: Kibenge
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis; D015870:Gene Expression; D007242:Herpesvirus 1, Bovine / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012661:Semen / Q000821:virology*; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D004279:DNA, Viral; D014764:Viral Proteins; C046998:bovine herpesvirus type-1 glycoproteins