title: Diagnostic dilemma encountered when detecting bovine viral diarrhea virus in IVF embryo production.
abstract: Routine quality controls in production of bovine embryos by in vitro fertilization (IVF) should include screening all materials of animal origin for the presence of bovine viral diarrhea virus (BVDV). Using a reverse transcription nested polymerase chain reaction (RT-nPCR) assay, we detected BVDV in primary cultures of uterine tubal cells (UTC) that had been used during IVF procedures. The goal of our ensuing investigation was to determine its source and assess risks associated with the identified contaminant. Sequencing of the amplified 5' nontranslated region (NTR) of the viral genome confirmed a Genotype I BVDV contaminant. This viral contaminant was also identified by RT-nPCR in multiple samples of the same lot of fetal bovine serum (FBS) that was used in transport media by the laboratory that harvested the UTC. Both routine and enhanced roller bottle methods for virus isolation failed to detect BVDV in the FBS. Furthermore, virus neutralization assays did identify antibodies to Genotype I strains of BVDV in the FBS. After 7 days of co-incubation, neither cultured, washed UTC nor exposed, washed embryos were RT-nPCR positive for BVDV. Eight embryos produced in the contaminated system were nonsurgically transferred into eight seronegative cows. None of the embryo recipients seroconverted to BVDV. Thus, contamination of cell culture medium with BVDV did not result in transmission of the virus when IVF embryos were transferred. Failure to transmit disease was likely aided by serendipitous control from anti-BVDV antibodies in the FBS. However, a diagnostic dilemma was created when the RT-nPCR assays used to screen for BVDV were positive, yet attempts to isolate the virus were negative. This case study illustrates that if molecular assays are to be used to confirm the pathogen-free status of IVF embryo production systems, media components of animal origin (e.g. FBS) should be screened with molecular assays for BVDV as well as traditional virus isolation techniques.
authors.affiliation: Sugg Laboratory, College of Veterinary Medicine, Auburn University, AL 36849-5519, USA. givenmd@vetmed.auburn.edu
authors.forename: M Daniel
authors.initials: MD
authors.lastname: Given
authors.forename: Kay P
authors.initials: KP
authors.lastname: Riddell
authors.forename: Patricia K
authors.initials: PK
authors.lastname: Galik
authors.forename: David A
authors.initials: DA
authors.lastname: Stringfellow
authors.forename: Kenny V
authors.initials: KV
authors.lastname: Brock
authors.forename: Naida M
authors.initials: NM
authors.lastname: Loskutoff
mesh_terms: D020121:5' Untranslated Regions / Q000737:chemistry / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000175:diagnosis / Q000635:transmission / Q000821:virology*; D002417:Cattle / Q000196:embryology*; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000662:veterinary; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics / Q000302:isolation & purification*; D004624:Embryo Transfer / Q000662:veterinary; D005260:Female; D005307:Fertilization in Vitro / Q000009:adverse effects / Q000662:veterinary*; D008297:Male; D008969:Molecular Sequence Data; D009500:Neutralization Tests / Q000662:veterinary; D009865:Oocytes / Q000821:virology; D011247:Pregnancy; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D012710:Serum Albumin, Bovine / Q000009:adverse effects
chemical_list: D020121:5' Untranslated Regions; D012367:RNA, Viral; D012710:Serum Albumin, Bovine