title: Quantity and infectivity of embryo-associated bovine viral diarrhea virus and antiviral influence of a blastocyst impede in vitro infection of uterine tubal cells.
abstract: In previous studies, bovine viral diarrhea virus (BVDV) remained associated with IVF embryos after viral exposure and washing. However, uterine tubal cells (UTC) were not infected when exposed embryos were washed and individually co-cultured with them. The objective of this study was to evaluate quantity and infectivity of embryo-associated virus and antiviral influence of a blastocyst as possible explanations for failure to infect the UTC in vitro. Morulae and blastocysts were produced in vitro and washed. A portion of the embryos were incubated for 2 h in medium containing 10(6) to 10(8) cell culture infective doses (50%, CCID50) of a genotype I, noncytopathic BVDV per milliliter and then washed again. Virus isolation was attempted on sonicated negative (virus unexposed) and positive (virus exposed) control embryo groups after washing. The influence of quantity and infectivity of embryo-associated virus was evaluated by transferring exposed, washed embryo groups (2, 5, and 10 embryos/group) or sonicate fluid of exposed, washed, sonicated embryo groups (2, 5, and 10 embryos/group) to cultures containing bovine UTC in IVC medium that was free of BVDV neutralizing activity. The antiviral influence of an embryo was evaluated by adding 1 to 10(5) CCID50 of BVDV to UTC in the presence or absence of a single unexposed blastocyst in IVC medium. After 2 d in co-culture, the UTC, IVC medium and washed embryos (when present) were tested separately for the presence of BVDV using virus isolation. Virus was isolated from sonicate fluids of all positive but no negative controls. Virus was not isolated from any UTC following 2 d of culture with virally exposed groups of intact embryos. However, virus was isolated from UTC cultured with sonicate fluids from some groups of 5 (60%) and 10 (40%) embryos. Infective virus also remained associated with some groups of 2 (20%), 5 (40%) and 10 (60%) intact embryos after 48 h of post-exposure culture. Finally, primary cultures of UTC were more susceptible to infection with BVDV in the absence of a blastocyst (P = 0.01). Results indicate that insufficient quantity and reduced infectivity of embryo-associated virus as well as an antiviral influence of intact IVF blastocysts may all contribute to failure of embryo-associated virus to infect UTC in vitro.
authors.affiliation: Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL 36849-5519, USA.
authors.forename: M D
authors.initials: MD
authors.lastname: Givens
authors.forename: P K
authors.initials: PK
authors.lastname: Galik
authors.forename: K P
authors.initials: KP
authors.lastname: Riddell
authors.forename: K V
authors.initials: KV
authors.lastname: Brock
authors.forename: D A
authors.initials: DA
authors.lastname: Stringfellow
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000821:virology*; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000517:prevention & control* / Q000635:transmission*; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D015925:Cryopreservation; D001908:Diarrhea Viruses, Bovine Viral / Q000302:isolation & purification / Q000472:pathogenicity*; D005187:Fallopian Tubes / Q000821:virology*; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods / Q000662:veterinary*; D005315:Fetal Diseases / Q000821:virology*; D008297:Male; D009028:Morula / Q000821:virology*; D009865:Oocytes / Q000166:cytology*; D011247:Pregnancy; D012662:Semen Preservation