title: Replication and persistence of different strains of bovine viral diarrhea virus in an in vitro embryo production system.
abstract: Recent studies have shown that exposed, in vitro-derived embryos remain contaminated with bovine viral diarrhea virus (BVDV) after washing. However, introduction of a Genotype II versus Genotype I strain of BVDV into an IVF system was reported to provide greater potential for transmission of disease. The primary objective of this study was to compare the potentials for different strains of noncytopathic BVDV to replicate in an IVF system, associate with IVF embryos and infect co-cultured cells via association with washed embryos. The secondary objective was to compare the effect of different strains of BVDV on embryonic development. Two Genotype I (SD-1 and NY-1) and 2 Genotype II (CD-87 and PA-131) strains of BVDV were evaluated. After IVM and IVF of oocytes, presumptive zygotes were washed and transferred into in vitro cultures containing uterine tubal cells (UTC) and medium that was free of BVDV-neutralizing activity. Immediately before addition of zygotes, the cultures were inoculated with 10(5) cell culture infective doses (50%, CCID50) of a strain of BVDV or maintained as a negative control. Cultures of zygotes were then incubated for 7 d. Embryonic development was observed on Days 3 and 7, and attempts were made to isolate BVDV from UTC and medium on Day 7. Also on Day 7, groups of intact, washed blastocysts were either transferred into virus-free secondary cultures containing UTC or sonicated with sonicate fluid assayed by both virus isolation and single-closed-tube reverse transcription nested polymerase chain reaction (RT-nPCR). After 3 d in secondary culture, hatched embryos were enumerated, and medium from the cultures, washed UTC and embryos were tested for BVDV by virus isolation. In addition, washed UTC and embryos were tested for BVDV using RT-nPCR. All strains of BVDV persisted and replicated in the embryo culture environment, but cleavage beyond the 4-cell stage, blastocyst development and hatching varied among cultures contaminated with different strains of virus. Further, the quantity of BVDV associated with washed embryos from both initial and secondary cultures varied among strains, but the variation was unrelated to difference in genotype (SD-1 and PA-131 greater than NY-1 and CD-87). Although all strains of BVDV replicated in UTC in the initial in vitro cultures and remained associated with washed blastocysts, susceptible UTC in the secondary in vitro cultures were seldom infected by any strain of virus.
authors.affiliation: Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL 36849, USA. givenmd@vetmed.auburn.edu
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 / Q000502:physiology; D002417:Cattle / Q000196:embryology*; D018920:Coculture Techniques; D046508:Culture Techniques; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics* / Q000254:growth & development* / Q000302:isolation & purification; D004622:Embryo, Mammalian / Q000502:physiology / Q000821:virology*; D005260:Female; D005307:Fertilization in Vitro; D005838:Genotype; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014599:Uterus / Q000166:cytology / Q000821:virology; D014779:Virus Replication; D015053:Zygote / Q000502:physiology / Q000821:virology
chemical_list: D012367:RNA, Viral