234 QUANTITATION OF BOVINE VIRAL DIARRHEA VIRUS ASSOCIATED WITH SINGLE ZONA PELLUCIDA-INTACT IN VIVO-DERIVED BOVINE EMBRYOS

Abstract

Quantitation of bovine viral diarrhea virus (BVDV) associated with individual transferable embryos is prerequisite to a thorough assessment of the risk for transmission of BVDV via embryo transfer. One objective of this study was to determine the proportion of in vivo-derived bovine embryos that remained virus-positive after artificial exposure to a high-affinity strain of BVDV and thorough washing. A second objective was to determine the quantity of virus associated with these individual embryos. A total of 87 zona pellucida-intact, Day 7, in vivo-derived bovine embryos were exposed to a type 1 noncytopathic strain of BVDV (SD-1) and washed according to International Embryo Transfer Society recommendations. Subsequently, individual embryos were sonicated, and the RNA was extracted from the sonicate fluids and stored at -80°C until assayed using a real-time quantitative polymerase chain reaction (QPCR). Twenty-six percent (23/87) of the embryos contained virus. The average quantity of virus associated with individual embryos after viral exposure and washing was 1.12 viral copies per 5 µL (SD = 1.57 copies 5 per µL^-1; SEM = 0.33 copies 5 per µL^-1). Assessment of data using tolerance intervals (P = 0.05) indicates that 90% of contaminated embryos will be associated with ≤4.64 viral copies per 5 µL, whereas 99% of contaminated embryos will be associated with ≤6.62 copies per 5 µL. Obviously, only extremely small quantities of virus were associated with less than one-third of the embryos tested. Based on previous research, it is presumed that this virus is associated with the outer layers of the zona pellucida. A logical next step in the risk assessment would be to determine if these quantities of zona-associated virus are sufficient to infect naïve recipients and/or embryonic cells after embryos are transferred. Further, similar efforts should be made to estimate the quantity of virus associated with in vitro-derived, zona pellucida-intact, bovine embryos after exposure to the same high-affinity strain of virus and washing.