214 USE OF VIRUS ISOLATION AND QUANTITATIVE POLYMERASE CHAIN-REACTION TECHNIQUES TO DETECT BOVINE VIRAL DIARRHEA VIRUS (BVDV) IN SINGLE OR SMALL GROUPS OF PRE-IMPLANTATION BOVINE EMBRYOS

Abstract

Because of its broad distribution among populations of cattle and its association with materials of animal origin used in embryo production, bovine viral diarrhea virus (BVDV) is a potential problem in applications of embryo technologies. While some isolates of BVDV are known to associate with both in vivo-derived and in vitro-produced bovine embryos, it has yet to be determined if the quantity of virus associated with exposed zona pellucida-intact embryos is sufficient to infect susceptible recipient cows via the intrauterine route. Techniques to detect and quantify BVDV associated with single transferable embryos are important to determine the risk of transmitting BVDV via embryo transfer. The objectives of this study were to define reproducible techniques to detect and quantify BVDV associated with single or small groups of bovine embryos contained in small aliquots of medium using virus isolation (VI) or real time quantitative polymerase chain reaction (Q-PCR) assays. In vivo-derived and in vitro-produced embryos were exposed for 2 h to approximately 10⁶-cell culture infective doses (50% endpoint) per mililiter of a high affinity strain of BVDV, SD-1, and then washed according to IETS guidelines. Embryos were assayed in groups of five or two embryos, or single. There were 5 replicates of the group of five embryos, 4 of the group of two embryos, and 3 of the single embryos for the in vivo-derived embryos undergoing VI; 5, 4, and 2 replicates, respectively, undergoing Q-PCR, and 2, 5, and 2 replicates, respectively, for the in vitro-produced embryo groups undergoing VI and Q-PCR. Those to be assayed by VI were sonicated and the sonicate fluids were layered onto Madin Darby Bovine Kidney (MDBK) cells and passaged to allow for viral replication; an immunoperoxidase monolayer assay was then used for viral detection. A Roche^® RNA/DNA extraction kit (Roche Diagnostic Systems, Inc., Somerville, NJ, USA) was used to extract RNA from virally exposed embryos, and extracted samples were assayed in duplicate Q-PCR reactions consisting of 100 ¼L. The primers used were L1 and U3 which are specific for conserved areas of the 5 prime nontranslated regions of the viral genome of BVDV. The PCR product was detected using hybridization probes s1 and s2 as in Struder et al. 2002 Biologicals 40, 289-296. In vivo-derived groups of five or two embryos, or single embryos, were positive for BVDV 100, 50, and 30% of the time, respectively, when VI was used and 100, 75 and 100%, respectively, when Q-PCR was used. The virus was detected in all of the in vitro-produced embryo groups of five, or two embryos, or single embryos, 100% of the time using VI, and in 100, 80, and 100% respectively, using Q-PCR. The virus isolation technique is highly sensitive but the need to destroy embryos by sonication to identify any embryo-associated virus precludes its use for embryos intended for transfer. Techniques for Q-PCR were sufficiently sensitive to detect and quantify 10 copies of RNA in a sample and to detect BVDV associated with single embryos.