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Most isolates of BVDV cause unapparent infections in cultured cells. Fetuses, postnatal animals or fetal bovine serum are possible sources of the virus for cultivated cells used as karyoplasts in cloning. Routine screening by veterinary diagnostic laboratories of 39 fetal fibroblast cell lines used in cloning research had revealed that 15 (39%) were positive for BVDV by various assays including RT-nPCR. As some were valuable transgenic cell lines, a rigorous protocol for evaluation of each line was undertaken to confirm infection with BVDV. A cryopreserved vial of each line was thawed, medium discarded and cells incubated (38.5°C in 5% CO2 and air) through 2 passages (6–10 days) in α-MEM supplemented with 10% equine serum. At the end of the second passage, cells were separated from medium, washed and assayed for presence of BVDV using virus isolation in 2 sequential passages in Madin Darby Bovine Kidney Cells and RT-nPCR. Available lots of fetal bovine serum and medium that had been used to culture the cells also were tested for BVDV. When the virus was detected, the RT-nPCR products were sequenced and compared. Also, an attempt was made to evaluate the earliest available cryopreserved passage of any positive cell lines. Results indicated that just 5 of 39 of the original cells tested (13%) were positive. Since cryopreserved earlier passages of 4 of the cell lines were available, they were assayed with the result that 2 of the 4 were not infected at the earliest passage. Further, BVDV was isolated from one lot of fetal bovine serum that was used to culture one of the cell lines. Sequence analysis verified that only 2 of these 4 cell lines were infected with the same isolate of BVDV, and one isolate was identical to the virus found in the fetal bovine serum used in medium to culture it. The discrepancy between our viral detection and that of the diagnostic laboratories is explained in part by the presumed test protocols. All BVDV-positive cells, as reported by the diagnostic laboratories, were positive by RT-nPCR. We presume that they did not separate medium from cells before assays. Thus, any noninfectious viral RNA that was in the medium (e.g. as would be expected in many lots of irradiated serum) would have been reported positive. The only possible sources for BVDV in these cell lines were the fetuses from which they originated or fetal bovine serum used in medium. Sequence analysis confirmed that serum was the source of viral infection in one line. The likely source of virus for 2 other lines was serum, since they were not infected at earlier passages. The 2 remaining cell lines were positive at the earliest available passages, so the fetuses from which cells were harvested could not be discounted as the source of BVDV. This report highlights the risks of introducing BVDV in embryo technologies and the difficulties that can be encountered in attempting accurate diagnosis of the presence of infectious virus.

198BOVINE VIRAL DIARRHEA VIRUS (BVDV) IN CELL LINES USED FOR SOMATIC CELL CLONING