Improved method for elimination of bovine viral diarrhea virus from contaminated cell lines using virus inhibitor DB772

Contamination of cell lines with bovine viral diarrhea virus (BVDV) derived from fetal bovine serum (FBS) is a major obstacle in cell biology experiments. Although the selection of BVDV-free FBS and constant viral checking are important to prevent the viral contamination of cells, they have been ineffective in the past contamination of several cell lines available from cell banks. Recently, a novel method to eliminate BVDV from one contaminated cell line, LFBK-α_vβ₆ cells, using BVDV inhibitor DB772 was reported. To confirm the reproducibility and possibility of practical use of this method in other cell lines, we attempted BVDV elimination using DB772 from five contaminated cell lines: BL3.1, EBTr, A26, FLK-BLV, and CC81. Treatment with DB772 significantly decreased viral genome counts in all BVDV-contaminated cells; however, cytotoxicity-like growth suppression was observed, in contrast to previously reported findings. Moreover, continuous cell passages in the presence of DB772, conducted in imitation of a previous study, resulted in the failure of elimination with rebound of viral genomes after several cell passages. Hence, we modified the method to combine limiting dilution of cultured cells with drug treatment and finally succeeded in BVDV elimination in four of the 5 cell lines, except for EBTr cells, which exhibited strong suppression of cell proliferation after DB772 treatment. Taken together, our findings will help to solve the problem of BVDV contamination in cell lines.