Mutational analysis of the SDD sequence motif of a PRRSV RNA-dependent RNA polymerase

Zhou, Yan; Zheng, HaiHong; Gao, Fei; Tian, DeBin; Yuan, ShiShan

BHK-21 cells were propagated in a 6-well plate (2×10 5 cells/35 mm well) and grown for 2 d to approximately 60%-80% confluence. The subconfluent BHK-21 cells were transfected with equal amounts (3.75 μg) of full-length mutant plasmid using Lipofectamine LTX&PLUS reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and incubated at 37°C with 5% CO 2 . The supernatant of the cell culture was harvested and inoculated onto MARC-145 cells 72 h posttransfection, and was monitored daily for cytopathic effect (CPE). When 80% CPE was observed, the supernatant of the cell culture was harvested and stored in a 70°C freezer as the primary rescued virus stock, designated as vGDD passage 0 (P0). The 1000-fold diluted P0 was used for inoculating fresh MARC-145 cells, and the supernatant was harvested at 4 dpi, and designated as P1. The P2-P5 virus stocks were prepared in the same manner.