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

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

To determine the multi-step growth curve of the P5 vGDD and vAPRRS, MARC-145 cells in 35 mm dishes were in-fected with 200 μL (0.01 MOI) of P5 viruses, repeated three times. An aliquot of the cell culture supernatant was harvested and replenished with an equal volume of EMEM containing 2% FBS at the indicated time points (6, 12, 24, 36, 48, 72, 84, 96 , 108, and 120 h) post-infection and stored at 70°C. Viral titration was performed by TCID 50 , and a growth curve was determined from the TCID 50 results, as described previously [29] (Figure 4 ). For detection of viral replication, an aliquot of the cell culture supernatant was harvested at 24 h after transfection to BHK-21 cells with pSDD and pAPRRS. Viral titration was performed by TCID 50 as described previously [29] (Table 1).