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

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

Passage experiments were carried out to detect the reversion of the S3050G mutant harvested from BHK-21 cells transfected with pGDD. The mutation specifying the SDD motif was sequenced from P1 to P5 of vGDD. The substitution of S3050G was found not revert (Table 1) , which indicated that vGDD was stable ( Figure 5B ). To investigate the influence of the S3050G mutation of RdRp on the vGDD genome, MARC-145 cells were infected with vAPRRS or vGDD. P0 viral supernatants were serially passaged through plaque purification five times, named P1-P5, and stored for further experiments. RNA was isolated from P1 to P5 virus particles and used for cDNA synthesis and subsequent PCR amplification (Table 3 , Figure 5A ). The sequence covering the regions of the vGDD genome was determined in at least three isolated cDNA clones. In all three sequenced cDNA clones, the original substitution had not reverted; however, two synonymous mutations, i.e., C3960U and U5277C had occurred ( Figure 5C ). The results indicated that mutant virus S3050G is stable and did not influence the fidelity of RdRp.