Effect of 5' Terminal Non Viral Nucleotide on the Rescue of PRRSV Infectious Clone

To determine the effect of non viral nucleotide on the rescue of infectious PRRSV and optimize the reverse genetics system of PRRSV, two full-length PRRSV plasmids were constructed containing the CMV promoter, and inserted ribozyme sequences in the downstream of CMV promoter in this study.Two non viral nucleotide sequences GG and GCTAGC were inserted into a position between the ribozyme sequence and the 5' UTR of the PRRSV genome, respectively, resulting two full-length PRRSV infectious cDNA clones, Pgg-PRRSV and PNhe-PRRSV.The plasmids were transfected into BHK-21 cells.The M protein of PRRSV was detected by indirect immunofluorescence 48 h after transfection.Two infectious viruses, Pgg-PRRSV and PNhe-PRRSV, had been successfully generated.The titers of the two viruses were determined to be 3.0 and 1.25 TCID₅₀/mL, respectively.And the titer of the two rescued viruses reached a peak as high as 6.5 TCID₅₀/mL after passaged for 3 generations in Marc-145 cells.The results demonstrated that the presence of the two non viral nucleotides (GG) could enhance the rescue in a higher level.Nevertheless, the effect of the two nucleotide sequences was indistinguishable when the viruses passaged for 3 generations.Together, our result indicated that the additional nucleotide sequence was effective only in the initial stage of rescue.