Fig. 1.

Construction of a recombinant HP-PRRSV expressing an additional transcription unit (rHV-GFP). (A) Strategy for the construction of the recombinant cDNA clone. Five overlapping fragments amplified from the HV genome were ligated into the modified pcDNA3.1 vector using the listed restriction enzyme cleavage sites. GFP gene and a copy of TRS6 were introduced into the non-coding region between ORF1b and ORF2a using restriction sites Stu I and Mlu I. TTAACC, the core hexanucleotide of TRS6; the sequence in italic indicates its flanking sequences. (B) The plasmid containing assembled HV genome was transfected into 293FT cells. The reused virus rHV was passaged on PAMs and immunofluorescence assay was performed for virus detection. Magnification, 200×. (C) The recombinant cDNA clone containing GFP gene and a copy of TRS6 was transfected into 293FT cells and directly examined for GFP expression at 48 h post transfection under an immunofluorescence microscope. Magnification, 200×. (D) rHV-GFP was serially passaged on PAMs and green fluorescence was directly examined under an immunofluorescence microscope. Magnification, 400×.