Generation of a porcine reproductive and respiratory syndrome virus expressing a marker gene inserted between ORF4 and ORF5a

In recent years, the availability of reverse genetics systems for porcine reproductive and respiratory syndrome virus (PRRSV) has created new perspectives for the use of recombinant viruses as expression vectors. Most of these recombinant PRRSV vectors express foreign genes through either an independent transcription unit inserted in ORF1b and ORF2, or in ORF7 and the 3' UTR. The aim of this study was to find an alternative site for foreign gene insertion into the PRRSV genome. Here, we constructed an infectious cDNA clone for a cell-adapted PRRSV strain, GXNN1396-P96. This cDNA-clone-derived recombinant virus (rGXAM) was comparable in its growth kinetics in MARC-145 cells to the parental virus, GX1396-P96. Using the infectious cDNA-clone, we inserted an independent transcription unit in ORF4 and ORF5a to generate a novel PRRSV-based recombinant virus expressing the green fluorescent protein (GFP) gene. Biological characterization of the recombinant virus, rGX45BSTRS-GFP, showed that it maintained similar growth characteristics but produced fewer infectious virions than the parental PRRSV. These data demonstrate that the ORF4 and ORF5a site is able to tolerate the insertion of foreign genes.