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.

中文翻译：

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表达在ORF4和ORF5a之间插入的标记基因的猪繁殖与呼吸综合征病毒的产生。

近年来，针对猪繁殖与呼吸综合征病毒（PRRSV）的反向遗传学系统的可用性为将重组病毒用作表达载体创造了新的前景。这些重组PRRSV载体中的大多数都通过插入ORF1b和ORF2或ORF7和3'UTR中的独立转录单位表达外源基因。这项研究的目的是找到一个外源基因插入PRRSV基因组的替代位点。在这里，我们为适应细胞的PRRSV菌株GXNN1396-P96构建了一个感染性cDNA克隆。这种cDNA克隆衍生的重组病毒（rGXAM）在MARC-145细胞中的生长动力学与亲本病毒GX1396-P96相当。使用传染性cDNA克隆，我们在ORF4和ORF5a中插入了一个独立的转录单位，以生成表达绿色荧光蛋白（GFP）基因的新型基于PRRSV的重组病毒。重组病毒rGX45BSTRS-GFP的生物学特性表明，与亲代PRRSV相比，它保持了相似的生长特性，但产生的感染性病毒颗粒更少。这些数据证明ORF4和ORF5a位点能够耐受外源基因的插入。