Laboratory of Animal infectious Diseases and molecular Immunology, College of Animal Science and Technology, Guangxi University, No. 100 Daxue Road, Nanning, 530004, China.
Arch Virol. 2020 Aug;165(8):1803-1813. doi: 10.1007/s00705-020-04679-3. Epub 2020 May 30.
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.
近年来，猪繁殖与呼吸综合征病毒（PRRSV）的反向遗传学系统的出现为重组病毒作为表达载体的应用提供了新的视角。这些重组 PRRSV 载体中的大多数通过插入 ORF1b 和 ORF2 或 ORF7 和 3'UTR 中的独立转录单元来表达外源基因。本研究旨在寻找将外源基因插入 PRRSV 基因组的替代位点。在这里，我们构建了一株适应细胞的 PRRSV 毒株 GXNN1396-P96 的感染性 cDNA 克隆。该 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。使用感染性 cDNA 克隆，我们在 ORF4 和 ORF5a 中插入了一个独立的转录单元，以产生一种新型的基于 PRRSV 的重组病毒，表达绿色荧光蛋白（GFP）基因。重组病毒 rGX45BSTRS-GFP 的生物学特性表明，它保持了相似的生长特性，但产生的感染性病毒粒子比亲本 PRRSV 少。这些数据表明 ORF4 和 ORF5a 位点能够耐受外源基因的插入。
