Generation of a porcine reproductive and respiratory syndrome virus expressing a marker gene inserted between ORF4 and O
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ORIGINAL ARTICLE
Generation of a porcine reproductive and respiratory syndrome virus expressing a marker gene inserted between ORF4 and ORF5a Yuxu Wang1 · Wei He1 · Qingqing Li1 · Xin Xie1 · Nian Qin1 · Hao Wang1 · Jiabin Huang1 · Siyuan Lin1 · Kang Ouyang1 · Ying Chen1 · Weijian Huang1 · Zuzhang Wei1 Received: 2 January 2020 / Accepted: 24 April 2020 © Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract 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.
Introduction Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases in the swine industry worldwide [27]. Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the family Arteriviridae, is an enveloped, single-stranded RNA virus whose genome is approximately 15.4 kb long and contains nine open reading frames encoding replicase polyproteins and seven structural proteins [9]. Approximately two-thirds of the 5′ end of the PRRSV genome encodes two polyproteins (pp1a and pp1ab) that are processed by viral proteases to form at least 16 nonstructural proteins (nsps), and these play a key role in viral replication and transcription Handling Editor: Sheela Ramamoorthy. Yuxu Wang and Wei He contributed equally to this article. * Zuzhang Wei [email protected] 1
Laboratory of Animal infectious Diseases and molecular Immunology, College of Animal Science and Technology, Guangxi University, No. 100 Daxue Road, Nanning 530004, China
[9]. Approximately one-third of 3′ end of PRRSV genome encodes the viral structural proteins, which are translated from a 3′–5′ co-terminal, nested set of subgenomic (sg) mRNAs. The transcription regulatory sequence (TRS), containing a six-nucleotide core sequence and flanking sequences, contributes to regulating the production of sg mRNA for the structural proteins [31, 36, 38, 49]. The seven structural proteins are t
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