Generation of a Recombinant Porcine Reproductive and Respiratory Syndrome Virus Stably Expressing Two Marker Genes

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) has been used as a gene expression vector in the development of vaccines. Most of these recombinant PRRSV vectors express only a single foreign gene through either an internal insertion in the hypervariable region of nsp2 or expression cassette and some of these recombinant vectors are genetically unstable. Here, we combined internal insertion in nsp2 and expression cassette methods to generate a novel recombinant PRRSV stably expressing the red fluorescence protein (RFP) and the green fluorescence protein (GFP) genes. Biological characteristic analysis of the recombinant PRRSV carrying the two marker genes, rGX-RFP-GFP, showed that it displayed similar growth kinetics and yet it yielded less infectious viruses when compared to the parental virus rGXAM. Co-expression of both the RFP and GFP was observed using confocal fluorescence microscopy when the rGX-RFP-GFP viruses infected MARC-145 cells. Furthermore, the PRRSV-based two-marker gene expression vector is genetically stable during 20 serial passages in MARC-145 cells. These data demonstrate that it is possible to express two interested immunogens from a single PRRSV vector.

One-Step Assembly of a PRRSV Infectious cDNA Clone and a Convenient CRISPR/Cas9-Based Gene-Editing Technology for Manipulation of PRRSV Genome

“…Previously reported methods for RNA virus genome manipulation usually face the challenge of selecting suitable restrictive endonucleases [ 23 , 47 ]. In contrast, the CRISPR/Cas9-based method improves the flexibility of editing the genome and saves time when modifying the viral genome [ 37 , 38 ].…”

“…Additionally, an immunogenically enhancing vaccine candidate that possesses a stronger ability to induce neutralizing antibody has been developed by mutating the glycosylation sites in GP5 [ 20 , 21 , 22 ]. Moreover, four regions in the PRRSV genome, including the interval between ORF1b and ORF2, the spacer between ORF4 and ORF5a, the position post-ORF7, and the highly variable region within nsp2, have been applied for carrying exogenous genes using a reverse genetics system, which provides the potential for the development of polyvalent vaccines based on PRRSV [ 23 , 24 , 25 , 26 ].…”

“…Therefore, it is necessary to improve this method. CRISPR/Cas9, a novel gene-editing technology derived from bacteria and archaea [ 27 ], significantly increases the selection freedom of the gene modification position [ 23 , 26 ] and is more time-saving [ 28 ]. CRISPR/Cas9 has been widely used to edit the genomes of various species [ 29 , 30 , 31 ].…”

Construction of a Porcine Reproductive and Respiratory Syndrome Virus with Nanoluc Luciferase Reporter: a Stable and Highly Efficient Tool for Viral Quantification Both In Vitro and In Vivo

“…The reporter proteins, including naturally occurring fluorescent proteins and genetically engineered derivatives, are usually inserted into the viral genome, and then the bioimaging of viral infection is achieved by the expression of reporter proteins, which is an effective tool to detect and quantify viral replication in vitro and in vivo ( 16 – 18 ). For PRRSV, both strategies fusion expressing and setting as an additional ORF have been utilized to express the foreign green fluorescent protein (GFP) and/or red fluorescence protein (RFP) ( 19 , 20 ). Considering the Nsp2 coding region could tolerate insertion and deletion, the GFP was first inserted into a unique deletion site located at amino acid positions 348 and 349 of the Nsp2 region in a genotype I PRRSV ( 21 ).…”

“…Initially, the reporter genes were not stable in the PRRSV genome, as the recombinant PRRSV with GFP fused in the Nsp2 region lost its GFP-expressing after passaging in cells seven times, due to partial deletion of the GFP gene ( 21 ). Recently, the coexpression of RFP and GFP in PRRSV genome has been reported to be genetically stable during 20 serial passages in MARC-145 ( 20 ). Although the stability of the foreign EGFP gene increased, as an individual ORF, the recombinant viruses cannot be rescued when a larger reporter proteins firefly luciferase (FLuc) was inserted at the same position ( 24 ).…”