A novel porcine reproductive and respiratory syndrome virus vector system that stably expresses enhanced green fluorescent protein as a separate transcription unit

Abstract

Here we report the rescue of a recombinant porcine reproductive and respiratory syndrome virus (PRRSV) carrying an enhanced green fluorescent protein (EGFP) reporter gene as a separate transcription unit. A copy of the transcription regulatory sequence for ORF6 (TRS6) was inserted between the N protein and 3′-UTR to drive the transcription of the EGFP gene and yield a general purpose expression vector. Successful recovery of PRRSV was obtained using an RNA polymerase II promoter to drive transcription of the full-length virus genome, which was assembled in a bacterial artificial chromosome (BAC). The recombinant virus showed growth replication characteristics similar to those of the wild-type virus in the infected cells. In addition, the recombinant virus stably expressed EGFP for at least 10 passages. EGFP expression was detected at approximately 10 h post infection by live-cell imaging to follow the virus spread in real time and the infection of neighbouring cells occurred predominantly through cell-to-cell-contact. Finally, the recombinant virus generated was found to be an excellent tool for neutralising antibodies and antiviral compound screening. The newly established reverse genetics system for PRRSV could be a useful tool not only to monitor virus spread and screen for neutralising antibodies and antiviral compounds, but also for fundamental research on the biology of the virus.

Generation of porcine reproductive and respiratory syndrome virus by in vitro assembly of viral genomic cDNA fragments

“…The transfected culture supernatant collected on day 3 post-transfection was infectious and caused CPE in the subsequent infection of MARC-145 cells. The observed incubation time for the virus recovery appeared similar to that of another reported system similarly utilizing a CMV promoter to generate vRNA in transfected cells (Wang et al, 2013). It is, however, slightly slower than a virus recovery system utilizing a direct transfection of T7 promoter-driven, in vitro transcribed vRNA into MARC-145 cells.…”

“…One of the most definitive ways to study the roles of specific sequences in a viral genome is to modify them and use them to generate infectious virus-that is, to 'rescue' virus with these modified sequences (Bridgen, 2012). Various approaches have been employed to generate PRRSV infectious clone, including the use of a bacterial artificial chromosome (Wang et al, 2013), a T7 promoter in the low copy-number plasmid pOK12 (Meulenberg et al, 1998;Nielsen et al, 2003), a CMV promoterdriven plasmid (Lee et al, 2005), and an RNA-launched system http://dx.doi.org/10.1016/j.virusres.2014.09.008 0168-1702/© 2014 Elsevier B.V. All rights reserved.…”

Attenuation of highly pathogenic porcine reproductive and respiratory syndrome virus by inserting an additional transcription unit

“…Recently, PRRSV gene expression vectors capable of expressing a foreign gene from an additional transcription unit were generated by inserting a copy of the transcription regulatory sequence for ORF6 (TRS6) [10][11][12][13]. This novel approach was used to construct recombinant viruses expressing antiviral cytokines as adjuvants to overcome immune subversion of PRRSV and enhance the viral specific immune response following vaccination [13], or expressing reporter proteins to monitor virus replication, as well as elucidating the role of host factors and screening for novel antiviral drugs [11,13]. This PRRSV reverse genetics system may also function as a vector for development of recombinant multivalent vaccines against swine diseases by encoding immunogenic antigens from other swine pathogens [10].…”

MicroRNA miR-24-3p Promotes Porcine Reproductive and Respiratory Syndrome Virus Replication through Suppression of Heme Oxygenase-1 Expression

“…Viruses used in this study were a highly pathogenic PRRSV strain, SD16 (GenBank accession number JX087437), and a recombinant PRRSV. The recombinant PRRSV was based on the genetic background of SD16 that expressed enhanced green fluorescent protein (EGFP) as an additional open reading frame (ORF) (rHP-PRRSV/SD16/EGFP) (32).…”

“…MARC-145 cells or PAMs were lysed, and the cellular proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was probed with one of the following primary antibodies: mouse anti-HO-1 polyclonal antibody at a 1:1,000 dilution (19), a monoclonal antibody (6D10) (32) to the PRRSV N protein at a 1:2,000 dilution, or an anti-␣-tubulin antibody at a 1:5,000 dilution (Abcam, Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG at a 1:2,000 dilution (Jackson, West Grove, PA, USA) was used to label primary antibody binding.…”