Abstract
Recombinant viruses possessing reporter proteins as tools are widely applied in investigating viral biology because of the convenience for observation. Previously, we generated a recombinant pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) with enhanced green fluorescent protein (EGFP) reporter for monitoring virus spread and screening of neutralizing antibodies. PRRSV with different kinds of reporters can support more application scenarios. Here, we described a new genetically stable infectious clones of a highly pathogenic PRRSV (HP-PRRSV) harboring the DsRed (a red fluorescent protein isolated from the coral Discosoma) gene. In the recombinant infectious clone, the transcription regulatory sequence 2 (TRS2) of PRRSV was inserted between the open reading frame 7 (ORF7) and 3′UTR to drive the transcription of DsRed gene, which makes it a separate transcription unit in the viral genome. Using the bacterial artificial chromosome (BAC) system and cytomegalovirus (CMV) promoter, the recombinant HP-PRRSV with the DsRed insertion was successfully rescued and showed similar growth and replication patterns compared with the wild-type virus in the MARC-145 cells. In addition, the DsRed protein was stably expressed in the recombinant virus for at least 10 passages with consistent fluorescence intensity and density. Using the recombinant HP-PRRSV with DsRed protein, the virus tracking in MARC-145 was observed by live-cell imaging. Meanwhile, quantification of the DsRed fluorescence positive cells by flow cytometry provides an alternative to standard methods for testing the level of PRRSV infection. This recombinant PRRSV with DsRed fluorescence protein expression could be a useful tool for fundamental research on the viral biology and shows the new design for stable expression of foreign genes in PRRSV.
Highlights
Porcine reproductive and respiratory syndrome (PRRS) is a widespread disease affecting domestic pigs, first reported in North America and Europe in the late 1980s (Wensvoort et al, 1991; Albina, 1997)
DsRed gene was flanked by the unique AsiSI and MluI sites for its insertion into the plasmid pBAC-SD16FL-AM, and its expression was driven by the transcription-regulating sequence of gene GP2a (TRS2) (Figure 1A)
An apparent DsRed fluorescence was observed in the MARC-145 cells after transfection with pBACSD16FL-transcription regulatory sequence 2 (TRS2)-DsRed (Figure 1B), which means the successful expression of indictor protein
Summary
Porcine reproductive and respiratory syndrome (PRRS) is a widespread disease affecting domestic pigs, first reported in North America and Europe in the late 1980s (Wensvoort et al, 1991; Albina, 1997). The symptoms of PRRS include reproductive failure, pneumonia, and increased susceptibility to secondary bacterial infection. The causative agent of PRRS is the PRRS virus (PRRSV), which is in the Arteriviridae family of the order Nidovirales. PRRSV is an enveloped, singlestranded positive-sense RNA virus of approximately 15 kb in length that contains 9 open reading frames (ORFs) (Fang and Snijder, 2010). In the early 2000s, the outbreak of highly pathogenic PRRS (HP-PRRS) inflicted serious economic losses, which characterized by high-grade fever with increased mortality in pigs of all ages (Tian et al, 2007). There is no remarkably effective vaccine for controlling PRRSV infection in pigs
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