Abstract
Pseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.
Highlights
Pseudorabies virus (PRV), belonging to the Herpesviridae family, subfamily Alphaherpesvirinae, genus Varicellovirus, is the aetiological agent of pseudorabies and was first described in 1813 [1–3]
A recombinant PRV with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs
A donor plasmid containing both EGFP and firefly luciferase expression cassettes flanked by homologous arms was constructed, in which the EGFP expression cassette was under the control of the CMV enhancer and chicken β-actin promoter (CAG) promoter, and the firefly luciferase expression cassette was under the control of the SV40 promoter (Figure 1B)
Summary
Pseudorabies virus (PRV), belonging to the Herpesviridae family, subfamily Alphaherpesvirinae, genus Varicellovirus, is the aetiological agent of pseudorabies and was first described in 1813 [1–3]. PRV is a neurotropic herpesvirus that can establish latent infection in nerve cells [4–6]. This virus can cause severe encephalitis in juvenile pigs [7] and various non-native hosts [8–10]. In non-native hosts such as mice, PRV infection is always lethal [11]. Owing to the use of the PRV vaccine strain Bartha-K61 from Hungary, which contains a complete deletion of the regions encoding glycoprotein E (gE) and US9 and partial. Viral reverse genetics technology plays an important role in PRV vaccine development and pathogenicity research [26–28]. The genome of PRV is a linear doublestranded DNA approximately 150 kilobases kb in length, encoding ~70 proteins [1]; it includes many nonessential
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