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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.

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BackgroundThe current vaccines for porcine reproductive and respiratory syndrome virus (PRRSV) have failed to provide broad protection against infection by various strains of PRRSV. Porcine Interleukin-4 (pIL-4) plays an important role in the regulation of the immune response and has been used previously as an immunological adjuvant. The objective of this study was to construct a recombinant PRRSV expressing pIL-4 and to evaluate the immune response of the recombinant virus in piglets.MethodsThe pIL-4 gene was inserted in the PRRSV (CH-1R strain) infectious clone by overlap PCR. Indirect immunofluorescence assay (IFA) and Western blotting were used to confirm the recombinant virus. The stability of the recombinant virus was assessed by DNA sequencing and IFA after 15 passages in vitro. Recombinant virus was injected into pigs and efficacy of immune protection was evaluated in comparison with the parental virus.ResultsThe recombinant virus (CH-1R/pIL-4) was successfully rescued and shown to have similar growth kinetics as the parental virus. The recombinant virus was stable for 15 passages in cell culture. Pigs vaccinated with CH-1R/pIL-4 produced a similar humoral response to the response elicited by parental virus, but IL-4 level in the supernatant of PBMCs from pigs vaccinated with CH-1R/pIL-4 was significantly higher than the parent virus at 28 days post-immunization (DPI). Flow cytometric (FCM) analysis showed that the percentage of CD4+CD8+ double positive T (DPT) cells in the CH-1R/pIL-4 vaccinated group was significantly higher than the parental virus at 3 and 7 Days Post-Challenge (DPC), and the IL-4 level in the blood significantly increased at 7 DPC. However, the viral load and histopathology did not show significant difference between the two groups.ConclusionsA recombinant PRRSV expressing porcine IL-4 was rescued and it remained genetically stable in vitro. The recombinant virus induced higher DPT ratios and IL-4 levels in the blood after HP-PRRSV challenge compared to the parental virus in piglets. However, it did not significantly improve protection efficacy of PRRSV vaccine.