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Abstract
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.
Highlights
The current vaccines for porcine reproductive and respiratory syndrome virus (PRRSV) have failed to provide broad protection against infection by various strains of PRRSV
The presence of recombinant PRRSV was identified by indirect immunofluorescence assay (IFA) and western blotting using the PRRSV N-specific antibody (6D10)
Porcine IL-4 cannot be used as immunological adjuvant since it was reported that IL-4 plays a different role in pigs than in mice and humans, and porcine IL-4 blocks antibody production and suppresses antigenstimulated proliferation of B cells [40]
Summary
The current vaccines for porcine reproductive and respiratory syndrome virus (PRRSV) have failed to provide broad protection against infection by various strains of PRRSV. 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. Two overlapping open reading frames (ORF 1a and ORF 1b) occupy two thirds of the genome at the 5′ end. These ORFs encode two large polyproteins that undergoes co-translational and posttranslational processing to give rise to at least 14 nonstructural proteins (nsps). According to the recently accepted replication model for viruses of the order Nidovirales , PRRSV and other arteriviruses synthesize a nested set of subgenomic (sg) mRNAs as a mechanism to regulate the expression of structural or accessory proteins [15]. Base pair interactions between the transcription-regulating sequences (TRSs) located at the 3′ end of the leader sequence and the complement of body TRSs presented upstream of each gene, have been shown to be essential for this process [16]
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