Construction PRRSV RecCH-1R Chimeric Virus And Cellular Immune Assessment Of PRRS And CSF Live Vaccine

Porcine reproductive and respiratory syndrome (PRRS) was characterized by reproductive disorders in pregnant sows and severe respiratory symptoms in piglets. Since it first appeared, PRRS has caused huge economic losses to the swine industry worldwide. The causative agent of PRRS-PRRS virus (PRRSV) belonged to family Arteriviridae, and genus Arterivirus, which was a single-stranded, positive-sense, enveloped RNA virus.A full-length cDNA infectious clone rCH-1R of PRRSV attenuated strain CH-1R, which was the171th generations of CH-la, was constructed succeeded in rescuing the virus in our laboratory, and named the virus as recCH-1R. In this study, the PCV2Cap gene was inserted into between ORFlb and ORF2a of the rCH-1R, and then, rCH-1R GP5and GP5+M genes were substituted for HP-PRRSV HuN4strain relative genes. Three chimeric viruses-recCH-1R-PCV2-Cap, recCH-1R-HuN4-GP5, and recCH-1R-HuN4-GP5+M were successfully constructed. RT-PCR, cytopathic effect observation, indirect immunofluorescent assay, electron microscopy and growth kinetics methods were used for identification of the viruses. The results showed that the chimeric virus recCH-1R-PCV2-Cap was similar to the parental virus recCH-1R in CPE, virus particles and reproductive characteristics. Furthermore, indirect immunofluorescent assay verified that the recCH-1R-PCV2-Cap virus could develop the immune reaction with PRRSV positive serum and PCV2Cap monoclonal antibody. The CPE observation of the chimeric viruses recCH-1R-HuN4-GP5, recCH-1R-HuN4-GP5+M were different from that of the parental virus, which the cells were agglomeration and detachment. All methods demonstrated that the three chimeric viruses were successfully rescued. When the viruses were passaged in Marc-145cells, PCV2Cap genes of chimeric virus recCH-1R-PCV2-Cap appeared different deletions, and chimeric viruses recCH-1R-HuN4-GP5and recCH-1R-HuN4-GP5+M were begun to occur reverse mutation from the8th and16th generations, respectively. Amino acid sequences of replacement of HuN4GP5or GP5+M proteins were mutated back for recCH-1R strain relative amino acid sequences. The results revealed that PRRSV recCH-1R had a strong self-repair effect.For detection of porcine cytokines IFN-y, IL-2and TNF-a from pig whole blood, the real time TaqMan RT-PCRs were developed in this study, and the established methods had a linear dynamic range from101copies/μL to108copies/μL, with the correlation coefficient R2of0.999. The assay was used to assess cellular immune response of classical swine fever (CSF) and PRRS attenuated live vaccine CH-1R and PRRS recCH-1R in pigs. Firstly, the results demonstrated that the IFN-y, IL-2and TNF-a mRNA expressions were significantly increased from3to10days (p<0.05), respectively; however, the higher expression of IFN-y lasted to14days when CSFV stimulated cells in comparing with unstimulated control cells in pigs post immunization with CSF attenuated vaccine. Nevertheless, those mRNA expressions had no significant difference between CSFV stimulated cells and control cells in unvaccinated pigs group. The proportion of CD4+CD8+subpopulations was gradually increasing post CSF attenuated vaccine vaccinated. So the results suggested that CSF attenuated vaccine could induce cellular immune response. Secondly, the results for detection PRRS CH-1R live vaccine and PRRSV recCH-1R showed that IL-2mRNA expression was significantly increased at7,21and28d (p<0.05), but significantly decreased at14d (p<0.05), and TNF-a mRNA expression was significantly increased from3d to28d expect for14d when PRRSV stimulated cells in comparing with unstimulated control cells in pigs post immunization with PRRS CH-1R vaccine and PRRSV recCH-1R. However, only some samples could detect the IFN-y mRNA expression of the two groups. The proportion of CD4+CD8+subpopulations was gradually increasing but CD4+CD8" subpopulations were gradually decreasing post PRRS CH-1R vaccine and PRRSV recCH-1R vaccinated. So the results suggested that PRRS CH-1R vaccine group and PRRSV recCH-1R group were both able to induce cellular immune response. The results suggested that IL-2and TNF-a could both serve as one of the indicators of cellular immune response assessment.