Construction And Immunization Efficacy Assessment Of Recombinant Porcine Reproductive And Respiratory Syndrome Virus Expressing Porcine GM-CSF Or IL-4

Porcine reproductive and respiratory syndrome(PRRS) is one of the important viral diseases in the world’s swine industry. It is classified as B statutory reporting disease by OIE and as a second animal disease in China. The aetiological agent, porcine reproductive and respiratory syndrome virus(PRRSV), belongs to a member of arteriviruses. PRRSV is an enveloped virus with a single-stranded, positive-sense RNA with a wide range of genetic diversity and a high degree of variability. PRRSV can suppress the innate immune response and induce relative lower level of neutralization antibody of the host, which pose a serious challenge to prevent and control of this disease. Prevention PRRS mainly rely on attenuated vaccine immunization currently, but now the immune failure occasionally occurred due to various reasons, it is necessary to the development of new prevention strategies. In this study, the recombinant viruses expressing different Body TRS and green fluorescent protein(EGFP) were constructed based on the infectious clone of PRRSV(CH-1R strain), and the efficacy of foreign gene expression was analyzed in this model. The recombinant viruses expressing Body TRS6 and Porcine Granulocyte-macrophage Colony Stimulating Factor(p GM-CSF) or/and porcine Interleukin-4(p IL-4) were constructed. Recombinant viruses were injected into pigs and efficacy of immune protection was evaluated in comparison with the parental virus. The obtained results were shown as follows:1. The recombinant infectious clones with different Body TRS and EGFP inserted between the N gene and 3’-UTR of PRRSV were constructed, and the viruses were rescued successfully after transfection of Marc-145 cells. The recombinant viruses had the similar growth characterization with the parental virus. Preliminary comparison showed the Body TRS6 had the highest efficiency of EGFP expression, followed by TRS2/5/7, and TRS3/4 had the lowest efficiency. The results of the genetic stability showed that the recombinant viruses were stable over at least 5 passages.2. The recombinant infectious clone with Body TRS6 and porcine GM-CSF or/and IL-4 inserted between the N gene and 3’-UTR of PRRSV were constructed, and the viruses were rescued successfully after transfection of Marc-145 cells. The recombinant viruses had similar growth kinetics as the parental virus. The results of the genetic stability showed that the recombinant virus expressing porcine GM-CSF was stable for 10 passages in Marc-145 cells and had the significant ability to stimulate bone marrow cells proliferation. The recombinant virus expressing porcine IL-4 was stable for at least 15passages. However, the inserted gene of the recombinant virus simultaneously expressing these two cytokines was gradually losted after passaged 3 or 5 in cell culture.3. The recombinant virus(CH-1R/p GM-CSF) was injected into piglets to evaluate its adjuvant effect for protection against HP-PRRSV infection. The results showed that pigs immunized with CH-1R/p GM-CSF produced a similar humoral response to that elicited using CH-1R. With regard to cell-mediated immunity assessed in peripheral blood, the recombinant virus induced higher proportion of CD4+CD8+double-positive T cells(DPT), higher level of IFN-γ at 0 and 7 days post-challenge(DPC), and lower viremia at 21 DPC than pigs immunized with parental virus. These results indicate that CH-1R/p GM-CSF can induce a significant cellular immune response and reduce the persistent infection compared with pigs vaccinated the parental virus.4. The recombinant virus(CH-1R/p IL-4) was injected into piglets to evaluate its adjuvant effect for protection against HP-PRRSV infection. The results showed that pigs vaccinated with CH-1R/p IL-4 produced a similar humoral response to the response elicited by parental virus, but Flow Cytometric(FCM) analysis showed that the percentage of CD4+CD8+double positive T(DPT) cells in the CH-1R/p IL-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 viremia, temperature change and clinical signs did not show significant difference between the two groups.