Construction And Charaterization Of The Recombinant Live Attenuated PRRSV Expressing Porcine GM-CSF

Porcine reproductive and respiratory syndrome (PRRS), which is characterized by severe reproductive failure and a high rate of late abortion and early farrowing in sows, and respiratory disease and mortality in young pigs, continues to be a huge threat for the pig industry. The causative agent of the disease is PRRS virus (PRRSV), a small, enveloped postive-stranded RNA virus, which suppresses and delays the development of neutralizing antibodies and the induction of cell-mediated immune responses of swine after infection. There are still no completely effective vaccines to control PRRSV. Currently available modified live vaccines have shown the most promising efficacy but have only been partially successful in protection against a wide array of heterologous viruses. Granulocyte-macrophage colony stimulating factor (GM-CSF) as a good immune adjuvants, which could activate dencritic cells (DCs) differentiation and improve the abilities of antigen-presenting cells (APCs), has been investigated in both subunit and modified live vaccines.To develop a more efficient vaccine, we constructed the recombinant plasmid pHuN4-F112-GM-CSF which porcine GM-CSF were inserted by overlapping PCR between the ORFlb and ORF2a of HuN4-Fl 12 vaccine strain derived from HP-PRRSV. To make sure that the inserted gene is stable in the recombinant virus, the transcription regulating sequence of PRRSV ORF6 (TRS6) between the gene GM-CSF and ORF2a, and the recombinant plasmid pHuN4-F112-GM-CSF-TRS6 was generated which the GM-CSF gene was expressed as a separate mRNA from an additional transcription unit. To introduce a deletion as marker, we further deleted the 110 amino acids in the Nsp2 region of HuN4-F112, and two to recombinant plasmids pHuN4-F112-△110-GM-CSF and pHuN4-F112-△110-GM-CSF-TRS6 were constructed. The four recombinant plasmids containing full-length viral cDNA were linearized by digestion of restriction enzyme Swa I. To rescue these viruses, capped RNAs of the viral genomes were transcribed in vitro from the full-length cDNA clones and transfected into BHK-21 cells, and then rescued and passaged on MARC-145 cells. The results showed that the rescued viruses could develop CPE on MARC-145 cells, and the viruses rHuN4-F112-GM-CSF-TRS6 and rHuN4-F112-△110-GM-CSF-TRS6 with TRS6 could express the insert gene GM-CSF without any mutation up to 20 passages identified by RT-PCR, DNA sequencing, Mlu I enzyming digestion and IFA. But the viruses rHuN4-F112-GM-CSF and rHuN4-F112-△110-GM-CSF without TRS6 were found mutated within GM-CSF gene after a few passages. Growth kinetics analysis in MARC-145 cells showed that the recombinant viruses had a similar replication rate as that of parental virus. The results indicate that TRS6 plays an important role to maintain the inserted gene stable in the recombinant viruses.To investigate whether the expression of GM-CSF from the recombinant viruses promotes maturation and/or activation of DCs in vitro, Infection of porcine bone marrow haematopoietic cells (BMHC) with the recombinant viruses was conducted. Both rHuN4-F112-GM-CSF-TRS6 and rHuN4-F112-△110-GM-CSF-TRS6 promoted better maturation and/or activation of DCs than the parental virus when BMHC were pretreated with or without GM-CSF, as shown by significantly more MHCⅠ+/CD80/86+ and MHCⅡ+/CD80/86+ doubly positive cells of bone marrow-derived DCs (BMDC)(P<0.05,P<0.01). Because the same effect on BMDC between rHuN4-F112-GM-CSF-TRS6 and rHuN4-F112-△110-GM-CSF-TRS6, we further investigated whether BMDCs in vitro treated with the recombinant virus rHuN4-F112-GM-CSF-TRS6 could secrete cytokines (mRNA or protein) modulating the Thl or Th2 response. BMDCs treated with the recombinant virus secreted significantly larger amounts of IL-12 and IL-1β than the parental virus from 24h to 96h post infection (p<0.01), and the secretion of IFN-y and IL-4 is significantly higher than parental virus by 96h post infection. The high expression of IL-12, IL-1β,IFN-γ and IL-4 by DCs treated by the recombinant virus would be used for regulating TH immune response, especially the secretion of IL-12 could promote TH1 response. Together, these data indicated that rHuN4-F112-GM-CSF-TRS6 and rHuN4-F112-△110-GM-CSF-TRS6 expressing GM-CSF protein could activate and maturate BMHC to DCs.Further investigation of the recombinant virus rHuN4-F112-GM-CSF-TRS6 in vivo showed that the replication ability of the recombinant virus in pigs was the same as parental virus. The level of PRRSV N antibody in pigs infected with the recombinant virus was significantly higher than that of pigs infected with the parental virus at 14 days post infection. At meanwhile, the percentage of activated CD8+ T cells and activated CD4+ T cells in the group of the recombinant virus was significantly higher than that in the group of the parental virus (P<0.05). These results indicated that the recombinant virus rHuN4-F112-GM-CSF-TRS6 could elevate the level of activated T cells in vivo, which would be better to regulate the immune response.Taking all together, the recombinant PRRSV rHuN4-F112-GM-CSF-TRS6 developed in the study could induce better immune responses in comparison with its parental vaccine virus HuN4-F112, and would be a potential new generation vaccine candidate against highly pathogenic PRRS.