Study On The Induction Of Interleukin-8 By Porcine Reproductive And Respiratory Syndrome Virus In Marc-145 Cells

Porcine Reproductive and Respiratory Syndrome, caused by porcine reproductive and respiratory syndrome virus (PRRSV), is an infectious disease characterized by severe reproductive deficiency in pregnant sows, respiratory symptoms in piglets and high lethal rate. Firstly reported in US in 1987, PRRS has been present in almost all of the pig-breeding countries and regions, and become one of the most serious infectious diseases that threaten pig industry. In the summer of 2006, the "untitled high-fever disease" broke out in several southern provinces of China, which was continuously prevalent, spread over more than ten provinces in China, and caused a considerable loss.Cytokines play a rather important role in virus immunity and pathogenesis. IL-8 was the first chemokine discovered by Yoshimur in 1987 that belongs to C-X-C chemokine ultra-family. IL-8 is a medium that regulates inflammation, and has an important regulatory function on the processing of inflammation and immunity caused by bacterial and viral infection. It has already been demonstrated that after infection PRRSV could induce the generation of IL-8 in vivo. However, the mechanism by which PRRSV induces IL-8 production is unclear. Based on this, we conducted the study on the mechanism of how PRRSV infecting Marc-145 cells induced the production of IL-8.1. The infection of PRRSV on Marc-145 cells activates IL-8Marc-145 cells were transfected with luciferase reporting plasmid pIL-8-luc promoted by interleukin-8(IL-8) promoter, and luciferase internal reference plasmid. The cells were inoculated with PRRSV CH-1a isolate 24 hours later and luciferase activity detection result showed that IL-8 was significantly activated 3 hours after PRRSV infection. The activation fold reached higher at 6 hours after infection and then gradually dropped. Thus PRRSV can activates IL-8 on Marc-145 cells, and 6 hours can be defined as the optimal activation point.2. The expression of PRRSV N protein and Nsp2 protein activates IL-8In order to determine which encoded proteins of PRRSV were involved in the induction of IL-8, the eukaryotic expression plasmids of PRRSV structural protein and non-structural protein were used to co-transfect Marc-145 cells respectively with pIL-8-luc, and pRL-TK was used as an internal reference plasmid. The luciferase activity was tested 36 hours later and it was found that both structural protein N and non-structural protein Nsp2 could activation of IL-8, but other encoded proteins do not have a significant activation effect.3 .The determination of required regions for IL-8 activation by PRRSV N protein and Nsp2 proteinThe expression of N protein and Nsp2 protein in Marc-145 cells can activate IL-8. To determine the exact domain where N protein and Nsp2 protein induce the production of IL-8, the eukaryotic expression plasmids of N protein and Nsp2 protein-deleted mutants were constructed. By using luciferase reporting system, N protein was found to act mainly on the region between the 30th to 73th amino acid, which contain NLS region, NoLs region and RNA-binding area for N protein, but no specific region was found for Nsp2 protein activating IL-8.4. The study on signal pathway of IL-8 activation in Marc-145 cells infected with PRRSVTo understand the signal pathway of IL-8 production in Marc-145 cells induced by PRRSV infection, Marc-145 cells were transfected with IL-8 complete sequence promoter reporting plasmid pIL-8-luc, the promoter reporting plasmid -133bp-luc which contains 133 bp upstream of IL-8 encoding region, and mutated plasmids NF-κBmut-luc,AP-1mut-luc,NF-IL-6mut-luc, which were obtained by site mutation of -133bp -luc on the binding sites of transcriptional factors, NF-κB,AP-1 and NF-IL-6, respectively, along with internal reference plasmid pRL-T. The cells were inoculated with PRRSV 24 hours later and luciferase activity was determined 6 hours after inoculation. The result showed that in Marc-145 cells transfected with NF-κBmut-luc and NF-IL-6mut-luc, IL-8 was not obviously activated. But pIL-8-luc,-133bp-luc and AP-1mut-luc could activate the activity of IL-8 promoter significantly. The expression of IL-8 could not be induced after the mutation of the transcriptional factor binding sites of NF-κB and NF-IL-6, indicating that these two transcriptional factors are essential for the activation of IL-8 by PRRSV.To find out whether MAPK signal pathway was engaged in the activation of IL-8, the Marc-145 cells transfected with IL-8 promoter luciferase reporting plasmids were processed with inhibitors of three MAPK signal pathways(p38,JNK and ERK), and luciferase activity was tested after PRRSV infection. It was found that the inhibitor of ERK pathway has some inhibitory effect on the activation of IL-8, but those of p38 and JNK do not exert an obvious effect, indicating that the ERK pathway of MAPK was involved in the induction of IL-8.