Study On The Mechanism Of Porcine Productive And Respiratory Syndrome Virus-increased PGE₂

Porcine reproductive and respiratory syndrome (PRRS) caused by pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) is characterized by respiratory disorders in piglets and reproductive failure in sows. In2006, a highly pathogenic strain of PRRSV (HP-PRRSV) was identified in China, characterized by high fever, high morbidity and high mortality. Prostaglandin E2(PGE2), a key factor causing fever, is synthesized by the limited enzymes cyclooxygenase type1/2(COX-1/2) enzymes in vivo. However, the mechanism underlying the fever induction of HP-PRRSV is still unknown. But whether PGE2and COX also play a role in HP-PRRSV induced fever remains inclusive.In this study, a significant inverse correlation between the high body temperature and survival days of infected pigs was oberved, indicating pigs with high temperature in late stage of infection die earlier. We found that HP-PRRSV infection increased the production of PGE2in plasma, bronchoalveolar lavage fluid (BALF) and PAMs. Then the mRNA and protein level of COX-1expression were obseved to be significantly increased in PAMs upon HP-PRRSV infection. Compared to COX-2inhibitor celecoxib treatment, the COX-1inhibitor sc-560treatment significantly retarded the induction of PGE2by HP-PRRSV infection, suggesting COX-1is responsible for the increasing of PGE2. In addition, PD98059(MEK inhibitor) significantly decreased the expression of COX-1and PGE2. As expected, ERKl/2phosphorylation increased rapidly after viral infection, demonstrating that ERKl/2and p-ERK are the key nodes to trigger COX-1expression after PRRSV infection. After the porcine COX-1promoter was cloned, a series of truncation mutants of the promoter region of porcine COX-1in the luciferase expression vector was constructed and tranfected to Marc-145cells. We found that a predicated a transcription factor binding motif located in-405/-215region of COX-1promoter. Meanwhile, bioinformatics approach and base mutation experiment furthter suggested a putative C/EBP-β motif existed in this area. Chromatin immunoprecipitation assay further confirmed that a p-C/EBP-β regulatory binding element existed at the site-404/-193. Furthermore, the fraction of nuclei and cytoplasmic protein and confocal experiment demonstrated that the expression of p-C/EBP-β were augmented and translocated into nuclei at early stage after HP-PRRSV infection. Moreover, the inhibition of ERKl/2significantly impaired PRRSV-induced p-C/EBP-β expression. We also found that the knock down of C/EBP-β suppressed the protein level of COX-1during HP-PRRSV infection. These results demonstrated that HP-PRRSV induced C/EBP-β activation through ERK1/2pathway. Then the phosphorylated C/EBP-β migrated to nuclei and bound to COX-1promoter region to initiate COX-1transcription.In summary, we hypothesised PGE2played a role in HP-PRRSV-induced fever. We find that the infection of HP-PRRSV promote the phosphorylation of ERK1/2, induces C/EBP-β activation and then the phosphorylated C/EBP-β migrates to nuclei and binds-404/-193region COX-1promoter to initiate COX-1transcription. The upregulated COX-1augments the production of PGE2, which might be the reason of HP-PRRSV-induced fever.