Porcine Pulmonary Alveolar Macrophages Transcriptional Responses Following In Vitro Infection With Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a serious infectious disease of swine. It is characterized by severe reproductive failure in sows, and respiratory distress in piglets and growing pigs. Since it was first reported in the United States in 1987, PRRS has already been one of the most economically important diseases of the global swine industry.Many evidences have proved that pulmonary alveolar macrophages (PAM) play an important role in PRRSV infection in pigs, the PAMs are the main target cells at the beginning of PRRSV invasion, the virus starts replication in PAM then release to the blood, causes the change of cell immunity. Macrophages are important immunocyte, study the influence of PRRSV infection to PAM is profit to explain the interaction between PRRSV and the host immune system and the mechanism of persistent infection immunosuppressive. Also it has significant meaning to elucidate the pathogenicity and pathogenesis of PRRSV.Now, microarrays have been more and more frequently used in the studies of the relationship between pathogens and hosts. Especially the high-flux characteristic of microarray, we can get a great quantity information after one experiment.Therefore, in this study, to help understand how PRRSV infection manipulates the function of PAM, we used a Affymetrix porcine cDNA microarray contains 23,937 probe sets that interrogate approximately 23,256 transcripts from 20,201 S. scrofa genes to investigate changes in PAM gene transcription that occur during in vitro infection with WuH1 stains. This study is a useful first step in screening a wide range of host genes for potential virus-induced changes in RNA levels and provides a preliminary identification of some of the host genes that may be differentially regulated. Data from the microarray experiments provide a foundation for further studies, at the functional level, into the molecular basis of PRRSV pathogenesis and virus mechanisms of immune evasion.The most research works were as following:We found that there were 2640and 3356 different genes in PAM at 6h and 24h after infection with WuH1 PRRSV strain, which were about 13.09% and 16.64% in the whole microarray probe sets. At 6h the up regulated genes and down regulated genes were 1235 and 1485,while the more than 2 fold un regulated and down regulated ones were 619 and 435.At 24h the up regulated genes and down regulated genes were 1746 and 1619, while the more than 2 fold up regulated and down regulated ones were922 and 773. Screening the different expression genes, especially related with immune response, inflammatory reaction , apoptosis and so on. Classify the different expression genes such as IFN genes,IFN effector genes,IFN signal genes ,CD molecular genes, HSP related genes , Chemokines and Toll like receptor genes. Use Genmapp software to draw gene pathway of Apoptosis. Check the microarray results with Real-time RT-PCR using 42 pair primers.By Real-time RT-PCR, we system analysis the expression of 15 Chemokines after infection by WuH1 PRRSV at 6h and 24h including CCL2-5, CCL8, CCL19, CCL20, CCL27, CCL28,CXCL2,CXCL8,CXCL10,CXCL12,CXCL14 and CX3CL1.In CC Chemokines family, the results revealed that the expression of RANTES, MCP-1 and MCP-2, which are functional in recruiting both Th1 and Th2 cells, was induced early after infection. CCL2 was at a low expression level in uninfected PAM at 6 hours and increased significantly at 24 hours, while after infection with PRRSV, the CCL2 mRNA production was increased quickly at 24 h in contrast to 6h.The mRNA expression of RANTES and MCP-2 are at a higher level when 6h while in contrast with that at 24h. Additionally, the expression level of MCP-1 was remained relatively up-regulated till 24h. Also the chemokine expression profiles of CCL19, CCL20, CCL27 and CCL28 were analyzed. CCL19 and CCL27 maintained no difference expression between infection group and mock one both at 6h and 24h, while CCL20 and CCL28 transcription levels gradually declined. Chemokine CXCL2 expression level was relative high at 24 hours after PRRSV infection. In contrast to 24 hours time point, CXCL8 and CXCL12 were significantly up-regulated at 6 hours. At both at 6 and 24 hours, CXCL14 mRNA level was descended contrast to the mock by Real-time PCR. Interestingly, CXCL10 expression level was down-regulated dramatically at 24h contrast to 6h both the infection group and mock group. Additionally, the up-regulate levels of CXCL2 and CXCL12 were more than 100 folds. The CX3C chemokine CX3CL1 mRNA levels in infected cells were not found to be statistically significant compared to those in the uninfected cells.