Identification And Analysis Of Differentially Expressed Proteins Of Host Cells Infected With Different Pathogenic Porcine Reproductive And Respiratory Syndrome Viruses

Porcine Reproductive and Respiratory Syndrome（PRRS）caused by Porcine Reproductive andRespiratory Syndrome virus (PRRSV) is one of the most important diseases destructing the swineindustry worldwide and can result in productive failure and stillbirth in pregnant sows, and respiratorycomplications in piglet. In1995, this disease attacked China for the first time. In2006, an outbreak ofhighly pathogenic PRRS (HP-PRRS) occurred in China and caused significant higher morbidity andmortality in different ages of pigs. So far the molecular mechanism of the increased pathogenicity ofHP-PRRSV is not well known. To elucidate the mechanism may be immensely contributed to thecontrol and elimination of PRRS. In our study, we applied proteomics approach to investigate themolecular mechanism of the different pathologic ability of different virulent PRRSV strains.First, the growth properties of high/low-virulent PRRSV in porcine alveolar macrophages (PAMs)were evaluated. The cytopathic effect (CPE) in HuN4infected PAMs was more severe than inHuN4-F112infected PAMs although both strains were able to infect PAMs as determined by CPE andimmuofluorescence assay (IFA). The growth curves of HuN4and HuN4-F112in PAM and Marc-145showed the similar result. These suggested that the cell tropism and replication ability of PRRSV havechanged during continuous passages in cell line Marc-145. The present study provided usefulinformation for further investigation into the relationship between virulence and growth properties ofhighly virulent and attenuated HP-PRRSV strains.Then we inoculated103TCID50HuN4and HuN4-F112with PAM cells respectively, and collectedthe total cellular protein to perform proteomics analysis. The results of two-dimensional differentialfluorescence electrophoresis(2D-DIGE) and image analysis showed that153DEPs was found for itssignificant different expression between two strains (p<0.01）. Mass spectrometry analysis revealed23significant differentially expressed cellular proteins between different strains. Then the Blast2GOsoftware was used for the gene annotation and functional classification, and it showed that these DEPsbelong to three sort functions, biological process (65.1%), molecular function (16.3%) and cellularcomponent (18.6%) by GO enrichment. Furthermore, we identified the transcription validation ofinteresting DEPs by fluorescent quantitative RT-PCR. The results showed that PKM2, HSBP1, PMSA6and PARK7differently induced by HuN4, and HuN4-F112. Expression level caused by HuN4andHuN4-F112are of significantly differences. PKM2, HSBP1, PMSA6and PARK7play an importantrole in the normal cells, may be involved in different pathogenic process of HuN4, and HuN4-F112.Marc-145is the most widely used cell lines to propagate HP-PRRSV in vitro. In order tounderstand Marc-145cells responses to highly pathogenic HuN4strain and attenuated HuN4-F112strain respectively, we use proteomics approach to investigate the differentially expressed cellularproteins of Marc-145infected by103TCID50HuN4and HuN4-F112respectively.2D-DIGE paired withthe mass spectrometry analysis, the results showed46significant differentially expressed cellular proteins were identified (p<0.01). And these proteins were analyzed for gene annotation and functionalclassification by the Blast2GO software. Further identification the validation of the differentialexpression of the dynamic changes of DEPs by western blot and obsevered that the CRT, hnRNPK, andHSPA9have different expression level between HuN4and HuN4-F112, and these proteins play animportant role in viral replication and immune response, may be involved in pathogenic process ofPRRSV.The current study verified the DEPs between HuN4-F112and HuN4infected PAM cells andMarc-145cells, analyzed the relationships between some DEPs and the virus pathogenicity. The data inthis research provide useful information to further study the molecular mechanism of pathogenicity ofhigh/low virulent PRRSV strains.