| CSF (also called Hog Cholera), caused by Classical Swine Fever virus,is a serious acute and fatal disease for pigs causing significant economic losses worldwidely for the pig industry. In recent years, due to the failure of imuunization for CSF, and the emergence of combined infection and secondary infection of CSF, the prevention and control of this disease is increasingly urgent. To study intensively the immune response and its molecular mechanism upon the infection with CSFV and further demonstrate the immune mechanism of CSF virus is of great significance for the development of novel vaccines to CSF as well as for more effective strategies for controlling and preventing the disease.Regulated upon Activation, Normal T cell Expressed and Secreted, RANTES is an important member of chemokine CC family, and is a chemokine with multiple activities. It not only has chemotaxis effect on different types of cells, but also activates lymphocytes, participate to the pathogenesis of inflammation and regulate the growth and differentiation of cells. Studies by now have revealed that RANTES plays a rather important role in the infection anf pathogenesis of many bacterial and viral pathogens. However, the relationship between CSF infection and RANTES expression is still not well understood. For this reason, we studied the dynamics of RANTES expression after CSF virus infection, and discussed the signal pathway of CSF regulation on RANTES expression. The main content are as follows1. The dynamics of RANTES mRNA expression after the PK-15 cells is infected with CSFVUsing Real-time PCR to analyze the expression of RANTES t different time points after PK-15 cells was infected with CSFV. We found no significant change of RANTES mRNA expression level in the early stage after CSFV infection on PK-15 cells. But 72 hours after infection, it was significantly lower than cells without infection, and CSFV infection significantly inhibited RANTES expression induced by poly (I:C). To further verify the result of real-time PCR, the luciferase reporter system of porcine RANTES promoter was constructed, and it was found that CSFV inhibited the activation of RANTES promoter.2. CSFV Npro is involved in the inhibition of RANTES expressionTo determine which proteins of CSFV play a vital role in the inhibition of RANTES, we built the eukaryotic expression plasmids of twelve CSFV proteins and analysed the affect on different protein on the activity of RANTES promoter by RANTES promoter luciferase reporter system. We found the transfection of expression plasmid for Npro could significantly inhibit the activity of RANTES promoter. Subsequently, mutation analysis was conducted on different transcription factor binding sites in RANTES promoter. It was shown that ISRE binding site played an important role in the inhibition of RANTES by CSFV Npro. To determine the essential region for Npro to inhibit RANTES, a series of mutants of Npro was constructed. The result indicated 21 amino acids on N terminal of Npro were not essential for the inhibition of RANTES expression. But the mutation of the 69th,112nd, and 136th amino acid related to the cleavage activity of Npro could partially or completely disable the Npro's inhibition on RANTES expression. It can be inferred that the cleavage activity of Npro is crucial for the inhibition of RANTES expression.3, Study on the Signal pathways of CSFV Npro on the inhibition of RANTESTo comprehend the upstream signal pathways of CSFV Npro on the inhibition of RANTES,The signal molecules and transcription factors activating RANTES signal pathway, including RIG-â… ,MDA5,IPS-1,IRF3,IRF7 were used for analyzing,and we found that the Npro inhibited the RANTES which is induced by the overexpresiion of RIG-â… ,MDA5,IPS-1,IRF3,IRF7.It demonstrated that Npro suppressed RANTES through the RIG-I/MDA5 signal pathway...
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