Screening And Testing Of Differential Proteins Of PAMs Infected With Highly Pathogenic And Attenuated PRRSVs

Porcine reproductive and respiratory syndrome virus(PRRSV) has remained a leading economically significant viral disease of swine worldwide for almost 28 years, and was first found in 1987, USA. In 2006, several large- scale, severe outbreaks of highly pathogenic PRRSV(HP-PRRSV) was reported in China. PRRSV is a member of the family Arteriviridae, together with Equine Arteritis Virus(EAV), Simian Hemorrhagic Fever Virus(SHFV), and the Lactate Dehydrogenase- elevating Virus of Mice(LDV). PRRSV is a positive- sense, single- stranded RNA virus, through a so-called ribosomal frameshift mechanism, pp1 a and pp1 ab are translated and hydrolyzed into more than 12 Nsps, and then they will help produce the structural proteins, with which participate the virus entry. PRRSV exhibits a highly restricted cell tropism both in vivo and in vitro. PAMs are the main target cells of PRRSV. PRRSV has also been shown to infect MARC-145, which is considered permissive cell lines for PRRSV. The infection effect between different virulent strains changes badly, and it is still largely unknown.HP-PRRSV(HuN4) and its attenuated PRRSV(HuN4-F112) were used as the backbone for constructing the recominant pHP-EGFP and pA-EGFP infectious clone. The membrane proteins of PAM were purified and performed for Shotgun MS. The membrane proteins of parental HP-PRRSV and APPRRSV infected PAMs were detected by Label-free MS. The results have great significance for the study of PRRSV’s pathogenesis.In order to screen the membrane proteins of PAMs interplayed with the PRRSV. The report gene of enhanced green fluorescent protein(EGFP) gene was inserted between ORF1 b and ORF2 a of the full length infectious clone of HP-PRRSV HuN4 and its attenuated PRRSV vaccine strain HuN4-F112 by SOE PCR(designated pHuN4-EGFP and pHuN4-F112-EGFP), and the recombinant PRRSV(vHuN4-EGFP and vHuN4-F112-EGFP) expressing EGFP was rescued by transfecting. Then, genetically stable test showed that the foreign gene was stable for passages. Moreover, the viral titer, plaque morphology and the growth ability of the recombinant virus were similar to the parental virus.After obtaining the recombinant vHuN4-EGFP and vHuN4-F112-EGFP, we sorted the positive cells infected with this kind of recombinant virus by FACS through detecting the green fluorescence signal and then the membrane protein of sorted cells was extracted and performed for the following Shotgun MS, some proteins were chosen and analyzed for further over expression in MARC-145 cells. Then the MARC-145 cells were infected with HP-PRRSV and AP-PRRSV again. Through Western Blotting, we can obtain the potential interactions between the target proteins and the HP-, AP-PRRSV respectively, we found that the Apolipoprotein M, JUP, Apolipoprotein A-IV and Kexin type 9 down regulated the PRRSV, while Clusterin isoform x1 up regulated the PRRSV.On the other hand, the HP-PRRSV(HuN4) and AP-PRRSV(HuN4-F112) were used for infecting PAM and the method of Label-free quantified MS were conducted for detecting with the the potential differential infection pathway of HP-PRRSV and AP-PRRSV. 95 differential proteins were detected and 26 of them were located on the membrane confidently, and we got KDEL receptor and Histone H3 to verify the Label-free MS by WB. Several different potential pathways between HP-PRRSV, AP-PRRSV were found by String analysis.