Proteome Proifling Of PRRSV-Infected MARC-145Cells And The Rescure Of ZCYZ Infectious Clone

Porcine reproductive and respiratory syndrome (PRRS) is a viral disease caused byporcine reproductive and respiratory syndrome virus (PRRSV). PRRSV mainly leads toreproductive disorder in sows and respiratory problems in pigs of all ages. The pigs infectedby PRRSV also show immnosuppresion and persistent infection. In vivo, PRRSV mainlyinfects porcine alveolar marcophages, however, it can replicate in the African green monkeykidney cells MA104and derived MARC-145in vitro. To better understand the host responseto viral infection and the pathogenic difference of different strains, we first analyzed thepathogenic of three PRRSV strains (SD1,SX-1and ZCYZ). The sequence MegAlign andanimal experiment results showed that SD1belonged to traditional strain with no deletion inNsp2, SX-1is high pathogenicity strain with30aa deletion in Nsp2, ZCYZ isolated fromhybrid wild boars is also a high pathogenicity strain with54aa deletion in Nsp2.The virulencefor SX-1, ZCYZ and SD1is high, moderate and mild to pigs, respectively, verified by animalinfection experiments.MARC-145cells were harvested at48hpi with SD1, SX-1and ZCYZ strains. First thesamples were digested and labeled with iTRAQ reagent, then the on-line2D LC–MS/MSanalysis was employed to make sure the differently expressed proteins. A total of233proteinswere identified form the three groups (SD1vs MARC-145; SX-1vs MARC-145; ZCYZ vsMARC-145), and the functions of these proteins can be classified into associated withmorphogenesis, protein synthesis and metabolism, response to stress and theubiquitin–proteasome pathway.Pathway-analysis was performed for the identified proteins to find out the distinguishedaccurate and targeted pathway. Based on the differently expressed proteins, the signaltransduction network was constructed to study the transduction course of protein-protein andfind out the central protein in the Signal-Net. Meanwhile, the up-regulations of HSPA8andthe down-regulations of ANXA2both existed in the three groups were validated byWestern-blot assay. ZCYZ strain was isolated from hybrid wild boars, also known as special wild pigs whichare a hybrid animal with75%wild boar lineage and25%domestic Duroc pig lineage. Hybridwild boars are genetically stable and retain the appearance of wild boars. In addition, theyhave less body fat, are strongly adaptable to various environments. In order to elucidate thegenetic characteristics of the strain and the deleted54aa in Nsp2for the influence of thepathogenicity, we sequenced the whole ZCYZ genome and constructed the infectious clone.The infectious clone was rescured successfully validated by IFA. Animal experimentindicated that the cloned virus possessed the weaker pathogenicity than the parental virus,which provided a good platform for developing genetically markered vaccines to differentiateinfection from vaccination.The study provided valuable information for the further study on molecular mechanismsand proteins associated with the pathogenicity of PRRSV.