Antiviral Activity Of Recombinant Collectins Against PRRSV In Vitro And Purification Of PRRSV With GEM Particles

Porcine reproductive and respiratory syndrome (PRRS) is an acute and highly contagious disease, which is caused by PRRSV. Until now, although researchers have made great efforts towards preventing and controlling PRRSV infection, it is also one of the most serious diseases for world pig industry and caused tremendous economic losses. Currently, the prevalent methods to control PRRSV infection include production management, Adaptation strategy of gilts and vaccination, but they fail to provide complete protection against this devastating disease. Thus it is an urgent need to develop new strategies to control PRRSV infection. In this study, SPA/Ficolin and PA-Ficolin/SPA were expressed, and antiviral activity of recombinant porcine SPA/Ficolin against PRRSV in vitro and purification of PRRSV with GEM-PA-Ficolin/SPA were investigated as below.1. Preparation of four kinds of recombinant proteinsSPA/Ficolin and PA-Ficolin/SPA was obtained using a Bac-to-Bac baculovirus expression system. The specific primers were designed by Primer Premier 5.0 including 8-histidine tag for protein purification and detection. The DNA of SPA, Ficolin and PA was amplified from recombinant plasmid constructed by our own laboratory. The amplified fragment was cloned into the PMD-I8T vector, then double enzyme digestion association sequence analysis. The positive plasmid was named as 18T-Ficolin, 18T-SPA and 18T-PA.18T-Ficolin,18T-SPA and pFastBacl plasmid were digested by EcoR I and Hind III respectively and linked by T4 DNA ligase and transformed into DH5a competent cells, yielding pFast-SPA and pFast-Ficolin; To save time pFast-PA-SPA and pFast-PA-Ficolin were prepared by one step as follows: Ficolin, SPA, PA and pFastBacl were digested by double enzyme digestion respectively, then Ficolin, PA and pFastBacl were linked by T4 DNA ligase; SPA, PA and pFastBacl were linked by T4 DNA ligase, then transformed into DH5a competent cells, yielding pFast-PA-SPA and pFast-PA-Ficolin.The pFast-SPA, pFast-Ficolin, pFast-PA-SPA, pFast-PA-Ficolin and pFastBacl plasmid DNA was transformed into MAX Efficiency(?) DH10BacTM competent E. coli for transposition into a bacmid. Blue/white selection was used to identify those colonies that contained a recombinant bacmid named as rBacmid-SPA, rBacmid-Ficolin、rBacmid-PA-SPA、rBacmid-PA-Ficolin, rBacmid-pFast and wild Bacmid from the blue bacteria。The above Bacmid DNA were then transfected into Sf9 insect cells using Cellfectin II reagent. The recombinant baculovirus was harvested 7 days post-transfection and was named as rBV-SPA、 rBV-Ficolin、rBV-PA-SPA、rBV-PA-Ficolin-、rBV-pFast, and wild BV。Then the proteins were expressed and detected by Western blotting analysis. The result showed that in addition to Ficolin, SPA, PA-Ficolin and PA-SPA were soluble and can be used for follow-up study.2. Antiviral activity of RpSPA against PRRSV in vitroRpSPA had potency against PRRSV on Marc-145 cells without causing cytotoxicity. Plaque assay results revealed that 30 μg·mL-1 of RpSPA reduced plaques by 77%(P<0.0001), whereas 15 μg-mL-1 RpSPA reduced plaques by 43%(P<0.05). Additionally, we found that RpSPA exerted potent anti-viral activity in a Ca2+-dependent manner, indicating that RpSPA may interact with PRRSV via the carbohydrate recognition domain. The virus entry assay showed that RpSPA could strongly depress virus attachment, but its effect on virus penetration was at most modest, moreover, our data indicated that the suppression on PRRSV penetration of RpSPA gradually weakened even disappeared as time of addition RpSPA increased. The data suggested that RpSPA might act on the stage of PRRSV entry into Marc-45 cells from the cell surface and once entry into cells it lost its action. Interestingly, our data also indicated that RpSPA had no effect on the total RNA level when the virus was allowed to perform only a single cycle of replication, while caused the total RNA level decreased greatly at 12 h,24 h and 36 h. This indicates that those virus progeny released from infected Marc-145 cells are blocked by RpSPA. A better understanding of the underlying mechanisms by which RpSPA influence viral entry might lead to new antiviral interventions. Taking the previous reported data and our own findings together, we hypothesize that RpSPA reduces PRRSV entry by binding to glycoprotein GP2, GP3, GP4 or GP5 on the surface of PRRSV, thereby preventing it from interacting with its receptors on Marc 145 cells. The nature of the interactions among PRRSV, RpSPA and cells is not yet known, so this model should be assessed in further studies.3. Purification of PRRSV with GEM particlesGEM (Gram positive enhancer matrix) display system is a novel surface display technolog. The design concept of this study is as follows:firstly, fusion protein with anchoring function can be loaded on the surface of GEM particle, then PRRSV can be loaded by collectins (Ficolin or SPA) depending on the specific binding capacity between collectins and kinds of microbes.The fusion protein with the anchoring effect is the key to our study. PA-Ficolin/SPA was harvested by Bac-to-Bac expression system and western blotting showed it had the espected activity. PA-Ficolin/SPA can bind to GEM particles through the anchor protein PA, and also can bind to PRRSV through Ficolin/SPA. Additionly, Ficolin/SPA has a widely microbial spectrum, also can bind to HIV, HCV and AIV, suggesting that GEM-PA-Ficolin/SPA system in our study can not only be used for PRRSV purification, but also for other viruses. Thus the GEM-PA-Ficolin/SPA display system in this study may be a candidate strategy for virus purification.