Construction And Analysis Of Transgenic Cell Lines And A Mouse Model Susceptible To Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome(PRRS),also well-known as "blue ear disease",is caused by porcine reproductive and respiratory syndrome virus(PRRSV).PRRSV infection can cause severe reproductive problems in sows and respiratory failure in pigs of all ages,which has resulted in significant economic losses in the swine industry globally.Currently,vaccination is the primary means for PRRSV prevention and control.However,due to the lack of robust cell lines and small animal models,the pathogenesis of PRRSV infection and mechanism of immunoprotection are still not yet well understood.Previous studies show that CD 163 is indispensable for PRRSV entry to cells and mediates PRRSV replication.Other receptors,such as CD 169 and CD151,also contribute to PRRSV infection.But the effect of combined action among different receptors have not been well studied.Here,we generated transgenic BHK-21 derived cell lines co-expressing different combinations of PRRSV receptors,which were co-transfected by porcine CD163(pCD163)alone/pCD163 and porcine CD169(pCD169)pCD163 and simian CD151(sCD151)/pCD163,pCD169 and sCD151 using PiggyBac transposon system.To eliminate the interference of pCD163 expression level,clones harbouring similar pCD163 level were selected to analyze the contribution of pCD169 and sCD151 to PRRSV infection.We found that all of the transgenic cells were susceptible to PRRSV and the synergistic reaction of pCD163,pCD 169 and sCD151 was important to improve susceptibility.Moreover,we demonstrated that the cell line co-expressing the triple receptors sustained viral infection and replication most efficiently,which was superior to the current cell platform used for PRRSV production,MARC-145 cells.In brief,we developed in vitro transgenic cell lines that could be intensively susceptible to PRRSV,which would have the potential to provide a useful tool for virus propagation,vaccine development and pathogenesis studies.In order to fill the blank of the PRRSV research on the small animal model,we carried out co-microinjection into the pronuclear of fertilized eggs to generate mouse models using the three PiggyBac expression vectors containing pCD163,pCD169 and sCD151 CDSs.We performed PCR,Southern blot and copy number detection to determine integration and copy number of exogenous genes.In further,expression of exogenous receptor genes in different tissues from transgenic mice was detected by qPCR and Western blot.To analyze whether the transgenic mice were susceptible to PRRSV,infection experiments were performed in vitro and in vivo,respectively.The amount of PRRS virus in the isolated macrophages,blood and various tissues were analyzed at different time points after infection.However,due to the low expression of the three exogenous receptor genes in AMs and other tissues,the infection efficiency was not as well as expected and no clinical signs appeared in transgenic mice.In summary,we developed transgenic BHK-21 cell lines that were susceptible to PRRSV using PiggyBac expression vectors containing pCD163,pCD169 or sCD151 CDSs.Based on our results,the triple transgenic cell line would be a useful tool for virus propagation,vaccine development and pathogenesis studies.Although the transgenic mice are not sensitive to PRRSV infection as desired,our results still provide a good data set for developing small animal models susceptible to PRRSV using similar approach in future.