Construction Of Infectious CDNA Clone Of PRRSV/GSWW/15 Strain And Deletable Regions In Non-structural Protein 2

Porcine reproductive and respiratory syndrome?PRRS?is an important infectious disease characterized by reproductive failure and respiratory disorder in sows and piglets.The causative agent is porcine reproductive and respiratory syndrome virus?PRRSV?.At present,PRRS is prevalent in the world.In 2006,the highly pathogenic porcine reproductive and respiratory syndrome virus?HP-PRRSV?emerged in the south of China and caused huge economic losses.PRRSV is highly variable and mainly infect the macrophage cells,which lead to the disfunction of immune system.The mechanism of low level protective immune response and long-last viremia are still not clear.PRRSV infectious clone is the most important tools to study the structure and function of viral genome.The establishment of the infectious clone of PRRSV epidemic strain will make a good foundation for study the mechanisms of virus infection and immunization.According to the whole genomic sequence of PRRSV/GSWW/15 strain,two half-length of genomic sequences?7636bp and 7774bp?were synthesized and cloned into pBR322 vector.And then the full length c DNA clone were constructed by ligation two half part molecules into pBR322 vector.After linearization of the recombinant plasmids,the whole genomic RNA were prepared by in vitro transcription.The first generation of viruses were rescued by electro-transfection of prepared whole genomic RNA into BHK-21 cells.The rescued virus were passaged on Marc145 cells,and the complete cytopathic effect?CPE?was observed within 36 h after inoculation of the first generation of the rescued virus.The rescued PRRSV GSWW virus were further confirmed by RT-PCR,indirect immunofluorescence assay?IFA?and sequencing for the present of virus antigen and molecular marker involved.The growth kinetics of the rescued virus are similar with that of the parental virus,and the rescued virus titer can reach 1×10¹⁰ copies/ml.This study show that field strain PRRSV infectious clone can be effectively established by long fragment DNA synthesis,and would provide a useful tool for study on the molecular mechanisms of PRRSV pathogenesis and development of novel genetically engineered vaccines.Because of the application of multiple attenuated vaccine strains,genetic recombination of virus and the emergence of new mutant strains are very common in field.This is a great challenge for PRRS prevention and control.Marker vaccines for differentiation of the vaccine strain and wild-type virus infected animals has become a key technical requirement.Research shows that Nsp2 is the most variable protein and contain multiple immunodominant B cell epitopes,which will be the potential region to make epitope-deleted marker vaccine strain.Therefore,based on the infectious clone of PRRSV/GSWW/15,a serial deletion in highly variable regions of NSP2 were involved by fusion PCR to explore the non-essential region in NSP2 protein.The results showed that the deletions in298-592?726-819?421-565?300-467?324-467?734-797 amino acid?aa?of NSP2 could not rescue the live virus.Deletion the 519-565 aa of NSP2 will greatly affect the virus replication.However,the deletion of421-467 aa in NSP2 do not affect the virus replication?designated GS-D4?,furthermore,the GS-D4virus show increase adaption to Marc145 cells as the virus titer of the eighth cell passage virus is higher than the parental virus.In conclusion,we constructed field strain PRRSV infectious clone and found that deletion of the421-467aa in Nsp2 afford the virus a better replication ability than parental virus in Marc145 cell.These results provide a sound basis for PRRSV epitopes-deleted marker vaccine.