Study On The Adaptive Capacity Of Porcine Reproductive And Respiratory Syndrome Virus For The Infection Of Marc145 Cells Mediated By Porcine Sialoadhesin

Porcine reproductive and respiratory syndrome(PRRS,also known as blue-ear disease)has been one of immunosuppressive diseases,caused by porcine reproductive and respiratory syndrome virus(PRRSV)and affecting the swine industry worldwide.Vaccination is one of the most important strategies for the prevention of PRRS.However,primary susceptible cells(such as porcine alveolar macrophages,PAM),are difficult to meet the demand for the mass production of vaccines against PRRSV,due to the restriction in cell tropism of the virus.In addition,some field isolates of PRRSV were unable to adapt to the culture cells(such as African green monkey kidney epithelial cell line,Marc145),due to the lack of sialoadhesin(Sn)receptor on the cell surface.These could hinder the sustainability to evaluate the immune responses and the abundance to improve the virus seed-lot systems of commercial PRRSV vaccines,which might be not conducive to epidemic prevention and control of its devastating outbreaks in the future.Thus,it is necessary to establish recombinant cell lines that facilitate for PRRSV infection in vitro.【Objective】To obtain a transgenic Marc145 cell line that stably expresses porcine Sn(p Sn),p Sn-Marc145,the genome of Marc145 cells was modified by using CRISPR/Cas9 technology.The knock-in of p Sn in Marc145 cells might contribute to the adsorption capacity and the adaptability and pathogenicity of PRRSV during the infection process,as a viable platform,which would be helpful to perform the understanding of PRRSV in the fundamental research and applications.【Methods】The intron region between RACGAP1 and ASIC1 in the 11 th chromosome of Marc145 cells was selected as a targeting gene locus.The single guide RNAs(sg RNA)were predicted and designed by using biological software.The Cas9 expressing plasmids was constructed and the most efficient sg RNA was determined by employing analysis software and Surveyor nuclease detection.To clarify whether the foreign gene could be integrated and expressed at the inter-gene locus,the donor plasmid harboring the gene of enhanced green fluorescent protein(e GFP)was used for homologous recombination repair which were determined by fluorescence microscopy,Junction PCR and Southern Blotting.Subsequently,the well-designed sg RNA plasmids were constructed by using different Cas9 variant vectors(p Sp Cas9,Cas9 n,e Cas9)and available for evaluating their cutting efficiency on the targeting DNA.p Sn-Marc145 cells were generated by the transfection of Cas9/sg RNA complex with lower off-target,higher cutting and homologous recombination efficiencies.The monoclonal cells were picked up and expanded in the presence of puromycin.The p Sn positive clones were screened using Junction PCR,Southern blotting,Western blotting and indirect immunofluorescence assay,following karyotype analysis,observation of cell morphology and assessment of the growth activity in cell life cycle.Finally,the comparison on biological properties of vaccine strains(VR2332,CH1 R,R98,JXA1)and wild-type strains(GSWW2018 and GSKL)of PRRSV was carried out in normal Marc145 and p Sn-Marc145 cells.【Results】5 sg RNA(sg RNA-3,-17,-23,-24,-33)were designed and synthesized,and sg RNA-24 showed the lowest off-target efficiency for the integration of e GFP at the targeting site in RACGAP1 and ASIC1 genes of the 11 th chromosome.The knock-in e GFP gene by CRISPR/Cas9 system was confirmed by the expression of specific green fluorescence in Marc145 cells.The cutting efficiency of Cas9n(double-sg RNA)combine sg RNA-24 was higher than that of Cas9 n and e Cas9 in Marc145 cells,and 2 p Sn-positive cell clones(24-15-7 and 24-15-9)were obtained by CRISPR/Cas9 system(Cas9n/doublesg RNA and p Sn donor plasmids).There were no genetic and phenotypic changes in p SnMarc145 cells,while had higher growth activity with that of normal Marc145 cells.Comparable to that of normal Marc145 cells,the increased adsorption capacity of high pathogenic PRRSV(HP-PRRSV,JXA1)was detected,despite no obvious effects on virulence and replication of all the indicated vaccine strains of PRRSV in p Sn-Marc145 cells,according the results from plaque forming assay,indirect immunofluorescence assay,virus growth curves,Western blotting and real-time quantitative PCR.Moreover,the results from genetic variation analysis,real-time quantitative PCR,plaque forming assay and virus growth curves,revealed that the adaptation of two wild-type strains of PRRSV was more rapidly and virulently in p Sn-Marc145 cells.【Conclusion】In this thesis,our experimental data demonstrated that(i)the intron region between RACGAP1 and ASIC1 of the 11 th chromosome could be served as one of the targeting sites by using CRISPR/Cas9 system in Marc145 cells and(ii)p Sn-Marc145 cells might be helpful for the production of HP-PRRSV vaccines and the isolation of field strains of PRRSV.