| Senecavirus A（SVA）,formerly called Seneca Valley virus,belongs to the genus Senecavirus of family Picornaviridae.S VA is the pathogen of Senecavirus A-associated vesicular disease（SAVD）,and the clinical signs of SVA infection are fluid-filled vesicles and erosions around their snouts,mouths and hooves.Depression,loss of appetite,lameness and lethargy also occur.From 2014 onward,occurrence of SAVD has been reported in USA,Brazil,Thailand,Colombia,Vietnam,China and other countries.Of these,it is noteworthy that a second explosive onset of SAVD emerged in Brazil in 2018,which was characterized by rapid spread and severe clinical manifestations in epidemiology.This second wave of infection suggested that persistent existence of SVA in pig herds may allow the virus to evolve toward being more virulent than ever.As a newly emerging pathogen of swine disease,the knowledge of SVA pathogenesis and interactions with host cells at early stage is warranted to be studied.Therefore,transcriptome analysis of Senecavirus A-infected cells was performed to analyzed the changes in host cells.Then,CRISPR/Cas9 technology was used to construct a group of innate immune gene KO cell lines,which were used to study the growth characteristic of SVA,Porcine sapelovirus and Getahvirus.1.Transcriptome analysis of Senecavirus A-infected cellsIn this study,we studied the gene expression profiles of LLC-PK1 cells,with or without SVA infection,for 6h and 12h,based on the interaction between virus and host cells.The results indicated that 1584 genes had a significant difference between GD056h group and MOCK6h group.These included 1072 up-regulated and 512 down-regulated genes.Compared with MOCK12h group,9785 DEGs were screened out at GD0512 h group,with 5238 being up-regulated and 4547 down-regulated.GO function and KEGG pathway enrichment analysis showed that several signaling pathways,such as innate immune and apoptosis were changed after SVA infection.Innate immume related-pathways were significantly modified,suggesting that immune mechanisms may play an important role in this process.To confirm the RNA-seq data,28 important different expression gene（DEGs）were selected for RT-qPCR assays.All DEGs exhibited expression patterns consistent with the RNA-seq results.Among them,type I interferons（including IFN-α and IFN-β）showed the largest upregulation,followed by RSAD2,DDX58,MX1 and and other antiviral innate immune genes.In contrary,ID2 and another 5 DEGs were down-regulated or unchanged.These results indicated that type I interferons play a critical role in immune responses against SVA infection at early stage,while other immune-related genes also participate in this process.2.Construction and application of innate immune gene and cellular receptor KO cell linesThe binding of virus with cellular receptor is the first step of virus invasion into host cells,and it is essential for virus infection.ANTXR1 has recently been identified as the cellular receptor for entry of SVU-001 into human cells.However,whether porcine ANTXR1 is the cellular receptor of SVA invasion remains to be studied.In addition,the transcriptome analysis results showed that the expression levels of type I interferons,MX1 and RSAD2 were activated subsequent to SVA infection,suggesting these antiviral factors play a critical role in the host immune responses.The production of type I interferon requires a variety of innate immune proteins,including RIG-I,IRF3,IRF7,NF-κB,TRIF and MAVS.In view of this,ST-R ANTXR1 KO and ANTXR1-overexpressed ST-R cells were constructed by CRISPR/Cas9 gene editing technology.Then,the cells were infected with SVA reporter viruses,and the fluorescent protein and luciferase activity were measured to detect the infection of the cells.The results showed that porcine ANTXR1 is the cellular receptor for entry of SVA GD05/2017 into porcine cells.Secondly,we selected several important immune genes in type I interferon signaling pathway,such as RIG-I,IRF3,IRF7,TRIF and antiviral factors RSAD2,MX1,and then constructed a group of innate immune gene KO cell lines by CRISPR/Cas9 gene editing technology.Subsequently,these cells were infected with three porcine viruses（SVA,PSV,GETV）,and their growth kinetics curves were measured.Results showed titer of SVA was significantly increased in ST RIG-I KO（knock out）,ST IRF7 KO and ST IRF3/RF7 KO cell lines,suggesting that RIG-I and IRF7 play an important role in the antagonize of SVA growth and replication.Different from the results of transcriptome analysis,titer of SVA had no significant changes in ST RSAD2 KO,ST MX1 KO and other cell lines.Therefore,we speculated that the ability of gene in antagonizing SVA growth and proliferation at the cellular level depend not only on its fold-changes in transcriptional level,but also on its function and its position in the immune related signaling pathway.At the same time,the results of PSV,GETV growth kinetic curves measured in immune gene KO cell lines showed TRIF and IRF7 play an important role in inhibiting the growth and replication of PSV and GETV.The purpose of this research is to study the growth characteristic of the virus by knocking out single innate immune gene at the cellular level,so as to illustrate the role of single innate immune protein in the antagonism of virus growth and replication.Also,this study provides practical cell materials for porcine virus infection,and also provides theoretical support for the study of viral pathogenesis. |
