Study On The Mechanism Of Knocking Out Porcine CALR Gene To Inhibit The Replication Of Japanese Encephalitis Virus

Japanese encephalitis is a zoonotic disease caused by Japanese encephalitis virus(JEV)infection.It is also called Japanese encephalitis for short.Swine encephalitis is a national second-class animal disease.Pigs are important intermediate hosts.After infection,they can be widely spread in the form of mosquito-pig-mosquitoes on the farm,causing huge economic losses to the livestock industry and threatening human health.Using molecular breeding methods to find effective disease resistance genes in the host,breeding diseaseresistant pigs is one of the effective ways to eradicate this disease.At present,the key disease resistance genes of JEV in pigs are still to be explored.In the early stage of this laboratory,the whole genome of porcine CRISPR/Cas9 knockout library was used to conduct high-throughput screening of host factors involved in JEV replication,and it was found that sgRNAs targeting CALR gene in surviving cells showed rich expression and ranked in the top five.In this study,the CALR gene knockout cell line was constructed by using CRISPR/Cas9 lentiviral technology,and the knockout effect was detected by Sanger sequencing identification and Western blot technology.And conducted functional studies to clarify the role CALR plays in JEV proliferation,laying an important foundation for finding possible viral receptors or functional genes that affect viral replication.The main results are as follows:(1)The CRISPR/Cas9 lentiviral technology was used to construct the CALR gene knockout cell line.Among the positives enriched by flow sorting using TIDE analysis,the mutation efficiency of CALR gene for base deletion or insertion was 39.0%,and the rate of one base insertion was as high as 27.2%.Sanger sequencing found that the CALR gene knockout monoclonal cell type was a frameshift mutation caused by the insertion of a single base.Western blot detection found that the gene’s protein was not expressed at all,indicating that the CALR gene knockout cell line was successfully constructed.(2)JEV infection experiments found that knocking out CALR gene can significantly inhibit the ability of JEV-induced cytopathic effect.After using JEV infected cells with a low MOI of 0.3 and a high MOI of 1 for 72h,it was found that the survival rate of CALR knockout cells was significantly higher than that of wild-type cells.Using the plaque experiment,it was found that the JEV titer of CALR knockout cells was significantly decreased after infection at 12h and 24h,proving that knockout of CALR gene can significantly reduce JEV replication;After 12 h,18 h,24 h,36 h and 48 h of JEV infection with a virus titer of 1,it was found that the expression level of JEV-encoded C gene mRNA in CALR knockout cell lines was significantly lower than that in control cells by absolute quantitative PCR,proving that knockout of CALR gene significantly reduced the number of JEV copies.After 12h,18h and 24h of infection with JEV(MOI is 1),immunofluorescence experiments revealed that the fluorescent signal of the nonstructural protein NS3 encoded by JEV in the CALR knockout cell line was significantly reduced,which proved that knockout of the CALR gene significantly inhibited expression of NS3 protein encoded by JEV.The cell survival status from 0h to 60h after JEV(MOI is 1)infection was monitored by real-time dynamic marker-free cell analysis technology,and it was found that the CALR knockout cells could resist JEV-induced cell death.Through the calcium content detection,it was found that knocking out the CALR gene can significantly reduce the calcium influx activated by JEV.The EdU cell proliferation experiment test showed that knocking out CALR gene does not affect the normal cell proliferation.(3)Transcriptome sequencing and bioinformatics analysis found that JEV infected CALR gene knockout cells can stimulate differential expression of antiviral genes,including multiple antiviruses including ISG15,MX1,MX2,CCL5,RSAD2,IFIT2,and CCL2 Genes were significantly np-regulated.Compared with wild-type cells,CALR knockout cells have 603 genes that are up-regulated and 723 genes that are down-regulated.Among them,several genes related to interferon signaling pathway were up-regulated,iincluding IFI6,IFIT1,IFIT2,IRF1,ISG15,MX1,MX2,OAS2,OASL,RSAD2,DDX58,etc.,showed that CALR gene knockout cells interferon signaling pathway is activated expression compared with wild-type cells,.(4)Using cluster analysis and PPI protein interaction network analysis,it was found that a large number of interferon-related signaling pathways were activated in the differentially expressed genes of JEV-infected wild-type cells and JEV-infected CALR knockout cells,including Interferon alpha/beta signaling,Interferon signaling,Interferon gamma signaling,Antiviral mechanism by IFN-stimulated genes,ISG15 antiviral mechanism and other nine interferon-related signaling pathways.In the interferon α/βsignaling pathway,the number of genes up-regulated in JEV-infected wild-type cells was 18,and the number of genes up-regulated in JEV-infected CALR knockout cells was 50.The interferon signal-related differentially expressed genes MXI,OAS2,STAT1,RSAD2,MX2,DDX58,and IFIT1 in CALR knock-out cells infected with JEV were significantly up-regulated than wild-type cells.CALR knockout cells stimulated by JEV can significantly enhance gene expression of interferon signaling pathway,indicating that knocking out the CALR genes may inhibit JEV proliferation by enhancing the expression of interferon signaling pathway related genes.