| Swine influenza virus(SIV)belongs to the genus Influenza virus of the Orthomyxoviridae family.Swine influenza(SI)caused by this virus is an acute respiratory infectious disease,which is highly contagious and often associated with Other infectious diseases are concurrent,increasing mortality.In the early stage of the laboratory,porcine kidney cells(PK-15)CRISPR/Cas9 whole genome knockout library(PK-15-Ge CKO)was used to screen the host genes related to swine influenza virus replication.Under this premise,this study looked for genes that have a greater impact on the life cycle of SIV among some candidate genes,and further studied the relationship between these genes and SIV replication.In this study,three pairs of sg RNA were designed for each candidate gene and constructed on the plasmid Lenti-sg RNA-EGFP.The PK-15 cell line stably expressing Cas9 protein was infected by lentiviral packaging,and a single gene knockdown cell line was constructed by CRISPR/Cas9 technology.Combined with TCID50 and cell survival,the effect of gene knockdown on SIV replication was detected,and two host factors,ALG5 and SLC35A1,were finally screened.In order to get closer to the state of natural infection,we constructed ALG5 and SLC35A1 gene knockout cell lines on neonatal pig tracheal epithelial cells(NPTr)that stably express Cas9 protein,and further studied the relationship between ALG5 and SLC35A1 and SIV replication.This study shows that ALG5 and SLC35A1 are important host factors that affect the replication process of SIV,laying a foundation for studying the replication and pathogenic mechanisms of SIV.The main contents are as follows:1 Effects of candidate gene knockdown on SIV replication in PK-15-Cas9 cellsAfter lentivirus infects PK-15-Cas9 cells,the efficiency of lentiviral infection was observed by green fluorescence,and the sg RNA knockout efficiency was verified by genome sequencing to determine the effective sg RNA of the candidate gene.Finally,single gene knockdown cell lines of each gene were obtained.Wild-type cells and single-gene knockdown cell lines were infected with SIV HB strains.The supernatant was collected 36 hours post infection to determine the virus titer and calculate TCID50.At the same time,cell survival was observed at 72 hours post infection.The results showed that among the candidate genes,except for A28,other genes knocked down PK-15-Cas9 cells to inhibit the replication of SIV to varying degrees.At 72 hours post infection,only the cell lines with knockdown of ALG5 and SLC35A1 had cell survival,and at the same time,the knockdown of these two genes had a significant inhibitory effect on viral replication.Therefore,ALG5 and SLC35A1 were selected to study the mechanism of SIV replication.2 Effect of ALG5 on SIV replicationAfter obtaining the ALG5 knockdown PK-15-Cas9 cell line,the monoclonal cell line knocked out of the ALG5 gene was screened using the limiting dilution method,cell colonies grown from single cells were selected,and the green fluorescence coverage was observed.It was verified that ALG5 was completely knocked out on PK-15-Cas9 cells by Western blot.The ALG5 knockout cell line on PK-15-Cas9 cells was obtained.At the same time,the ALG5 gene was cloned into the p3×Flag-cmv14 vector.ALG5 knockdown on PK-15-Cas9 cells significantly inhibited SIV replication,and overexpression promoted SIV replication.In order to get closer to the state of natural infection,an ALG5 gene knockout cell line was constructed on NPTr-Cas9 cells.The sg RNA was constructed on the Lenti Guide-puro vector.After lentivirus infection of the cells,positive cells were selected with Puromycin and monoclonal cell lines were picked.Finally,the ALG5 knockout cell line on NPTr-Cas9 cells was obtained.It was verified that ALG5 knockout on NPTr-Cas9 cells significantly inhibited SIV replication,and overexpression promoted SIV replication combined with TCID50 and Western blot.In order to determine the effect of gene knockout on cell viability,it was verified that there was no significant difference in cell activity between the knockout cells and wild type cells by CCK-8 experiments.After SIV infection of wild-type NPTr-Cas9,the expression of ALG5 protein was up-regulated as the virus proliferated.In addition,it was confirmed that ALG5 knockout can also inhibit the replication of PR8,H9N2,and F26 influenza strains,and has no strain specificity,and the inhibition of H9N2 strain replication is most obvious.3 Effect of SLC35A1 on SIV replicationBy the same method,we constructed a SLC35A1 knockout monoclonal cell line on NPTr-Cas9.SLC35A1 knockout inhibited SIV replication more significantly than ALG5.At the same time,72 hours after the SIV-infected gene knockout cell line was confirmed by cell count,the survival rate of the knockout cell line was greater than 80%.In addition,SLC35A1 knockout can inhibit the replication of PR8,H9N2 and F26 strains,no strain specificity.In order to verify that SLC35A1 affects the stage of SIV replication,we confirmed by viral adsorption experiments that after SLC35A1 knockout,the adsorption of SIV on the cell surface was significantly hindered,indicating that SLC35A1 knocked out on NPTr-Cas9 inhibited SIV by affecting the adsorption of viral particles of proliferation. |
