| Pseudorabies virus（PRV）can cause infectious pseudorabies（PR）in pigs,causing fever in adult pigs,abortion and stillbirths of sows,and death of piglets.At present,PR has not been completely eradicated in China,which has brought economic losses to China’s pig industry.Previous studies on PRV-infected cell transcriptomes have focused more on differentially expressed genes after 12h of infection,while few have studied cell transcriptome changes during early viral infection.Genes related to adhesion and invasion are also unknown.In this experiment,total RNA of PRV-infected PK-15 cells in 0hpi,2hpi,6hpi,12hpi,24hpi,36hpi,48hpi were transcribed.The transcription data were analyzed by bioinformatics,and the differentially expressed genes at 0hpi,2hpi and 6hpi were screened.Subsequent functional annotation of these differentially expressed genes was conducted,including GO functional analysis,KEGG pathway analysis,PPI network analysis,to help us understand the biological events occurring in the early stages of viral infection,looking for possible viral receptor or a functional gene that affects viral adhesion and invasion.In addition,this study also used the CRISPR/Cas9 technology to knock out CYP1A1 which was significantly differentially expressed in RNA-Seq result and the receptors NECTIN-1 and NECTIN-2 of the PRV reported in PK-15 and 3D4/21cell.The results show that:（1）The time and dose effect of PRV infection on PK-15 cells,that is,the CPE of cells appeared more obvious with the increase of MOI and infection time.When PK-15was infected with PRV-Eαstrain with MOI of 10-6,death of a large number of cell was still observed in 84hpi.PRV-Eαstrain was a strong lethal virus for PK-15 cells.（2）Transcriptome sequencing was conducted of total RNA of PK-15 cells infected with PRV-Eαstrain in 0/2/6/12/24/36/48hpi.Compared the RNA-seq data with porcine and PRV genomes respectively,PK-15 cells and PRV genomes transcriptional levels showed a significant negative correlation with prolonged infection time.The PRV gene began to express after 6 hours of infection,while the gene expression level of PK-15 cells began to decrease.Afert 6hpi of PRV-GFP virus strain in PK-15 cells,GFP protein began to be expressed in the cells.The above results indicate that the virus enters the host cell within 2 h-6 h of infection and begins the replication cycle,consistent with the data of RNA-seq.（3）There were 830 differentially expressed genes（DEGs）after 2 hours of PRV infection on the condition of|log2FC|≥2,p≤0.001.After 6h of PRV infection,the DEGs were 848.There were 381 DEGs within 2hpi to 6hpi of PRV infection.8 genes were differentially expressed at 0-2 hpi,0-6 hpi and 2-6 hpi.Both at 0-2 hpi and 0-6hpi,410 genes are differentially expressed.Both at 0-6hpi and 2-6hpi,137 genes were differentially expressed.Both at 0-2hpi and 2-6 hpi,65 genes were differentially expressed.（4）Functional annotation of DEGs at three time points of PRV-infected PK-15 cells:GO analysis enriched with immune response,negative transcriptional regulation of RNA polymerase II promoter,inflammatory response,lipopolysaccharide response,endocytosis and pH adjustment of vesicles.KEGG was enriched into pathways such as the p53 signaling pathway,cytokine-cytokine receptor interactions,and synaptic vesicle cycling.Protein function classifyshowed association with hydrolase,transport and receptor functions.PPI network analysis enriches the interaction between multiple differential genes.（5）sgRNAs designed to target CYP1A1,NECTIN-1,and NECTIN-2 have been verified to be active.The NECTIN-1 was successfully knocked out in PK-15 cells by lentiviral infection.And NECTIN-2 was successfully knocked out in 3D4/21 cells.In this study,the transcriptome sequencing of PRV-infected PK-15 cells at 0,2,6,12,24,36,48 hpi revealed changes in the transcriptome levels of virus and host cells after PRV infection in host cells and key genes and important pathways involved in viral adsorption and invasion.At the same time,the related genes CYP1A1,NECTIN-1 and NECTIN-2 of PRV adhesion and invasion were knocked out by CRISPR/Cas9 technology,which provided a reference for the study of the pathogenesis of PRV and provided some theories for breeding of pigs. |
