Porcine deltacoronavirus(PDCoV)infection can cause watery diarrhoea and death in piglets,which is a serious health hazard for pigs.In order to study the changes of gene transcription level in host cell caused by PDCoV infection,total RNA was extracted from ST cells that were infected with PDCoV and negative control group not infected with the virus,and their cDNA libraries were constructed and then subjected to transcriptome sequencing(RNA-Seq)by using the Illumina HiSeq 6000 high-throughput sequencing platform.The results showed that the sequencing error rate of all samples was≤0.03％;Q20％and Q30％were above 98％and 94％,respectively;the GC content ranged from 51.09％to 55.15％;the correlation analysis results showed that the R of each group was＞0.9,which indicated that the sequencing data were reliable and could be used for the subsequent analysis of transcriptomics data.The ggplot2 software was used to analyse the correlation of the sequencing results within each group,and the accuracy of the sequencing data was determined by the correlation coefficient R＞0.9;DESeq2 software was used to screen for significantly differentially transcribed genes that met the requirement of log2(Fold change)＞1 and P＜0.05;ClusterProfiler R software was used to perform GO function and KEGG signaling pathway enrichment analysis on genes with significant differences in transcription;pheatmap software was used to analyse the clustering of transcriptionally significantly different genes that related to immune response in each group;nine transcriptionally significanfly different genes were randomly selected to validate the results of RNA-Seq by RT-qPCR.The screening results showed that compared with the cells in the negative control group,a total of 5719 transcriptionally significantly different genes were screened,including 3573 transcriptionally significantly up-regulated genes and 2146 transcriptionally significantly down-regulated genes.The results of GO function and KEGG signalling pathway enrichment showed that the transcriptionally significantly up-regulated genes were related to the immune response and defence response of ST cells,and involved in the signalling pathways of cytokine receptor interactions,natural immune response,and tumour necrosis factor,etc,while the transcriptionally significantly down-regulated genes were related to the redox and metabolism of cells,and were related to cellular metabolism signalling pathways,including amino acid metabolism,lipid metabolism,glyoxylate and dicarboxylic acid metabolism signalling pathways.The clustering analysis of transcriptionally significantly different genes associated with immune response showed that compared with the negative control group,the transcriptional levels of interleukin genes(IL1R1,1L-6,IL-10RB,IL-15 and IL-12A,etc.),antiviral genes(MX1,OAS)and interferon genes(INFAR1,IFN-ALPHAOMEGA)were significantly up-regulated in the PDCoV-infected group.These results indicated that the natural immune response of ST cells was activated after PDCoV infection,but the metabolic response of the cells was reduced,which was more conducive to weakening the replication of the virus in ST cells and exerting a certain antiviral effect.The RT-qPCR results of the nine transcriptionally significantly different genes were largely consistent with the RNA-seq results,indicating the high reliability of the transcriptome sequencing data.This study provides a theoretical basis for further analysis of the infection mechanism of PDCoV.
porcine deltacoronavirusRNA-seqtranscription significantly different genes
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