Simultaneous High-resolution Analysis Of Human Foreskin Fibroblast Cells And Orf Virus Transcriptomes

The orf virus(ORFV)can cause an acute,contagious dermatitis not only to sheep and goat,but also human,cow,and other vertebrates.Because of the safety,repeat infections even in the presence of anti-body,and its strong immunomodulation,it has been utilized as a safe and efficient viral.vector against diverse infectious diseases and tumors.The effect to inhibit cancer cell is obvious in animal model.However,the nature of the genes triggered by the vector in human cells and the interaction between virus and human cells are poorly characterized.We found that orf virus can replicate and propagate in human foreskin fibroblast cells(HFF-1)and the viral protein can express normally in the cells.Therefore,we compared specific changes in the transcriptomic profiles in HFF-1 cells and orf virus following infection using RNA sequencing technology.HFF-1 cells were stimulated for 0,3 or 8 hours with live ORFV at a multiplicity of infection(MOI)of 5.Following the incubation,total RNA was extracted and its quantity and quality of the samples were eligibal(RIN>9.5,28S/18S>=1.6).The cDNA library were constructed and pooled to clustering.Single-end read sequencing was performed on an Illumina HiSeq2500 sequencer.After trimming the adapters,PCR primer sequences,ribosome RNA and low quality base reads,the clean reads were mapped to the hg19 human reference genome.Genes with a q-value(corrected p-value)lower than 0.05 and the fold change over 2 were categorized as differentially expressed.All the identified differentially expressed genes(DEGs)were mapped to the terms in GO database to analyze functional significance.The DEGs were also mapped to terms in the KEGG PATHWAY Database for pathway analysis.The principle component analysis(PCA),cluster and trend analyses were performed using the OmicShare tools.At 3 and 8 h.p.i,82 and 489 genes were up-regulated,respectively,while 180 and 85 genes were down-regulated,respectively,comparing to uninfected samples.From the time course,the expression levels of the majority of genes descended before rising.ORFV specifically upregulated and downregulated a variety of genes of HFF-1 cells,including genes involved in antiviral immune response,apoptosis,cell cycle and a series of signaling pathways,such as the IFN and p53-signaling pathways.Orf virus regulated immune genes for chemokines,chemokine receptors,cytokines,cytokine receptors,and molecules involved in antigen uptake and processing after infection.Specifically,expression of pro-apoptotic genes TP73,APAF1,E2F1,TNFSF10,PMAIP1 and BCL2L11 increased at 8 hours pos-tinfection.The p53 signaling pathway was activated to induce apoptosis at the same time.However,the cell cycle program was promoted after infection,which may be due to the immunomodulatory genes of ORFV.This presents the first description of transcription profile changes in HFF-1 cells after orf virus infection and provides an in-depth analysis of the interaction between the host and orf virus.The results of sequencing were verified by quantitative real-time PCR.For virus mRNA mapping,the remaining reads were mapped to the reference genome of ORFV OV-GO.In terms of overall ORFV transcription,59 genes were transcribed more than 10 read counts in three experiments at 3 hour after infection,and another 18 genes were transcribed at 8 h.p.i.Transcripts from 14 ORFs were never detected in this analysis.Moreover,the coverage of transcribed plus strand were higher than the minus ones at 3 or 8 h post infection.Obviously,the majority of transcripts concentrated on both terminal of the genome,a pattern similar to that occurring at early times after VACV or MOCV infection.The viral gene expression is increasing from the samples at 3 h.p.i.to the ones at 8 h post infection.The function of the genes expressed at 3 and 8 h.p.i.mainly related to viral DNA replication and host interaction.There is hardly any expression for the genes whoes function related to the virion association at 3 h.p.i.But it has small expression at 8 h.p.i.We predicted the early promoter motif according to the transcription profile of ORFV and it is a 15-nt consensus sequence(AAAA-TGAAAA—A).In summary,we constructed the model of human foreskin fibroblast cells for ORFV infection and presented the transcriptomic profiles of HFF-1 cells and orf virus after infection.This study provides an in-depth analysis of the differences in genome expression profiles of HFF-1 cells infected with orf virus.The function and signaling pathways for thoses differentially expressed genes of host cells were analyzed.At the same time,the viral gene function classification and its promoter category were analyzed.Moreover,early promoter motif of orf virus is predicted.The study will provide important information about the interaction between human cells and orf virus for further study.Furthermore,it will aid in the development of therapeutic oncolytic ORFV vaccine.