Analysis Of Immune Responses In HSV-1 Infected Mouse And Tree Shrew Based On Transcriptome Sequencing

Objectives:1.To establish the models of HSV-1 infection in mouse and tree shrew at different time points;2.Transcriptome sequencing analysis of trigeminal ganglion at different infection time points;3.To explore difference immune mechanisms between mouse and tree shrew following HSV-1 infection.Methods:60 adult tree shrews were randomly divided into control group(n=6)and experimental group(n=54).100 BALB/c mouse were randomly divided into control group(n=6)and experimental group(n=94).The experimental group of mouse and tree shrews were operated in both eyes and were subjected to a corneal scratch virus infection model.In the control group mouse and tree shrews,only scratches in both eyes were not inoculated with the virus.At the time of infection,3dpi,5dpi,7dpi,10dpi,14dpi,28dpi,58dpi,the corneal slit lamp was photographed and TG was obtained.Guarantee at least 6 animals at each time point.Photographs of the pathogenesis of mouse and shingles keratitis were taken with a slit lamp camera;A portion of the trigeminal ganglion was snap frozen in liquid nitrogen,extracted tissue RNA was sent to Wuhan Huada Corporation for RNA-seq sequencing,another part of the trigeminal ganglion was extracted RNA,and viral DNA was detected by PCR.Data analysis possible genes and signaling pathways that mediate the difference immune responses between mouse and tree shrew following HSV-1 infection.Results:PCR detection of viral DNA in trigeminal ganglia of mouse and tree shrew:Two pairs of primers detected HSV-1 positive products in both the mouse and tree shrew 7 dpi trigeminal ganglia,confirming the establishment of mouse and tree shrew HSV-1 infections models.Slit-lamp observation of pathological changes in mouse and tree shrew simplex keratitis:Dendritic and map-like ulcer lesions were observed in the cornea of HSV-1 infected mouse and tree shrew,and ocular surface inflammatory responses in the acute phase of mouse herpetic keratitis were Strongly,scars remained on the corneas of most mouse after 58dpi,and the inflammatory responses of the tree shrew herpetic keratitis were mild.There was no significant difference in inflammatory response between the two eyes.After 58dpi,most of the tree shrews returned to normal.Analysis of HSV-1 virus gene expression in trigeminal ganglia of mouse and tree shrew:The expression and expression of viral genes in mouse acute HSV-1 infection were higher than those in tree shrew.Among the genes that were expressed in the acute phase of HSV-1 infected mouse but not in the acute stage of tree shrew,there were 12 genes such as ICP4,UL3,UL4.LAT was only expressed in HSV-1 infected mouse during the latent period,in addition to LAT,the latent period of tree shrew has 10 viral genes such as ICP0,ICP22,UL6,UL34.Results of host gene expression analysis of HSV-1-infected mouse TGs involved in immune responses:Most genes are up-regulated.Among them,CXCL9,CXCL10,CXCL11,CD8A,CD8B and KLRD1 genes are involved in the regulation of CD8+T cell signaling pathway,activated CD8+T cells play a cytotoxic role by secreting granzyme B(GZMB),inhibiting HSV-1 infection and reactivation of latency.The viral genes IL18BP,IFNG and JCHAIN are encoded in the CD4+ T cell signaling pathway.Results of host gene expression analysis of HSV-1-infected tree shrews TGs involved in immune responses:Most genes were up-regulated and a small number were down-regulated.The genes involved in the regulation of CD8+ T cell signaling pathways include CXCL9,CXCL10,CXCL11,CD8B,and others.The HLA-DPA1,HLA-DQA2,HLA-DRA,and HLA-DQB1 expressed throughout the infection cycle participate in the coding of MHC class Ⅱ and exert immune regulation through the CD4+ T receptor signaling pathway.Conclusions:1)ICPO,which are highly expressed during HSV-1 infection in tree shrew,may be important genes involved in the regulation of latent virus reactivation;2)The high expression of ICP4 in mouse acute infection may be the main gene mediating the death of TG neurons;3)The immune response of tree shrew HSV-1 infection is the combined immune response of CD4+ T cells and CD8+ T cells;4)Tree shrew is an animal model of herpes simplex keratitis better than mouse.