The Study On Mechanism Of Bovine And Equine Infected By Foot-and-Mouth Disease Virus Based On Transcriptome Of Nasopharynx

Foot-and-mouth disease(FMD)is a highly infectious disease which is caused by foot-and-mouth disease virus(FMDV).The infected livestock suffer from serious production losses because of the outbreak of FMD,which has a devastating effect on the economy.It has become the main obstacle of the international trade in animals and animal products.At present,there are more than 100 countries around the world are affected by the disease,the disease has been listed as a major multinational global elimination plan of animal epidemics and the key inspection object of safe convention of biological weapons.FMDV is with host tropism,including all cloven-hoofed animals,wild ruminants and pig.They can been infected and carry the virus for a long time,however,perissodactyla are not infected with FMD,the horse is highly resistant animals.Even if they were contaminated by the virus,there is not the symptoms of the disease and serum type characteristics.In this study,a comparative study was conducted before and after FMDV infection on the bovine susceptible parts,the nasopharynx and the horse's nasopharynx by the whole transcriptome sequencing technique,in order to clarify the genetic basis of susceptibility of livestock.The present study can provide new ideas about the effective immune control methods of foot-and-mouth disease and using advanced gene manipulation techniques to reconstruct the specific genes in livestock,so as to improve the disease resistance of livestock.The article's results are as follows:1.The receptor of the FMDV were detected in the nasopharynx of cattle and horse,including ?v?1,?v?3,?v?6,?v?8,by immunohistochemical method.2.RNA-seq was performed on the bovine nasopharynx before(n=3)and after(n=3)the infection.The total data of the six samples(clean data)were above 12 G,between the infection group and the control group,differential expression mRNAs were a total of 5,967 and 143 differential expression lncRNAs.The GO results showed that genes involved in biological processes,cell component and molecular functions were 4,705,4,788,5,173 respectively.KEGG results showed that pathways were related to physiological and biochemical reactions such as the transport of amino acid,cell metabolism,immune response,amino acid metabolism,protein transport,lysosomes.3.Between the infection group(n=3)and the control group(n=3)of equine nasopharynx,differential expression mRNAs were a total of 8,658 and 576 differential expression lncRNAs.The GO results showed that genes involved in biological processes,cell component and molecular functions were 1,369,1,440,1,598 respectively.KEGG results showed that pathways were related to adhesion connections,immune responses,immune diseases and virus-host reactions,lysosomes.4.RNA-seq was used for small RNA sequence in the bovine nasopharyngeal tissue before and after infection.We identified known miRNA(mature):611,Novel mi RNA(mature):187.The number of differential expression mi RNA between the infection group and the control group were 56(total),30(up),26(down)respectively.By the cluster analysis,miRNAs could be divided into 41 cluster and 231 families,and by prediction software of animal miRNA target gene,we got the corresponding relationship between the miRNAs and target genes.The biological function of target genes were mainly involved in proteins transport,cell metabolism,extracellular vesicle,bacterial invasion of epithelial cells,phagocytosis.We got the association analysis results of miRNA,lncRNA and mRNA.We drawn the regulatory networks of key gene of IRF8,CYCS and CASP3.5.RNA-seq was used for small RNA sequence in the equine nasopharyngeal tissue before and after infection.We identified known miRNA(mature):414,Novel miRNA(mature):175.The number of differential expression mi RNA between the infection group and the control group were 180(total),93(up),87(down)respectively.By the cluster analysis,miRNAs could be divided into 41 cluster and 207 families,and by prediction software of animal miRNA target gene,we got the corresponding relationship between the miRNAs and target genes.The biological function of target genes were mainly involved in the bacterial invasion of epithelial cells,antigen processing and presentation,the regulation of the cytoskeleton of actin cytokines,and the proteolysis of ubiquitin.We got the association analysis results of miRNA,lncRNA and mRNA.We drawn the regulatory networks of key gene of TLR3?TLR4?TLR2?TRAF3?ATG9 and GADD45 A.6.The DEGs were analyzed by bioinformatics.When the bovine nasopharynx is in response to the foot-and-mouth disease,the signal is transmitted into the cell by the integrin receptor,then focal adhesion signal,cell skeletal signal and MAPK signaling pathway activate NF-?B,resulting in the release of inflammatory mediators and the immune response.Cytoskeletal pathways increase cell permeability,leading to intracellular protein leakage.In the case of virus removal,cattle mainly rely on mitochondrial pathway apoptosis,Capase3 can activates Gasdermin E(DFNA5),the member of the Gasdermin family,apoptosis converts into cell pyrolysis which cause cell lysis.At the same time,the virus can block the transcriptional and translation system of the host,so the secretion of antiviral substances reduced,and the virus can evade the immune response and then replicate and spread.All these factors contribute to the bovine susceptibility and clinical symptoms of the disease.Although there are also integrins in the equine nasopharynx,the mechanism of the removal of the virus and fighting against the virus is mainly as follows: The TRAM-TRIF-IRF3 pathway to participate in the antiviral reaction is activated by the pattern recognition receptor TLR4 and TLR3.When the nasopharyngeal tissue is stimulated by the virus,beta-defense(BD-1)prevents the virus from entering the horse.After TLR binding ligand,its protein conformation changes and the autophagy of cells is activated.Autophagy can be used as a defense mechanism to remove pathogens.The protein synthesized by the virus in cells may be degraded and discharged by autophagylysosomal pathway.The cells of the horse did not occured the inflammatory death and the virus did not block the transcriptional and translation initiation system of the horse.TLR2 and TLR3 all can activate the secretion of TSLP,in most cases,TSLP can combine with receptors TSLPR(CRLF2)on the surface of the dendritic cells,and induce Th2 mediated inflammatory reaction.But equine nasopharynx after exposure to the virus did not express CRLF2.Because of the TSLPR silence,the inflammatory response has not been induced.So we speculate that the combination of all these factors lead to the horse being a domesticated animal with foot-and-mouth disease resistance.Therefore,in the level of transcriptome,because of the above differential expression gene and their non-coding RNA regulatory factors,result in bovine and equine have different coping mechanisms in contact with the FMDV,different susceptibility mechanisms and disease resistance.In addition,in order to verify the reliability of sequencing data,some key genes were verified by q-PCR,and the results were consistent with the sequencing results.