Analysis Of RNA-Seq And TLRs Distribution In Small Intestinal Mucosa In Piglets Infected With PEDV

Porcine Epidemic Diarrhea(PED)is an acute infectious disease caused by Porcine Epidemic Diarrhea Virus(PEDV)that causes atrophy of the intestinal villi and impairs intestinal function,causing a serious block on the steady development of the pig industry.Since 2010,the variant PEDV strains have emerged and become prevalent,making the clinical vaccine efficacy of existing vaccines unsatisfactory.In order to be able to effectively control the occurrence of PED,it is necessary to further explore the molecular pathogenic mechanism of PEDV.With the rapid development of high-throughput sequencing technology,the data after sequencing discovered more functional genes in the study of PEDV infection in the piglet's intestinal tract,not only important for the study of PEDV infection mechanism,but also for the intestinal immune response of piglets.Functional genomics studies provide new ideas and methods.(1)6 healthy piglets of 3 days old were inoculated orally with 10 ml of lab-preserved PEDV virus fluid as a test group,and 4 piglets with 10 ml of PBS buffer solutions as a control group.After 6 h,the small intestinal mucosa tissues samples of two groups were collected for transcriptional analysis.(2)Pathogenic mechanism and host immune response after PEDV infection.High throughput sequencing technology was used to analyze the differentially expressed genes in small intestinal mucosa after PEDV infection,to understnd the interaction between PEDV and the host.By establishing a PEDV infected piglet model,a total of 10 sets of small intestine mucosal tissues from two groups of piglets were collected and whole gene transcriptome sequencing was performed using the BGISEQ-500 sequencing platform.The results showed that a single sample yielded an average of 6.55 Gb of data;after reassembly of the transcript,more than 65,552,000 clean reads and 74,222,000 more raw reads were obtained,and the sample's comparative genome average comparison rate was 93.55%,and the average alignment ratio of the comparison set was 75.76%,the total number of genes detected was 22,605,of which 22,248 were known genes and 371 new genes were detected;20,894 new transcripts were detected,of which 17,864 belonged to new genes known to encode proteins.Among the alternative splicing isoforms,371 are transcripts of novel protein-coding genes,and the remaining 2659 are long-chain non-coding RNAs.Of these genes,3168 genes were up-regulated and 3876 genes were down-regulated.GO functional enrichment analysis of differentially expressed genes in transcriptome sequencing.Differentially expressed genes were analyzed from three functional categories: molecular function,cellular components,and biological processes.The results showed that the significantly enriched GO items were mostly associated with intracellular and intracellular signal transduction,enzyme activation,and biological metabolism.By analyzing the KEGG pathway of differentially expressed genes,it were significantly enriched in these four pathways,metabolic pathways,PI3K-Akt signaling pathways,cancer signaling pathways,and adhesion plaques.We found that these differentially expressed genes are mainly involved in the body's metabolic processes,signal transduction and adhesion,and further speculated that these differentially expressed genes may be involved in PEDV infection in the small intestine mucosa to regulate the immune response and apoptosis.Verify the reliability of transcriptome sequencing results.In this study,GAPDH was selected as the reference gene,and the intestinal mucosa of infected piglets with PEDV and healthy piglets were used as samples.28 genes with differentially expressed genes related to biological significance were selected from transcriptome sequencing data and primers were designed and qRT-PCR was performed.The results was basically the same to that of transcriptome sequencing.(3)Toxic status of organs in PEDV-infected piglets.PEDV RT-PCR was performed by collecting hearts,lungs,spleen,and salivary glands of experimental piglets.The results showed that the intestinal,mesenteric lymph nodes and heart of piglets were PEDV positive,and all other organs were negative.The detection rate of intestinal and mesenteric lymph nodes was 100%,and the heart was 16.7%.It can be preliminarily demonstrated that PEDV can be distributed in other organs than intestinal tissue after infection.(4)Expression of TLR4 and TLR6 receptors in PEDV-infected piglets.The heart,liver,spleen,lung,kidney,and lymph node tissues of two groups of piglets were collected,primers were designed,and ?-Actin was used as an internal reference gene.Real-time fluorescence quantitative PCR was used for detection.The results showed that the expression levels of TLR4 and TLR6 were generally in a decreasing trend,but the contents of TLR4 and TLR6 were higher in spleen and kidney,and the difference was significant.It can be suggested that there are differences in the immune responses of TLR4 and TLR6 on different organs after PEDV infection.Taken together,this study investigated transcriptome sequencing of small intestine mucosal tissues of piglets infected with PEDV,obtained differential genes and annotated the genes involved in viral infection,and studied distribution of PEDV infection and Toll-like receptor immune responses..It provides data support for the initial exploration of the pathogenic mechanism and immune response of PEDV,and provides reference for further study of PED clinical diagnosis and prevention and control measures.