Study On The Expression Profile Of MRNA And MiRNA In Spleen Of SPF Chicken Infected With Avian Reticuloendotheliosis Virus

Avian reticuloendotheliosis virus(REV)is the causative agent of avian reticuloendotheliosis(RE).RE is a tumor and immunosuppressive disease that is seriously harmful to birds.RE,Marek's disease(MD)and avian leukemia(AL)are also known as three viral tumor diseases that endanger poultry.miRNAs play an important role in the interaction between the host and the pathogen.Most miRNA genes are located in fragile loci that are closely related to tumor formation,and play a key role in tumor-like genes or tumor suppressor genes in various aspects of tumor development.Studying and analyzing the endogenous miRNAs of REV-infected cells has important theoretical significance and application prospects for further revealing the molecular mechanism of REV pathogenesis and exploring cell anti-REV molecular targets.In view of this,this study used the SPF chicken artificial infection model to select spleen tissue as the research object,because the spleen is an important peripheral immune organ of chicken,and it is the place where mature T and B lymphocytes settle and immune response.High-throughput sequencing technology and qRT-PCR detection method were used to identify the interactions between REV and chicken at the level of miRNA regulation by identifying major differential mRNAs and miRNAs related to immune pathogenesis.The main research contents are as follows:1.This study used a REV-SNV strain to intraperitoneally inject 1 day old SPF chicks.Three immune organs(spleen,thymus,and bursa)were collected at 7,14,21,28,35,and 42 days after infection,combined with clinical symptoms,ocular pathological changes,and analyzed for immune organ index.The results showed that after REV infection of SPF chickens,compared with the control group,chicks in the infected group showed signs of apathy,feathers and sparseness,loss of appetite and weight loss.The necropsy and thymus atrophy and spleen enlargement were observed.At the same time,the bursal and thymus index decreased,and the spleen index increased.2.In this study,a specific probe and primer were designed based on the conserved sequence of the gag gene of REV,and the TaqMan probe qRT-PCR detection method was established.The standard curve of this method has a good linear relationship.The linear regression equation is Y=-3.4171X+41.92,and the correlation coefficient is R~2=0.9965.The specificity is good,only REV can be detected,no cross-reflection phenomenon;sensitivity is good,and the minimum detection template concentration is about It is 10 copies/?L,which is 1000 times that of ordinary PCR;the repeatability is good,and the intra-and inter-assay coefficient of variation is less than 2%.Using this method,the viral load and pre-viral load in the spleen,thymus and bursa of the chicks at 7,14,21,28,35 and 42 days after REV infection of SPF chickens were tested.The results showed that at each time point Both viral RNA and proviral DNA can be detected.The overall trend of viral load in the spleen,thymus and bursa of Fabricius was the same,and the lowest(pre)viral load was detected at 28 dpi.The highest(pre)viral load was detected at 14 dpi in both the spleen and the thymus except for the highest(pre)viral load detected at 21 dpi.3.Spleen tissue samples at 7,14 and 21 dpi were selected and transcriptome sequencing was performed on both samples using high-throughput sequencing.Through transcriptome data analysis,1507 differential mRNAs were obtained,which involved innate immune responses(TLR,MAD5,TRIM25),adaptive immune responses(LY6E,CD36,LAG3),apoptosis and autophagy(IRF1,PDCD1,WNT5A).And inflammation(CCL4,TNFRSF18,CDKN2)and so on.In addition,pattern recognition receptors,T cell receptors,JAK-STAT,TNF,and NF-kappa B signaling pathways play important roles during REV infection.Finally,33 immune-related mRNAs were verified by qRT-PCR.The expression levels were positively correlated with the sequencing data.The correlation coefficients at three time points were:R~2=0.961,0.9681 and0.9467,indicating that the transcriptome sequencing results were accurate and reliable.4.miRNA expression profiling results showed that 63 differential miRNAs(30 known miRNAs and 30 novel miRNAs)and 7373 candidate target genes were obtained at 7,14 and 21dpi.Functional enrichment analysis showed that miRNAs can play important biological functions at different time points and play important regulatory roles in various types of signaling pathways.In addition,miRNAs have slightly different regulatory effects in different growth stages of SPF chickens.Finally,15 miRNAs were verified by qRT-PCR,and their expression levels were positively correlated with the sequencing data.The correlation coefficients at three time points were:R~2=0.9384,0.9718 and 0.9788,indicating that the small RNA sequencing results were accurate and reliable.5.Integration analysis of miRNA and mRNA showed that 482 differential target genes were obtained at 7,14 and 21 dpi,and 886 known miRNA-mRNA interactions were predicted to interact with 580 novel miRNA-mRNA pairs.Functional enrichment analysis revealed that differentially expressed target genes were significantly enriched in immune-related signaling pathways,such as the immune system,cell growth and apoptosis,signaling molecules and interactions,and signal transduction categories.Fourteen pairs of immune-related miRNA-mRNA pairs were further verified by qRT-PCR.The results showed that miRNA can regulate the changes of inflammatory cytokines(CCR2,CCR4,CCR8,STAT1)and affect the expression of T,B lymphocyte regulatory factors(CTLA4,CASP10,RAPGEF4).At the same time,the expression of apoptotic factors(FOS,JUN,TNFSF6)and tumor regulatory factors(MAPK10,TNFRSF13C)also changed.These results broaden the understanding of the mechanisms of immune suppression and induction of tumorigenesis induced by REV infection,but the specific mechanism of action needs further study.