Host Innate Immune Response Induced By Avian Reticuloendotheliosis Virus And Transcriptomics Study Of Virus Infected Bursa

Reticuloendotheliosis(RE)is an important tumorigenic and immunosuppressive disease caused by the avian retrovirus,avian reticuloendotheliosis virus(REV).Immunosuppression caused by REV infection in the clinic has caused significant losses to the poultry industry.In recent years,most of the REV infections have been found co-infection with other immunosuppressive viruses such as Marek’s disease virus and avian leukosis virus subgroup J,which increasing the difficulty of prevention and control this disease.Therefore,the control of the disease has an important impact on the healthy development of the poultry industry and the improvement of the quality of biological products.To date,the mechanism of host immune response caused by virus-infected hosts and the pathogenic mechanism of the virus are still unclear.In this study,REV HA1101 strain was used to infect chicken peripheral blood lymphocytes and artificially inoculated SPF chickens.The real-time quantitative PCR,transcriptomics and bioinformatics methods were used to explore the possible role of viral infection.Cytokines and related signaling pathways provide important clues for further understanding of the immunosuppression and pathogenesis caused by hosts infected by REV.I.Effect on innate immune-related genes in peripheral blood lymphocytes infected with REVIn order to clarify the effect of REV infection on the innate immune response in chicken peripheral blood lymphocytes,real-time PCR was used to detect the expression level of innate immune-related genes in REV-infected SPF chicken peripheral blood lymphocytes,which including Toll-like receptors(TLR3,TLR4,TLR7,TLR15 and TLR21),signaling pathway-associated genes(IRF7,MDA5,MyD88,MDA5,NF-κB,IRAK4,TRAF6,TRIF and TAK1),inflammatory cytokines(IL-1p,IL-6,IL-8,IL-10 and IL-18),and antiviral-associated cytokines(IFN-α,1FN-β,IFN-y,iNOS,MHC1,and OASL)at 12h,24h,48h,and 72h post infection.The results showed that the mRNA transcription levels of TLR3,TLR4 and TLR7 were significantly decreased within 72 h after REV infection in SPF chicken peripheral blood lymphocytes.But the TLR15 and TLR21 were not effected.The expression levels of IRF7,IRAK4,NF-κB,TRAF6,TRIF,TAK1 and MHC I were consistent with the trend of TLR3;while MyD88,IL-10,IL-18,MDA5,IFN-β,iNOS and OASL were up-regulated at 72h infection;IL-1β,I-L6,and IL8 increased significantly,getting the peak at 72h;IFN-a was slightly inhibited at 48h after infection,and IFN-y was significantly increased at 12h after infection.These results indicate that REV can induce the inhibition of innate immune receptors TLR3,TLR4 and TLR7,and the inhibition of Toll-like receptor signaling pathway-related cytokines in different stages of infection;inflammatory cytokines and anti-virus related cytokines are upregulated in different degrees.Taken together,these results provide a basis for further elucidation of the immunosuppressive mechanism of REV2.Expression of immune-related molecules in the major immune organs of virus infected SPF chickensIn order to understand the early innate immune response of REV-infected hosts,this study conducted a REV artificial inoculation of SPF chicken experiments.The chickens were divided into the infection group and the control group.The major immune organs such as peripheral blood,thymus,spleen,bursa and cecal tonsil were collected at the 1st,3rd,5th,7th,14th,21st,28th and 35th day after infection.Detection of viral replication,innate immune receptors(TLR3 and TLR7),inflammatory factors(IL-1β and IL-6),and interferons(IFN-α,IFN-β,and IFN-γ)was carried out by real-time PCR.After REV infection,the trend of virus replication appeared to rise first and then fall repeatedly.In the thymus,the virus content reached the highest at the 14th day;in the peripheral blood lymphocytes,spleen,bursa and cecal tonsils,the virus content reached the highest at the 7th day;at the 28th day,the virus content began to increase again Innate immune receptors(TLR3 and TLR7)and type II interferon(IFN-y)also increase in gene expression when the virus replicates in large amounts;gene expression of inflammatory factors(IL-6 and IL-1β)changes in peripheral blood lymphocytes and spleen.The trend in the bursa is consistent with viral replication;the expression of type I interferon(IFN-α and IFN-β)is up-regulated at day 28th.These results indicate the early immune response of REV infected host immune organs,and provide a basis for the further study of the mechanism of immune suppression caused by REV infection3.Transcriptional profiling of gene expression in chicken bursa of fabricius infected with reticuloendotheliosis virusREV-infected chickens can cause immunosuppression,co-infections and secondary infections,resulting in higher mortality.However,little is known about the interaction between viruses and hosts.This study aimed to reflect the interaction between the virus and the host through REV-infected bursal tissue gene transcriptomics.According to the results of the second part of this study and related references,the virus content of REV infected SPF chickens reached the highest value in the bursa,so the bursa was selected as the research object of transcriptomics analysis.Bioinformatics analysis showed that there were a total of 15296 differentially expressed genes after REV infection.These differentially expressed genes involved in cellular processes,binding,biological regulation,metabolic processing,stimuli,and immune system.Through KEGG signaling pathway analysis,differentially expressed genes are mainly involved in immune,tumor and metabolic related signaling pathways,such as Toll-like receptor signaling pathway,cytokine-cytokine receptor interaction,JAK-STAT signaling pathway,NOD-like receptor signaling pathways and metabolic signaling pathways.The REV HA1101 strain infected chicken bursa transcriptome provides a theoretical basis for better explanation of the molecular pathogenic mechanism of REV infection.4.Effects of ligands of innate immune receptors on REV replicationToll-like receptors are based on the identification of pathogen-associated molecular patterns,sensing the presence of pathogenic microorganisms,activating intracellular signaling,then leading to activation of downstream signaling pathways,by producing inflammatory cytokines,I interferon and other inflammatory mediators to induce immune response.Therefore,Toll-like receptors play an important role in inducing host responses to pathogens.Ligand stimulation of Toll-like receptors can elicit a corresponding immune response,preventing infection by bacterial and viral pathogens.Based on the results of previous studies,ligands of TLR3 and TLR7 were used to treat cells to study the ligands effect on REV replication in CEF cells.The results showed that the ligands of TLR3 and TLR7 stimulated CEF cells to inhibit the replication of REV in cells,and the effect of TLR3 ligand was more obvious.The inhibition of virus replication might related to the high expression level of antiviral cytokines in cells.