Transmission Route Of Infectious Bursal Disease Virus In Chicken And Virus Influenced MicroRNAs Effect On Host STING/TBK1 Pathway

Infectious bursal disease（IBD）is an immunosuppressive disease caused by an infectious bursal disease virus（IBDV）.IBDV mainly infects Bursa of Fabricius（BF）and causes massive destruction of B cells in the chicken,which leads to enormous economic loss to the poultry industry worldwide.The nasal cavity is the main entrance for pathogens,but how IBDV breaking through the nasal barrier and transfer to the bursa is still unclear.Although the nasal mucosa of chickens is intact,consisting of a multi-layer defense barrier,including the epithelial cilia of the vestibule of the nasal cavity,the pseudostratified epithelium,and lymphatic tissue of the posterior nasal cavity,and the nasal-related lymphoid tissue existing near the posterior nostril cleft.IBDV still could effectively break through the nasal mucosal barrier and spread in chickens.Thus,this research focused on the mechanism of how IBDV spreads from nasal to bursa,which might lay a solid theoretical foundation for the development of poultry vaccines.Our study mainly includes three parts.In first part,in-vivo experiments were performed to unveil the transmission mechanism of airborne IBDV from nasal to bursa.While the other two parts were based on in-vitro experiments to explore the importance of STING related DNA andRNA sensing pathways.For this purpose,IBDV and its induced miRNA’s were used as a target to studyRNA sensing pathway,while on the other hand CpG and its induced miRNA’s were used to elaborate DNA sensing pathway.Briefly,in the first part,we studied the transmission route of IBDV in poultry and found large number of viral particles accumulated in the posterior part of nasal,of which the lymphocytes and olfactory cells are the main target cells for virus infection.After passing through the barrier of the nasal cavity,IBDV is transferred to immune organs such as the bursa and spleen through peripheral blood circulation and severely affects the Ig M⁺B lymphocytes in these peripheral immune organs.In the second part,our studies confirmed that IBDV might promoteRNA sensing pathway and blockRNA interference for its replication in DF1 cells.Finally,in the third part,we found that the microRNA induced by CpG can effectively promote the cytosolic cGAS/STING DNA sensing pathway.In summary,this study,on the one hand,clarified the transmission mechanism of IBDV in chicken.On the other hand,it illustrated the underline mechanism of IBDV’s replication.The detailed research for each part is listed as follows:Experiment-1 The transmission route of Avian infectious bursal disease virus and its effect on B lymphocytesNasal cavity is considered to be the main gateway for pathogens invasion.But the detailed transmission route of IBDV from nasal to bursa and its influence on the immune cells are still unclear.In this study,IBDV classical virulent strain BC6/85 was inoculated intranasally into two-week-old and three-weeks-old chicks.To begin with,we found that the nasal mucosa of chicken was divided into interior and posterior regions according to their structure and function.The interior part is mainly composed of flat epithelium,while the posterior region is rich in pseudostratified epithelium and lymphoid tissue.Moreover,immunohistochemistry（IHC）results found that IBDV mostly entered and infected the posterior region of the chicken’s nasal and transmitted into the blood at the early stage of infection.By the way,we also found that the lymphocytes and olfactory cells of the posterior region might be the main entrance for virus infection.Furthermore,we found viral traces in blood as early as 1hpi,but the Ig M⁺B-cell destruction occurs after 6hpi.Finally,we found that IBDV can infect immune organs such as the bursa of Fabricius and spleen through blood circulation and severely destroy Ig M⁺B lymphocytes of these immune organs.The IHC and immunofluorescent assay（IFA）results showed that IBDV severely damages the bursal follicular with the passage of time,independent of the age difference of the host chicken.Together,our study comprehended the airborne IBDV transmission via intranasal route into the BF and explained its distribution in PBMC and spleen,together with the B-cell generation of antibodies repertoires after immunosuppressive disorder in an age-dependent manner.Experiment-2 Effect of infectious bursal disease virus and poly I:C onRNA receptors andRNA interference in DF1 cellsAfter billion years of evolution,the antiviral strategy for host has changed from short palindrome repeats with regular intervals in prokaryotic toRNA interference（RNAi）in plants and invertebrates,and evolute to interferon system on vertebrates.However,when vertebrates evolved the antiviral defense system fromRNAi to interferon,someRNAi systems（Drosha and Dicer）remains unchanged.Drosha and Dicer are the key enzymes in microRNA production,and it works the same in the antivirusRNAi mechanism.Recently,several avian double-strandedRNA viruses are reported to causeRNAi antiviral in mammals cells and insect cells.However,whether chicken cells can also useRNAi to resist the virus remains to be explored.The present study was conducted to evaluate the efficiency ofRNAi in response to double-strandedRNA（dsRNA）virus（IBDV）replication in-vitro.First of all,we found that the moderate-virulent vaccine strain of IBDV（B87）can induce apoptosis on chicken fibroblast（DF1）cells in-vitro,and the virus replication is dependent on the time course of infection.The IBDV strain B87 was sensed by TLR3 perform downstream activation of MDA5/LGP2 and STING/IRF7 pathways which further promote IFNβproduction.Secondly,IBDV strain B87 downregulates dicer and influences the miRNA biogenesis pathway on DF-1.To investigate whether chicken miRNAs are mediate with host or IBDV,we selected miRNAs（miR-1635,miR-1644 and miR-6675-3p）having the ability to bind IBDV-VP2 protein.Thirdly,miR-1635 binds to IBDV-VP2 and block the viral replication,whereas miR-6675-3p promotes the replication of the virus on DF1 cells.The miR-1635 promotes TBK1 regulation while miR-6675-3p activated IFN-βproduction via STING pathway after IBDV infection on DF1 cells.Finally,these miRNAs can also regulate the Wnt/Ca²⁺pathway on DF1 cells.Interestingly,in addition to the ability of miR-1635 and miR-6675-3p to bind IBDV-VP2,they are also involved in the downregulation of dicer.Thus,these results provide sufficient evidence to illustrate the mechanism of IBDV inRNAi regulation in DF-1cells.Experiment-3 miR29a and miR378b influence CpG-stimulated dendritic cells and regulate cGAS/STING pathwayThe Cytosine-phosphate-guanosine（CpG）motif is well-known adjuvant used in combination with vaccines against various diseases.CpG can be recognized intracellularly by dendritic cells（DCs）.DCs are the potent antigen-presenting cell and play a crucial role in pathogen recognition and regulation of innate immune responses.The cyclic guanosine monophosphate-adenosine monophosphate（GMP–AMP）synthase（cGAS）and the stimulator of interferon genes（STING）signal pathway is the cytosolic DNA sensing pathway involved in foreign or self-DNA recognition and innate immune response activation.So,whether and how CpG activates the cGAS/STING signal pathway still needs further clarification.MicroRNAs（miRNAs）are small non-codingRNAs of 22 nucleotides in size.Besides their ability to bind messengerRNAs（mRNAs）and block their activity,miRNAs can strongly influence the antigen-presenting ability of DCs.In this study,we examine the action of miRNAs on CpG-stimulated and na（?）ve DCs and their effect on cGAS/STING signal pathway.Firstly,we selected miRNAs（miR-29a and miR-378b）based on the expression of CpG-stimulated bone marrow-derived dendritic cells（BMDCs）.Secondly,we investigated the immune response of DCs after CpG-stimulation and transfection of miR-29a and miR-378b.The miR-29a and miR-378b increased the expression of both the immunoregulatory DCs surface markers（CD86 and CD40）,as well as the immunosuppressive molecules CD273.Thirdly,cytokine detection revealed that both miR-29a and miR-378b enhanced interferon-β（IFN-β）expression while suppressed tumor necrosis factor-α（TNF-α）production.Finally,dual luciferase assay confirmed that miR-378b could bind TANK-binding kinase binding protein 1（TBKBP1）and activate the cytosolic cGAS/STING signaling pathway.In contrast,miR-29a targeted interferon regulatory factor 7（IRF7）and promoted the expression of STING.To our knowledge,this study is the first to provide insight into the molecular mechanism of miRNAs induced by CpG,activating cytosolic cGAS/STING pathway and regulate the innate immune response.