Host Innate Immune Response Induced By Duck Tembusu Virus And The Interaction Between The Virus And RLR-mediated Signaling Pathway

Duck Tembusu virus(DTMUV)is an enveloped,positive-sense single-stranded RNA virus,belonging to the genus Flavivirus.The virus was first isolated from the sick ducks of Fujian and Zhejiang in April 2010,characterized by decline in egg production of laying ducks,growth retardation and neurological symptoms of commercial ducks.This disease spreads quickly and has caused huge economic loss of duck industry in China.The host innate immune response plays an important role in the early stage of pathogenic microbial infection.Pattern recognition receptors(PRRs)are the key components of the innate immune system,which mainly recognize the conserved element of pathogenic microorganisms,such as viral nucleic acid,bacterial lipopolysaccharide etc.RIG-I like receptors(RLRs)are a class of cytoplasmic PRRs that are mainly distributed in nonimmunized cells,this class consists of three receptors,including the retinoic acid induced gene-I(RIG-I),melanoma differentiation associated gene 5(MDA5)and laboratory of genetics and physiology-2(LGP2),which can recognize the RNA virus.Upon recognition,RLRs will initiate a downstream signal cascade through mitochondrial antiviral signaling protein(MAVS)to synthesize a series of cytokines such as Interferon(IFN)and inflammatory cytokines.Finally,the antiviral innate immune state is established.To date,the pathogenicity of DTMUV is unclear,host immune response induced by DTMUV and the role that RLRs played in the process of viral infection are a lack of understanding.In this study,the pathogenicity of DTMUV in Cherry Valley ducks and the innate immune response induced by infection were observed by histopathological analysis and real-time quantitative PCR(qRT-PCR).The effects of RIG-I,MDA5 and MAVS on viral infection were analyzed by using overexpression,RNA interference and dual-luciferase reporter gene assay.The inhibitory effect of DTMUV on RLRs signaling pathway was also discussed.The experimental results are divided into the following four aspects:1.The pathogenesis of DTMUV in the different aged Cherry Valley ducksThe effect of host age on the out come of DTMUV infection was studied in ducks.Three groups of Cherry Valley ducks at 1,3,and 7 weeks of age were intramuscularly infected with DTMUV to systematically observe the clinical symptoms,pathological changes,tissue viral loads,and immune responses.Severe clinical symptoms and neurological dysfunction were observed in 1-week-old ducks as early as 2 days posti nfection(dpi)and some died at 5–7 dpi.Three weeks-old ducks showed similar but milder symptoms and no deaths.However,7-weeks-old ducks showed only transient loss of appetite.Gross lesions gradually reduced in severity as ducks matured.One-week-old ducks showed endocardial hemorrhage,splenomegaly,swelling in the lymphfollicles of the ileum,liver,and kidney swelling with degeneration,and meningeal hyperemia.Three-weeks-old ducks showed only mild pathological lesions.No visible lesions were observed in 7-weeks-old ducks.However,pathological histologyanalysis demonstrated all infected ducks displayed viral encephalitis.DTMUV could be detected in the brains of 1-week-old ducks as early as 1 dpi and virus titers of most organs in 1-week-old ducks were significantly higher than that of 3-and 7-weeks-old ducks at 3–5 dpi.The patterns of IFN-?,IL-2,and serum neutralizing antibodies were similar,and there were significant difference between the youngest ducks and the older ducks at early infection stage(P < 0.05).More important is that although the antibody titers of all infected ducks were similar from 9 to 17 dpi,reduced clearance of virus was observed in the youngest groups comparing with the other two groups,indicating that immune system maturity was more important than the presence of neutralizing antibody.In summary,this study demonstrates that viral pathogenesisis strongest in 1-week-old ducks and the age-related immune response plays an important role in the pathogenesis of DTMUV in ducks.2.Immune response of ducks and DEFs infected with DTMUVIn order to investigate the innate immune response to DTMUV,1-week-old duck was infected with DTMUV,the spleen and brain were sampled at the indicated time,the relative qRT-PCR was used to measure the expression of the innate immune-related genes.The results showed that RIG-1,MDA5,and TLR3 are significantly upregulated after DTMUV infection(P < 0.05),indicating that they were involved in the host immune response to DTMUV,and the expression of proinflammatory cytokines(IL-1?,IL-2,IL-6,IL-8)and antiviral proteins(Mx,OAS,etc.)are also upregulated early in infection.The expression of IL-6 increased most significantly in the tissues tested.The expression of I-and II-IFNs was different in both spleen and brain,they were upregulated to different degrees in the spleen but not in the brain.In addition,we also detected the expression of RIG-I,MDA5,I-IFNs and some proinflammatory cytokines,the results were similar to those of the animal experiment,RIG-I and MDA5 were activated by DTMUV,leading to the expression of IFN-?,IFN-?,ISGs,IL-6and IL-8.Our study suggests that the host immune responses are activated early in infection,but it is unable to defense against the quick replication of DTMUV,the overexpression of cytokines may damage the host immunity.3.RIG-I and MDA5 are involved in the defense against DTMUVIn order to further clarify the role of RIG-I and MDA5 in the process of resistance to DTMUV infection,two receptor-mediated IFN-? production was analyzed by using overexpression and dual-luciferase reporter gene assay in DTMUV-infected DEFs.Firstly,it was found that the mRNA expression of RIG-I,MDA5,MAVS and IFN-? was significantly increased after DTMUV infection through the previous experiments.Furthermore,the full length of RIG-I and MDA5 and their effect domain were pre-expressed on DEFs,and then were infected with DTMUV.The results showed that both of the receptors and their effect domains were able to inhibit the proliferation of DTMUV.Subsequently,we cloned and expressed the duck MAVS gene(duMAVS),overexpression and RNA interference confirmed that duMAVS was necessary in the DTMUV infection.In addition,overexpression of RIG-I and MDA5 could promote the production of transcription factors NF-?B and IRF-7 induced by DTMUV infection,indicating that RIG-I and MDA5 could activate downstream IFN-?through NF-?B and IRF-7 signal pathway upon recognizing DTMUV.This part of study preliminary determined that RIG-I/MDA5-MAVS-IRF-7/NF-?B signaling pathway mediated IFN-? production to participate in the innate immune response against DTMUV.4.The non-structural NS1 protein of DTMUV inhibits the RIG-I and MDA5-mediated signaling pathwayIn this study,we found that the DEFs infected with DTMUV in advance were transfected with RIG-I or MDA5,the induction of IFN-? was significantly lower than negative control group,which indicated that DTMUV had a certain inhibitory effect on RIG-Iand MDA5-mediated IFN-? production.In order to further clarify which nonstructural protein of DTMUV plays a key role in antagonizing the IFN-?,seven nonstructural proteins of DTMUV(NS1,NS2 A,NS2B,NS3,NS4 A,NS4B and NS5)were cloned and their eukaryotic expression plasmids were expressed,respectively.The results of study showed that the expression of IFN-? in cells with overexpressed NS1 protein was significantly lower than that in the control group,indicating that the NS1 protein of DTMUV played a major role in antagonizing the of RIG-I and MDA5 signalinng pathways.Furthermore,NS1 and duMAVS recombinant plasmids were co-transfected into DEFs,24 h post transfection,the cells were prepared for laser confocal experiments,the results showed that co-localization of NS1 and duMAVS in cells,which determined that the NS1 protein of DTMUV might inhibit the RLR signaling pathway by interaction with duMAVS.However,the direct interaction between the NS1 protein and duMAVS still requires further evidence by immunoprecipitation and other assays.