Molecular Cloning Of Duck RIG-Ⅰ Gene And Its Function In The Process Of Duck Reovirus Infection

In 2011, an unidentified disease in Peiking duck was reported in China. The infection caused 40%morbidity at different age and 10%-20% mortality in different flocks. It has been reported that the pathogens of this disease was new duck reovirus(DRV), which is non-enveloped, with icosahedral symmetry and a double capsid containing 10 double-stranded RNA segments which can be separated by polyacrylamide gel electrophoresis(PAGE) into three size classes: large(L1–L3), medium(M1–M3) and small(S1-S4). However,so far the pathogenecity mechanism of DRV hasn’t been clear, especially, little study was done in the field of innate immunity of ducks and the interaction between DRV and host.RIG-I like receptor is the part of pattern recognition receptors.It recognize RNA located in cytoplasm,and quickly start signal transduction,active the inflammatory cytokines. It has an effect on antiviral infection.RIG-I could identify ds RNA,including DRV.However,Its role in resisting DRV infection is not clear.In this study,we cloned duck RIG-I.Using Real-time PCR to detect the function of RIG-I in resisting DRV infection.To lay the foundation for further study of duck antiviral infection mechanism.1.Cloning duck RIG-IWe designed a pair of primer to amplify duck RIG-I. after sequencing analysis,we found it full-length gene is 2802 bp and has a reading frame.encoding 933 aa. RIG-I had some typical characteristics of RLR family, for example, there were two caspase activation sites and a recruit domain structure within the N-terminal of RIG-I. In the C-terminal of RIG-I, the regions of RNA helicase, ATP binding site, RNA binding site and repression domain were also present. The 806-929 of the amino acid are highly conserved regions.The results of phylogenetic tree showed that the sequence of duck RIG-I was highly conserved among the same population, which shared 98.4%-99.8 % homology with the published duck RIG-I sequences. But the homology between duck and other species was lower, for example, the identities between duck and goose,homo, Mus, Ctenopharyngodon idella and susscrofa were 95.9%, 62.3%, 61.5%, 61.2% and54.4%,respectively.2. Standard Curve Generation of DRV for Absolute Quantification using Real-time PCRAfter quantification PET-30a-DRVδC, we dilute it ten times.eight dilution degrees and every dilution degree repeat three times.after Real-time PCR,A liner regression equation(Y =-3.458 x +42.19) was obtained and the regression coefficient( R2) of the standard curve is 0.998. Minimum detection the substrate concentration is 100 copies/mcl.we consider the method is reliable.3. Standard Curve Generation of duck RIG-I gene using Real-time PCRAfter quantification PEASY-T5-RIG-I, we dilute it ten times.eight dilution degrees and every dilution degree repeat three times.after Real-time PCR,A liner regression equation(Y=-3.327 x +33.01) was obtained and the regression coefficient( R2) of the standard curve is 0.998. Minimum detection the substrate concentration is 10 copies/mcl.Referred to the dissociation curve analysis, we consider the method is reliable.4.The influence of DRV replication after transfect with exogenous duck RIG-ITo study whether duck RIG-I recognizes DRV and induces an antiviral response, DEF cells were transfected with duck RIG-I ORF and duck RIG-I CARD, infected with DRV, we found the duck IFN-a and Mx were all enhanced. To shut out the interference of endogenous duck RIG-I in DEF, DF1 cells which are free of RIG-I were transfected with duck RIG-I ORF and duck RIG-I CARD, and infected with DRV, we found the IFN-β were increased. The result show that exogenous duck RIG-I functions against DRV infection.5.The function of endogenous duck RIG-I against DRV infectionTo study whether endogenous duck RIG-I bring about innate immune response on DRV infection,Quantitative real-time PCR(q RT-PCR) assay identified the expression of du RIG-I m RNA in the liver, spleen,lung, kidney, glandular stomach, pancreas, cecum, thymus, air sac, brain, skin, and muscle in ducks. The du RIG-I m RNA level in the bursa of fabricius, heart, and harderian gland in ducks was relative low. We analyzed the expression of du RIG-I and a number of key cytokines including RIG-I, Mx, and IFNα in ducklings infected with duck reovirus(DRV) and found that the expression of du RIG-I in the spleen and kidney tissues in infected ducks was significantly increased, suggesting the activation of du RIG-I in the process of viral infections. We also detected the expression of RIG-I, Mx and IFNα in duck embryo fibroblast(DEF) cells inoculate with DRV at different time points. Our results showed that their expression profiles were similar to those in the spleen and kidney tissues in the DRV-infected ducks.Taken together, these results suggest that du RIG-I plays an important role in innate immune responses against DRV infection.