Molecular Cloning, Tissue Distribution Of Chicken TRAF-3 And TRAF-6 Genes, And Their Roles In Chicken’s Ativiral Immune Response

As the first line of defence of the body, innate immunity has been extensively studied in response to infection of pathogens and acquired immunity. TRAF3 and TRAF6 are two key members of TRAFs that play an important role in maintaining innate immunity of the mammals. However, so far the role of them on innate antiviral immune pathways in chickens remains unanswered and need be futher elucidated. In this study, the full-length cDNA coding sequences of chicken TRAF3 and TRAF6 genes were cloned firstly, and their differential expression in several tissueswere also determined by real-time PCR.In addition, the chick embryo fibroblast(CEF) were infected with different doses of the Poly(dA:dT) and Poly(I:C), and the differential expression of TRAF3, TRAF6 and their down-stream effective genes in CEF resist exogenous DNA’ and RNA’s expression were determined by real-time PCR at each time points or doses.Furthermore, the CEF were also infected with different doses of the virus virulence, so it could promote us to further determine their exact actions in the process of chicken antiviral immunity. Finally, eighty ten-days-old chicks were randomly divided into three groups:30 chicks infected with the moderate virulence NDV strain 30 chicks with the high virulence NDV strain, and 20 chicks mock-infected. Five chickens were euthanized from both infected and four age-matched non-infected groups at 24,48, and 96 hours after infection, respectively. In the meatime, bursas, spleens, and thymuses were sampled from each chicken. The response of TRAF3, TRAF6 and their down-stream effective genes to Newcastle disease virus (NDV) infection were also investigated. The results are listed as follows: 1) The full-length cDNA sequences of chicken TRAF3 and TRAF6 genes were successfully cloned. The results showed that there were two TRAF3 putative sequences and one newsequence, while only one putative sequence and one new sequence were found for TRAF6. Additionally, the pupative sequencesof chicken TRAF3/6 were similar to that of human, indicating they maybe play the similar role in the innate immunity in chicken as that in human.2) The differential expression of TRAF3 and TRAF6 genes were determined by real-time PCR in several tissues. It showed that they were highly expressed in the spleen, lung, and thymus,they were highly significant than others(p<0.01), suggesting that TRAF3/6 maybe mainly participate in the innate immunity through the chicken’s spleen, lung and thymus.3) The CEF were infected with different doses of Poly(dA:dT) and Poly(I:C).The results were found that the expression of TRAF3, TRAF6 and down-stream genes were increased in both dose- and time-dependent manner. It means that TRAF3 and TRAF6 participated in the work of recognitioned bind DNA patterns and modes of doublestranded RNA viruses. It also means that TRAF3 and TRAF6 have the time-and concentration-dependent.4) The CEF were infected with different MIO post-infection and different virus virulence, the expression of TRAF3, TRAF6 and their down-stream genes was increased as the dose of virulence increases.And they were highly expressed at MOI=1 and 8or12h, they were highly significant than others(p<0.01). That means TRAF3 and TRAF6 participate in the innate immunity.It also shows that TRAF3 and TRAF6 have the time-and concentration-dependent.5) At different NDV post-infection (dpi) in the spleen, thymus and bursa of fabricius of the ten-day-old chicks, the results means that expression of TRAF3, TRAF6 and their down-stream genes were up-regulated in the spleen, bursa and thymus. So it could be speculated that TRAF3 and TRAF6 participate in the innate immunity of spleen, bursas and thymus. And it also shows that TRAF3 and TRAF6 were leaned to the different NDV.