| Toll-like Receptor 3 (TLR3) is one of the TLRs whose ligand is double strand RNA (dsRNA). Infectious bursal disease virus (IBDV), which is a dsRNA virus, could be recognized by TLR3. Infectious Bursal Disease (IBD) causes significant losses to the poultry industries, such as high mortality, and immunosuppression in young chickens. Up to now, little is known on the signaling pathway, mechanisms, and determines the function of chTLR3 on IBDV infection in vivo. In this study, it is important and essential to study the relationship of ChTLR3 signaling pathway with IBDV-infection in commercial chickens.As previously, our group was successfully cloning chicken’s toll like-receptor3 gene (GX-sh-chTLR3). In this study, the polyclonal antibody against ChTLR3 was prepared by immunization of BALB/c mice with GX-sh-chTLR3 protein, which was expressed in E-coli. An Indirect Enzyme Link-immunosorbent Assay (ELISA) was developed for detection of the antibody level in mice serum. The results showed that among various serum dilution,1:800 immune serum dilution with 1:400 antigen (chTLR3-LRR recombinant protein) dilution are the best condition for ELISA detection, whereas 1:100 immune serum dilution with 1:400 immune serum dilution showed the lowest positive (2.21 ratio). This result suggests that the polyclonal antibody prepared in this study has a high sensitivity for detecting of recombinant chTLR3-LRR by ELISA.The relationship of chTLR3 and IBDV infection was carried out in non-vaccinated 4-week-old commercial chickens by challenging with wIBDV strain NN1172 and attenuated vaccine strain B87. At 1dpi,2dpi,3dpi,5dpi,7dpi, the bursa cells were isolated, and the viral RNA genomes, mRNA of chTLR3, IFN-β, IL-8 were examined by real-time RT-PCR. The result showed that viral load in the bursa of NN1172-infected group was highest at 3dpi showed 1.5X103/μl whereas B87 infected B87 showed 0.651×103/μl, and the two groups went down afterwards as compared to those in uninfected group. Meanwhile, the expression level of chTLR3 is significant different between NN1172-infected and B87-infected group. We noted upregulation expression of TLR3 in very virulent group (NN1172) at post infection-day 3 by 5.365 fold (P<0.05). However, in attenuated vaccine strain (B87)-infected bursa, TLR3 gene expression was significantly downregulated at 1dpi by 0.23 fold (P<0.05). This result demonstrated that NN1172-infected induced TLR3 signaling pathway in the bursal is more pronounced than attenuate vaccine (B87) infected bursal.Moreover, mRNA expression level of downstream molecules of TLR3 signaling pathway, e.g. IFN-β and IL-8 also were detected by real time PCR at each appointed time post infection. In the present study, we noted that the expression of IFN-β in the bursa of B87-infected group was down-regulated at 1dpi; however, IFN-β in NN1172, B87-infected group were significantly increased at 3 dpi and they showed to be decreased at 5 dpi and strong significantly decreased at 7dpi. The result suggested that the enhancement of antiviral activities was activated at the early time point and then inhibited by the host after 3dpi. Gene expression of Chemokine IL-8 in the bursa of very virulent strain (NN1172) chickens was significant upregulated at 3 dpi. And at 1dpi and 5dpi the transcription of attenuated vaccine strain (B87) were normal or a bit decrease as compare to NN1172. This result suggested that both viral strains activate proinflamatory cytokine and chemokine responses which are more pronounced in very virulent IBDV-infected.In summary, the activation of TLR3 signaling pathway induced by very virulent IBDV (NN1172) is stronger than attenuated vaccine IBDV (B87). The signaling strength was closed related to virulence and replication rate of IBDV. This work is not only served as the foundation for further research on pathogenic mechanism of IBDV-infected in commercial chickens, but it also provides a platform for IBDV prevention. |
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