| The Toll-like Receptors (TLRs) are a subset of innate receptors commonly referred to as pattern recognition receptors (PRRs). The host uses these receptors to detect the presence of infection by pathogen-associated molecular patterns (PAMP) that are present on microbial organisms. TLRs play a key role not only in the initiating immediate innate immune responses to prevent spread of infection, but also in the potentiating the responses of adaptive immunity. 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, can cause severe immuno-suppression in poultry industry. At present, the mechanism of IBD is still unclear. Little is known on the interaction between virus and host. In this study, we sought to clone chicken TLR3 (chTLR3) and determine the function of chTLR3 signaling pathway on IBDV infection in vitro.Firstly, full-length of chicken Toll-like receptor 3 gene (chTLR3) from the PBLs of Guangxi "Sanhuang" chicken (GX-sh-chTLR3) was amplified by touchdown reverse transcription-polymerase chain reaction (TD-RT-PCR). Sequential analysis and structure prediction of GX-sh-chTLR3 were also carried out. The results showed that full-length of GX-sh-chTLR3 was 3036bp and shared 96.5～99.7% homology with other chTLR3 sequences. The amino acid sequences showed that the mutations were mainly happened in 5ˊend of extracellular region (from 1-173 amino acids). GX-sh-chTLR3 shared a low homology (68%～74.8%) with Bos taurus, Mus musculus, Ovis ories, Suis scrofa, Equus caballus, Oryctolagus, Pan trogldytes, Homo sapiens and Gorilla. In the phylogenetic tree, the TLR3 of avian, mammals and rats were divided into two clades with avian TLR3 in one clade, mammals and rats TLR3 in another clade. The structure prediction showed that GX-sh-chTLR3 was a transmembrane protein whose extracellular region showed a horseshoe-shaped solenoid structure, consisted of a lot ofα-helix on outside andβ-sheet on the concave surface inside and they arranged parallely and alternately. The polyclonal antibody against chTLR3 was prepared by immunizing mouse with a recombinant protein from GX-sh-chTLR3 domain (304aa-582aa) which was expressed in E.coli. The polyclonal antibody could recognize TLR3 protein expressed in chicken embryo fibroblast cells stimulated by IBDV. These results suggested that GX-sh-chTLR3 domain (304aa-582aa) had good immunogenicity and the polyclonal antibody had good specificity. These results may serve as a basis for further research on the structure and function of GX-sh-chTLR3, and provide important bedding material for study on the role of its initiating disease-immunoregulation of the chicken.Secondly, the function of chTLR3 on PBLs infected with IBDVs was assessed by real-time RT-PCR. A quantative PCR based on IBDV-VP2 was established to detect IBDV. In this system, the results of sensitivity test and repeated trials suggested that the standard curve had a good linear range, high sensitivity, specificity and stability, which could be used in the quantitative detection of IBDV.Then, PBLs was infected with IBDV, and the dynamics of viral loads in the cells, the expression of TLR3 mRNA and the downstream cytokines IL-8 and IFN-βwere detected by real time RT-PCR. The results showed that the viral load in PBLs was significantly higher than any other time-point in 6 hours after infection. The expression level of chTLR3 was also the highest at this time-point. However, the dynamics expression of the TLR3 downstream cytokines was different. The highest expression level of IFN-βmRNA was begun at 3h post-infection (pi), and went down afterwards, while the expression trend of IL-8 was the same as chTLR3. The results showed that chTLR3 did have relationship to IBDV infection. The different expression pattern of IL-8 and IFN-βsuggested that the immune respond in the cells infected by IBDV might activated by different TLR3 pathways.Finally, we selected the 6h pi time-point to assess the function of chTLR3 on PBLs infected with different pathotypes of IBDV. PBLs, which were infected with vvIBDV, intermediated-plus virulent IBDV and intermediate virulent vaccine strain, were harvested at 6h pi, respectively. The viral load, expression of chTLR3, IFN-βand IL-8 were detected by real time RT-PCR. The results showed that the expression of chTLR3 and the downstream cytokine IFN-β/IL-8 in PBLs infected with NN0704 strain (vvIBDV) was strikingly higher compared to the intermediate-plus virulent strain (NN040124) and vaccine strain (B87(in)). The viral load of of different pathotype of IBDV also had the same trend. Our results indicated that in immune cells, the strength of TLR3 signaling pathway was correlated to the virulence of IBDV: the higher virulent, the stronger signaling.In summary, in PBLs, IBDV infection could activate TLR3 pathway and resulted in the accordingly immune response. The signaling strength was closely related to virulence and replication rate of IBDV. The results may not only serve as a basis for further research on pathogenic mechanism of IBDV and molecular mechanism of chTLR3 in IBDV infection,but also provide a new scientific exploring area in effective IBDV prevention. |
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