The Effect Of DHV-Ⅰ On TLRs Signaling Pathway In Duck Macrophages

Duck viral hepatitis (DVH) is caused by duck hepatitis virus (DHV), the serotype of DHV divided into DHV-Ⅰ, DHV-Ⅱ, DHV-Ⅲ, and new DHV. DHV-Ⅰ susceptibly infected ducklings and caused acute death because of breakthrough in the blood-brain barrier by replicated rapidly. Some Papers indicated that interferon(IFN) and interleukin(IL) could inhibit replication of virus. Toll-like receptor7(TLR7) recognized ssRNA viral and activated dependented-MyD88signaling pathway, and then activated cells to release IFN-Ⅰ, IL-6, IL-12and other cytokines, the role of TLR7has been attached imptortance. Studies have shown that increasing TLR7expression could inhibit the replication of ssRNA viruses, such as hepatitis C virus(HCV), respiratory syncytial virus(RSV) and West Nile virus(WNV), but there the effect of DHV-I on TLR7has not been reported.In this study, we analysised expression and distribution of TLR7, MyD88, NF-κB, IL-6and IFN-α in ducklings tissues, and detected expression of TLR7, MyD88, NF-κB, IL-6and IFN-α in duck macrophages infected with DVH-I in different period, Oh,2h,8h,24h and32h, then used siRNA to interference expression of TLR7in duck macrophages, exploring the impact of DVH-I on TLRs signaling pathway in duck macrophages.1Tissue expression profile and sequence analysis of TLR7, MyD88, NF-κB, IL-6and IFN-α in duckExpression of TLR7, MyD88, NF-κB, IL-6and IFN-α in seven tissues (heart, liver, spleen, lung, kidney, brain and bursa)of duck was detected by RT-PCR. The tissus expression profiles showed that mRNA expression of TLR7, NF-kB, IL-6and IFN-α were detected in these seven tissues in duck, MyD88was not detected in the kidney and brain but could be detect in other tissues, and expression of TLR7, MyD88, NF-κB, IL-6and IFN-α mainly distributed in the peripheral immune organs, speculated that the reason was due to these genes involved in immune regulation an inflammatory processes. The sequences of MyD88and NF-κB gene in duck have not been reported. We cloned part of duck MyD88and NF-κB were sequenced and used BLAST for similarity. The results showed sequences of duck MyD88/NF-κB had high similarity (about90%) with chicken, pearl birds and other birds, and with human, mouse similarity in about80%. Then we derivated the nucleic acid sequence into amino acid sequence to compare homology. It showed the amino acid sequence of duck MyD88and NF-κB were highly similar(97%～100%) with chicken, pearl birds and other birds, the homology between duck MyD88and mammal MyD88was84%, but the homology between duck NF-κB and mammal NF-κB was about30%, it indicated that the part gene of MyD88was conserved between different species, and the conservative of NF-κB was not high.2Expression of TLR7, MyD88, NF-κB, IL-6and IFN-α in duck macrophages infected with DHV-ⅠWe used CFX Manager1.6software to analyze TLR7, MyD88, NF-κB, IL-6and IFN-α of SYBR Green Ⅰ qRT-PCR. The results showed that Melting temperature of TLR7, MyD88, NF-κB, IL-6and IFN-α gene product were80℃,85℃,83.5℃,91.5℃and82℃, the gene amplification efficiency of TLR7, MyD88, NF-κB, IL-6and IFN-α gene were102.2%,101.9%,99.4%,105.1%and94.4%, the correlation coefficients of TLR7, MyD88, NF-κB, IL-6and IFN-α gene were0.999,0.993,0.999,0.997,0.989. These conformed the standard of construction in qRT-PCR, it indicated that SYBR Green Ⅰ qRT-PCR were specificity in TLR7, MyD88, NF-κB, IL-6and IFN-α of duck. Duck macrophages was infected with DHV-Ⅰ in2h,8h,24h and32h, and relative expression of TLR7, MyD88, NF-κB, IL-6and IFN-α gene in duck macrophages was detected by SYBR Green Ⅰ qRT-PCR, the results was analyzed by duncan methods. The results showed the mRNA expression of TLR7in duck primary macrophages increased in8hour to24hour, the mRNA expression of MyD88, IL-6and IFN-α were no significant changes in2hour to8hour, MyD88, NF-κB, IL-6and IFN-α mRNA expression was significantly increased in24hour, It indicated that the DHV-Ⅰcould induce mRNA expression increasing of TLR7, IFN-α and IL-6in duck macrophages. TLR7, MyD88, NF-κB and IL-6mRNA expression reduced in32hour, it inferred that TLRs signaling pathway negative regulation mechanism has been started for reducing the overall expression of gene in signal pathway due to the amount of TLR7expression in cell.3Inhibition of TLR7expression by RNAi affects production of cytokines in duck macrophagesFluorescently labeled negative control of siRNA was designed to transfect in duck primary macrophages by Lipofectamine2000, and optimized the transfection reagent metering an siRNA concentration of RNAi experiments. The results showed that Lipofectamine2000measurement (1.0μl) and siRNA concentration (50pmol) transfection could reach75%transfection efficiency in duck macrophages. Three pairs siRNA (siRNA500, siRNA1681and siRNA2957) of duck TLR7gene were designed for RNAi. Inhibition mRNA expression of TLR7in duck macrophages was detected by qRT-PCR to select efficacious siRNA, and we found siRNA1681could effectively silence mRNA expression of TLR7in duck macrophage, the expression level of TLR7was decreased about71%, in line to the standard of interfere70%mRNA expression by siRNA transfection. Using siRNA1681silent TLR7expression, detection of TLR7, MyD88, NF-κB, IL-6and IFN-α mRNA expression in duck macrophages infected with DHV-Ⅰ in24h. The results showed mRNA expression of TLR7, MyD88, NF-κB, IFN-α and IL-6in duck macrophage was significantly lowered by siRNA1681inhibited, and increased without siRNA inhibited. It suggesting that TLR7mediated dependented-MyD88signaling pathway to transfer immunization information in duck macrophages infected with DHV-Ⅰ.