The Preliminary Study On The Effect Of Avian IBV Infection On The Expression Of Innate Immune Related Genes In Trachea Mucosa

Avian infectious bronchitis (IB) which results in great economic losses to the poultry industry is an acute, highly infectious and contagious respiratory disease, caused by avian infectious bronchitis virus (IBV). IBV is a typical mucosal pathogen, which initiates infection of respiratory epithelium, and then spreads to epithelial surfaces throughout the body. The affected areas are mainly epithetlial surfaces of various organs.Therefore, it is imperative to carry out research on IBV mucosal immunity.The study on the IBV pathogenic mechanism is one of the main direction for the prevention and control of IB.And the research on the mucosal immunity is the priority of IBV pathogenesis research. Innate immunity plays an important role in the non-specific immunity to infection and the stages of initiation, regulation and effect in specific immune responses.The research on the effect of IBV infection on the expression of innate immune related genes in trachea mucosa is limited.This present study was designed to investigate the expression dynamics of pattern recognition receptors (TLR2, TLR3, TLR6, TLR7), cytokines (IL-1β, IL-6, IL-10, IFN-α, IFN-β, Mx), chemokines and immune cell transport factor (CXCR4, CCR6, MMP3, MIP-1β)a total of14innate immune related genes mRNA in chicken tracheal mucosa after IBV (GX-YL5) infection using the SYBR Green Ⅰ real-time fluorescence quantitative PCR detection method. Meanwhile, the research on the best reference genes selection was conducted among5candidate reference genes of ACTB, TBP, GAPDH,18S rRNA and28S rRNA.The specific primers for TLR2, TLR6, TLR7, IL-1β, IL-6, IL-10, IFN-β, CXCR4, CCR6, MMP3and MIP-1β gene were designed and synthesized according to the conserved region of individual gene sequences in GenBank. The primers of TLR3, IFN-α, Mx, ACTB, TBP, GAPDH,18S rRNA and28S rRNA were referenced those of the published.19gene fragments were amplified by RT-PCR and the standard curvewas established using the purified product as a standard. The analysis of melting cure was also carried out by SYBR Green I of real-time fluorescence quantitative PCR. The results showed that, the standard curve curve correlation coeffieients (R2) were in between0.992and1.000, and the amplification efficiency were in between94%and102%. All the melting cure showed a single peak. The results demonstrated that the established quantitative RT-PCR detection method was highly specific and reliable.On the basis of establishing real time fluorescent quantitative PCR detection method, the expression of candidate reference genes28SrRNA,18S rRNA, GAPDH, ACTB, and TBP in the trachea mucosa were detected at defined time points after infection of chickens with IBV (GX-YL5). Then4analysis methods of geNorm, NormFinder, BestKeeper and comparative delta Ct were used to analyze fluorescence quantitative PCR data, and expression stability value of the gene was obtained. ACTB, GAPDH and TBP were screened out as suitable reference genes to normalize mRNA levels in the trachea mucosa at defined time points after infection of chickens with IBV (GX-YL5).On the basis of establishing real time fluorescent quantitative PCR detection method, the expression of pattern recognition receptors (TLR2, TLR3, TLR6, TLR7), cytokines (IL-1β, L-6, IL-10, IFN-α, IFN-β,Mx), chemokines and immune cell transport factor (CXCR4, CCR6, MMP3,MIP-1β a total of14innate immune related genes in the trachea mucosa were detected at defined time points after infection of chickens with IBV (GX-YL5). And ACTB, TBP and GAPDH were used as reference genes, relative quantitative analysis was performed by pfaffl method. The results showed that Mx, TLR2and TLR7showed marked upregulated at1day post-infection(dpi); IFN-P RNA amounts were more markly upregulated, CXCR4and MIP-1β RNA amounts were markly upregulated at3dpi; IFN-β RNA amounts were the most markly upregulated, IL-1β RNA amounts were markly upregulatedat5dpi; IL-1β and MIP-1β RNA amounts were markly upregulated at8dpi; TLR2and IL-6RNA amounts were markly upregulatedat12dpi.IFN-β RNA amounts showed significant increase of97.51-fold and165.54-fold at3and5dpi, respectively, implying that IFN-β is particularly important in the early phase of the innate immune response to IBV infection.In conclusion, original data of innate immunity related genes expression dynamics in chicken trachea mucosa after IBV (GX-YL5) infection were obtained in the present study, the effects and pathways of avian IBV infection on the trachea mucosa innate immune were analysised at the molecular level, revealing the molecular mechanisms of IBV infection effect on trachea innate mucosa immunity, enriching the research of mucosal immune data and system, providing scientific basis for the study of mechanisms of mucosa immunization and the induction adjuvants of mucosa immunity.