Effect Of Infectious Bronchitis Virus M41 Infection On The Key Molecules In MDA5 Signaling Pathway In Chicken

Avian infectious bronchitis(IB)is an acute highly contagious respiratory diseases caused by av:ian infectious bronchitis virus(IBV),a member of coronavirus,which can infect different species,all ages of chickens,causing respiratory tract and kidneys and other organs with varying degrees of damage,resulting in decrease of production efficiency in broiler and reduction of egg quality and quantity in layer,seriously affecting the development of poultry industry.IBV has numerous serotypes,and the cross-protection between different serotypes is poor.Thus it is a great challenge to prevent and control IB.Therefore,fiurther studying the pathogenesis and immune mechanisms of IBV is of great significance for prevention of IB.Innate immune system provides the first line of defense against various of viruses infection for the host,and plays an important role in non-specific immune process and the initiation,regulation and effects of specific immune response.Its defense function mainly depends on pattern recognition receptors(PRR).RIG-I-like receptors(RLRs)are an important coniponent of PRR,including three members,such as retinoic acid inducible gene I(RIG-I),melanoma-associated gene 5(MDA5)and laboratory of genetics and physiology gene 2(LGP2).They can recognize invasive intracellular RNA and induce the production of type I interferon.Unlike mammals,chickens lack RIG-1 gene and just have MDA5 and LGP2.The study on the effect of IBV infection on chicken MDA5 signal pathway is limited.The present study applied the established real-time fluorescence quantitative PCR method to detect the mRNA transcription levels of key molecules in MDA5 signaling pathway(MDA5,LGP2,MAVS and STING),downstream joint proteins(TRAF3 and TRAF6),associated kinases(TBKl and IKK。),transcription factors(NF-kB and IRF7),cytokines(IL-8,IL-12,IFNa and IFN p)and interferon stimulated genes(Mx,PKR,OAS,Viperin,IFITM3 and IFIT5)as well as IBV viral load in tracheae and kidneys of SPF chicken at 1,3,5,8,11,14,21 and 28 days post-infection(DPI)with IBV M41.At the same time the IL-8,IL-12,IFNa and IFNp concentrations in tracheae and kidneys and serum IgG antibody were detected by ELISA.It aimed to explore the influence of IBV infection on the MDA5 signaling pathway in the SPF chicken.The results showed that the real-time fluorescence quantitative PCR method for detecting the expression of MDA5,LGP2,MAVS,STING,IL-8,IL-12,PKR,OAS,Viperin,IFITM3 and IFIT5 mRNA were successfully established.The standard curve amplification efficiency were between 0.956～1.093,correlation coefficients(R2)were between 0.9963～0.9999,indicating high amplification efficiency,good linearity,and quantitative accuracy of standard curve.All melting curves showed single peak and lack of non-specific products and primer dimmers,indicating the good specificity of methods.Sensitivity test results showed that when the plasmid standard was diluted to 1 × 10 copies/μL,the specific amplification can still be detected,indicating the methods were sensitivity.Intra and inter reproducibility tests results showed that coefficients of var:iation(CV)were less than 3.05%,indicating the methods were repeatability and stability.The artificial infection results showed that viral load in tracheae and kidneys of the experimental group maintained at high level from 1 DP I to 11DPI.The peak of viral load in the tracheae and kidneys were at 5DPI and 3DPI,respectively.And the viral load in the tracheae was higher than that of kidneys.The results indicated that IBV M41 strain have different tropism to different tissues and organs.IBV specific antibody levels in the peripheral blood did not change significantly from 1DPI to 3DPI,but began to rise at 5DPI,and reached a peak a 14DPI,then decreased.IgG antibody levels and viral load showed negative correlation,indicating that specific anti-IBV antibodies play an important role in the protection against and remove viruses.During 3DPI-14DPI,MDA5,LGP2,MAVS,STING,TRAF3,TRAF6,TBK1,IKKs,NFκB,IRF7,IL-8,IL-12,IFNα,IFNp,Mx,PKR,OAS,Viperin,IFITM3 and IFIT5 mRNA transcription levels in tracheae were significantly(P<0.05)or highly significant(P<0.01)up-regulation at multiple time points.IL-8,IL-12,IFNa and IFNp protein concentrations of the experimental group was significantly(P<0.05)or highly significant(P<0.01)higher than those of control group.These indicated that these genes may be involved in the anti-IBV innate immune response in tracheae.The mRNA transcription levels of MDA5,LGP2,TRAF3,TRAF6,IRF7,IL-8,Mx5 PKR,OAS,Viperin,IFITM3 and IFIT5 in kidney were significantly(P<0.05)or highly significant(P<0.01)up-regulation at multiple time points.But MAYS,STING,TBK1,IKKε,NFκB,IL-12,IFNa and IFNp mRNA transcription levels were significantly down-regulation,and IFNa and IFNp protein concentrations of the experimental group were also lower than those of control group at multiple time points.The possible reason is the existance of some unknown factors blocking the virus signals from MDA5 and LGP2 to MAYS and STINGb inhibition of MAYS and STING activation,and reducing the production of I interferon.But the transcription factor IRF7,IL-8 and interferon stimulated genes Mx,PKR,OAS,Viperin,IFITM3 and IFIT5 significantly increased.Possibly there might be some RLR-independent signaling pathways or regulation in the kidney and they clear IBV cooperating with adaptive immunity finally.In summary,IBV M41 initial infection activated the MDA5 signaling pathway of the tracheae to induce a variety of anti-viral gene expression,and cooperate with adaptive immunity to clear the viruses from the site of infection.There might be some unknown factors blocking the MDA5 and LGP2 contacting with MAYS and STING in kidneys,inhibition of virus signaling transmission to the downstream through MDA5 signaling pathways.Therefore,IBV M41 strain infection has a significant impact on MDA5 signaling pathways of the chicken trachea and kidney,but their influencing mechanisms are not exactly the same.This study was first time to focus the effect of IBV infection on host MDA5 signal pathway and explored the molecular mechanisms of IBV infection effect on innate immune signaling pathways.It will provide new ideas for the studies of IBV pathogenesis,prevention and treatment as well as antiviral drug.