Study On The Effect Of Fowl Adenovirus Serotype 4 And Its Main Encoding Proteins On The MRNA Transcription Levels Of Toll-like Receptors

Fowl adenovirus serotype 4（FAdV-4）is highly pathogenic to broilers of 3to 6 weeks of age and usually causes hydropericardium hepatitis syndrome（HHS）,the disease broke out in Henan,Shandong and Guangxi of our country since 2015,causing severe economic losses to poultry industry.Toll-like receptors（TLRs）are typically Pattern recognition receptor to recognize conserved molecular patterns of pathogens when they invade the body,and then induce a cascade of responses,it can induce the expression of type I interferon and inflammatory factors to resist and eliminate the pathogen.It has been proved that the expression of TLRs mRNA will change after the pathogen invades the body.Monitoring the expression of TLRs is helpful to elucidate the pathogenesis of the pathogen.However,there is no systematic study on the effect of FAdV-4 infection on TLRs mRNA expression.In this study,FAdV-4 Guangxi isolate（FAdV-4-GX2019-010）was used to infect chicken hepatoma cell（LMH）and SPF chickens,subsequently,the effects of FADV-4 major coding proteins（fiber1,Penton,22K,52K）on the mRNA expression levels of 10 TLRs and 8 signaling pathway-related molecules were examined to lay a foundation for further study on the pathogenesis of Fadv-4.The specific researchcontents are as follows:1.Effect of Fadv-4 infection on Toll-like receptor mRNA transcription in LMH cellsLMH cells were infected with FAdV-4-GX2019-010 at MOI=0.01,and cell samples were collected at different time points including 12 h,24 h,36 h,36 h,48 h,60 h,72 h,84 h,96 h,108 h and 120 h.The results showed that the cell became bright and round at 72h,and the virus rapidly proliferated at 12～48 h,then slowly proliferated,and reached its peak at 96 h.The expression of TLRs mRNA was up-regulated at 72～120 h post infection in all 10 avian species,and the up-regulated fold of chTLR21 was the largest（56.71 fold）,followed by chTLR4,chTLR1a and chTLR1b.These results suggest that they are important receptors for FAdV-4 resistance in vitro.2.Effect of FAdV-4 infection on Toll-like receptor mRNA transcription in tissues and organs of SPF chickensSPF chickens were infected with FAdV-4-GX2019-010 by intramuscular injection.The hearts,livers,spleens,thymus and bursa of fabricius were collected at 1,3,5,7 and 10 days after infection,the viral load and TLRs mRNA expression of 10 avian species were detected by quantitative real-time PCR.The results showed that the characteristic pathological changes suchas pericardial effusion and rhubarb stain were found in the infected group.The viral load in liver was the highest,reaching 10^7.65copies/μL on the 5th day,followed by heart,spleen,Bursa of Fabricius and thymus.From 5 to 7 days after infection,,the mRNA expression levels of 10 kinds of avian TLRs were up-regulated in 5kinds of tissues:9 kinds of TLRs were up-regulated in liver,among which,the up-regulated fold of chTLR4 was the largest（31.86 fold）,followed by chTLR2a,chTLR3;5 kinds of TLRs up-regulated in the heart,among whichchTLR4 was the most up-regulated（30.26 fold）,followed by chTLR15,chTLR1b and so on,and 4 kinds of TLRs up-regulated in the spleen,among whichchTLR21 was the most up-regulated（8.92 fold）,followed by chTLR4,chTLR3 and so on;9 TLRs up-regulated in thymus,among whichchTLR4 was the most up-regulated（10.37fold）,followed by chTLR21,chTLR3 and so on,and 8 TLRs up-regulated in Bursa of Fabricius,among whichchTLR7 was the most up-regulated（11.49fold）,followed by chTLR4,chTLR21 and so on.It is suggested that chTLR4,chTLR7 and chTLR21 are important receptors involved in FAdV-4 resistance in vivo.3.Effect of FAdV-4 protein on the mRNA transcription level of Toll-like receptor and signaling pathway related moleculesThe encoding proteins fiber1,penton,22K and 52K of FAdV-4 were cloned into the p EF1α-HA eukaryotic expression vector.After successful expression of the target proteins were verified by indirect immunofluorescence and Western blot,they were transfected into LMH cells,and cells were infected with FAdV-4-GX2019-010 at 24 h after transfection,further cultured for 48 h to collect cell samples,and the mRNA expression levels of TLRs and related molecules were detected by fluorescence quantitative PCR.The results showed that the expression of fiber1 protein up-regulated chTLR1b,chTLR3,chTLR7and chTLR21,as well as pathway-related molecules MyD88,IRF3/7 and IFN-β;The expression of Penton protein up-regulated chTLR21 and down-regulated NF-κB and IL-1β;However,the mRNA expression of TLRs and signaling pathway-related molecules after the expression of 22K and 52K proteins was not significantly different compared with the control group.It is suggested that the protein Fiber1 and Penton may play an important role in the pathogenesis of FAdV-4.In conclusion,FAdV-4-GX2019-010 infected LMH cells and SPF chickens showed higher up-regulation of chTLR4 and chTLR21,suggesting that they are important receptors for FAdV-4 resistance.It was further found that Fiber1could promote the signaling pathway mediated by chTLR1b,chTLR3,chTLR7and chTLR21,and up-regulate the expression of MyD88,IRF3/7 and IFN-β.Penton could affect the signaling pathway mediated by chTLR21,the expression of NF-κB and IL-1βwere inhibited.It is suggested that Fiber1 and Penton may play an important role in the pathogenesis of FAdV-4.In this study,the effects of FAdV-4-GX2019-010 infection on the mRNA expression of ten avian TLRs were analyzed in vivo and in vitro.Four major FAdV-4 coding proteins（fiber1,Penton,22K and 52K）were expressed in LMH cells,to investigate the effect of FAdV-4 on the mRNA expression of TLRs and pathway-related molecules,and to lay a foundation for further study on the pathogenesis of FAdV-4.