The Effects Of ALV-J Infection On Structure And Function Of Chicken Embryo Intestinal Epithelial Cells

Avian leukosis(AL)is a type of avian infectious diseases caused by avian leukosis virus(ALV),which can reduce the production performance,tumor occurrence and death of diseased chickens.At present,it is found that ALV infected chickens can be divided into A,B,C,D,E,J and K,among which J subgroup(Subgroup J avian leukosis virus,ALV-J)is the main epidemic subgroup in China.ALV-J is highly pathogenic,often accompanied by multiple infections and secondary infections,which directly affects production performance and causes huge losses to the poultry industry.The intestinal tract of poultry is susceptible to pathogenic microorganisms,which can lead to decreased production performance or even death,and intestinal epithelial cells can form a barrier to prevent pathogenic microorganisms from invading the body.In order to understand the effect of ALV-J infection on chicken intestines,this study intends to explore the effect of ALV-J infection on the structure and function of chicken embryo intestinal epithelial cells(IECs)in vitro.In this study,IECs were isolated and cultured by 18 to 19-day-old SPF(Specificpathogen-free)chicken embryo duodenum,and the specific genes cytokeratin 19(CK19),alkaline Sex phosphatase staining,anti-E-cadherin immunofluorescence staining,etc.were used for identification,and the activity of the isolated IECs was detected using CCK-8reagent.The results showed that the cell suspension containing crypt cell clusters grew into a single layer of polygonal cells after 1 d of culture.PCR detected a specific band of about144 bp CK19.After alkaline phosphatase staining,the cells contained brownish black particles.Cells after anti-E-cadherin immunofluorescence staining are arranged in a "paving stone" network and produce specific green fluorescence.CCK-8 cell activity test showed that IECs significantly proliferated(p <0.05),indicating success IECs with good cell activity were isolated.ALV-J HN06 isolate was infected with IECs after 1 day of culture with 0.01 MOI,PCR amplification was performed on the key regions of the ALV-J receptor gene(ch NHE1),and PCR and fluorescence were performed using the IECs c DNA from 1 to 5 days after infection Quantitative PCR detected ALV-J.After 3 days of infection,IECs were positive by p27 antigen ELISA,and then used ALV p27 and JE9 monoclonal antibodies to perform indirect immunofluorescence and Western blot tests,respectively.The results show that PCR can amplify a specific band of about 153 bp,which has been verified by sequencing as the key region sequence of the ALV-J cell receptor ch NHE1.PCR detected 545 bp ALV-J specificity from the IECs c DNA after infection The bands were positive and ALV-J was positive by fluorescence quantitative PCR.Only infectious IECs had specific green fluorescence in the indirect immunofluorescence test,and only ALV p27 protein bands in the western blot test.The above studies indicate that ALV-J can infect chicken embryo intestinal epithelial cells and replicate effectively.In order to analyze the effect of ALV-J infection on the structure and function of IECs,the ALV-J HN06 isolate was cultured with 0.01 MOI for IECs after 1 day of cultivation,and the changes in cell activity were detected by CCK-8 reagent.Annexin V-FITC and TUNEL cell apoptosis detection kit staining,and detect the expression of Caspase3 and Caspase9 m RNA by fluorescence quantitative PCR to analyze the changes in apoptosis,and detect the tight junction proteins of IECs(Zo-1,Occludin),Toll-like genes by fluorescent quantitative PCR(TLR1,TLR2,TLR4,TLR5,TLR7,TLR15,TLR21)and cytokine(IFN-α,IFN-β,IL-2,IL-6,IL-8)m RNA expression level changes.The results showed that,compared with the control group,the activity of IECs cells was significantly reduced after ALV-J infection(p<0.05).After Annexin V-FITC staining,cells showed green specific fluorescence,and after TUNEL staining,cells showed red specificity Fluorescence,after being infected with Annexin V-FITC,the apoptosis rate of ALV-J IECs was increased by flow analysis,the expression levels of Caspase3 and Caspase9 increased,and Caspase9 increased significantly on the second,fourth,and fifth days after infection(p<0.05);The m RNA expression levels of Occludin and Zo-1 increased significantly only on the 4th day after infection(p <0.05);on the 2nd to 5th days after infection,the expression levels of TLR1,TLR2,TLR5,TLR15 and TLR21 increased significantly(p<0.05),TLR7 significantly decreased only on the first day after infection(p<0.05),TLR4 significantly decreased on the first and fifth days after infection(p<0.05);meanwhile,the expression levels of IFN-α and IL-6 did not change Obviously,but IFN-β increased significantly on the 4th day(p<0.05),IL-2 and IL-8 increased significantly(p<0.05)and extremely significantly(p<0.001)on the 2nd day after infection.In summary,based on the isolation and cultivation of chicken embryos IECs,an ALV-J model of chicken embryos IECs infection in vitro was established.ALV-J infection promotes the apoptosis of IECs and the decrease of cell activity,which leads to the destruction of intestinal epithelial structure integrity,stimulates IECs to produce antiviral factors and inflammatory cytokines,and promotes the expression of Toll-like receptor genes.The results of this study provide data support for further exploration of the impact of ALV-J infection on intestinal health,and will help to comprehensively study the pathogenic mechanism of ALV-J.