Regulation Of Heme Oxygenase-1 On H9N2 Avain Influenza Virus Proliferation In Chicken Oviduct Epithelial Cells

H9N2 subtype avian influenza viruses(AI)belongs to low pathogenic avian influenza,its infection rate is high but fatality rate is low.The infection can results in immunity decline,respiratory and genital tract lesions of poultry,it also infects the layers’ production and egg quality,and increase the number of shell egg,deformed egg,rubber eggs and soft shelled egg.However,the molecular mechanism of H9N2 subtype avian influenza virus infection in chicken oviduct epithelial cells has not been reported.Heme oxygenase-1 is an important anti-oxidative stress protein,which plays an important role in anti-inflammation,anti-oxidation,anti-apoptosis and anti-viral infection.Therefore,the chicken oviduct epithelial cells(COEC)were isolated and cultured in vitro to study the effect of H9N2 AIV infection on the expression of HO-1 in cells,and to explore the mechanism of HO-1 regulating H9N2 AIV replication.The main results are as follows:1.The study of H9N2 AIV proliferation in primary chicken oviduct epithelial cel s.The primary chicken oviduct epithelial cells were isolated and cultured successfully.The cells showed typical epithelioid shape with good growth status,and the detection of cytokeratin 18 was positive.The third generation of COEC was infected with H9N2 AIV,and obvious CPE was observed in the cells which characterized by cell shrinkage,thinning,elongation.Some cells become round,even crack to death.Western Blot could detect the expression of viral NS1 protein in host cel s.2.The expression change of HO-1 after H9N2 AIV infect COEC.The expression of HO-1 at different times after the infection of H9N2 AIV was detected by Real time-PCR and Western Blot.The results showed that the infection of H9N2 AIV significantly increased the expression of HO-1 both in m RN A and protein level.3.The role of HO-1 in the replication of H9N2 AIV.In order to study the role of HO-1 in the course of H9N2 AIV infection,we treated COEC with the HO-1 inhibitor Zn PP first,then detect the infection of Zn PP to H9N2 AIV replication by Western and blot.The results showed that Zn PP both inhibited the expression of HO-1 and the replication of H9N2 AIV.Then we specifically interfere the expression of HO-1 in COEC with si RNAto reduce the basal expression level of HO-1,the protein expression of HO-1 and H9N2 AIV was then detected.The results showed that the expression levels of HO-1 and H9N2 AIV in COEC transfected with si RNAdecreased significantly.In conclusion,this study demonstrated that H9N2 AIV infection can up-regulate the expression of HO-1 in COEC,and down regulation of HO-1 expression could significantly inhibit the infection of H9N2 and AIV.These results provide important reference to the targets of anti H9N2 drugs,and also provide new ideas for the prevention and control of avian influenza viruses.