Study On The Expression Patterns And Mechanism Of Antiviral Proteins IFITM1 And IFIT1 Induced By Avian H9N2 Influenza Virus In Endothelial Cells

Avian H9N2 influenza virus(H9N2 AIV)is widely prevalent in poultry and its host range has expanded from poultry to mammals.Endothelial cells are identified to participate in the innate immune response through expressing multiple proteins during virus infection.Interferon-inducible proteins IFITM1 and IFIT1 can be induced by virus infection,and inhibit virus replication through a series of mechanisms.Therefore,it is important to investigate the expression patterns and mechanisms of IFITM1 and IFIT1 induced by H9N2 AIV,and the effect of IFITM1 and IFIT1 on the antiviral response in endothelial cells.The expression patterns and mechanisms of IFITM1 and IFIT1 induced H9N2 AIV,and the effect of IFITM1 and IFIT1 on antiviral response in endothelial cell are still unknown.To solve these problems,H9N2 AIV A/Chicken/Hebei/4/2008(Ck/HB/4/08)was selected as the research strain and used to make inactivated virus particles.Human umbilical vein endothelial cells(HUVECs)were used to investigate the expression patterns and mechanisms of IFITM1 and IFIT1 and their effects on the antiviral ability of cells.Human bronchial epithelial cells(BEAS-2Bs)were selected as reference cell type.BALB/c mouse model was used to investigate the expression of IFITM1 in vivo.First,we used plaque formation assay to study whether H9N2 AIV infect two kinds of cells.Secondly,the expression levels of IFITM1 and IFIT1 induced by H9N2 AIV or viral particle inoculation were detected by RT-PCR and Western blot.Results showed that H9N2 AIV can infect and proliferate in HUVECs and BEAS-2Bs,both virus infection and viral particle inoculation increase the mRNA and protein levels of IFITM1 and IFIT1,and the expression patterns of IFITM1 and IFIT1 between HUVECs and BEAS-2Bs are different.To analyze the expression mechanism of IFITM1 and IFIT1 induced by H9N2 AIV and viral particle,we used ELISA kits to detect the levels of interferon-?/?.RT-PCR analysis was used to detect the effects of HA and NA protein on the expression of IFITM1 and IFIT1.We found that both virus infection and viral particle inoculation increased the levels of interferon-?/? in BEAS-2Bs,however,the levels of interferon-?/? in HUVECs were not significantly changed.HA or NA protein treatment could not induce the expression of IFITM1 and IF1T1 in both two kinds of cells.Immunofluorescence was used to locate viral particles by staining nucleoprotein(NP)within cells.NP-positive cells were observed at 8h after inoculated with viral particles in HUVECs and BEAS-2Bs.To investigate effects of IFITM1 and IFIT1 induced by viral particles on antiviral responses in cells,we transfected HUVECs and BEAS-2Bs with IFITM1 or IFIT1 specific siRNA and CRISPR activation plasmid to knock down or overexpress the two proteins.Then cells were infected with virus,and the antiviral effects were evaluated by virus titers in cell culture supernatants.The results showed that the IFITM1 decreased virus titers in culture supernatant of HUVECs,while IFIT1 had no effect on virus titers in culture supernatants of HUVECs and BEAS-2Bs,indicating that IFITM1 induced by viral particle mediates the antiviral response of HUVECs.We selected BALB/c mouse model to investigate the expression of IFITM1 induced by virus particle inoculation in vivo.Mice in each group were inoculated with viral particles through trachea,IFITM1 mRNA and protein levels were detected at different time points after inoculation.To observe the protective effect of viral particles on lung tissue of mice infected by H9N2 AIV,mice in each group were inoculated with viral particles followed by infection with H9N2 AIV at 12h after inoculation.Levels of IL-6 and TNF-a were detected at 1d,3d,5d,7d post infection,and pathological changes in the lung tissue were detected at 5d post infection.The results showed that virus particle inoculation could decrease the levels of IL-6 and TNF-a and extenuate the lesions in lung tissue of challenged mice.In conclusion,our results revealed the different kinetics of IFITM1 and IFIT1 expression induced by H9N2 virus infection and viral particle inoculation between HUVECs and BEAS-2Bs.IFITM1 induced by viral particle may be independently of interferon-a/p and significantly enhanced the antiviral response in HUVECs.One-time inoculation of virus particles could increase the expression of IFITM1 in lung tissue,and partially reduce the damage of lung tissue caused by virus infection.