3-anhydro-6-hydroxy-ophiobolin A Displays High In Vitro And In Vivo Efficacy Against Influenza A Virus Infection

Influenza is still a significant cause of mortality and morbidity worldwide,and the emerging drug resistance is increasing challenge to the treatment of influenza A virus(IAV)infection.Therefore,development of novel anti-influenza agents has become an urgent task to combat against the viruses.Here,we describe a novel compound that displays potent activity against IAV in vitro and in vivo.The compound is 3-anhydro-6-hydroxy-ophiobolin A(named as L435-3),a new derivative of ophiobolins.This study explored the role of L435-3 in influenza A virus replication by in vitro and in vivo experiments.A549 human alveolar epithelial cells and the Balb/c mice were used in this project.To search for potential anti-influenza agents,we screened a library of new bioactive compounds derived from fungi.A549 cells were infected with influenza virus strain A/WSN/33(H1N1)(WSN)or A/PR/8/34(H1N1)(PR8)and then treated with or without the small molecule compounds.Hemagglutinin assay was used to determine whether any these compounds had inhibitory effects on IAV infection.Strikingly,we found that treatment with L435-3 decreased the viral titers at low concentration indicating that it is the most potent inhibitor among these compounds.Thus,L435-3 was selected for further studies.The A549 cells were infected with WSN or PR8 and treated with L435-3.Hemagglutinin assay,plaque assay and western blotting were used to determine L435-3 effect on influenza virus replication after different post-infection time.Female BABL/c mice were infected with influenza virus strain A/WSN/33(H1N1)(WSN)or A/PR/8/34(H1N1)(PR8)and then treated with or without L435-3.All mice were monitored for body weight change and survival rate and posterior ischic optico-neuropthy to analysis the effect of L435-3 for influenza virus replication.Plaque assay and western blotting were used to detect the L435-3 effects on influenza virus replication of mice.cDNA microarray analysis was performed to determine the differentially expressed genes in IAV-infected A549 cells in response to L435-3 treatment.To verify the cDNA microarray data and detect the mRNA expression level of lung of mice,RT-PCR and quantitative real-time PCR were employed.Finally,immunohistochemical studies were used to determine whether treatment with L435-3 could affect expression of SIgA in mouse lung after IAV infection,and white blood cells in mouse peripheral blood were analysed by blood routine examination.The results are shown as follows:(1)We found that the viral titers and IAV protein synthesis were significantly reduced after L435-3 treatment of A549 cells.(2)The symptoms were remarkably less severe,a less of the weight loss and the mortality,the viral titers and IAV protein synthesis were significantly reduced after L435-3treatment of IAV infection mice.(3)We observed that L435-3 treatment significantly increased the expression of type III interferons,several critical interferon stimulated genes,and the result also be confirmed L435-3-mediated inhibition of IAV replication in mice.(4)In this study,we found that L435-3 treatment suppressed IAV-induced could enhance SIgA expression in lungs of infected mice and increase white blood cells in peripheral blood of IAV infection mice.