Inhibitory Effect And Underlying Mechanism Of Ativirals On Influenza A Virus Infection

Aim:The high morbidity and mortality caused by Influenza virus (IFV) infection and difficulties in the development of efficient vaccine highlight the urgent need to develop agents with novel pharmacological strategies. To find available compounds which can reduce both viral replication and inflammation, ARB was investigated in the first part of this study for its inhibitory effect and underlying mechanism on IFV infection. We also study the anti-inflammatory effect of ARB on the inflammation induced by IFV or poly I:C. In the second part of this study, we evaluated the inhibitory effect of an empirical Chinese-medicine-formula Jiawei-Yupingfeng-Tang (JYT) on IFV infection in vitro and in vivo.Methods:1) The cytotoxicity of ARB and the sensitivity of ARB to different stains of influenza were determined by MTT assay on MDCK, Hep-2and A549cells. Body-weight-loss, lethality, median survival time, viral titer and lung-index were used to evaluate the effect of ARB against lethal pneumonia in an IFV infected mice model. The levels of inflammatory cytokines in peritoneal macrophage and mice exposed to IFV or poly I:C, were tested by real-time RT-PCR and ELISA. The vRNA, cRNA and mRNA of IFV were reverse-transcirpted by specific primer with tag and the levels of RNA were determined realtime RT-PCR. The effect of ARB on the accumulation of viral protein and celluar i?B were determined by western blot. The effect of ARB on vRNP exportation and phagocytic activity of peritoneal macrophage were determined by the immuno-fluorescent assay. Finally, we evaluate the inhibitory effect of ARB on the cytokine dysregulation induced by poly I:C in an acute inflammatory mice model.2) The cytotoxicity of JYT on A549cells was determined by MTT assay. The effect and mechanism of the JYT against IFV was tested by plaque reduction assay in the low respiratory tract cell lines A549. A lethal influenza infected mice model developing into interstitial pneumonia was used to evaluate the effect of JYT in vivo.Results:1) The TC50values of ARB on MDCK, Hep-2and A549cells were61.6,65.9and67.5?M, respectively. The EC50values of ARB on IFV A/Yamagata/56/93(H3N2), IFV A/PR/8/34(H1N1), IFV A/FM/1/47(H1N1) and IFV A/Hubei/74/09(H1N1) were17.3.17.2,18.4and18.8?M, respectively. The EC50values of ARB on IFV infected A549and MDCK cells were17.2and19.5?M, respectively. In vivo. post-treatment with ARB decreased the mortality of infected mice by alleviating virus-induced inflammation, mitigating lung lesion and reducing the viral titers. The survival rate of the ARB-treated groups at dosages of180.0and90.0mg/kg/d were58.0%and40.0%, higher than those of the viral control group (P<0.01), while the lung index of treated groups reduced76.0%and63.0%, and viral titers decreased33.3%and26.1%, respectively (allp<0.01). Furthermore, ARB can efficiently inhibit the exportation of vRNP, differently regulate the transcription of vRNA, cRNA and mRNA of IFV, and inhibit the phosphorylation and degradation of i?B, suggesting the post-treatment of ARB can inhibit the production and propagation of progeny virus by inhibiting the activqtion of NF-?B. Especially, the vRNA levels of IFV infected cells reduced52.0%and80.0%with the treatment of20.0and40.0?M of ARB (p<0.01). In addition, ARB can also protect mice and macrophages from IFV or poly I:C-induced inflammation through suppressing IL-1?, IL-6, IL-12, TNF-? and elevating IL-10secretion. The expressions of IFN-a and IFN-y were not significantly affected by ARB, but IFN-?1was reduced at5th d.p.i. in the murine lung. And the phagocytic activity of peritoneal macrophage was not effect by ARB.2) The TC50of JYT on A549cells was1373.3?g/mL. The IC50of JYT on IFV A/PR/8/34(H1N1) and IFV A/FM/1/47(H1N1) were215.1and264.2?g/mL, respectively. JYT extract dose-dependently inhibited IFV when given before, simultaneous and after viral infection. And it was more effective to block the entry of virus when JYT was given1h after or simultaneously with viral infection, the IC50values of the two manners were215.1and264.2?g/mL. Further investigation indicated that JYT could inhibit both the attachment and penetration processes of virus with the IC50values of287.5and228.7?g/mL, respectively. In vivo, the survival rates of lethal influenza-infected mice were40%,50%and70%with treatment of JYT extracr in the concentration of35,90and180mg/kg/d, respcetively. And the lung index of the treated groups decreased from22.4%to38.2%. The virus-induced lung lesion could also alleviated by JYT.Conclusion:1) ARB can efficiently inhibit all of the four IFV stains. The inhibitory effect of ARB may due to the inhibitory effect of ARB on the NF-?B activation which is responsible for the retention of vRNP and vRNA. ARB can also diminish both IFV and poly I:C-induced acute inflammation through modulating the productions of cytokines, suggesting the potential use of ARB in virus induced cytokine dysregulation.2) JYT extract dose-dependently inhibited1FV when given before, simultaneous and after viral infection. And it was more effective in blocking the entry of virus. Importantly, JYT extract increased the survival rate of lethal influenza-infected mice, prolonged survival time and alleviated the virus-induced lung lesion. These data support JYT as an alternative modality to be used in the treatment of respiratory viral infection induced by IFV.