Inhibitory Effect Of Dendrobine On Influenza A Virus Replication

Objectives:Influenza,an infectious disease,poses a threat to public health and has provoked worldwide pandemic of influenza for many times in history and lead to a high mortality.With the antigenic drift and shift,influenza virus possesses a high variability and the appearance of novel strains has caused a serious impact on the survival of mankind.There is an urgent need for the prevention and treatment of influenza,especially drug therapy.Two classes of anti-influenza drugs which targeted M2 channel or neuraminidase(NA)are approved for clinically treatment of influenza until now,but the ongoing threat of resistance and adverse effects of these drugs limits their development and application.Because the chemical synthetic drugs to treat influenza virus infection are extremely numbered,it is crucial to develop low toxic and effective anti influenza drugs from natural products.In the Heat-Clearing Liquid-Safeguarding Formula recorded in Treatise on Seasonal Diseases,Dendrobium decoction can treated heat and sweat,wind-heat,consumption of body fluid caused by febrile disease,and so on.Dendrobium has multiple pharmacological effects,mainly including tonifying stomach and promoting fluid,moistening lung to arrest cough,nourishing yin and clearing heat,and is commonly used in various symptoms,such as dry mouth and polydipsia,consumption of body fluid caused by febrile disease,asthenia fever after disease.Dendrobine is a main active alkaloid isolated from Dendrobium nobile.In this project,we aim to study the anti-influenza activity of denrobine and its mechanism of action.This may provide invaluable information for the fully application of "Shihu",lay a foundation for the development of new anti influenza,and provide a new idea for the study of the pathogenesis of influenza virus.Methods:The MTT assay and plaque assay were used to determine the vitro cytotoxicity of dendrobine and evaluate its activity against influenza A virus;Western blotting,immunofluorescence Microscopy and qRT-PCR to explore the inhibitory effect of dendrobine on influenza A virus replication;Haemagglutination inhibition assay,SPR,H5N1 pseudovirus assay and neuraminidase inhibition assay to study the mechanism;Mini-replicon assay,site-directed mutagenesis,confocal and docking method to investigate the target of anti-virus.Results:1.Dendrobine clearly displayed the inhibition effect against influenza A virus infection,including A/Aichi/2/68(H3N2),A/FM1/47(H1N1)and A/Puerto Rico/8/34 H274Y(H1N1)with IC50 value of 5.32±1.68,3.39±0.32,2.16±0.91 μg/mL,respectively.2.Dendrobine inhibited multiple round of virus replication and its activity was effective up to 72 h.3.Dendrobine could not prevent the entry of influenza virus.4.Dendrobine could significantly decrease the expression of HA protein in the 0-4 h post infection.5.Dendrobine disturbed the catalytic activity of vRNP,inhibited the export of NP protein and decreased its expression via binding with NP.6.Dendrobine could bind to NP via R267,V270,F338 and E339,which showed>99%conservation in more than 30,000 NP sequences.Conclusions:1.Dendrobine possessed antiviral activity against influenza A viruses and effectively inhibited multiple round of virus replication.2.Dendrobine performed antiviral activity in the early stage of the viral replication.3.The target of dendrobine is viral NP protein.4.The amino acids responsible for dendrobine binding are the conservative functional sites of NP;5.Dendrobine could interfere with the nuclear export of NP and its oligomerization by binding to NP,leading to the inhibition of viral replication.