In Vitro And In Vivo Anti-influenza A Virus Activity Of The Sesquiterpene Lactone Brevilin A

Background and purposeInfluenza A virus（IAV）is an important pathogen of respiratory infections,and it causes not only seasonal influenza but also influenza pandemics.IAV is prone to antigenic variants,which can lead to the generation of novel influenza viruses and poses global threats to human health.Vaccination is an effective intervention to prevent and control influenza.However,Vaccination have a short effective duration and low potency.Thus,antiviral drugs are a vital option for the treatment of influenza.Until now,three types of antivirals have been approved by the FDA for influenza prevention and treatment,but the potency of antiviral drugs are limited by the emergence of drug-resistant influenza virus,therefore,it is urgent to develop anti-influenza drugs with novel mechanisms of action.We have previously shown that brevilin A,a sesquiterpene lactone isolated from C.minima,suppresses the infection of influenza A/PR/8/34 H1N1（PR8）in vitro.Here,we further investigate the antiviral effect of brevilin A against influenza virus in vivo,the antiviral spectrum and mode of action of brevilin A against IAV in vitro based on previous research.Methods and resultsWe firstly established a mouse model infected with PR8 and tested the effect of brevilin A on the weight and survival rate of infected mice.The results showed that brevilin A significantly improved the body weight of mice and increased the survival rate to 50%（Vehicle-treated mice showed 100%mortality）.We next investigated the effect of brevilin A on inhibition of several IAV strains in vitro by plaque reduction assay,these results show that brevilin A significantly inhibits different IAV subtypes,the EC₅₀ values of brevilin A obtained with PR8,A/FM/1/47H1N1（FM1）,A/Hong Kong/498/97 H3N2（HK498）and A/chicken/Guangdong/1996H9N2（Gd1996）were 2.96±1.10,1.60±1.14,3.28±1.09 and 2.07±1.12μM,respectively.In order to evaluate the effect of brevilin A on the growth curves of various influenza viruses or virus titers,we determined the virus titers at different time points in multiple replication cycles through the plaque experiment.We found that brevilin A inhibits progeny virus production in various virus-to-cell ratios,with a maximum reduction by about 10-fold.The plaque experiment and immunofluorescence method were used to investigate the stage of viral cycle where brevilin A exhibits its activity against PR8 virus,these data suggest that brevilin A blocks the intermediate stage（s）of the influenza virus life cycle between approximately 4 to 6 hpi.We further performed RT-qPCR to evaluate the effects of brevilin A on viral mRNA,v RNA and c RNA synthesis,compared to vehicle control,v RNA in the brevilin A treatment group was significantly reduced.At the same time,western blotting analyses were conducted to examine the effect of brevilin A on expression of viral protein,these results indicated that in the brevilin A treatment group,the protein levels of M1,M2,NS1 and NS2decreased to varying degrees as compared with virus control,among them,M2 and NS2protein expression was reduced to 70%.The M1,M2,NS1 and NS2 proteins are produced by the alternative splicing of M and NS mRNA.We next performed RT-qPCR to determine the effect of brevilin A on the mRNA expressions of M1,M2,NS1 and NS2,compared with the virus control group,we found that brevilin A reduces the mRNA levels of viral M1,M2,NS1 and NS2 to varying degrees,the M2 mRNA was markedly reduced following treatment with brevilin A,with a reduction of 55%–60%compared to vehicle.We next performed laser confocal immunofluorescence to investigate the effects of brevilin A on the nucleocytoplasmic trafficking of vRNPs.These results show that brevilin A induces vRNPs aggregation in the nucleus compared to vehicle.ConclusionBrevilin A has anti-IAV activity in vivo and in vitro,furthermore,brevilin A inhibited influenza viral replication in three aspects,including v RNA synthesis,expression of viral mRNA,and protein encoded from the M and NS segments,and nuclear export of viral vRNPs.Together,these results are encouraging for the exploration of sesquiterpene lactones with similar structure to brevilin A as potential anti-influenza therapies.