Anti-influenza A Virus (H3N2) Activities Of Extracts From The Medicinal Plant Bletilla Striata

Objective Influenza A virus(IAV)infection is a major public health threat leading to significant morbidity and mortality.The emergence of drug-resistant virus strains highlights the urgent need to develop novel antiviral drugs with alternative modes of action.In this study,we examined the anti-influenza virus effects of extracts from the medicinal plant Bletilla striata,and investigated its possible mechanisms of blockage of viral attachment by inhibition of viral hemagglutinin(HA)protein or sialic acid(SA)receptors on host cells,blockage of viral replication,and release by inhibition of neuraminidase(NA).Methods 1.The aqueous extract and ethanol extract from a were screened for influenza virus A/Sydney/5/97(H3N2)inhibitory activity by embryonated chicken eggs assay.The Bletilla striata extracts were mixed with virus and incubated at 37℃ for 1 h,and the mixtures were inoculated into nine-day-old embryonated chicken eggs via the allantoic route.The levels of influenza A virus in the allantoic fluid was determined by the hemagglutination test and real-time reverse transcription-PCR after 48 hour’s incubation.The HA titer and Ct value were used as measurement indicators.2.In order to test the ability of Bletilla striata extracts in blocking the viral invasion to Madin Darby canine kidney(MDCK)cells,we used and analyzed the pretreatment and simultaneous treatment assays.In the pretreatment assay,Bletilla striata extracts treated the MDCK cells and incubated for 18 h before virus infection.In the simultaneous treatment assay,the influenza virus were pretreated with Bletilla striata extracts for 1 h,and then the mixtures were added into the cell monolayers.The cytopathic effect(CPE)was evaluated by MTT assay to detect the inhibitory effects against viral invasion.3.To understand how Bletilla striata extracts prevents virus invasion to host cells,we examined whether Bletilla striata extracts competitively inhibit influenza virus-mediated hemagglutination using the hemagglutination inhibition(HI)assay.The guinea pig red blood cells(gRBCs)were pretreated with Bletilla striata extracts for 1 h before agglutinated by influenza virus.The hemagglutination pattern was read following the incubation of the plates for 1 h at 4℃.The assay was used to evaluate whether Bletilla striata extracts inhibit viral attachment onto gRBCs by interfering with SA receptors.We also evaluated whether Bletilla striata extracts directly interfer with viral haemagglutinin to block binding of viruses to cell receptors.The influenza virus was pretreated with Bletilla striata extracts for 1 h,and the solution was mixed with an equal volume of a 1.5%gRBCs suspension and incubated for 1 h at 4℃.4.To evaluate the anti-influenza activity after virus infection,we employed the post treatment assay,quantitative real-time PCR to test the effect of Bletilla striata extracts on viral replication.In post treatment assay,MDCK cells were inoculated with virus at 37℃ for 1 h,followed by removal of the medium and addition of the tested Bletilla striata extracts.MTT assay was carried out to evaluate whether Bletilla striata extracts are able to inhibit replication of influenza virus.RNA extraction was performed at 18 h after influenza virus infection and the levels of intracellular influenza RNA were measured by quantitative real-time PCR.5.The neuraminidase inhibition(NAI)assay was employed to evaluate the effects of Bletilla striata extracts on the neuraminidase activity using the neuraminidase inhibitors screen kit.The IC50 of Bletilla striata extracts on neuraminidase was determined used as a measurement indicator.Results 1.The results of embryonated chicken eggs assay showed that aqueous Bletilla striata extract treatment at a dose of 80.0 mg/egg to 20.0 mg/egg decreased significantly both the HA titre and Ct value(P<0.01)compared to the non-treated infected eggss.The two indicators were significantly decreased after the embryonated chicken eggs were treated with ethanol Bletilla striata extract ranged from 1.4 mg/egg to 0.4 mg/egg(P<0.01).2.In pre-treatment assay,the aqueous Bletilla striata extract and ethanol extract did not show significant antiviral activity.The simultaneous treatment assay showed that both aqueous extract and ethanol extract showed effective inhibition of virus infectivity against influenza virus.The 50%inhibitory concentrations(IC50)of them were 18.3 mg/mL and 235.7 μg/mL,respectively.3.The gRBCs pretreated with Bletilla striata extracts could not prevent the binding of viruse to gRBCs in this assay.These findings suggest that Bletilla striata extracts do not block binding of viruses to cell receptors by directly interfering with SA receptors on host cells.By the haemagglutinin inhibition assay,pretreatment with aqueous Bletilla striata extracts could prevent the binding of virus to gRBCs at a concentration as low as 80.0 mg/mL.These results suggest that aqueous Bletilla striata extract block binding of viruses to cell receptors by directly interfering with viral haemagglutinin.4.The aqueous extract and ethanol extract were showed effective antiviral activity in the post treatment assay.The values of IC50 were 20.6 mg/mL and 358.6 μg/mL,respectively.Real-time reverse transcription-PCR analysis showed that 40.0-20.0 mg/mL of aqueous extract or 360.0 μg/mL of ethanol extract significantly reduced level of RNA transcription in the inoculated cells at 18 h after viral infection compared to the non-treated infected cells(P<0.01).These results indicate that Bletilla striata exerts antiviral effects by the mechanism of blockage of virus replication.5.The results of NAI showed that ethanol Bletilla striata extract exerts exhibited an inhibitory effect on the NAs with IC50 values of 16.0 mg/mL.Our results suggest that Bletilla striata exerts inhibit virus release by inhibiting neuraminidase activity.Conclusion Our results indicated that Bletilla striata exerts had strong anti-influenza virus activity.We found that aqueous Bletilla striata extract inhibited influenza virus infection suggesting two possible ways of viral inhibitions,1)blockage of viral attachment by inhibition of viral HA protein,2)blockage of viral replication.The ethanol Bletilla striata extract showed blockage of viral replication,may be block viral release by inhibition of NA.These findings will provide important information for new drug development for the treatment of influenza virus infection.