A Study On Antiviral Activities Of Betulonic Acid Against Influenza A Virus In Vitro

Influenza is a kind of infectious respiratory diseases caused by influenza viruses.It can be divided into four serotypes: A,B,C,and D.Among them,influenza A virus is the most widely spread and most influential one.Influenza A viruses are characterized with highly morbidity,rapid transmission and strong infectivity.Some highly pathogenic zoonosis virus subtypes are prone to cause high mortality and pose seriously threats on human and animal lives worldwide.Vaccination is the most widely used strategy for preventing influenza epidemics clinically.However,due to the rapid variation of influenza viruses,the development of vaccines usually falls behind newly emerging virus subtypes,and thus it is hard to provide timely and effective protection for susceptible population.At present,clinically used anti-influenza drugs mainly include two categories: M2 ion channel inhibitors(rimantadine and amantadine)and neuraminidase inhibitors(zanamivir,oseltamivir and peramivir).M2 ion channel inhibitors have been clinically used for many years and are resistant to most of circulating virus strains,and resistant strains to neuraminidase inhibitors are also increasing.Existing vaccines and chemical drugs are insufficient to deal with constantly changing influenza epidemics.Therefore,it is urgent to develop novel,broad-spectrum,and highly effective anti-influenza virus drugs.Betulonic acid(BA),a pentacyclic triterpenoid compound,is extracted from Chinese herbal medicine Frucyus Liquidambaris.It has been reported that BA has good anti-tumor,anti-inflammatory,anti-viral and other pharmacological activities,but it's anti-influenza virus activities have not been investigated.In the present dissertation,a model of Madin-Darby canine kidney cells(MDCK)infected with A/PR/8/34(H1N1,PR8)influenza virus strains was established.The activity of 30 pentacyclic triterpenoids against PR8 infection in MDCK cell cultures was screened with Indirect Immunofluorescence Assay(IFA).BA showed a remarkable inhibition on replication of PR8 in MDCK cell cultures,which was confirmed by qRT-PCR assay.Furthermore,a model of A549 cells infected withPR8 was established,and antiviral activities of BA against PR8 in A549 cell cultures were confirmed through comprehensive antiviral assesments,including virus titer titration with CPE assay and determination of NP with IFA and NP mRNA with RT-PCR.The results showed that BA exhibited significant and persistent inhibition on PR8 replication at concentration ranging 2.5~10 ?M in a dose-dependent manner.At concentrations less than or equal to 20 ?M,BA did not show cytotoxicity on A549 cells.For determining that which stages were inhibited by BA treatment during PR8 replication cycle,three BA treatments including pretreatment with A549 cells,co-treatment with viruses and post-treatment post virus infection were conducted.The results showed that BA exhibited significant inhibition on PR8 replication in the pretreatment and the treatment post virus infection assays.It is also verified that BA did not affect attachment of PR8 to A549 cells through hemagglutinin inhibition assay.The results of this paper provide a preliminary basis for further studies of BA as a novel anti-influenza drug or a lead compound.