Antiviral Activities Of Artesunate And Dihydroartemisinin Against Influenza A Viruses In Vitro And In Vivo

Influenza A viruse(IAV)infection is the most common cause to induce human respiratory infections.Clinically,classic symptoms of influenza include sudden onset of high fever,cough,headache,malaise,prostration,and inflammation of the upper respiratory tract and trachea.With characters of strong infectivity,rapid transmission,high morbidity and mortality,and transmissibility between different species,influenza pandemics have been heavy threaten on human being's and animal health and lifes.Currently,vaccination and antiviral drugs are the commonly used strategies for preventing and controlling influenza.Vaccination act as the primary role for preventing influenza,however,it's application and efficacies are limited by the character of highly variation of IAVs and time-delay for developing a vaccine against newly emerging strain.To date,two classes of anti-influenza drugs have been commonly used,including inhibitors of the M2 ion channel(amantadine and rimantadine)and Neuraminidase(NA)inhibitors(oseltamivir and zanamivir).Inhibitors of the M2 ion channel can block the ion channel formed by the M2 protein,but circulating IAVs are seriously resistant to M2 inhibitors.NA inhibitors can interfere with the enzymatic activity of the NA protein.Although NA inhibitors have advantage of being less prone to select resistant influenza viruses than M2 ion channel inhibitors,they are used for the prevention and treatment of influenza infections with high cost.Therefore,it is imperative to develpoe effective drugs with novel targets against IAV infections.Traditional Chinese medicine(TCM)has been used to prevent and treat influenza for a long history in China,however,it's efficacies are limited by TCM's characters of complicated chemical components,unclear mechanisms of actions and inconsistent quality.Seeking for novel antiviral compounds from TCMs and natural products is one of the basic pathways to develop antiviral against influenza.Artemisinin,a sesquiterpene lactone containing an internal peroxide bridge,is isolated from the Chinese medicinal herb Artemisia annua.Artesunate(AS)and Dihydroartemisinin(DHA)are the derivatives of artemisinin.which have been widely used for the treatment of different malaria clinically.These compounds have been also reported to have activities of anti-cancer,antiinflammatory property and immunoregulatory functions by modern pharmacological studies.Up to now,the activity of AS and DHA against IAV infection has not been reported previously.In the present study,in vitro IAV infection models were established by infecting lung adenocarcinoma epithelial cells(A549)with IAV strain A/Guangzhou/03/2009(panH1N1).The inhibition of AS and DHA on proliferation of progeny virus in A549 cells was assessed by titrating virus titer with the endpoint dilution assay.To elucidate the underlying mechanisms,a series of experiments were conducted.In order to determine which stages were inhibited by AS and DHA in a single replication circle of IAV,drugs were added at different time-points post infection(time-of-addition assay)and virus titers were analyzed by the endpoint dilution assay.Hemagglutinin(HA)inhibition(HI)test and neuraminidase(NA)inhibition(NAI)assay were performed to evaluate the effects of AS and DHA on HA and NA of IAV.BALB/c mouse model infected with A/Guangzhou/03/2009(panH1N1)was used to evaluate protective effects of AS in vivo.Body weigh change and survival were used as parameters for efficacy evaluation.Our results demonstrated that AS and DHA in the concentration ranged 5~1.25 ?g/mL strongly inhibited H1N1 replication in a dose-dependent manner.AS and DHA showed inhibition on production of viral nucleoprotein(NP)mRNA and NP proteins in the qRTPCR assay and Immunofluorescence assay(IFA)and Western blot assay,respectively.In the time-of-addition assay,AS and DHA were found to exhibit inhibition on viral multiplication when added during 0~6 h post infection.Whereas AS and DHA had no effect on the absorption and release of H1N1 towards/from infected A549 cells based on results of HI and NAI assay.Intraperitoneal injection of AS,at dosages of 80 mg/kg/day or 40 mg/kg/day,remarkably increase the survival rate and the mean survival days,and also inhibit the body weight lose of infected mice,indicating that AS has significantly protective effects on H1N1 infected BALB/c mice.To our knowledge,this study is the first time to reveal the antiviral activities of AS and DHA against IAV infection in vitro,and AS against IAV in vivo.These findings have provided the primary basis for further studies of the development of AS and DHA as novel antivirals against IAV infection.