Screening And Antiviral Assessment Of Butene Lactone Compounds Against Influenza A Virus

Influenza,as a contagious respiratory disease,was primarily caused by influenza virus.Influenza induced viral pneumonia,which threatened the safety of human health and life.The antigenic drift or shift in influenza A virus could induce epidemics locally or pandemics worldwide.Its high morbidity and mortality have attracted worldwide attention.Currently approved anti-influenza drugs were M2channel blockers,such as amantadine,rimantadine,and neuraminidase inhibitors,such as oseltamivir,zanamivir.The antiviral drugs have met several limitations because of the high frequency of emerging variants and undesirable toxic side effects.Therefore,it is urgent to find new antiviral compounds with less cytotoxicity and efficient antiviral effects.The purpose of this research is to screen a compound with low cytotoxic effects and high antiviral activity from series of butene lactone compounds and evaluate the activity and potential antiviral mechanism.Firstly,91 butene lactone compounds were screened by a combined method of MTT and CPE.The half inhibition concentration(CC₅₀)of the compounds to MDCK cell was determined.The antiviral activity was measured by 100 TCID₅₀ and the half effective concentration(EC₅₀)was calculated.SI is the ratio of CC₅₀ and EC₅₀.The potential antiviral compound was screened out from butene lactone compounds compared with the CC₅₀,EC₅₀ and SI of ribavirin.Secondly,the antiviral activity was confirmed in vitro.MDCK cells were treated with compound to detect the inhibitory effects before,at the same time of,or after influenza A virus inoculation.The ratios of apoptotic cells were determined by Flow Cytometry after compound incubation for 48 hours.The inhibition of influenza neuraminidases was determined by Neuraminidase Inhibitors Screen Kit.Finally,the antiviral activity was confirmed in vivo by establishing mouse infection model.The mice were randomly grouped into seven groups:normal,virus control,oseltamivir,ribavirin,compound-treated high,medium and low dose group.At 2 hours post-infection,the compounds were orally administered once daily for 5days.During the experiment,mice were observed daily for body temperature,body weight and clinical symptom.On 1,3 and 5 days post-inoculation,mice were sacrificed to collect their blood and lungs,detecting cytokine levels,calculating lung index,observing lung lesion,testing the virus load by the ELISA,qRT-PCR,HE and IHC.These results indicate that?f was screened out from 91 butene lactone compounds and has potential antiviral activities against H1N1 compared with the CC₅₀,EC₅₀0 and SI of ribavirin.The time-of-addition assay reveals that a post-adsorption step of influenza A virus replication was inhibited by?f.The apoptosis and NA activity were suppressed after?f incubation,which could reduce the release of progeny virion.In mice model,the clinical symptoms of inactivity,anorexia,laboured breathing was alleviated.The body temperature recovered to normal and the weight decreased slowly.The lung index,inflammation and pathological injury in lung were reduced by?f.The expression of virus NP antigen and M gene in lungs were decreased,which reveal the virus load was reduced by?f.The level of IgG in mouse plasma was increased and the level of TLR-3,RIG-I,IRF-3 and iNOS were reduced after?f administration.The expression of pro-inflammatory cytokines,including TNF-?,IL-1?,IL-6 and IL-8 were down-regulated,whereas the expression of IL-10 and IL-13 were up-regulated in?f-treated mice groups.All of these data suggested that?f is helpful to suppress inflammatory,enhance immune response and reduce lung injury.Thus,?f has potential effect in the treatment of the influenza A virus infection.