Mechanism Of Surfactin Inhibiting The Infectivity Of Porcine Intestinal Enveloped Viruses

Porcine epidemic diarrhoea virus(PEDV)and transmissible gastroenteritis virus(TGEV)belong to the envelope virus.They need to fuse the membrane of the virus and the cell membrane in the process of invading the intestinal epithelial cells.Membrane fusion inhibitors can inhibit the membrane fusion of the viral envelope,and becoming a new direction for the development of antiviral drugs.Inverted-cone lipid membrane fusion inhibitors are effective against many viruses by changing the physical properties of viral envelopes.In this study,we found that surfactin molecule conformed to the structure of inverted cone membrane fusion inhibitor,so we carried out the study on the mechanism of anti-virus infection of surfactin.Firstly,it was confirmed that surfactin played a role before virus infected cells,inserted into the lipid molecular layer of the capsule in a way that did not destroy the integrity of the virus envelope,and decreased the tendency of negative curvature of the outer lipid of the capsule,thus inhibiting the fusion of the virus envelope and the cell membrane.Secondly,after the safety evaluation of oral surfactin in mice,it was proved that oral surfactin could effectively prevent diarrhea symptoms caused by PEDV in newborn piglets.Finally,ten kinds of synthetic lipopeptides with similar chemical composition and molecular structure to surfactin were obtained by chemical synthesis.They all have different degrees of antiviral effects,one of which has lower cytotoxicity.The content of this study is divided into the following three parts:1.Mechanism study of surfactin inhibits viral membrane fusion during invasionSurfactin from Bacillus subtilis could completely abolish the replication of porcine epidemic diarrhea virus(PEDV)and transmissible gastroenteritis virus(TGEV)in epithelial cells at a relatively lower concentration range(15-50 μg/ml),without cytotoxicity or viral membrane disruption.Membrane fusion inhibition experiments showed that surfactin treatment significantly reduced the fusion rate of virus to the cell membrane.Meanwhile,thermodynamic experiments showed that the incorporation of a small amount of surfactin hinders the negative curvature formation of lamellar phase lipids,verifying surfactin as a membrane fusion inhibitor.In addition,fluorescent lipopeptide similar to surfactin was synthesized,and its viral membrane lipids insertion ability was confirmed by spectroscopical experiment.The results further indicate that the surfactin could direct insert into the viral envelope and inhibits its membrane fusion with epithelial cells.In conclusion,surfactin is a membrane fusion inhibitor for enveloped viruses.2.Screening of surfactin analogues with better PEDV performance by synthetic lipopeptide methodTen surfactin analogues were obtained by chemical synthesis.Their anti-PEDV activity,hemolytic activity,and physicochemical properties of critical micelle concentration were detected.In order to develop safer drugs,higher anti-PEDV activity and lower hemolytic activity are the main goals.Compared with surfactin,SLP 5 in this research shows a lower hemolysis activity,with antiviral activity maintained,increasing the selectivity index from 4 to 52.In other words,the safe and effective concentration range of SLP5 has increased by 12 times.In addition,SLP 5 has a direct antiviral effect on PEDV,the same as surfactin.Structurally,SLP 5 is a linear lipopeptide with three carboxyl groups.surfactin derivatives similar to SLP 5 could be obtained by lactone bond hydrolyzation.3.Subchronic experiment of oral surfactin in mice and the anti-PEDV activity of its intestinal contentThe mouse model was used to explore the possibility of oral route administration for the treatment of digestive tract envelope virus disease.The mice were divided into 4 groups with 10 mice in each group.Different concentrations of surfactin(0,4,20,100 mg/kg bw)were orally administered every day.In the highest dose group,the surfactin concentration is above the hemolytic threshold if the daily dose was evenly distributed in the body.After 28 days of sub-chronic experiment,none of the four groups of mice died.After being sacrificed,samples were taken and tested.The results showed that the highest dose group loss weight significantly,liver injury indicator increased significantly,the small intestine slices show a certain degree of mucosal injury.The remaining indicators of four groups of mice were normal.The anti-PEDV activity of the non-protein components of the jejunal contents of the four groups gradually increased with the oral surfactin dose,and reached a significant level in the medium and high dose groups.The results showed that oral administration of surfactin was feasible,and daily administration of 20 mg/kg bw could significantly increase the anti-PEDV effect of the digestive tract contents.In this study,surfactin as the first reported natural membrane fusion inhibitor,shows a potent anti-PEDV and anti-TGEV activity.And a preliminary exploration was made on its modification and oral administration schedules,which provided a research direction for the development of surfactin family enteric envelope virus therapeutic drugs.