Screening,Identification And Action Mechanism Study Of Antiviral Active Substances From Bacillus Subtilis Metabolites

Infectious diseases caused by viral infections pose a serious threat to both animal and human health.Antiviral drugs are crucial strategies for treatment of viral diseases.However,antiviral drugs available in veterinary clinic are limited.With the novel structure and high biological safety,natural antiviral drugs have emerged as important sources of novel antiviral drugs.Microbial metabolites,particularly those derived from Bacillus subtilis(B.subtilis),exhibit extensive antibacterial and anti-inflammatory activities.Recently,microbial metabolites have also been reported to have antiviral activity,suggesting that these metabolites are also source of candidates for natural antiviral agents.Therefore,identification of novel antiviral agents with high antiviral activity from microbial metabolites are of great significance.Previously,we have isolated and identified a strain of B.subtilis-L2 with significant antiviral activity from broiler chicken fecal samples.However,the components and mechanisms responsible for its antiviral activity remain unclear.To investigate whether the metabolites of B.subtilis-L2 have antiviral activity,metabolites of B.subtilis(SMBS-L2)were generated by bacterial fermentation and centrifugal filtration.We found that SMBS-L2 significantly inhibits the replication of pseudorabies virus(PRV-GFP)in various cell lines including PK-15 cells,with antiviral effects showing dose-dependency(P<0.001).Further studies revealed that SMBS-L2 did not affect the expression of interferon synthesis-related genes,but significantly inhibited the binding,entry,and replication of PRV.In vivo experiments showed that oral administration of SMBS-L2 in mice significantly countered PRV-infection induced mortality and weight loss,reduced viral loads in tissues,and alleviated tissue damage caused by PRV infection,indicating that SMBS-L2 also exhibits significant antiviral activity against PRV in vivo.To identify the molecular structure of the active components with anti-PRV activity in SMBS-L2,SMBS-L2 was extracted with organic solvents of different polarities.The results show that the antiviral active components are present in the acetone extract.Moreover,by using eluents of gradually increasing polarity in silica gel column chromatography,eight different components were separated by thin-layer chromatography.Among them,five components(S1,S5,S7,S10,and S13)showed antiviral activity in vitro,with components S5,S7,and S13 exhibiting dose-dependent antiviral activity.By using nuclear magnetic resonance(NMR),S5 was identified as uracil and S7 was identified as 4,4’-azanediylbis(3,3-dimethylbutan-2-one),respectively.These results indicate that SMBS-L2 contains at least two antiviral active substances,uracil and 4,4’-azanediylbis(3,3-dimethylbutan-2-one).Given that S7 is a newly identified small compound with antiviral activity,we next attempt to investigate its antiviral mechanisms.The results indicated that S7 did not affect PRV adsorption and entry but significantly inhibited PRV replication,indicating that S7 primarily exerts antiviral activity during the viral replication stage.Preliminary research suggested that S7 may inhibit the expression of PRV DNA polymerase UL42 protein,thereby affecting PRV replication.In summary,this study identifies that SMBS-L2 exhibits good anti-PRV activity and five anti-PRV active components from SMBS-L2 were isolated by acetone extraction and thin layer chromatography.Moreover,NMR analysis shows that anti-PRV active substances S5 and S7 are uracil and 4,4’-azanediylbis(3,3-dimethylbutan-2-one),which is a novel antiviral compound.In addition,4,4’-azanediylbis(3,3-dimethylbutan-2-one)might impair PRV replication via targeting PRV DNA polymerase UL42.Our results thus lay an important foundation for the development of novel antiviral drugs from B.subtilis metabolites.