Antiviral Activities Of Carbazole Derivatives Against Porcine Epidemic Diarrhea Virus In Vitro

Porcine epidemic diarrhea virus（PEDV）belongs to coronavirus,Alphacoronavirus genus.PEDV can infect small intestinal epithelial cells,causing acute diarrhea,vomiting,dehydration and high mortality in newborn piglets,posing huge losses to the pig industry.In recent years,due to the emergence of PEDV mutants,there is a lack of effective therapeutics to inhibit infection by and spread of PEDV,except for a few vaccines.Therefore,finding efficient medicines to control PEDV infection is of great importance.Carbazole alkaloids are a kind of nitrogen-containing aromatic heterocyclic compounds,which are mainly distributed in coal tar and Rutaceae.Carbazole alkaloids and the derivatives shared similar biological functional properties such as antibacterial,anti-protozoan,insecticidal,anti-inflammatory,anti-oxidation,anti-platelet aggregation,antiviral and neuroprotective activities.However,it is not clear whether carbazole alkaloids and the derivatives have any anti-coronavirus activities such as anti-PEDV.In this study,the inhibitory effect and antiviral mechanism of carbazole alkaloid on PEDV replication were studied in vitro.The results and contents are as follows:In this study,to screen potent anti-PEDV drugs,18 carbazole alkaloid derivatives（No.1-No.18）were synthesis and evaluated by quantitative real-time PCR（RT-qPCR）and fluorescence microscopy to monitoring PEDV N gene copy number and the fluorescence signal of PEDV.No.5,No.7 and No.18 carbazole derivatives showed potent anti-PEDV activities in Vero 81 cells.To confirm the inhibition,flow cytometry（FCM）was used to calculate the fluorescence signal of Vero-81 cells infected with PEDV 24 hours after treatment with No.5,No.7 and No.18 carbazole derivatives,respectively.It was found that the fluorescence value decreased significantly in the cells pre-treated with No.5,No.7 and No.18 carbazole derivatives,indicating that No.5,No.7 and No.18 poses potent anti-PEDV infection activities.Among them,No.7 and No.18 carbazole derivatives were proved to be less than 80%toxic in Vero-81 cells by cytotoxicity test.Based on these results,the anti-PEDV activities of the No.7 and No.18 carbazole derivatives were further confirmed.To check if the carbazole derivatives inhibit the replication of PEDV is dose-dependent or not,western blot（WB）,fluorescence,RT-qPCR and tissue culture infective dose(TCID₅₀)were used to assess the expression and transcriton of PEDV N and virus titer.And the data showed that both No.7 and No.18 carbazole derivatives can inhibit PEDV replication in a dose-dependent manner.To explored the underline mechanism,the effect of carbazole derivatives on the replication cycle of PEDV was detected.The results showed that the copy number of N gene and virus titer decreased significantly when carbazole derivatives were added in the attachment stage.In addition,to explore the broad-spectrum inhibitory effect of carbazole derivatives on porcine enteric coronaviruses,porcine delta coronavirus（PDCoV）and PEDV,swine acute diarrhoea syndrome coronavirus（SADS-CoV）infected LLC-PK1 cells or Vero-81 cells treated with No.7 and No.18 carbazole derivatives respectively.It was found that mRNA of PDCoV N or PEDV N or SADS-CoV M,as well as virus titer,were significantly lower than the cells in the absence of carbazole derivatives,indicating that both No.7 and No.18 carbazole derivatives had broad-spectrum inhibitory effect on porcine intestinal coronavirus replication.This study not only lays a foundation for the development of anti-PEDV drugs,but also provides a new idea for the research of broad-spectrum anti-coronavirus drugs.