Establishment Of Anti-Dengue Virus Screening Model And Anti-Dengue Virus Activity Of FNC

Dengue fever caused by Dengue virus(DENV)is an acute infectious disease.In recent years,because of the increase of floating population and global warming,the epidemic of dengue fever has become more and more serious.Dengue is included in the World Health Organization as one of the neglected tropical diseases,and so far no special anti-dengue virus drugs are available.This study was to screen out compounds with anti-dengue virus.We choose ribavirin as the positive drugs,Preliminary screening of drugs by detection the luciferase activity of DENV-Ⅱluc~+virus replicon.The screened effective compounds were rescreened by plaque assay,and then the effectiveness of the compound was verified by Western blotting.Finally,the cytotoxicity of the compound was detected by MTT assay.This is the drug screening model of dengue virus in vitro.The EC50 of ribavirin inhibited the DENV-Ⅱis 40.78±1.02μM,which was close to other reports.The stable and reliable anti-dengue virus drug screening model was established successfully.Using this model,807 compounds were screened and found FNC has a effective anti-DENV-Ⅱactivity.Secondly,to evaluate the inhibitory effect of azvudine(FNC),hydrochloride salt of azvudine(FNC-HCL)and triphosphate azvudine(FNC-TP)on dengue virus(DENV)in vitro.Method is same as above.The results showed that EC50 of FNC,FNC-HCL and FNC-TP in inhibiting DENV replication were from 0.54±0.29 to25.42±6.70μM.The EC50 of the positive drug ribavirin,which inhibits DENV-Ⅱ,was 40.78±1.02μM.Western blotting and real time quantitative PCR results showed that FNC,FNC-HCL and FNC-TP inhibited the expression of DENV E protein,and significantly inhibited the replication of DENV viral RNA.FNC,FNC-HCL and FNC-TP can significantly inhibit the replication of DENV at cellular level and protein level,the inhibitory activity of DENV is superior to ribavirin.It is expected to become a candidate for anti-dengue virus drugs.Through the time of drug addition and time of drug elimination assay,we found that FNC acts on the synthesis stage of dengue virus RNA.Finally,the anti-dengue virus activity in vivo of FNC was researched.First of all,4-6-week-old BaBb/c mice were intracerebral injection of infection with DENV-ⅡD1090 strain,symptoms such as weight loss,arch back,and hind limb paralysis were observed.Mice infection model was successfully established.BaBb/c mice infected with DENV at a dose of 2 mg/kg per day gavage administration.The results showed that FNC in this condition did not play a role in inhibiting DENV in mice.This result may be related to the toxicity in vivo of FNC.