Study On The Anti-Japanese Encephalitis Virus Activity Of Sofosbuvir

Objective:Japanese encephalitis virus(JEV)standard quality granules were constructed,and the anti-japanese encephalitis virus activity of sofosbuvir in vitro was analyzed by the standard quality granules detection system.Methods:1?Continuous blind culture of JEV was conducted by using BHK-21 cells.The full genome sequence of JEV was found and downloaded from the National Center for Biotechnology Information(NCBI)(https://www.ncbi.nlm.nih.gov/).BioEdit software was used to compare the download sequence,and the relatively conservative region was selected to design specific primers.The target fragment was amplified by polymerase chain reaction(PCR).2?1%the agarose gel was used to electrophoretic target strip.The target strip was cut under the gel imager UV lamp,then recovered by agarose gel Recovery Kit,and the product was cloned onto the pMD-19T vector.The products were then transformed into DH5? and coated on LB solid plate containing 100ug/ml ampicillin(AMP)for overnight culture in 37?incubator.Single bacteria on LB solid plate were selected and cultured in LB liquid medium containing AMP.Plasmids were extracted and identified by double enzyme digestion(sal1,bamh1).3?Japanese encephalitis virus standard quality granules were diluted by 10 fold gradient without nuclease and recorded as 10-1,10-2,10-3,10-4,10-5,10 6,10-7,10-8 and 10-9.Each gradient concentration was set with 3 parallel multiple pores.At the same time,Japanese encephalitis virus stock solution and 10 fold gradient dilution virus solution(virus stock solution,10-1,10-2,10-3,10-4)were added.DdH2O blank control group was set up.The stability,specificity and sensitivity of the standard quality granules were detected by real time fluorescence quantitative PCR(RT-qPCR)SYBR method with the designed specific primers.4?CCK8 reagent was used to detect the proliferation of human hepatoma cell line(Huh-7)in different concentrations of sofosbuvir(500?M,100?M,20?M,4?M,0.8?M,0.16?M,0.032?M,0.0064?M),and a blank control group was set up.5?The direct killing effect,adsorption blocking,intracellular proliferation inhibition and replication of JEV were studied on Huh-7 cells at the selected concentration.Relative quantitative analysis of antiviral effects of different routes by RT-qPCR.Results:1?The culture supernatant of Japanese encephalitis virus was obtained after it was cultured on BHK-21 cells for several times.According to the JEV sequence alignment downloaded from NCBI database,a relatively conservative prM structural protein region was selected.Using the JEV download sequence(GenBank:MH258853),the specific primers were designed to amplify the target fragment of prM region,which was identified as the correct 441 base pairs by sequencing.2?The target fragment was obtained by 1%agarose gel electrophoresis,then recovered and cloned into pMD-19T vector and cultured in a monoclonal way.The plasmid was identified by enzyme digestion.3?The results of RT-qPCR showed that there was little difference between the values of the multiple pores(<0.5),the fusion curve was specific and the peak value was single.At the same time,the standard curve equation of JEV standard quality grain is obtained by taking the Ct value of standard quality grain as the X-axis and the copy number log10 of standard quality grain as the Y-axis:y=-0.2889x+11.516,R2=0.993.In addition,the virus copy numbers of stock solution,10-1,10-2,10-3 and 10-4 concentrations were 1184130.27,120912.86,10736.84,1053.45 and 121.25copies/ul,respectively.4?Compared with the control group,there was no significant difference in the activity of sofosbuvir(500?M,100?M,20?M,4?M,0.8?M,0.16?M,0.032?M,0.0064?M)on Huh-7 cells(P>0.05).5?The direct killing effect of sofosbuvir on JEV showed that different concentrations of sofosbuvir had statistical significance compared with the control group(P<0.05),indicating that sofosbuvir solution had a certain degree of direct killing effect on JEV.But there was no difference between different concentrations of sofosbuvir(P>0.05).The adsorption and blocking experiment of sofosbuvir on JEV showed that the different concentration of sofosbuvir was significantly different from that of the control group(P<0.01),indicating that sofosbuvir solution had an inhibiting effect on the adsorption of JEV.There was no statistically significant difference between the group of 500?M and the group of 50?M(P>0.05).There was significant difference in the absorption and blocking effect on JEV between the other concentrations of sofosbuvir(P<0.01).The intracellular inhibition of sofosbuvir on JEV showed that there was a statistically significant difference(P<0.01)between sofosbuvir at different concentrations and the control group,indicating that sofosbuvir solution had an inhibitory effect on the intracellular proliferation of JEV.There was no statistically significant inhibitory effect of sofosbuvir(500?M,50?M)on intracellular proliferation of JEV(P>0.05).There was significant difference in the inhibitory effect of other concentrations of sofosbuvir on JEV cell proliferation(P<0.01).The replication effect of sofosbuvir on JEV showed that there were statistically significant differences(P<0.01)between the groups of sofosbuvir(500?M,50?M,5?M,0.5?M)and ribavirin(10?g/ml)compared with the control group(P?0.01),indicating that sofosbuvir solution(500?M,50?M,5?M,0.5?M,)and ribavirin(10?g/ml)could inhibit the replication of JEV.There was no significant difference between the group of 500?M and the group of 50?M and ribavirin(P>0.05).There was no statistically significant difference between the group of 50?M and the group of 5?M and ribavirin(P>0.05).Compared with 500?M,0.5?M and ribavirin group,sofosbuvir 5?M had statistical significance(P<0.05).There was a significant difference in the inhibition of JEV replication among other concentrations of sofosbuvir(P<0.01).Conclusion:Based on the prM region of JEV,the standard quality granules have good stability,specificity and sensitivity,which are suitable for quantitative detection of JEV copy number.Sofosbuvir has a direct killing effect on JEV to a certain extent.The effects of adsorption blocking,intracellular proliferation inhibition and replication on virus were significant.