Study On Host MiR-499-5p Which Inhibits Japanese Encephalitis Virus Replication By Targeting NS2A And Modulates NF-?B Pathway

JEV is a severely endangered zoonotic neurotropic virus that can cause human death or leave permanent neurological sequelae,and it has a tremendous impact on humans.Pigs play as storage and amplification hosts for JEV,JEV infection have also brought huge losses to the pig industry.In addition,the ecology of JEV is complex and it is generally believed that mosquitoes must be used as a medium for inter-individual transmission,however,recent studies have shown that pigs can be spread by direct contact,and the noses and mouths are highly susceptible.This finding suggests that the prevention and control of JEV infections which have no specific drugs or therapies will be more difficult in addition to vaccination and mosquito control,and the research on the prevention and treatment of JEV has important practical application significance.miRNA is a non-encoding small RNA that exerts gene regulatory functions at the post-transcriptional level and participates in cell growth,differentiation,apoptosis,and metabolism,and it plays an important regulatory role in virus infection and host cell anti-virus.miR-499-5p,which has been initially identified as having a strong inhibitory effect on JEV replication and no related reports of mechanisms that regulate viral infection,was selected as the study object.Firstly,JEV SC-2 strain was cultured and TCID₅₀0 was measured,and JEV qRT-PCR detection method was successfully established.The method was applied to the determination of JEV SC-2 strain proliferation in different cells.In the experiment,miR-499-5p was overexpressed or inhibited by transfection of miRNA mimics or inhibitors in cells,and the effect of miR-499-5p on JEV was verified by qRT-PCR and TCID₅₀0 methods.The results showed that the TCID₅₀0 of the JEV SC-2 strain infected with BHK-21 was10^-5.68/100?L,and the TCID₅₀0 of the PK-15 strain infected with the JEV SC-2 strain was10^-5.46/100?L,the JEV SC-2 strain began to enter the logarithmic phase after 12 h of infection with BHK-21 cells,but 24 h to PK-15 cells.Experiments have also demonstrated that overexpression of miR-499-5p results in a significant decrease in JEV gene levels,and the inhibition of miR-499-5p has the opposite result;overexpression of miR-499-5p also leads to a decrease in JEV virus titer.It was confirmed that miR-499-5p can inhibit the proliferation of JEV.In order to find out the reason why JEV is regulated by miR-499-5p,firstly,the potential target sites of miR-499-5p target JEV genome were predicted by the target prediction software.Successfully constructed the wild-type and mutant recombinant plasmids for dual-luciferase reporter gene vectors which include the target sites,and the dual luciferase reporter gene assay system was used to detect and found that the relative luciferase activity of the recombinant wild-type plasmid pEZX-NS2A-WT was significantly reduced by miR-499-5p,and the recombinant plasmid pEZX-NS2A-mut was not Significantly affected,further using the competitive inhibition method,JEV infection can competitively bind or consume miR-499-5p transfected into cells,thereby indirectly weakening its inhibitory effect on pEZX-NS2A-WT luciferase activity,it is more certain that miR-499-5p can target JEV genomic RNA to inhibit its replication.To explore the further relationship between miR-499-5p and JEV,bioinformatics analysis was used to enrich the potential target genes of miR-499-5p.Combining with published studies,we started with the NF-?B pathway and apoptosis pathways of inflammation,the relative qRT-PCR was used to detect the differences in gene levels of NF-?B1?p50?,REL-A?p65?,Bcl-XL,Mcl-1,and PUMA in two important pathways in cells which overexpressed miR-499-5p,the results show that the miR-499-5p species are highly conserved and participate in many cellular biological processes,it can inhibit the expression of NF-?B1?p50?from the level of genes and proteins,and can significantly inhibit the upregulation of inflammatory factor IL-6 and IL-8 caused by JEV.Experimental studies have shown that miR-499-5p may serve as a potential prevention and control target for JEV.It provides a new thought for the prevention and control of JEV and enriches the theoretical basis for the interaction between miRNA and viruses.