Part ? Complete Genome Sequencing And Molecular Characterization Analysis Of Imported Dengue Virus Serotype ? And Serotype ? Part ? Screening And Mechanism Of MiRNA Regulating HAV Antagonizing LFN-? Induction

Background:In recent years,Dengue Virus(DENV)infection caused by frequent international tourism has become a public health concern in China.Although sporadic imported cases of DENV have been documented in Yunnan,China,since 2000,the whole genome sequences and molecular characteristics of imported DENV-3 from Laos and DENV-4 from Thailand have not been reported.Objective:To interpret the complete genome sequences and molecular characteristics of two imported dengue virus strains.Methods:Viral isolation from the patient,s serum was carried out in mosquitoes C6/36 cells.Reverse transcriptase polymerase chain reaction(RT-PCR)was used to identify and serotype the two viruses.The complete sequencing of DENV-3 and DENV-4 was determined by Sanger dideoxy sequencing and Illumina Hiseq 4000 platform,respectively.Similarity analysis was implemented by NCBI-BLAST.Multiple sequence alignment was performed by MegAlign module of the Lasergene 7 software package DNASTAR.Phylogenetic analysis,which based on envelope gene and complete coding sequence,was performed by Maximum-likelihood method.Recombination events were analyzed using RDP4 software.Results:The virus imported from Laos belonged to dengue virus serotype ? and named YNPE3 strain;the YNPE3 strain has a total length of 10627 nucleotides(nt)with an open reading frame(ORF)encoding 3390 amino acids.Strain YNPE3 shared a nucleotide similarity of 98.6%-98.8%with the highly associated DENV-3 strain from India(JQ922556.1,KU216209.1,KU216208.1).Phylogenetic analysis indicated that the YNPE3 strain belonged to genotype ? of DENV-3 and might have a closely relationship with Laos and India genotype ? strains.The strain imported from Thailand is dengue virus serotype IV,named 2013JH285 strain;the 2013JH285 strain has a full length of 10,772 nucleotides,it has 99.0%nucleotide and 99.7%amino acid similarity with the isolate CTI2-13 from Thailand in 2013.Phylogenetic analysis revealed that the 2013JH285 strain belongs to genotype ? of DENV-4 and might have a high correlation with CTI2-13 strain from Thailand.Recombination analysis suggested that the 2013JH285 strain originated from inter-genotypic recombination of DENV-4 strains.Conclusions:This research first reported the full genome sequence and molecularcharacteristics of DENV-3 and DENV-4 imported from Laos and Thailand isolated fromYunnan Province,these results will contribute to further disease surveillance,and epidemiological and evolutionary research of DENV-3 and DENV-4 in Yunnan province of China.Background:Hepatitis A virus(HAV)does not produce cytopathic effects when cultured in vitro,and inhibits the production of IFN-? in cellular antiviral innate immunity to establish a persistent infection,suggesting that hepatitis A virus has formed strategies to escape host immune responses during the long evolutionary process.Numerous studies have elucidated the molecular mechanism by which HAV antagonizes the production of IFN-? in host cells.MicroRNAs(miRNAs).as regulators of gene expression,are widely involved in the regulation of a variety of physiological and pathological processes including virus-host interactions.Whether miRNA mediates the antagonistic effect of HAV on IFN-? induction has not been elucidated.Objective:To investigate the role of miRNAs in HAV escaping host immune defense mechanisms from the perspective of RNA.Methods:Human lung diploid fibroblastic cell(KMB17)was used as an infection model for the HAV live attenuated vaccine strain H2.Firstly,the differential expression profiles of miRNAs in KMB17 cells infected with HAV virus were constructed by miRNA microarray experiment,and the results were verified by qRT-PCR.Bioinformatics is then used to predict the target genes of miRNAs,and select candidate miRNAs related to IFN-? induction from up-regulated miRNAs from the perspective of participating in intracellular IFN-?-induced signal regulation.Phenotypic,effector miRNAs that inhibit IFN-? production and promote HAV replication are screened by functional deletion and functional acquisition experiments.Mechanistically,target gene prediction was performed on the selected effect miRNA,and the target gene of the miRNA was identified by the Dual-Iuciferase reporter gene system,and the target gene of the miRNA was determined by qPCR and Western blotting in the HAV-infected cell model.Results:miRNA microarray experiments screened out 457 HAV-induced dysregulated miRNAs.Using the key adaptor protein in the IFN-?-inducible signaling pathway RIG/MAVS and TLR of RNA virus as a potential target gene for differentially expressing miRNAs as a screening criterion,a total of 13 miRNAs which target the key adaptor protein in the IFN-?-inducible signaling pathways were screened.By qRT-PCR,it was found that when hsa-miR-146a-5p was overexpressed,the expression level of IFN-? in the overexpression group was 0.5-fold that of the control group(P<0.01),and the copy number of HAV was 2.2-fold that of the control group(P<0.05).While silence hsa-miR-146a-5p expression,IFN-? production was increased(P<0.05)and HAV copies significantly decreased(P<0.001),they were 2.9-fold and 0.6-fold that of the control group,respectively.These results indicate that hsa-miR-146a-5p promotes the antagonism of HAV to the host cell IFN-? signaling pathway.Further target gene prediction displays that TRAF6 is an adaptor protein molecule that miR-146a-5p targets in the IFN-? inducible signaling pathway.The results of the Dual-luciferase reporter system showed that compared with the pmirGLO plasmid control group,mi-nc and wild-type plasmid pMIR-TRAF6-3UTRl co-transfected group,The fluorescence signal of the miR-146a-5p and wild-type plasmid pMIR-TRAF6-3'UTR1 co-transfection group were significantly attenuated(P<0.0001),they were 0.26-fold and 0.30-fold that of the two control group,respectively,indicating that TRAF6 is the target gene of has-miR-146a-5p;At the same time,when hsa-miR-146a-5p was overexpressed,the expression of TRAF6 was significantly decreased at mRNA and protein levels(P<0.05,P<0.01),which were 0.64 and 0.55-fold of the control group,respectively;After silencing has-miR-146a-5p,TRAF6 increased significantly at mRNA level and protein level(P<0.05),which were 3.5-fold and 2.85-fold higher than that of the control group.These results indicate that hsa-miR-146a-5p inhibits the expression of TRAF6 at the transcriptional level.Conclusions:The expression of hsa-miR-146a-5p was up-regulated after HAV-infected cells,and HAV-induced hsa-miR-146a-5p inhibited the IFN-?-induced signaling pathway by inhibiting the expression of TRAF6 at the transcriptional level.These findings provide new insights into the mechanisms of interaction between small RNA viruses and host cells.