| Objective:Dengue fever is the most widespread mosquito-borne disease caused by dengue virus(Dengue Virus,DV)infection and is a major threat to world health.Therefore,there is an urgent need to develop vaccines and antiviral drugs against dengue virus.Antibody-dependent infection enhancement effect(ADE)directly affects the effectiveness of neutralizing antibody production of atypical dengue virus.Dengue virus infection can induce monocytes to produce type I interferon(IFN-I)and inhibit virus replication.In this study,human peripheral blood monocytes THP-1 cells from patients with acute monocytic leukemia were used as the object of study to establish an in vitro model of antibody-dependent infection of dengue virus.The changes of intracellular innate immune system caused by dengue virus infection were studied to explore the differentially expressed genes in IFN-I signaling pathway in DENV-ADE effect,and to further screen out the key signal molecules and predict their possible regulation.Methods:The infectious titer of DENV-3 was stable after being isolated from the serum of patients with clinical infection and sub-cultured on C636 cells.The gene sequences traced to the origin of the virus were analyzed by MEGA6.0 software,and the homology and mutation between the virus and some closely related sequences were analyzed by BIO-EDIT,and the homologous recombination analysis was carried out by SimPlot software.The anti-dengue serotype virus 2(DENV-2)prM antibody was diluted and mixed with DENV-3 of different MOI to infect THP-1 cells.The total RNA of the cells and the viral RNA viral load in the cell supernatant were detected.To explore the best conditions for establishing the best in vitro model of dengue virus antibody-dependent infection enhancement effect,including the optimal antibody dilution and virus infection of MOI.Then the DENV-ADE cell model was established under the best conditions.The total RNA,of control group,virus infection group(DENV)and antibody dependent infection enhancement group(DENV-ADE)cells were collected at 2 h,6 h,12 h,24 h and 48 h after virus infection,respectively.Using IFN-I response signal pathway PCR array chip to three groups at these five time points.The total RNA of cells was detected.The differentially expressed genes were analyzed online through the PCR Array data analysis website,the results were verified by Q-PCR and Western Blot,and KEGG was used to explore the IFN-I response signal pathway and signal molecules that may be regulated in the DENV-ADE effect.Results:A stable strain of DENV-3,was isolated and identified from the serum of dengue patients in Xishuangbanna,Yunnan Province in 2013,which can be used in the next experiment.Sequence analysis showed that the strain was closely related to the epidemic strains in other parts of China in the same year,such as YN01,HN/2013/22 and MN1302,homology analysis showed 99%,base analysis and amino acid analysis also showed low mutagenicity.Homologous recombination analysis showed that the virus strain was highly consistent and there was no recombination signal.By exploring the modeling conditions of different antibody dilution and different virus MOI,it was found that the dengue virus ADE effect could be observed in cells treated with different virus MOI,and from the change of virus copy number inside and outside the cell,it was found that when the antibody dilution was 1 to 256.the results showed that the dengue virus ADE effect could be observed by different antibody dilution and virus ADE treatment.The best viral enhancement effect can be obtained.After the antibody dilution was 1/256 and the virus was treated on THP-1 cells for different time,the expression of 7 genes was significantly changed by PCR array microarray detection of IFN-Ⅰ response signal pathway.After verification by Q-PCR and Western Blot,the gene IL6 was not detected,and the expression results of five genes were consistent with the results of microarray:after 2 hours,the expression of gene NOS2 increased in DENV group and decreased in DENV-ADE group.The expression of gene IFNB1 and IFI27 in DENV group decreased at 6 hours,while that in DENV-ADE group decreased slightly,and the expression of gene MET and DENV-ADE in DENV group decreased at 12 hours.After 24 hours,the expression of gene IFNB1 decreased and DENV-ADE decreased in DENV group.After 48 hours,the expression of IFNA2 and DENV-ADE in DENY group decreased.Conclusion:A stable dengue virus serotype 3 strain was isolated and sub-cultured,and the in vitro model of ADE was established on THP-1 cells by using prM antibody and DENV-3 virus.When the dilution of MOI=0.3,and antibody was 1 to 256,It is the best condition to enhance the replication and proliferation of virus in this study.After THP-1 cells were treated with DENV and DENV-ADE,the expression of EFN-I related genes increased and weakened,and the expression of most of them showed a downward trend.Such as IFNB1,IFI27,MET,IFNA2.The changes of gene expression were different at different time points,such as the change of NOS2 expression at 2h,but there was no difference after that,indicating that the related genes of IFN-I did play a certain role in the enhancement of dengue type III virus antibody-dependent infection.And there is a difference between the early and late stages.The above results show that some of the key signal molecules in the IFN-I signaling pathway are one of the main mechanisms of DENV-ADE. |
PDF Full Text Request