| Background:Dengue fever is an acute infectious disease caused by dengue virus infection and transmitted by Aedes albopictus.At present,dengue fever has become the most widely distributed and frequently occurring arbovirus disease in the world.However,the pathogenesis of dengue fever is not fully understood,and it is currently widely believed that severe dengue fever is related to antibody dependent enhanced infection(ADE).Although there has been increasing attention and research on antibody dependent enhanced infection mediated by dengue virus in recent years,the specific molecular mechanism remains unclear.Due to the lack of suitable ADE animal models and the incomplete elucidation of the specific molecular mechanisms of virus host interaction in ADE effects,the exploration of the pathogenesis of dengue fever has been seriously hindered,becoming one of the main bottlenecks in drug and vaccine development.Methods:The differential expression profiles of long non-coding RNA(lncRNA)in THP-1 cells after DENV-3 direct infection(DENV group)and antibody dependent enhanced infection(ADE group)were detected and analyzed by RNA Seq.At the same time,in order to better study the pathogenesis of severe dengue fever,a dengue fever infection model and an antibody dependent enhanced infection animal model were established in IFNAR mice with type I interferon deficiency.The survival rate,body weight,clinical score,viral nucleic acid load in serum and tissue,whole blood cell count,and organ damage were monitored in the infected mice,Detect the expression of cytokines and changes in lymphocyte and neutrophil related antigens.On the basis of successful modeling,64425 single cells from mouse liver samples of two NC groups,two DENV groups,and two ADE groups were sequenced by single cell RNA sequencing(scRNA seq)and spatial transcriptome sequencing.By combining the more detailed cell group results obtained by single cell sequencing with the spatial location information obtained by spatial transcriptome sequencing,we further understand the function of cells and further reveal the pathogenesis of dengue fever.Results:A large number of differentially expressed lncRNAs(DE lncRNAs)were found in NC group,DENV group and ADE group by RNA Seq.Eight DE lncRNAs were verified among the three groups by qRT-PCR.It was found that their expression trend was consistent with the sequencing results of the transcriptome.In addition,it was found that IncRNA not only participates in the regulatory network of CeRNA,but may also encode small peptides.Based on the successful establishment of animal models in the DENV and ADE groups,single cell sequencing was conducted and six samples were analyzed to screen for 8 completely different cell types,which can be further divided into 24 cell subtypes.In NC group,DENV group and ADE group,T cells,portal pericyte and B cells increased in turn,among which portal pericyte(mainly Apoc1+portal pericyte)increased most significantly;Functional enrichment analysis revealed that these cells are mainly.involved in complement activation,cascade amplification,and the PRAR signaling pathway.In addition,the comparison also found that in the NC group,DENV group,and ADE group,neutrophils and macrophages decreased sequentially,with the most significant decrease being in macrophages(mainly P1a2g7+macrophages);Functional enrichment analysis revealed that these cells are mainly involved in the migration of white blood cells.By combining the results of scRNA seq with spatial transcriptome,we found that the distribution of sppl+epithelial cells(cluster9 and cluster21)in ADE increased.Functional enrichment analysis revealed that these cells are mainly involved in injury response and repair,cell adhesion,and vascular development regulation.Through cell communication analysis,it was found that sppl+epithelial cells(cluster9 and cluster21)can interact with neutrophils,macrophages,B cells,and T cells,with the most significant interaction with neutrophils and macrophages.Moreover,spp1+epithelial cells(cluster21)can specifically participate in the regulation of the EGF signaling pathway.Conclusion:This study analyzed the differential lncRNA expression profile after DENV-3 direct infection and antibody dependent enhanced infection of THP-1 cells.RTqPCR was performed to verify the differential lncRNA among groups,and the Functional verification of differential lncRNA against DENV-3 infection was performed.In this study,we established a non fatal ADE mouse model with IFN-α/β Receptor deficiency and IFNy This is the first report on a mouse model of low-dose infection and ADE infection with complete receptor signaling pathways.Compared to the traditional method of infecting pregnant mice with one serotype to immunize them,transmitting maternal antibodies to offspring,and then infecting newborn mice with another serotype to establish a dengue virus ADE infection model,this study used commercialized dengue virus antibodies to incubate with another serotype virus stock and infect mice.The modeling conditions were more stable and reliable,and the repeatability was strong.And through single cell sequencing and spatial transcriptome sequencing,it was found that the portal vein cells mainly expressed in the liver of NC group were Ttr+portal pericyte(cluster7),but the distribution of Hba-a2+portal pericyte(cluster6)increased during dengue virus infection,and the distribution of Spp1+Col3a1+epithelial cells(cluster9)and Spp1+Cluster+epithelial cells(cluster21)increased during antibody dependent infection,And during the process of dengue virus infection and antibody dependent enhanced infection,changes in the spatial release of the aforementioned cells may occur.These changes do not exert biological functions through a single pathway,but rather through interactions with various immune cells to exert biological functions. |
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