In Vitro Model Study Of Dengue Virus Infection In Human Bone Marrow Mesenchymal Stem Cells

Dengue virus（DENV）belongs to the family of flaviviridae RNA virus,which is transmitted by Aedes aegypti mosquitoes and infects people with mild symptoms such as fever,myalgia and rash,and even dengue haemorrhagic fever（DHF）,and may also appear with the risk of shock Dengue shock syndrome（DSS）.Since its discovery in1779,DENV outbreaks have been characterized by high outbreak frequency and wide spread,and have gradually developed into a global problem.The virus is not only a threat to human health,but also a huge disruption to economic development.Antiviral drugs and preventive vaccines for DENV are still not available,and it has been classified as a major global public health problem.The current cellular models for DENV research are mainly of mosquito origin and murine origin,and there is still a deficiency in human in vitro models.However,human bone marrow mesenchymal stem cells（h BMSC）are not only a hematopoietic organ but also an important immune organ,and using h BMSC as a cellular model for DENV infection is a good way to study the mechanisms of hemorrhagic fever and strong immune response caused by DENV.The use of h BMSC as a cellular model of DENV infection has superior applicability for studying the mechanism of the development of clinical symptoms such as hemorrhagic fever and strong inflammatory response caused by DENV.Currently,there is no study on DENV infection model on h BMSC,based on this,this thesis carried out an in vitro model study of DENV infection on h BMSC.The main results obtained in this thesis are as follows:The four representative DENV serotypes prevalent in Yunnan Province were firstly selected and isolated from plasma using C6/36 cells.The genotypes of DENV-1,2,3 and 4 serotypes were type I,Asian type I,type III and type I,respectively;RT-q PCR analysis showed that the viral load increased from 10⁷copies/m L to 10⁹-10¹⁰copies/m L after three generations of blind transmission;indirect immunofluorescence assay showed that all four serotypes could The titers of DENV-1,2,3 and 4 serotypes were 1.5x10⁷PFU/m L,3.75x10⁷PFU/m L,2x10⁶PFU/m L and5.562x10⁷PFU/m L,respectively,as measured by the phospho-spot method.The next step was to establish an in vitro infection model for h BMSC.Next,a cellular model of h BMSC infection by DENV four serotypes was established.By observing the cytopathic lesions,it was found that all four DENV serotypes caused cytopathic lesions,among which serotypes DENV-2 and 4 caused severe cytopathic lesions in h BMSC with a lesion rate of over 90%at 96 hpi,and serotypes DENV-1 and 3 had a lesion rate of 70%and 50%at 96 hpi,respectively;RT-q PCR analysis showed that the viral load of all four DENV serotypes in the supernatant The RT-q PCR analysis showed that the viral load of all four DENV serotypes in the supernatant entered a plateau at 48 hpi;the positive and negative chain assays showed that the positive and negative chains were detected simultaneously at all time points and the trend was consistent;the virus titers in the supernatant were measured by the phlebotomy method showed that the virus titers of DENV-2 and 4 serotypes peaked at48 hpi and then decreased,while the virus titers of DENV-1 and 3 serotypes increased with time;the indirect immunofluorescence assay showed that all four DENV serotypes stably expressed DENV-NS1 protein in h BMSC;inflammatory factor assays showed that the expression of inflammatory factors IL-4,IL-6,IL-8,IL-10,IL-Iβ,IFN-α,IFN-β,IFN-γ,TNF-α,and MCP-1 were all elevated after DENV infection,with DENV-2and 4 infection caused the most significant changes.This experiment confirmed that all four DENV serotypes were susceptible to h BMSC,especially DENV-2 and DENV-4,and established an in vitro infection model of h BMSC infection by four DENV serotypes.Finally,the analysis of differentially expressed proteins after h BMSC infection with DENV-2 showed that a total of 272 differentially expressed proteins were identified,of which 158 proteins were up-regulated and 114 proteins were down-regulated;GO functional analysis showed that differentially expressed proteins mainly act on cellular anatomical entities and protein complexes,through cellular processes,metabolic processes,biological regulation and stimulation of response and signaling KEGG analysis showed that a total of 195 signaling pathways were enriched,mainly lysosomal,EBV infection,HPV infection,RIG-I-like receptor signaling pathway and TNF signaling pathway,indicating that differentially expressed proteins were mainly concentrated in response to viral invasion and immune response pathways;protein interaction network analysis showed that DENV-2infection,the upregulation of SOD2and the downregulation of CTSB contributed to apoptosis in response to the pathogenicity of DENV-2;three other key proteins,TNFAIP6,THBS1 and CCN2,were also identified,which may be associated with DENV infection resulting in clinical symptoms such as hemorrhagic fever and strong immune response,and have not been reported in the relevant literature.In summary,in this study,through the isolation and culture of four DENV serotypes and the establishment of a cellular infection model on h BMSC,and the study of proteomic differences after DENV-2 infection of h BMSC,we revealed the genotypes of the four serotypes,the infection characteristics of h BMSC and the differential expression between the proteins caused,and more comprehensively understood the process of DENV infection with the host This provides an important reference for elucidating the mechanism of DENV-induced hemorrhagic fever and strong immune response,identifying drug targets associated with DENV infection,and searching for new antiviral strategies.