Studies On The Role And Mechanism Of Exosomes During The Infection Processes Of Rabies Virus In MRC-5 Cells And Vero Cells

Rabies virus infection caused about 17,400 deaths in 2015 and it is still a serious public health problem.In recent years,the potential use of cell culture techniques for vaccination against rabies and other viruses has been explored extensively.Human diploid cells(HDCs),and Vero cells are all widely used as substrates for rabies vaccine production.Vaccines derived from HDCs and Vero cells are relatively safer than nerve tissue vaccines,embryonated egg-based vaccines,primary hamster kidney cells,purified chick embryo cells.As early as 1974,the production process of rabies vaccine based on MRC-5 was established through complex domestication and optimization of infection conditions.Owing to MRC-5 cell expansion trains are long duration,cell culture procedures are complex,and cell yields are inefficient.These factors all contribute to the prohibitively high cost of rabies vaccine production in HDCs,which limits its use in developing countries.Vero cells are an immortalized cell line from African green monkey kidney cells,which are deficient in the synthesis of type I IFN(IFN-I)and are a cell strain sensitive to RABV.In 1985,the rabies vaccine based on Vero cells was firstly used for vaccination.In this study,the interaction mechanisms of RABV with host cells(MRC-5 and Vero cells)were investigated to reveal the detailed mechanisms of MRC-5 cells in resistance to RABV infection and Vero cells in sensitivity to RABV infection,so as to provide theoretical basis and technical reference for the production of rabies vaccine based on MRC-5 cells.Exosomes are cell-derived vesicles that are secreted by many mammalian cells and have ability to deliver a variety of active molecules.In recent years,the function of exosomes in virus-host interaction has attracted more and more attention.Therefore,in this study,the investigation on the interaction mechanisms between RABV and host cells mainly focused on the roles and mechanisms ofexosomes in RABV infection of MRC-5 and Vero cells.Our study found that exosomes played a role in the RABV infection of MRC-5 and Vero cells,but their functions and mechanisms were different.The main findings are as follows.(1)OptiPrepTM density gradient centrifugation was used to isolate exosomes from rabies virus-infected cell culture supernatants.(2)Rabies virus infection could induce exosome secretion from MRC-5 and Vero cells.(3)Blocking exosomal release promoted rabies virus infection in MRC-5 cells;conversely reduced rabies virus infection in Vero cells.(4)RABV infection up-regulated the expression of mircoRNA(miR423-5p)and IFN-? in MRC-5 cells,thereby inhibiting RABV infection.(5)Exsomes could mediate miR423-5p for intercellular transmission,and thus enabling adjacent MRC-5 cells to resist RABV infection.(6)Rabies virus infection increased the expression of IFN-? and the IFN signaling associated genes in MRC-5 cells.(7)Rabies virus need or hijack the endosomal sorting complexes required for transport(ESCRT)pathway for their release and the ESCRT pathway has been shown to play a role in the formation and secretion of exosomes.Based on the above findings,this study draws some conclusions:(1)OptiPrepTM density gradient centrifugation can isolate and purify exosomes from the supernatant of RABV-infected cells.(2)RABV infection can induce MRC-5 cells to secrete exosomes,which can carry up-regulated miRNA-423-5p and deliver miR-423-5p from RABV-infected MRC-5 cells to neighbor cells and that miR-423-5p can abrogate the inhibitory effects of SOCS3 on the IFN signaling pathway by targeting the SOCS3 gene,thus resulting in enhancing the ability of MRC-5 cells to resist RABV.(3)RABV infection can induce Vero cells to secrete exosomes,which can further promote the infection of Vero cells by RABV.This study preliminarily reveals the molecular mechanism of MRC-5 cells in resistance to RABV infection and enriches people's understanding of the interaction between RABV and host cells.Therefore,miR-423-5p,exosomes or the IFN-I signaling pathway can be used to improve the production of MRC-5 cell-based rabies vaccine.