| Tembusu virus(TMUV),a new flavivirus emerged in China since 2010,spreads quickly and mainly infect waterfowls,including egg duck,breed duck and goose and the like with a morbidity nearly 100%.Now,TMUV has become one of the major diseases harming China's waterfowl breeding industry.Exosomes are membrane vesicles produced by cells,participating in a variety of pathophysiological processes and mediating intercellular substance transport and information transmission.Some virus-infected cells-derived exosomes contain viral proteins and nucleic acids,which could transmit the virus infection toward other cells and help the virus escape the immune response,playing an important role in the infection,but whether TMUV-infected cell-derived exosomes have the same characteristics is still unclear.In this study,exosomes extraction scheme for TMUV infected cells-derived exosomes was optimized,specifically from three aspects of cell types,maintenance time and extraction methods.Firstly,three commonly used cells,HEK293 cells,C6/36 cells and DEF cells,were selected for comparing the output of exosomes after infection.BCA protein concentration determination showed the highest output of exosomes in HEK293 cells,which was then determined as the cells used in following analysis.Next,after inoculation of TMUV in HEK293 cells,supernatant was collected at 36 h and 48 h for exosomes isolation,respectively,and BCA analysis showed that the exosomes isolated after 48 hour's maintenance was slightly more than 36 hour's,but difference was insignificant.Meanwhile,considering the increased probability of cell death,injury or exosomes disruption with maintenance time,the 36 hour's was finally selected as the optimal maintenance time for exosomes isolation.After then,this study also compared the exosomes isolation rate of traditional overspeed centrifugation method,kit and ultrafiltration combined with the kit.Results showed that the concentration of the samples extracted by the first two methods is about 1 ?g/?L,while the last method was about 12 ?g/?L,significantly higher than the other two methods,and similar conclusions were also appeared in Western Blot analysis.Finally,this study optimized the exosomes extraction scheme for TMUV infected cells-derived exosomes,and established a method of extracting exosomes from HEK293 cells infected with TMUV and using ultrafiltration combined with kit after 36 hours' maintenance.Then,immunomagnetic beads labeled with anti-cd63 antibody were used to screen exosomes for purification.And,the purified exosomes samples were used in Western Blot analysis,flow cytometry analysis and photographed by transmission electron microscopy.Results showed the CD63,CD9 and CD81-positive of those samples,while the positive rates of CD63 and CD81 are between 56.5%~84.2%,and TMUV E protein were detected in the TMUV infected cells-derived exosomes;transmission electron microscopy showed a complete structure about 20~200nm and classic saucer like structure with no contamination of other components,demonstrating that the exosomes prepared by the optimized scheme in this study have a large number and good purity,which can be used for follow-up research.Twelve primers were designed according the whole genome of TMUV SDMS strain and used in the detection of TMUV genome in exosomes.Results showed that TMUV infected cells-derived exosomes contain the whole genome of virus,and the genome variability is also small.After then,C6/36 cells were inoculated with TMUV infected cell-derived exosomes,which induced obvious cytopathic changes,while IFA assay and q PCR viral load assay also showed that those exosomes shared similar infection ability as virus.In addition,hyper-immune serum was also produced in this study for exosomes and TMUV neutralization.The results of IFA showed that the infection ability of neutralized TMUV decreased significantly,with only weak fluorescence being observed,but the infection ability of the neutralized exosomes was the same as that of the original exosomes or virus before neutralization,indicating that the exosomes mediated infection can effectively avoid the neutralization effect of antibodies and assist the virus to complete the immune escape. |
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