外泌体介导小反刍兽疫病毒疫苗株N75-1在细胞间的传播

Role of exosomes in mediating the intercellular transmission of peste des petits ruminants virus vaccine strain N75-1

本研究旨在探究外泌体介导小反刍兽疫病毒(peste des petits ruminants virus,PPRV)在细胞间传播的作用。通过免疫荧光、Western blot和TCID50测定等方法检测PPRV疫苗毒株N75-1感染Vero细胞源外泌体(PPRV-exosome)共培养对细胞(正常Vero细胞)PPRV感染和复制的影响。结果显示,分离纯化到典型的"杯托状"外泌体,且PPRV-exosome中含有完整的PPRV基因组。外泌体共培养结果显示,与PPRV-exosome共培养的细胞中出现明显细胞病变;与正常细胞源外泌体(Mock-exosome)共培养细胞相比,PPRV-exosome共培养组可见PPRV阳染细胞且病毒增殖水平显著增加(P<0.05)。进一步研究表明,外泌体抑制剂(GW4869)可导致外泌体介导的PPRV在细胞中的复制呈现剂量依赖性降低。以上结果表明,外泌体能够介导PPRV感染性RNA在细胞间传递并建立增殖性感染。

Peste des petits ruminants(PPR)caused by peste des petits ruminants virus(PPRV)infection has the characteristics of rapid onset and high fatality rate,which is currently affecting the development of animal husbandry.Exosomes are intraluminal vesicles formed by late endosomes or multivesicles.They are important carriers for cell-to-cell transport and can be"held and used"by viruses during virus transmission.This study aimed to explore the role of exosomes in mediating the spread of PPRV between cells.In this study,the virus infection and replication in Vero cell incubated with the exosomes(PPRV-exosome)isolated from PPRV-infected Vero cells were analyzed by immunofluorescence,Western blot and TCID_(50) assay.The results showed that the typical"cuplike"exosomes were isolated and purified,and the exosomes from PPRV infected cells contained complete PPRV genome.Moreover,the obvious cytopathic effect(CPE)was observed in the cells co-cultured with PPRV-exosome.As compared with the cells incubated with exosomes purified from normal cells(Mock exosome),PPRV staining cells and significantly increased virus levels were observed in the cells incubated with PPRV-exosome(P<0.05).Further study showed that pretreatment of cells with GW4869(exosome inhibitor)caused a dose-dependent decrease in the level of exosome-mediated PPRV proliferation in cells.These results demonstrate that PPRV-infected cells derived exosomes can carry infectious PPRV nucleic acids to establish proliferative infections in recipient cells.