In vivo dynamic distribution of multivesicular bodies and exosomes in spleen of DTMUV infected duck.

Exosomes are vesicles secreted by the multivesicular bodies (MVBs), which have been shown to mediate immunity regulation and virus transmission. In this study, the dynamic distribution and function of the MVBs and their exosomes was investigated through morphological characterization and molecular analyses in duck spleens infected with duck Tembusu virus (DTMUV) at different times post infection (1hpi, 2hpi, 12hpi, 24hpi). CD63, the marker of MVBs and exosomes, was distributed in the sheathed capillaries and the periellipsoidal lymphatic sheaths (PELS) of the white pulp. The numbers of MVBs and their exosomes were dramatically increased at 2 hpi, and with the increasing infection time, the numbers of MVBs and their exosomes were gradually decreased. DTMUV proteins were associated with exosomes according to double label immunofluorescence results. Ultrastructural characterization by transmission electron microscopy revealed four developing stages of MVBs containing exosomes were detected in high endothelial cells of the sheathed capillaries, lymphocytes and the ellipsoid-associated macrophages in PELS. Free exosomes were observed in the extracellular matrix and the blood vessels. Genes and proteins related to the endocytosis pathway were obviously up-regulated at 2 hpi as confirmed by RT-qPCR and western blotting. We speculated that DTMUV mediates host invasion through the endocytosis pathway by utilizing MVBs and their exosomes. The in vivo distribution pattern of MVBs and their exosomes in DTMUV infected spleens is shown for the first time in this study. This report could lay the foundations for understanding the infection mechanism of DTMUV.