ABSTRACT
Objective:To explore the effect of duck Tembusu virus infection on secretion of exosomes in BHK-21 cells and the pathogenesis of the Tembusu virus.Methods:The exosomes were collected and purified from the culture supernatant of BHK-21 cells infected with duck Tembusu virus AH-F10 strain and the control BHK-21 cells by PEG precipitation method respectively.The purified exosomes were identified by electron microscopy,Western blot assay and mass spectrometry.Results: The classical exosome particle morphology was observed with hyperchromic cup-shaped vesicles and average particle size of 30-160 nm in diameter under transmission electron microscopy.The mean size of the exosome from the infected cells were bigger than the mean size of the exosome from the unin-fected.Western blot assay demonstrated that CD9 and CD63 were detected in purified exosomes as exosome marker molecula.A total of 106 proteins were identified by mass spectrometry assay,84 proteins of infected BHK-21 cells exosome,49 proteins of the uninfected, and the infected and the uninfected BHK-21 share 27 common proteins on exosomes.Conclusion:Duck Tembusu virus infection affect the exosome secretion of cells in connection to the particle size and protein molecular composition.This experiment can lay the foundation for further research of Tembusu virus infection and pathogenesis.
ABSTRACT
In order to determine the amino acid sequence of functionthat play a role of methyltransferase (MTase) activity in duck Tembusu virus (DTMUV),we have made sequence analysis and structure prediction of the MTase in DTMUV by bioin formatics methods,as well as using the sequence and structure of MTase in other flaviviruses which have been reported.The bioinformatical analysis results showed that the MTase and the MTase of other three kinds of flaviviruses had the higher homology in nucleotide sequence and amino acid sequence,for 64.76％,64.55％,67.33％ and 74.71％,68.45％,75.15％ respectively,and the same basic structural characteristics which contained SAM binding site and had a typical of 4 alpha screw and 7 beta folding.At the same time,there were classical conservative sites K-D-K-E of flavivirus MTase in DTMUV MTase sequence.In conclusion,they imply that the MTase of DTMUV might belong to MTase family of flaviviruses.