The Mechanistic Research On Antibody-Dependent Enhancement Of Dengue Virus Infection In Vitro

Objective:To research the mechanism on antibody-dependent enhancement of dengue virus infection in vitro. Methods:The virus-Antibody complexes were prepared by mixing anti-dengue premembrane (PrM) antibodies with viruses (MOI=3) for90min before adding to the K562cells suspension for infection. Then the infection of dengue virus in K562cells were detected by indirect immunofluorescence stain and Real-Time PCR.The differentially expressed proteins and function on antibody-dependent enhancement of dengue virus infection were analyzed by2-D electrophoresis and identified by MALDI-TOF MS technology and G-O.Then we selected differentially expressed protein to testify the enhancement by Western Blot.At last we detected the the infection of dengue virus on antibody-dependent enhancement by RNAi and Real-Time PCR.. Results:It was found that the ADE effect was concentration-dependent. The strongest antibody-dependent enhancement (ADE) of dengue infection occurs when antibodies dilution were at1/2560, but not at others. The infection of dengue virus was significantly enhanced according the results of indirect immunofluorescence stain and Real-Time PCR. The analyses of multiple2-DE gels and MALDI-TOF MS indicated that a total of15protein spots differentially expressed during the process of ADE, including2up-regulated proteins,PFDN1and MYL3,which were confirmed by Western Blot.G-O Database search and identification of functional protein analysis showed that these altered proteins mainly related to protein folding.actin filament-based process,circulatory system process,regulation of ATPase activity.regulation of heart and striated muscle contration. The results of RNAi and Real-Time PCR about PFDN1showed that there was a significantly descent of expression level of dengue virus on antibody-dependent enhancement. Conclusion:We established the model of antibody-dependent enhancement of dengue infection in vitro successfully, and found the related proteins during the process of ADE,which will provide reliable experimental basis for the research on the mechanism of ADE of dengue virus.