Screening And Mechanisms Studies On Chemicals Against Severe Fever With Thrombocytopenia Syndrome Virus Infection

An emerging infectious disease, the severe fever with thrombocytopenia syndrome(SFTS), was reported in rural areas of some provinces in Central China. The pathogen of thisdisease, Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV), was isolated andidentified in2009by Chinese Center for Disease Control and Prevention. SFTSV is asegmented negative sense single-stranded RNA virus, belongs to phlebovirus within thefamily Bunyaviridae. There are no specific treatments available for SFTS at present, andmuch remains unknown about how SFTSV interactions with the host cell and leads to asuccessful infection. Understanding the virus infection process and pathogenesis are criticalfor developing effective interventions of the prevention and treatment of the disease. In thisstudy, we initiated our study by screening of a set of chemicals modulating SFTSV infection,we then focused on the relationship between receptor tyrosine kinase (RTKs) and SFTSVinfection, and we attempted to explore the molecular mechanism of SFTSV infection.We first characterized the viral replication and release of SFTSV in human umbilicalvein endothelial cells (HUVEC) by immunofluorescence staining and qRT-PCR. Duringscreening a set of chemicals, we found two of them can inhibit the infection of SFTSV. Thesetwo chemicals are inhibitors of RTKs and its downstream PI3K/AKT-mTOR pathway, wetherefore analyzed the alternations of phosphorylation status of cell RTKs upon SFTSVinfection. A set of small interfering RNA (siRNA) for different RTKs were also tested fortheir effect on viral infection. We found there were no significant changes between normaland infected cells in morphology. The amount of virus detected in cell pellet increased as atypical "S" shape growth curve over infection time, and the virus titer in culture supernatantincreased to high levels and presented as a "wave" growth mode. These data are consistentwith SFTS typical clinical symptoms thrombocytopenia observed in patients. We foundchemicals BEZ235and Sunitinib can effectively block SFTSV infection when the chemicalwas added along with the virus or2hours after viral innoculation. BEZ235is the inhibitor ofphosphatidylinositol-3-kinase (PI3K) P110subunit and mammalian target of rapamycin(mTOR). Sunitinib is the inhibitor of platelet-derived growth factor receptor beta (PDGFR β)and vascular endothelial growth factor receptor (VRGFR). These results indicated that the SFTSV entry is regulated by the RTKs-PI3K/AKT-mTOR pathway. By analyzing RTKsphosphorylation status, we found that the level of some RTKs (AXL, TrkA, VEGFR, FGFR)phosphorylation was enhanced after infection. Moreover, we demonstrated that knockdown ofNeurotrophic tyrosine kinase receptor type1(TrkA) on HUVEC significantly reduced SFTSVinfection, similarly, silencing of PDGFRβ on Hela cells and VEGFR1on cos7cells can alsoinhibit the infection to some extent. In contrast, the viral infection was increased by knockingdown FGFR1on HUVEC. These results suggested that SFTSV infection may activate someRTKs like TrkA, PDGFR, VEGFR and modulate the RTKs-PI3K/Akt-mTOR signal trans-duction pathway in host cell to complete viral genome replication, transcription, viral proteinsynthesis, assembly and progeny virus release.Our study shed new light on the process of SFTSV infection and help to understand thepathogenesis of the virus. It will also be useful in developing more effective prevention andtreatment against the viral infection.