Study On The Mechanism Of Splenic Fibroblastic Reticular Cells And Autophagy Induced By Severe Fever With Thrombocytopenia Syndrome Virus Infection

Background and objective: Sever fever with thrombocytopenia syndrome virus（SFTSV）is a tick-borne negative-strand RNA virus that can cause SFTS with a case fatality rate of6.18%-27%.Autophagy is an evolutionarily conservative process that maintains balance in the host through the degradation of damaged proteins or organelles.Autophagy can exert an antiviral response when different viruses invade cells.Our team found that the target cells of SFTSV fatal infection are mainly splenic fibroblastic reticular cells（FRCs）,indicating that FRCs virus infection plays an important role in severe illness and death.Therefore,to explore the situation and mechanism of spleen FRCs autophagy after SFTSV infection is helpful to reveal the mechanism of antiviral response and virus escape at the main target cell level.Methods: In this study,we established a target cell line FRCs in vitro to study the mechanism of SFTSV infecting FRCs and inducing autophagy.After SFTSV was infected with FRCs,the viral load of virus replication at different time points was detected by quantitative real-time PCR,and the changes of autophagy levels at different time points were detected by Western blot.The plasmids of nonstructural protein NSs and nucleocapsid protein NP of S segment of SFTSV,namely pCAGGS-HA-NSs and pCAGGS-HA-NP,were constructed in vitro.Western blot,quantitative real-time PCR and immunofluorescence were used to study the change trend of autophagy in FRCs infected with SFTSV,inactivated SFTSV or FRCs transfected with viral NSs and NP proteins.Western blot and confocal microscopy were used to detect the change of autophagy flux caused by SFTSV infection or virus NSs and NP proteins,and to further study the mechanism of autophagy.We continued to study the effect of SFTSV protein plasmid transfection into FRCs on autophagy from the autophagy pathway.Western blot and immunofluorescence were used to study how SFTSV proteins pCAGGS-HA-NSs and pCAGGS-HA-NP affect autophagy by regulating the autophagy signal pathway ERK1/2-mTOR.Results: We found that after SFTSV was infected with FRCs,the viral load of SFTSV increased significantly 8h after infection,and the virus replicated rapidly.The autophagy level of FRCs increased at 8 h,12 h and 24 h after virus infection,but decreased significantly at 48 h and 72 h after virus infection.After heat-inactivated SFTSV was infected with FRCs 12 h,the level of autophagy increased.In addition,the plasmids pCAGGS-HA-NSs and pCAGGS-HA-NP of SFTSV were successfully expressed in vitro after transfection of FRCs,and autophagy could be induced at 24 h after transfection.The results showed that autophagy flux increased 12 h after SFTSV virus infection or 24 h after virus protein transfection into FRCs,and SFTSV and NP proteins were co-located with autophagy marker LC3,respectively.Further study on autophagy signal pathway showed that the level of ERK1/2 phosphorylation increased and the level of mTOR phosphorylation decreased in FRCs transfected with NSs and NP proteins.After the addition of pathway inhibitor U0126,the level of ERK1/2 phosphorylation decreased,the level of mTOR phosphorylation increased,and the level of autophagy decreased.Conclusions: All in all,the virus-induced autophagy we studied in FRCs mainly occurred in the early stage of viral infection and decreased in the late stage.The results showed that both SFTSV and heat-inactivated SFTSV induced autophagy in the early stage of infection and 24 h after SFTSV protein plasmid transfection.And SFTSV NSs and NP protein plasmid transfection can affect autophagy by regulating ERK1/2-mTOR signal pathway.These findings suggested that autophagy occurred in FRCs in the early stage of infection after SFTSV infection of target cell FRCs,and autophagy disappeared with the prolongation of infection time.These results lay a foundation for the subsequent study of the pathogenesis and the role of autophagy in the immune response to virus replication and virus infection.