Pyroptosis Induced By SFTSV Infection And Effect Of Pyroptosis On Virus Proliferation

ObjectiveTo investigate the death pattern of Vero(African green monkey kidney cells)and THP-1(Human myeloid leukemia mononuclear cells)caused by severe fever with thrombocytopenia syndrome virus(SFTSV)infection,verify the existence of pyroptosis,and regulate pyroptosis to explore its effect on virus proliferation,so as to provide new ideas for studying the pathogenic mechanism of SFTSV and the treatment of infection.Methods1.Cell death caused by SFTSV infectionCytopathic effect(CPE)was observed at different time points after SFTSV infection at different multiplicity of infection(MOI).CCK-8,trypan blue exclusion test and LDH release test were used to reflect the cell viability,mortality rate and cell membrane damage.2.PCD forms in Vero and THP-1 cells infected with SFTSVThe cells were pretreated with different death inhibitors,and then infected with virus.The cell viability of different treatment groups was detected by CCK-8 kit,and the results of flow cytometry with Annexin V-FITC and PI double staining after cell infection and the proportion of TUNEL positive cells were detected to reflect the possible cell death form.The levels of related markers in the pyroptosis pathway were detected by qRT-PCR,Western blot,and ELISA to confirm the existence of pyroptosis.3.Effect of pyroptosis on viral proliferationVX-765 was used to inhibit the key marker of pyroptosis caspase-1,or lentivirus transfection was used to silence or overexpress caspase-1.The virus titer in the cell culture supernatant of different treatment groups was detected by plaque formation assay to reflect the effect of pyroptosis on virus proliferation.4.Statistical AnalysisSPSS 26.0 software was used to analyze the experimental data.Each group of experiments was repeated 3 times,and the differences between groups were compared by t test or one-way ANOVA,and multiple comparisons were performed by Dunnett-t test,α=0.05.Image rendering was performed using GraphPad Prism 9.0 software.Results1.SFTSV infection caused cell deathSFTSV infection could cause CPE in Vero and THP-1 cells,which gradually appeared cell shrinkage or swelling,lysis,increased cell debris,increased refractive index and detachment of Vero cells with lesions,increased adhesion of THP-1 cells,cell clumping and extending pseudopodia.The cell viability and cell death rate were significantly changed at 48h and 72h after infection,and the cell membrane damage could be detected at 12h after infection.2.PCD forms in Vero and THP-1 cells infected with SFTSVAfter SFTSV infection,the viability of Vero cells treated with different death inhibitors increased to varying degrees.With the increase of MOI and the persistence of infection,the proportion of early apoptotic cells in Vero cells showed a significant increase trend,but the proportion of pyroptosis and late apoptotic cells did not increase significantly.The proportion of pyroptotic cells and late apoptotic cells increased significantly in the early stage of THP-1 cell infection.At a MOI of 10,the proportion of early apoptotic cells increased significantly from 48h to 72h after infection,but the proportion of pyroptotic cells and late apoptotic cells remained unchanged.After the infection of Vero and THP-1 cells,the proportion of TUNEL positive cells increased significantly,and pretreatment with VX-765 could inhibit it.After infection,the mRNA and protein expression levels of pyroptosis-related genes were increased in both cells.The release of IL-1β in Vero cells was increased,and the release of IL1β and IL-18 in THP-1 cells was increased,which could be inhibited by VX-765.3.Effect of pyroptosison viral proliferationVX-765 could down-regulate the expression of caspase-1 protein and inhibit the proliferation of SFTSV in Vero cells infected with SFTSV.The mRNA level of caspase-1 in Vero cells was down-regulated by LV-CASP1-RNAi,and the virus titer in the cell supernatant was decreased.The transfection of LV-CASP1 in THP-1 cells could induce the overexpression of caspase-1 and significantly increase the virus titer in the supernatant of THP-1 cells.Conclusion1.SFTSV infection can cause pyroptosis of Vero and THP-1 cells,and it is speculated that there is mutual transformation with other forms of death.There were apoptosis,pyroptosis and necroptosis simultaneously in Vero cells,and apoptosis was the main one.Pyroptosis occurred in THP-1 cells in the early stage of infection,and the proportion of apoptosis increased in the late stage.2.Pyroptosis can affect the proliferation of SFTSV,and the virus titer in the cell culture supernatant is positively correlated with the expression of caspase-1.