Effects Of Severe Fever With Thrombocytopenia Syndrome Virus On TLRs Of THP-1 Cells

Objectives:To investigate the effects of severe fever with thrombocytopenia syndrome virus on toll-like receptors expression of THP-1 cells.Methods:1.SFTSV JS14 was cultured in well-grown Vero cells.Culture supernatant was collected to test copies of viral genomes every 24 h and the optimal harvest time was selected to harvest virus.SFTSV was condensed with Millipore centrifuge tube and conserved at-80? for next use.2.A series of 10-fold dilutions of SFTSV were added into well-grown Vero cells with 200 ul every well.Vero cells were overlaid with 3 ml autoclaved methylcellulose-DMEM covering every well,the covering was removed gently by PBS after 9 days.Then,the Vero cells were fixed with 4 % pre-cooling formaldehyde and stained with crystal violet.After washing three times by PBS,the plaques were counted.Unit:PFU/ml.3.SFTSV was inoculated onto THP-1 cells in 6 well plates with 0.1 multiplicity of infection(MOI).Experiment was divided into control group and SFTSV group.cells were collected at 24 h,48 h and 72 h,respectively.The m RNA levels of TLR2,TLR3,TLR4,TLR7 and TLR9 was measured by fluorescence quantitative PCR.In addition,the protein expression of TLR2,TLR3,TLR4,TLR7 and TLR9 on THP-1was detected by flow cytometry.Results1.SFTSV was cultured in Vero cells.(1)During the process of severe fever with thrombocytopenia syndrome virus culture,virus reached the peak of replication at 4th day.Virus supernatant could harvest after 4 days.(2)Plaques were clearly visible by methylcellulose plaque assay.2.Expression of TLRs:(1)The expression of TLR2 protein and m RNA on the surface of THP-1 cells of SFTSV group was increased compared with the control group and there are statistical differences(P<0.05),of which the TLR2 increased obviously at 48 hours.In SFTSV group,the expression of TLR2 at 48 h showed significantly difference compared with 24 h and 72 h(P<0.05)but no statistical difference was found between 24 h and 72 h.(2)TLR4 increased higher than TLR2.Expression of TLR4 protein and m RNA on the surface of THP-1 cells of SFTSV group was increased compared with the control group and there are statistical differences(P<0.05),of which the TLR4 increased obviously at 48 hours.In SFTSV group,the expression of TLR4 at 48 h showed significantly difference compared with24 h and 72 h(P<0.05)but no statistical difference was found between 24 h and 72 h.(3)TLR3 protein and m RNA in the THP-1 cells of the SFTSV group were not significantly increased at 24 h compared with the control group,but increased at 48 h and 72 h and there are statistical differences(P<0.05).In SFTSV group,the expression of TLR3 at 24 h showed significantly difference compared with 48 h and72 h(P<0.05).(4)TLR7 protein expression lower in THP-1 cells.Compared with the control group,TLR7 protein and m RNA in SFTSV group did not increase significantly at 24 h,but increased at 48 h and 72 h and there are statistical differences(P<0.05).In SFTSV group,the expression of TLR7 at 24 h showed significantly difference compared with 48 h and 72 h(P<0.05).(5)There was no significant change in TLR9.Conclusions:1.The expression levels of TLR2 and TLR4 on the surface of THP-1 cells,TLR3 and TLR7 in THP-1 had all increased.TLR4 increased most significiantly.There was no significant change in TLR9.