Research On ShRNA Targeting Non-structural Protein(NSs)Inhibits The Replication Of SFTSV

ObjectiveTo preliminarily explore whether the short hairpin RNA（shRNA）targeting non-structural proteins（NSs）of severe fever with thrombocytopenia syndrome virus（SFTSV）inhibits SFTSV replication,and further study the functions of NSs and its importance in virus replication.This study of sh RNA-mediated antivirus effects may be a potential new tool for inhibiting SFTSV infection.Methods1.The virus was amplified by the infection of Vero cells.The indirect immunofluorescence method and Western Blotting were used to detect the amplified SFTSV.Viral titers were detected by indirect immunofluorescence.2.Construct the sh RNA plasmids targeting SFTSV-NSs,and the constructed plasmids were verified by company sequencing and Eco RI/Nco I double digestion.3.The sequenced SFTSV NSs-shRNA1,SFTSV NSs-shRNA2 plasmid were respectively co-transfected with SFTSV NSs-Flag plasmid to verify whether the plasmid worked by Western Blotting.In the subsequent experiments,the plasmid SFTSV NSs-shRNA1 with good inhibitory effect was selected.4.Vero cells were infected with SFTSV after transfected with Non-Target sh RNA and SFTSV NSs-shRNA1 respectively.Immunofluorescence and Western Blotting were performed to verify whether silencing NSs could inhibit virus infection.5.Vero cells were transfected with 0.8 μg of SFTSV NSs-sh RNA andinfected with SFTSV with the same MOI value.The supernatant was collected at different time（24 h,48 h,72 h）after infection,and the virus titers of the supernatant was measured by TCID₅₀（use 0.8 μg of Non-Target sh RNA as a control and do the same as the interference group）.Vero cells were transfected with SFTSV NSs-sh RNA of different concentrations（0.4 μg,0.8 μg,1.2 μg）and then infected with SFTSV with the same MOI value.Supernatants were collected 24 hours after infection,and the virus titers in the supernatant were measured by TCID₅₀（Non-Target sh RNA is used as a control,the amount and treatment method are the same as those in the interference group）.Vero cells were transfected with 0.8 μg of SFTSV NSs-sh RNA and infected with SFTSV with different MOI values（0.1 MOI,1 MOI,5 MOI）.Supernatants were collected 24 hours after infection,and the virus titers of the supernatants were measured by TCID50（use 0.8 μg of Non-Target sh RNA as a control and do the same as the interference group）.Results1.SFTSV virus was successfully amplified which was confirmed by indirect immunofluorescence and Western Blotting detection.The titer determined by indirect immunofluorescence method after 16 h of virus infection was 2.33×10⁸ FFU/m L.2.After sequencing and agarose gel electrophoresis detection,sh RNA plasmids targeting SFTSV NSs were successfully constructed.3.Western Blotting test results showed that the band of co-transfected plasmids group was weaker than the SFTSV NSs-Flag alone,and the SFTSV NSs-shRNA1 plasmid has a good inhibitory effect.4.Compared with the control group（infected with SFTSV after transfected with Non-Target shRNA plasmid）,the SFTSV group infected with SFTSV NSs-shRNA1 plasmid was transfected with indirect immunofluorescence.The interference of histones weakened,indicating that the constructed SFTSVNSs-shRNA1 plasmid can inhibit the replication of SFTSV.5.Use TCID50 to measure the titers of supernatants at different interference time（24 h,48 h,72 h）,different concentrations of sh RNA（0.4 μg,0.8 μg,1.2 μg）and after interference SFTSV infection with different MOI values（0.1 MOI,1MOI,5 MOI）.Compared with the control group,the results showed that the virus titer of each group was reduced,and the difference was statistically significant（P<0.05）,indicating that the constructed SFTSV NSs-shRNA1 plasmid can inhibit the replication of SFTSV.And it was concluded that SFTSV NSs-shRNA1 can inhibit virus replication for at least 72 h;the higher the plasmid concentration（1.2 μg）,the stronger the inhibitory effect;suppression rate slightly decreases as MOI value increases.ConclusionsThe plasmid SFTSV NSs-shRNA1 was successfully constructed.The sh RNA targeted to SFTSV NSs can inhibit SFTSV replication which can provide an experimental basis for further research on potential new tools to control SFTSV infection.