Cell culture and virus

Human cervical cancer cell line (Hela) was routinely maintained in our lab. Human hepatoma cell lines Hun7.0 and Huh7.5.1 cell lines were generously provided by Dr. Ian McGilvray (University of Toronto, Canada). Hela cells were cultured in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% fetal bovine serum(FBS) and 100μg/ml penicillin and 100 μg/ml streptomycin sulfate at 37°C in 5% CO₂ incubator. Human liver cancer cell lines Huh7.0 and Huh7.5.1 were cultured in DMEM supplemented with 10% fetal bovine serum, 100μg/ml penicillin and 100 μg/ml streptomycin sulfate,1% nonessential amino acid.

SFTSV isolated from the plasma sample of a SFTS patient in Xinyang 154Military hospital was propagated in Vero cells. Viral RNA from supernatants was extracted with a special RNA extraction Kit (Qiagen, German). The viral RNA equivalents (copy/ml)of SFTSV was measured by a reverse transcription (RT) real-time quantitative -PCR Kit for SFTSV RNA (Da An Gene Inc., Guangzhou., China) with 5μL RNA with the condition:50°C, 15min; 95°C, 15min; 94°C, 15s and 55°C, 45s for 40 cycles. The PCR amplification cycle numbers (Cts) were measured and converted into SFTSV RNA equivalents (copy/ml) using SFTSV RNA standards (recombinant pseudotyped virus, copy/ml). The supernatants from Vero cells infected by SFTSV were collected at 72 hours after inoculation. Hela, Huh7.0, Huh7.5.1 cells and Vero cells were infected by supernatants mentioned above at 48 hours before harvesting the cells and supernatants for further detections.